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Mapping the euchromatic long arm of the human Y chromosome

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Mapping the euchromatic long arm of the human Y chromosome
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MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS



















By

BEVERLY STEELE ALLEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1992














ACKNOWLEDGEMENTS


I have been privileged to know a number of individuals

during my graduate education, and would like to acknowledge

them here. First, I thank my mentor, Dr. Harry Ostrer, for

his guidance, support, enthusiasm, and encouragement. My

education and training under his direction have been a

valuable and rewarding experience. Thanks also go to my co-

chairman, Dr. Tom Yang, and the members of his lab, for

helpful discussions and guidance. Special thanks go to Dr.

Harry Nick for his efforts on my behalf in coordinating my

position in the Division of Genetics with that in the

Department of Biochemistry and Molecular Biology. I would

like to thank my other committee members Dr. Philip Laipis,

Dr. Sue Moyer, and Dr. Ward Wakeland for helpful discussions

and encouragement.

In addition, I would like to acknowledge the financial

support of the Division of Genetics in the Department of

Pediatrics, and the R.C. Phillips Research and Education

Unit. I would like to thank Dr. Charles Williams, Chief of

Genetics, for his support and for the use of laboratory

space after Dr. Ostrer's departure to New York University.

Finally, I would like to acknowledge the unfaltering

love, support, encouragement, and occasional bullying of my

ii









husband, Bert, during this time. His intercession and

spiritual leadership were also greatly appreciated. I thank

our families for their love and encouragement over the

years. I also thank our sisters and brothers in Christ, the

Lees, Ahns, Barnards and many others for their prayers,

love, and understanding. My thanks go also to Dr. Catherine

Ketcham for her friendship and long-distance encouragement,

and to my shooting' buddy Kathy Mercer for fun times on the

range.


iii
















TABLE OF CONTENTS



ACKNOWLEDGEMENTS . . . .

LIST OF TABLES . . . . .

LIST OF FIGURES . . . . .

ABBREVIATIONS . . . . .

ABSTRACT . . . . .

CHAPTERS

1 INTRODUCTION . . . .

2 DELETION MAPPING OF THE EUCHROMATIC LONG
ARM OF THE HUMAN Y CHROMOSOME . .

Introduction . . . .
Materials and Methods . . .
Results . . . .
Discussion . . . .

3 EVOLUTIONARY COMPARISONS OF SEQUENCES
MAPPING TO THE HUMAN Y CHROMOSOME .

Introduction . . . .
Materials and Methods . . .
Results . . . .
Discussion . . . .

4 LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM OF
THE HUMAN Y CHROMOSOME . .

Introduction . . . .
Materials and Methods . . .
Results . . . .
Discussion . . . .

5 CONCLUSIONS AND FUTURE DIRECTIONS ..


* vii

* viii

. ix


8
1


8


77
80
86
102


S. 106









REFERENCES . . . . .. .ll

BIOGRAPHICAL SKETCH . . . .. .121















LIST OF TABLES


Table 2-1. Y chromosome linkage of BAY clones . 32

Table 2-2. Genome Data Base D-number assignments 48

Table 3-1. Interspecies comparisons of Southern blot
data . . . . . .. 65

Table 4-1. High molecular weight restriction fragments
detected by distal Yqll probes . . .. 88















LIST OF FIGURES


Figure 2-1. NIGMS Y chromosome translocations and
deletions .. . . . . 25

Figure 2-2. Y Chromosome Mapping Panel . .. 28

Figure 2-3. Southern blot analyses of Y regional
mapping panel . . . . .. 37

Figure 2-4. Hybridization of probes to DNA from
regional mapping panel . . . .. 42

Figure 2-5. Summary of Yq interval mapping . .. 46

Figure 3-1. Conservation of Y-linked sequences
homologous to BAY1-8b in great apes . .. 64

Figure 3-2. Conservation of Y-linked sequences
homologous to BAY3-8 in great apes . .. 68

Figure 3-3. Conservation of Y-linked sequences
homologous to BAY2-lla, 1F5, and 49f in great
apes . . . . . .. 69

Figure 3-4. Conservation of GMGYl-like sequences 70

Figure 3-5. Comparative evolutionary analysis of Hind
III fragments in great ape males . .. 72

Figure 4-1. Hybridization of distal Yqll probes to
PFGE Southern blots . . . .. 91

Figure 4-2. Hybridization of distal Yqll probes to
PFGE Southern blots produced from digests with two
restriction enzymes . . . .. 93

Figure 4-3. Proposed long range map of interval 7 on
the long arm of the Y chromosome . .. 98


vii















ABBREVIATIONS


bp Base pair(s)
BSA Bovine serum albumin
dCTP Deoxycytidine triphosphate
DMSO Dimethylsulfoxide
dNTPs Deoxynucleotide triphosphates
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetate
Kb Kilobases
Mb Megabases
nt Nucleotide(s)
PBS Phosphate-buffered saline
PMSF Phenylmethylsulfonylfluoride
rpm Revolutions per minute
SDS Sodium dodecyl sulfate
SSC Saline sodium citrate
SSPE Saline sodium phosphate EDTA
TE Tris-EDTA buffer


viii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS

By

Beverly Steele Allen

May, 1992

Chairman: Dr. Thomas P. Yang
Major Department: Biochemistry and Molecular Biology

A deletion map of the human Y chromosome was produced

from a novel panel of Y translocation-and deletion-bearing

cell lines assembled for the purpose of mapping the

euchromatic long arm. Southern blot data from 35 cloned

sequences indicate an ordered arrangement of eight

intervals, six of which are located in the euchromatic long

arm. Twenty-nine of the cloned sequences were novel

isolates from a Y chromosome-specific library. The most

distal interval was chosen for comparative evolutionary and

pulsed-field gel electrophoretic analyses because of its

likely proximity to the region whose deletion is associated

with abnormal spermatogenesis.

Twelve regionally mapped probes were chosen for studies

of their conservation in the genomes of the great apes. All

of the sequences that mapped to the most distal euchromatic

ix









interval were included in the analyses. These were used as

probes for Southern blots of genomic DNA generated from

rhesus monkeys, cattle, and mice. Only GMGY1 was conserved

in all species studied, and may represent a newly arrived

pseudogene, as it was Y-linked only in humans. All of the

other sequences were conserved on the Y chromosomes of the

great apes, but were absent from rhesus monkey genomes. The

association of these sequences with the Y chromosomes of the

great apes is hypothesized to be related to their marked

cytogenetic similarities.

A long-range molecular map of the most distal

euchromatic interval was produced that covers approximately

1600 kb, of which some 1000 kb are from that interval. The

map was oriented by mapping a probe from the next most

proximal interval such that the centromeric and telomeric

ends were distinguished. No HTF islands were observed over

the entire length of the map. Instead, sites for

infrequently cleaving restriction endonucleases were

randomly distributed throughout the region. No sites for

the enzyme Not I were noted. The long-range mapping

analyses indicated that this region of the Y chromosome is

heavily methylated.















CHAPTER 1

INTRODUCTION

The Y chromosome is the heterogametic sex chromosome of

mammalian males. Normally, the Y chromosome is present in

the haploid state. The human Y consists of two

cytogenetically distinct regions. The genetically active

euchromatic region comprises the short and proximal long

arms. The adjacent heterochromatic region comprises the

distal long arm and is considered to be genetically inert.

Based upon electron microscopic measurements (Golomb and

Bahr, 1971), the length of the human Y chromosome is

estimated to be approximately 30 Mb (Goodfellow et al.,

1985; Smith et al., 1987). The pseudoautosomal region,

consisting of X-Y homologous sequences that pair and

exchange during meiosis (Burgoyne, 1982; Buckle et al.,

1985; Cooke et al., 1985; Simmler et al., 1985), begins at

the telomere of the short arm and extends about 2.5 Mb

toward the centromere (Pritchard et al., 1987; Brown, 1988;

Petit et al., 1988; Rappold and Lehrach, 1988; Petit et al.,

1990). The border between the pseudoautosomal region and

sequences specific to the Y chromosome contains Alu repeated

sequences (Ellis et al., 1989). The testis determining

gene, SRY, resides within this Y-specific area approximately








2

14 kilobases proximal to the pseudoautosomal boundary

(Sinclair et al., 1990). Other Y chromosome-specific

sequences extend to the centromere then distally to the

heterochromatic region. DNA sequences are interspersed

among the Y-linked sequences that exhibit varying degrees of

homology with the X chromosome and/or the autosomes (Bishop

et al., 1984; Cooke et al., 1984; Page et al., 1984; Wolfe

et al., 1984; Geldwerth et al., 1985; Koenig et al., 1985;

Bickmore and Cooke, 1987). DXYS1 and related single-copy

sequences have been demonstrated to share blocks of homology

with the human X chromosome (Page et al., 1982; 1984; Koenig

et al., 1984; 1985; Cooke et al., 1984) that in one case

extend for 50 kb (Bickmore and Cooke, 1987). Alu- and Kpn-

related repetitive elements are also found along the Y

chromosome (Smith et al., 1987). The heterochromatic region

of the human Y chromosome lies distal to these predominantly

low-copy number sequences and extends to the telomere of the

long arm. The heterochromatic region, constituting 50-70%

of the total length of the human Y chromosome (Golomb and

Bahr, 1971), contains two sets of Y-specific repeats. These

repeats appear in agarose gels as 3.4 and 2.1 kb bands in

male DNA digested with the restriction endonuclease Hae III

and are absent from similarly prepared female DNA (Cooke,

1976). As a consequence of varying numbers of copies of

these Y-specific repeat families, the heterochromatic region









3

may vary greatly in size without phenotypic effects (BUhler,

1985).

Few genes have been assigned to the human Y chromosome

on the basis of their phenotypic effects, and the existence

of most of the Y-assigned genes remains contested. SRY, the

candidate gene for testis determination, and ZFY, the

earlier candidate gene for that phenotype, are the only Y-

linked genes that have been cloned on the basis of their

chromosomal location (Sinclair et al., 1990; Page et al.,

1987). A few genes that encode proteins have been cloned

without previous suspicion of their Y-linkage. MIC2, the

first such gene cloned, encodes a cell surface antigen, 12E7

(Goodfellow et al., 1986), and represents the first cloned

pseudoautosomal gene. Other genes include TSPY, a testis-

expressed sequence (Arnemann et al., 1987), RPS4Y, a

ribosomal protein gene that has been proposed to be involved

in the etiology of Turner syndrome (Fisher et al., 1990),

and GM-CSF, the granulocyte-macrophage colony stimulating

factor gene (Gough et al., 1990). Genes provisionally

mapped to the Y on the bases of imparted phenotypes include

growth control Y (Human Gene Mapping 10, 1989; Henke et al.,

1991), tooth size (TSY) (Alvesalo and de la Chapelle, 1981),

H-Y antigen (Simpson et al., 1987), and the azoospermia

factor, AZF (Tiepolo and Zuffardi, 1976; Fitch et al., 1985;

Hartung et al., 1988). The amelogenin gene, recently

reported to be expressed from both the X and Y chromosomes,








4

may represent the TSY locus (Salido et al., 1992).

Pseudogenes for gamma actin (Heilig et al., 1984), steroid

sulfatase (Yen et al., 1988), argininosuccinate synthetase

(Daiger et al., 1982), and several retroviral sequences

(Silver et al., 1987) also have been found to reside on the

human Y.

The molecular model of the human Y chromosome

necessarily derives from analysis of anonymous cloned

sequences rather than from classical genetic studies that

depend upon recombination between linked loci. Despite the

dearth of genes, the chromosome is well suited to molecular

genetic mapping. Most sequences have only a single Y-linked

allele because of the hemizygous nature of the chromosome.

Three methods have been used to isolate unique sequence

probes for the Y chromosome. Some Y-linked probes have been

fortuitously isolated from human cDNA or genomic DNA

libraries. While not well suited to generating large

numbers of Y-linked probes, the first unique sequence probe

shared by the sex chromosomes was isolated in this manner

(Page et al., 1982). Genomic libraries constructed from DNA

from Y chromosomes separated from the other chromosomes by a

fluorescence activated cell sorter have proven to be a more

efficient means of obtaining Y chromosome-specific probes

(Affara et al., 1986; Oosthuizen et al., 1990; Nakahori et

al., 1991). The third technique involves screening genomic

libraries constructed from human-rodent somatic cell hybrids









5

containing the human Y as the only detectable human

chromosome (Bishop et al., 1983; Wolfe et al., 1984).

Y-linked sequences isolated from these libraries have fallen

into several categories: some are exclusively Y-linked, and

some have homologues on both the X and Y chromosomes. Some

have homologues on the Y and autosomes, whereas a small

minority have homologues on the X, Y, and autosomes (Bishop

et al., 1984; Affara et al., 1986; Oosthuizen et al., 1990;

Nakahori et al., 1991).

The mammalian X and Y chromosomes are thought to have

originated from a homologous pair of chromosomes (Polani,

1982). The terminal short arms of the X and Y chromosomes

pair and exchange sequences during meiosis, possibly a

remnant of that homology. These pseudoautosomal sequences

in humans are conserved on the sex chromosomes of the great

apes (chimpanzees, gorillas, and orangutans) (Weber et al.,

1987; 1988). Nearly half of randomly isolated single-copy

Y-linked sequences share homologies with the X chromosome

outside the pairing region. A number of human Y-linked

sequences from both the short and long arms have been

studied and most are conserved in the great apes, but are

not Y-linked (Page et al., 1984; Bickmore and Cooke, 1987;

Koenig et al., 1985; Erickson, 1987; Burk et al., 1985).

The human male-specific repetitive elements from the

heterochromatic region of the long arm are conserved in

these hominid primates, but are autosomally-linked rather








6

than Y-linked (Cooke et al., 1982; Kunkel and Smith, 1982;

Cooke et al., 1983). These findings suggest a recent origin

for a significant portion of the long arm of the human Y

chromosome and some part of the proximal short arm as well

(Page et al., 1984; Burk et al., 1985; Bickmore and Cooke,

1987). The observation that some of the randomly isolated

human Y chromosomal sequences share homology with the X

chromosome and are located on the primate X but not Y

chromosomes, suggests that the human Y is the result of

recent sequence rearrangements (Page et al., 1984; Bickmore

and Cooke, 1987). The possibility of functional

significance has been suggested for 4B-2, the first human Y-

linked sequence found to be Y-linked in the higher primates

(Burk et al., 1985). Since then, only two other anonymous

sequences from the human Y, p69/6 and 1F5, have been shown

to be conserved on the Y chromosomes of the great apes

(Erickson, 1987; Whisenant et al., 1991).

The small size of the human Y chromosome makes it

amenable to the newly developed long-range molecular mapping

techniques. Pulsed-field gel electrophoresis (PFGE) used in

conjunction with enzymes that cleave infrequently in

mammalian genomes, allows the analysis of DNA fragments up

to 9,000 kb (Schwartz and Cantor, 1984; Barlow and Lehrach,

1987). Because of the concentration of sites for the

infrequently cleaving restriction endonucleases in regions

termed HTF (Hpa II tiny fragment) islands, the process of








7

physically mapping a chromosomal region using PFGE may

provisionally locate genes (Brown and Bird, 1986; Bird,

1987). These islands are rich in the dinucleotide CpG, non-

methylated and usually associated with the 5' ends of

housekeeping genes (Bird, 1986; Bird, 1987; Bird et al.,

1987; Pontarotti et al., 1988; Sargent et al., 1989). Long-

range restriction mapping of other areas in the human genome

has been used to locate a number of genes (Hardy et al.,

1986; Bird, 1987; Bird et al., 1987; Estivill et al., 1987;

Sargent et al., 1989; Henke et al., 1991) and has not yet

been applied to the distal euchromatic long arm of the Y

where it may facilitate the localization of genes in the

region, especially the AZF gene(s) associated with

spermatogenesis (Human Gene Mapping 10, 1989).

In the experiments described in Chapter 2, anonymous

sequences isolated from a flow-sorted Y chromosome library

were characterized for linkage to the Y or other

chromosomes, and regionally mapped on a panel of cell lines

containing deleted or translocated Y chromosomes. A number

of probes, including those mapping to the most distal region

of the euchromatic long arm, were analyzed for their

conservation in several mammalian species. The results of

these experiments are presented in Chapter 3. Chapter 4

presents a description of the experiments used to prepare a

long-range restriction map of sequences mapping to the

distal euchromatic long arm of the Y chromosome.















CHAPTER 2

DELETION MAPPING OF THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME

Introduction

A physical map has been developed for the distal short

arm of the human Y chromosome. This area includes the

pseudoautosomal region, where sequences are exchanged

actively with the X chromosome (Cooke et al., 1985; Simmler

et al., 1985; Goodfellow et al., 1986), and the next most

distal interval where the testis-determining gene lies

(Sinclair et al., 1990). Other genes including GM-CSF, ZFY,

RPS4Y, TSPY, and amelogenin have been cloned from the short

arm region (Gough et al., 1990; Page et al., 1987; Fisher et

al., 1990; Arnemann et al., 1987; Salido et al., 1992). At

the time that these studies were undertaken, a restriction

map had not yet been constructed for the rest of the Y

chromosome. Deletion maps have been constructed with novel

Y probes, but different reference translocations and

deletions were used in each case, making comparisons

difficult (Affara et al., 1986, Vergnaud et al., 1986,

Oosthuizen et al., 1990, Nakahori et al., 1991). Other

studies have addressed the presence or absence of Y

chromosomal sequences in various patients exhibiting

abnormalities in fertility or sexual development (Andersson

8








9

et al., 1988; Johnson et al., 1989; Skare et al., 1990;

Kotecki et al., 1991; Nakahori et al., 1991), again

providing no composite picture for comparative purposes.

HTF islands are located in the pseudoautosomal region

(Goodfellow et al., 1988; Henke et al., 1991) as well as in

the proximally located Y-specific region near the ZFY gene

(Page et al., 1987). At the time this study was undertaken,

no information existed regarding either the presence or

distribution of these gene-associated islands on the long

arm of the Y. Identifying HTF islands represented one means

to locate genes on the long arm of the Y chromosome.

These experiments were undertaken to map the

euchromatic long arm of the human Y chromosome, Yqll. The

goals were to characterize new Y chromosomal probes, derive

a regional map for these new and previously described Y

chromosomal sequences, and to address the distribution of

HTF islands on the long arm. The anticipated result of

these mapping studies was the generation of a long-range

restriction map of the most distal interval defined by the

mapping panel. Such a restriction map might facilitate the

localization of the spermatogenesis-related gene(s) that

were provisionally mapped to the region (Tiepolo and

Zuffardi, 1976; Bobrow, 1985; Hartung et al., 1988). An

additional aim of this project was to test a newly assembled

mapping panel for the Y chromosome long arm.









10

This chapter describes the isolation and regional

mapping of 29 novel Y-linked sequences randomly isolated

from a Y chromosome-specific library. Most of the probes

chosen from the library exhibited no highly repetitive

sequences; any sequences containing such repeats were

preannealed to total human genomic DNA to facilitate their

use in the mapping analyses. Several attempts were made to

isolate CG-rich clones that might contain HTF islands

(Lindsay and Bird, 1987). Six probes that were previously

mapped to the long arm of the Y were included to correlate

the mapping data with existing information about the region.

A regional mapping panel was assembled from the Human

Genetic Mutant Cell Repository (NIGMS), in Camden, NJ. Two

cell lines that were already available in the laboratory

were included in the panel. Mapping of new and existing Y

chromosomal probes against a standard panel, such as that

introduced here, presented a means to facilitate the

production of a common physical map of the long arm of the

human Y chromosome and help in localizing genes that are

postulated to reside on Yq.

Materials and Methods

Mapping Panel Cytogenetics

A group of 10 fibroblast or lymphoblast cell lines was

purchased from the National Institute of General Medical

Science (NIGMS) Human Genetic Mutant Cell Repository,

Camden, NJ. Two additional lymphoblastoid cell lines








11

previously prepared in the laboratory were added to the

panel. Each cell line was cultured, and chromosomes were

prepared by standard techniques (Verma and Babu, 1989).

Air-dried slides with mitotic chromosomes were aged several

days before staining. For G-banding, slides were treated

with 1 ml of 0.025% trypsin-EDTA (GIBCO) in 100 ml Tyrodes

buffer (8 g/L NaCl, 1 g/L glucose, 1 g/L sodium bicarbonate,

0.05 g/L sodium phosphate monobasic, 0.2 g/L KCl) for 1-2

minutes prior to staining for 1-3 minutes in 2% Giemsa

(Harleco). Using another method for producing G-bands, some

slides were pretreated by immersion for 20 minutes in 0.1 N

HCl, and rinsed in water. The slides were then immersed for

90 minutes in 2X SSC, 50% formamide at 37C. After rinsing

in distilled water, the slides were dehydrated in 95%

ethanol. Air-dried slides were stained with Wrights stain

(Verma and Babu, 1989) mixed 1:3 with 0.6 M phosphate

buffer, pH 6.8 for 1-4 minutes. Y chromosomal

translocations and deletions were verified for each cell

line and compared to the documentation provided by NIGMS.

Only those cell lines that retained the reported Y

chromosomal translocations or deletions were used for the

mapping panel.

DNA Extraction

At least ten confluent T25 monolayer cultures or

approximately 20 ml of actively dividing lymphoblastoid

cells were harvested for DNA extraction. Lymphoblastoid









12

cells or trypsinized fibroblasts were pelleted and

resuspended in 3-5 ml of DNA extraction buffer (10 mM Tris,

pH 7.8, 2 mM EDTA, 400 mM NaCl). After addition of SDS to

0.5% and predigested Pronase (Boehringer Mannheim) to a

final concentration of 1 mg/ml, the solution was incubated

overnight at 37C. The solution was extracted with 24:24:1

phenol:chloroform:isoamyl alcohol, pH 8.0, followed by one

to two extractions with 24:1 chloroform:isoamyl alcohol.

DNA was precipitated with ice cold 95% ethanol. The DNA was

spooled onto a glass pipette and rinsed briefly with 70%

ethanol before air drying. The DNA was resuspended in 300-

500 4i TE, pH 8, with a drop of chloroform, then rotated

overnight at 4C. DNA concentration was calculated from the

absorbance at 260nm. Purity was also calculated from the

ratios of absorbances obtained at 260nm and 280nm

(A260/280), with a ratio of 1.8-2.0 considered to be

optimal.

Normal male and female donors provided blood samples

from which DNA was extracted. These provided controls for

the presence and absence of a Y chromosome. Blood obtained

by venipuncture into 10 ml Vacutainer tubes (Becton

Dickinson) containing 0.1 ml of 15% EDTA was centrifuged to

separate plasma and cells. The plasma was removed to just

above the buffy coat. The buffy coat and some of the

underlying red cells were transferred to 15 ml conical

centrifuge tubes. DNA extraction buffer was added to 5 ml,









13

SDS to 0.5%, and predigested Pronase to 1 mg/ml. Tubes were

incubated overnight at 37C and extracted twice with

phenol:chloroform:isoamyl alcohol and twice with

chloroform:isoamyl alcohol before ethanol precipitation as

above.

Library Screening

The methods used for phage library screening were

generally as described in Maniatis et al. (1982). Phage

were isolated from a Y chromosome-enriched library that was

obtained from the Lawrence Livermore National Laboratory

(LLOYNS01). The library was prepared from flow-sorted human

Y chromosomes that were cut to completion with the

restriction endonuclease, Hind III. The fragments were

inserted into the Hind III cloning site of the cloning

vector, lambda Charon 21A. The insert sizes ranged up to

9 kb. The yield of independent recombinants was 2.5 x 105,

and the amplified library contained 27 genome equivalents.

The library was plated at approximately 20,000 pfu/plate on

150 mm petri dishes in three separate experiments. Host

bacteria, E. coli LE392, were cultured in NZY medium

supplemented with 0.2% maltose. Appropriate numbers of

phage were mixed with host cells and incubated at 37C for

20 minutes. Infected cells were mixed with molten (47C)

0.7% agarose, poured over the surfaces of 1.5% agar plates

and incubated for 12 to 16 hours at 37C. Plates were

allowed to cool to room temperature prior to transferring









14

phage plaques to nitrocellulose filters (Schleicher and

Schull). Marked filters were placed on plates until

thoroughly wet and plate bottoms were correspondingly

marked. A duplicate filter was laid on the plate after the

first was removed and allowed to remain for 30 seconds to 1

minute longer than the previous filter to equalize the

quantity of phage adhering to the two filters.

Corresponding marks were also made on the plate bottoms for

the second filters. Filters were dipped into denaturing and

neutralizing washes as described in Maniatis et al. (1982),

then allowed to air dry. The filters were placed between

sheets of Whatman 3MM paper and baked at 80C in a vacuum

oven for 2 hours. Duplicate filter pairs were sequentially

hybridized as described below.

Phage Selection

Duplicate filters were first screened for the presence

of clones containing GC-rich sequences. A series of

oligonucleotides containing the recognition sequence of the

restriction enzyme Not I were synthesized by the Health

Science Center DNA Core Facility. The oligonucleotides were

radiolabeled using T4 kinase (Maniatis et al., 1982) and

hybridized to duplicate filters. The first oligonucleotide

used for screening was 8 nt in length and consisted of only

the recognition sequence for the restriction endonuclease,

Not I (GCGGCCGC). Hybridization for this oligonucleotide

was performed in 6X SSC (IX SSC is 0.15 M NaCl, 0.015 M








15

sodium citrate, pH 7.0), 0.1% SDS at 27C overnight,

followed by washing at 4C in 4X SSC, 0.1% SDS, and 2X SSC,

0.1% SDS, and finally at 27C in 2X SSC, 0.1% SDS (Melmer

and Buchwald, 1989). Two additional screenings were

performed with 12 nt degenerate oligonucleotides:

TNGCGGCCGCNN; and a mixture of oligonucleotides

ANGCGGCCGCNN, TNGCGGCCGCNN, GNCGCGGCCGCNN, CNGCGGCCGCNN.

Hybridization conditions were the same except that the

hybridization and final washes were at 37C, room

temperature, and 37C, respectively (Melmer and Buchwald,

1989). Filters were wrapped in plastic wrap and exposed to

Kodak XAR-5 film at -70C. After autoradiography, plates

were aligned with films and positively hybridizing plaques

were removed and placed in 1 ml of SM (0.1 M NaCl, 0.01 M

MgSO4, 0.05 M Tris, 0.01% gelatin, pH 7.5) as described in

Maniatis et al. (1982). The filters were stripped of

oligonucleotide probe in a solution of 0.1X SSPE (0.15 M

NaCl, 0.01 M NaH2PO4, 1 mM EDTA, pH 7.4) and 0.5% SDS at

90C, and were rehybridized with radiolabeled total human

genomic DNA in 6X SSPE, 0.2% SDS, 2% Blotto (10% (w/v)

Carnation Non-Fat Dry Milk in water, plus 1% (w/v) sodium

azide as a preservative) at 60C overnight. Final washes

were at 60C in 2X SSPE, 0.1% SDS, followed by

autoradiography at -70C. Duplicate filters were aligned

again with their plates and only those plaques with no

hybridization signal (designated BAY1-1 to BAY4-12) or with








16

only a faint gray signal (designated from BAY1 to BAY50)

were removed and placed in 1 ml SM at 4C. Plate lysates

were prepared from 10 A. of the SM solution as described in

Maniatis (1982). Following overnight incubation, 5 ml of SM

was poured on to the surfaces of the plates. These were

incubated overnight at 4C, then tilted and the phage-

containing SM was removed. After centrifugation to remove

bacterial particulates, the supernatants were treated with

0.3% chloroform and stored at 4C prior to insert

amplification. Aliquots of the supernatants were stored at

-70C with DMSO added to 7%.

Insert Amplification from Phage

Human DNA inserts in recombinant phages were directly

amplified using the polymerase chain reaction (PCR). Single

stranded DNA primers 24 nt in length were constructed that

would anneal specifically to the immediate right and left

sides of the single Hind III cloning site in lambda Charon

21A; thus the PCR reaction would amplify the human DNA

insert flanked on either side by a short stretch of lambda

DNA. The primer sequences were synthesized by the DNA Core

Facility at the University of Florida Health Science Center.

The sequences were: 5'AGA AGA GTT AGT TGA CTA TAC AGC-3'

(right side of Hind III site), and 5'ATG TTT GAA TGT GAT AAC

CGT CCT-3' (left side of Hind III site). PCR reactions were

performed in a Perkin Elmer Cetus Thermal Cycler in 0.5 ml

microcentrifuge tubes in volumes of 100 Ai under mineral








17

oil. Reaction mixtures contained 500 ng of each primer,

Triton X-100 to 0.01%, 1/10 volume of Perkin Elmer Cetus 10X

Reaction Buffer, MgCl2 added to 3 mM, 200 AM dNTPs, 5 gl of

phage lysate solution and 2.5 units Taq DNA polymerase

(Perkin Elmer Cetus). Mixtures were heated to 95C for 5

minutes to break open the phage heads prior to thermocycling

at 94C for 30 seconds, 52C for 2 minutes annealing time,

and 72C for 5 minutes extension time for 35 cycles. A

final 10 minute extension at 72C was performed to extend

any incomplete reaction products. The reaction mixture was

chilled at 4C until collected. Reaction products were

extracted once with chloroform to eliminate mineral oil

overlays. The contents of each tube were separated by

electrophoresis in 0.8% low melting temperature (LMT)

agarose gels. Individual inserts were extracted from the

agarose or used directly in LMT agarose for radiolabeling.

Multiple bands amplified from a single lysate were labeled

from highest (a) to lowest molecular weight (b, c, etc) with

lower case letters (see Table 2-1).

Southern Blotting and Hybridizations

10 to 15 Ag aliquots of DNA were digested with Hind III

or other appropriate enzymes according to the manufacturer's

recommendations. Digestion products were separated by

electrophoresis in 0.75% agarose gels overnight. Following

electrophoresis, gels were stained with 0.25 gg/ml ethidium

bromide, destined for 30 minutes to 2 hours in water, then








18

photographed. The DNA was acid hydrolyzed with 0.25 M HC1

for 20 minutes, then transferred to nylon membranes

(Zetaprobe [BioRad], Genescreen Plus [NEN], or Hybond N+

[Amersham]) in 0.4 M NaOH overnight. After blotting,

membranes were rinsed with 2X SSPE, air dried for several

hours or vacuum dried at 80C for 30 minutes to 2 hours.

Zetaprobe membranes were prewashed at 65C for one hour in

0.1X SSPE, 0.5% SDS prior to the first use.

Probe DNAs were radiolabeled with 32P dCTP (3000

Ci/mmol, ICN) by random primer extension (Feinberg and

Vogelstein, 1984) using a BRL kit. The unincorporated

nucleotides were removed using a NACS PREPAC column (BRL).

Hybridizations were performed for 16-24 hours using

conditions recommended by the manufacturers or in Church

buffer (1% BSA, fraction V [Sigma], 1 mM EDTA, 0.5 M sodium

phosphate, pH 7.2, 7% SDS) (Church and Gilbert, 1984).

Post-hybridization washes were in 2X SSPE, 0.1% SDS at room

temperature for 20 minutes, and twice in 0.1X SSPE, 0.1% SDS

at 65C for 10-30 minutes each. Membranes were blotted to

remove excess fluid, wrapped in plastic wrap then exposed to

Kodak XAR-5 film at for 1-6 days at -70C. After

autoradiography, the probes were stripped from the blots

without allowing membranes to dry in a solution of 0.1X

SSPE, 0.5% SDS at 95-100C for 20-30 minutes. Wet blots

were sealed in plastic and refrigerated until the next use.









19

Probes containing repeated sequences were preannealed

to total genomic human DNA by the method of Sealy et al.

(1983). NACS-purified probes were ethanol precipitated at

-70C for 20 minutes, pelleted, and briefly air dried.

Pellets were resuspended in TE, pH 8 and heated to 65C for

2 minutes. 25 jl of 20X SSC and 0.4 mg of human placental

DNA sheared to 300-500 bp were added in a final volume of

0.1 ml. The mixture was boiled for 10 minutes, chilled one

minute, and incubated for 25 minutes at 65C to a Cot of

100. This mixture was added directly to the

prehybridization solution and hybridization was performed

for 16-24 hours. Washes were performed as already

described.

Placental DNA Extraction

Freshly delivered placenta was placed on ice and

transported to the laboratory where it was washed several

times in cold PBS. One inch pieces were cut off and washed

in PBS until somewhat clear and cut to about 1 cm2.

Approximately 3-4 grams of tissue pieces were placed in a

blender with 200 ml of homogenization buffer (0.25 M

sucrose, 3 mM CaCI2, 10 mM Tris, pH 7.9, 1% Triton X-100

(v/v)) and blended on lowest speed for about 5 minutes.

Blender contents were filtered through six pieces of gauze

and the foamy solids on top were added back to the blender

along with the next batch of tissue. Blending and filtering

were repeated until the entire placenta was processed. The









20

filtrate was kept on ice throughout the processing. The

filtrate was poured into 50 ml conical centrifuge tubes and

the nuclei pelleted at 4C, 1500 rpm. Nuclei were washed

once in homogenization buffer without Triton and pooled.

Pellets were resuspended in DNA extraction buffer. SDS was

added to 0.5%, and Pronase to 1 mg/ml. This solution was

incubated overnight at 37C. The solution was extracted

once with phenol:chloroform:isoamyl alcohol and twice with

chloroform:isoamyl alcohol. RNase was added to a final

concentration of 50 Ag/ml. The solution was incubated at

37C for 2 hours. The solution was reextracted, ethanol

precipitated, and spooled onto a glass pipette tip, dipped

into 70% ethanol, and air dried briefly. The DNA was

resuspended in TE, pH 8, sonicated to about 300-500 bp, then

ethanol precipitated and resuspended to 10 or 20 mg/ml.

Y Chromosome Probes Provided by Other Investigators

In order to correlate the data produced in this study

with published information about the region, probes that

mapped to the euchromatic long arm of the human Y chromosome

were obtained from other investigators. All probes

described below are subclones into plasmids.

Probe 49f, a 2.8 kb EcoR I insert in pBR322, was

derived from a Y-enriched cosmid library (Bishop et al.,

1983, 1984). It detects a 2.8 kb Y-specific band in EcoR I-

digested DNA at high stringency ("cognate band"). At low

stringency, it detects two autosomal bands as well as









21

numerous Y-specific bands in Taq I-digested DNA. It defines

locus DYS1. 49f was kindly provided by Dr. Jean

Weissenbach.

Probe GMGY1, a 0.8 kb EcoR I insert in pUC19, was

derived from a library constructed from a hamster-human

hybrid cell line containing the Y chromosome as the only

identifiable human chromosome (Affara et al., 1986). In

MspI-digested DNA, it detects a 2.4 kb Y-specific band and 2

autosomal bands of 3 kb and 1.7 kb. It defines locus DYS12.

GMGY1 was kindly provided by Dr. N. A. Affara.

Probe 1F5, a 4.1 kb EcoR I insert in pBR325, was

derived from a XCharon 21A human Y chromosome library

(LAOYNS01, ATCC) (Whisenant, et al., 1991). It detects a

cognate 4.1 kb Y-specific band in EcoR I-digested DNA. It

defines locus DYS128. 1F5 was kindly provided by Dr. Mohan

Bhatnagar.

Probe 4B-2, a 3.3 kb EcoR I insert in pBR322, was

derived from a library constructed from a hamster-human

hybrid cell line containing the Y chromosome as the only

identifiable human chromosome (Burk et al., 1985). It

detects a cognate 3.3 kb Y-specific band in EcoR I-digested

DNA and defines locus DYS15. 4B-2 was kindly provided by

Dr. Kirby Smith.

Probe MIAY, a 2.9 kb EcoR I-Hind III insert in pBR322,

was derived from a genomic library constructed from purified

human X chromosomes (Koenig, et al., 1984, 1985). It









22

detects a 3.6 kb Y-specific fragment in EcoR I-digested DNA

and defines locus DYS22. MIAY was kindly provided by Dr.

Michael Koenig.

Probe Y3.4 is a 3.4 kb Pst I insert in pBR322. It was

derived from the 3.4 kb Hae III repeat seen in male, but not

female DNA. It defines the Y-specific repeat family DYZ1

(Cooke, 1976). Y3.4 was kindly provided by Dr. Kirby Smith.

These plasmids were transformed into appropriate host

bacteria, amplified, purified, and restricted with the

appropriate enzyme(s), separated in low melt agarose, and

extracted by standard procedures (Maniatis et al., 1982).

Results

Mapping Panel Selection

Ten cell lines were initially obtained from the Human

Genetic Mutant Cell Repository (NIGMS) in Camden, NJ. The

karyotype of each line was verified prior to inclusion of

the cell line into the mapping panel. The Y chromosomal

fragments present in each NIGMS cell line used in the panel

are shown in Figure 2-1. Two of the ten cell lines

originally obtained from NIGMS, GM 3774 and GM 3595, were

excluded from the panel. GM 3774 reportedly contained an

isochromosome Yq, but this was not verified after receipt of

the culture. Two separate cultures of GM 3595 exhibited

extremely poor growth, resulting in its exclusion from the

panel even though its reported karyotype was confirmed.

GM 7970, reported by NIGMS to contribute only band Yql2, was








23

chosen for the panel for two reasons. First, if GM 7970

truly retained only Yql2, it would serve as a control for Y

heterochromatin. Conversely, should GM 7970 retain a

cytogenetically indistinguishable portion of band Yqll, the

cell line would be a source of distal Yq euchromatin. Two

deletion cell lines already available in our laboratory (Pf

and Si) were added to the panel and will be submitted to

NIGMS. The final mapping panel consisted of ten cell lines:

six unbalanced translocations and four terminal deletions.

All of the translocations and one deletion, Pf, exhibited

cytogenetically detectable heterochromatin. The Y

chromosomal fragments represented in each line are depicted

in Figure 2-2. Breakpoint locations are approximations

based on the molecular data obtained in this study and do

not represent the cytogenetic resolution used to verify the

karyotypes in the various cell lines (compare Figure 2-1).

Isolation of Probes

Most of the Y chromosomal fragments utilized in this

project were novel isolates from a Y chromosome-specific

library, and six were obtained from other investigators.

Approximately 100,000 pfu were screened initially from

plates with well-isolated plaques. Duplicate filters were

hybridized first with the 8 bp oligonucleotide consisting of

the recognition sequence for the restriction endonuclease,

Not I. Fewer than ten positive signals were obtained, and

only one appeared as a duplicate signal on the second
























Figure 2-1. NIGMS Y chromosome translocations and deletions. Metaphase chromosomes from
cultures of each NIGMS cell line were G-banded. Translocations and deletions are
indicated by t or d, respectively, below the chromosome. The arrowheads indicate
translocated or deleted Y chromosomal material.















CO CV) 0) CO 0 0 CV) CV
0 CD CD mV 0
I- CN 0
0 N N N N 0C 0)







t t t d d t t t









26

filter. All putative Not I-containing phages were subjected

to PCR to amplify the inserts, and aliquots of the inserts

were restricted with Not I and electrophoretically separated

next to uncut controls. None of the inserts was cleaved by

Not I, indicating a relatively high production of false

positives by the screening procedure.

Rehybridization of the filters with radiolabeled total

human genomic DNA was intended to distinguish between

plaques containing highly repeated sequences and those with

few or no repetitive elements. In order to visualize

signals from repeated elements on the Y chromosome, the

stringency of washing conditions was reduced by raising the

salt content to 2X SSPE and decreasing the hybridization and

wash temperatures to 60C. In the initial screening,

plaques producing a faint gray signal on autoradiographs

were removed. These clones were designated BAY1 to BAY50.

In the second screening, 80,000 pfu were plated and screened

with the single degenerate 12 bp Not I oligonucleotide in

another attempt to detect clones with infrequently cleaving

restriction sites. As in the initial screening with the

shorter oligonucleotide, fewer than ten putatively positive

clones were detected, and none was determined to contain

Not I sites after digestion of the purified inserts with

that enzyme. Selecting only those plaques producing no

signals, twelve clones were removed from each of four























Figure 2-2. Y Chromosome Mapping Panel. Representations of the Y chromosomal fragments
present in each cell line as judged by hybridization mapping are indicated by the
horizontal lines. The positions of the breakpoints are relative and do not represent the
resolution used in cytogenetic analyses. Karyotypes are indicated above the lines.









45, X / 46,X,del (Y) (pter >q11:)

45,X/46,X,del (Y) (pter>q11.2:)

46,X,del(Y) (pter>q11:)


Deletions


46,X,del(Y)(pte r q12:)


GM 2730

GM 2668

Si

Pf


9403 46,X,-X,+der(X;Y)(Xqter>Xp22.3::Yq11.2>Yqter)

8773 46,X,t(X;Y)(XqterXp22.1:'Yq11.2 Yq ter)

2103 46,X,t(X;Y)(Xpter> Xq11::Yq11>Yqter)

2469 46,X,t(X;Y)(Xpte rXq22::Yq11>Yqter)

118 46,XX.-15,+der(15),t(Y;15)(15qter>15p1::Yq11>Yqter)

7970 47,XX,+der(9),t(Y;9)(9pter> 9q13::Yq12,Yq ter)


Translocations


GM

GM

GM

GM

GM

GM








29

plates, designated BAY1-1 to BAY4-12. A third screening for

clones with infrequently cleaving restriction sites was

performed on another 80,000 pfu and filters were hybridized

with the mixed degenerate 12 bp Not I oligonucleotides.

Again, Not I sites were not detected in six putatively

positive plaques.

Human inserts in the recombinant phage were amplified

directly by PCR thereby avoiding difficulties associated

with growing and purifying large quantities of phage.

Because there were no commercially available primers

specific to the cloning site of lambda Charon 21A, primers

were designed that annealed on either side of the Hind III

site and not elsewhere in the vector. Plaques did not

necessarily contain a single population of recombinant

phage. PCR products amplified from a single plaque might

demonstrate as many as five separate bands (for example,

BAY35a-e, Table 2-1).

Characterization of Probes

Y chromosome linkage of sequences was ascertained by

hybridization of radiolabeled probes to Southern blots of

Hind Ill-digested genomic DNAs. Since the library was

produced from a complete Hind III digest, amplified inserts

should recognize genomic Hind III fragments of the same

sizes as the probes ("cognate" bands). Genomic DNA was

obtained from human male and female donors; 3E7, a mouse

hybrid cell line containing from one to four copies of the









30

human Y chromosome (Goodfellow et al., 1983); and Rag, the

mouse parental cell line from which 3E7 was produced (Klebe

et al., 1970). The optimal pattern indicating Y-linkage

consisted of one or more identical bands in the male and 3E7

lanes that were absent in the female and Rag negative

control lanes. Another pattern seen with some Y-linked

sequences demonstrated bands in female, male and 3E7 lanes,

indicative of X chromosome or autosomal linkage. Such

probes were usable for regional mapping only if at least one

band of a different molecular weight was seen in the male

and 3E7 lanes than was present in the female lane (for

example, BAY4 detected a 2.3 kb male and 3E7 Y-linked band,

as well as 3 and 1.6 kb male and female X-/autosome-linked

bands, Table 2-1). In some cases, identically sized bands

were seen in male, female and 3E7 lanes, indicating Y- and

X-/autosome-linkage. This pattern could sometimes be

resolved into different sized Y and X/autosome bands by

hybridizing the probe to genomic Southern blots generated

from other restriction enzymes such as Taq I, Msp I, or

EcoR I. One probe from this study, BAY33c, was analyzed in

this manner, as was GMGY1 (Affara et al., 1986).

Inserts from 64 recombinants produced different

patterns when hybridized to these Southern blots. Table 2-1

summarizes the observations made about the recombinants from

these preliminary analyses. Nine inserts detected no bands

in the genomic digests (BAYl-llc, BAY2-4c, BAY4-5, BAY2,









31

BAY6b, BAY9, BAY14, and BAY22a and BAY22b), whereas six

inserts produced background signals of such intensity that

the presence of non Y-linked bands could not be ascertained

(BAYl-7a, BAYl-7b, BAYl-8a, BAY3-2, BAY3-9, BAY34a). Two

recombinants, BAYl-8b and BAY2-7a, contained internal

Hind III sites and therefore detected two genomic fragments

that mapped to the same interval. The sizes of the genomic

fragments added up to the sizes of their respective cloned

genomic inserts. In the case of BAY2-2, an intense

background smear was observed in the male and female lanes

while a clear 1 kb signal was detected in the 3E7 hybrid

lane (not shown). After the repetitive elements in the

probe were preannealed to total genomic DNA to a Cot of 100,

the background smear was eliminated from the human lanes,

allowing for the unambiguous detection of the 1 kb band in

both the male and 3E7 lanes. This band was absent in the

female and Rag lanes. With the exceptions of BAY33a,

BAY33b, BAY33c, BAY34a, and BAY41 all clones numbered BAY1

to BAY50 were preannealed to a Cot of 100 prior to

hybridization to compete out the repetitive elements that

had appeared as faint gray signals in the library screening.

Eleven inserts detected Y and non-Y fragments of the same

sizes in Hind III-digested genomic DNAs of males, females,

and 3E7 (BAYl-lla, BAY2-7b, BAY2-9b, BAY2-lla, BAY 2-11b,












Table 2-1. Y chromosome linkage of BAY clones.


SIZE COGNATE NONCOGNATE
Y Y
3.5 -
1.2 -
4 -
2.1 1.2, 0.9 kbt
2.0 + -
0.8 +
0.5 -


NON-Y REPEATS


+


3, 1 kb*


+

7 kbt






2.5 kbB


1.3
* 1.5
* 2.3
* 1.4
* 5
0.6
3
5
* 0.6
3
* 3
* 3


2.3kb

+


(3, 1.6kb)
(1.2kb)
(3, 1.6kb)

+

N


5kb


BAY
CLONE
l-7a
l-7b
l-8a
l-8b
1-lla
1-11b
l-llc
l-llc


*


Cot
100


2-2 *
2-4a *
2-4b
2-4c
2-5 *
2-7a *
2-7b
2-9a *
2-9b
2-10 *
2-11a*
2-11b

3-1 *
3-2
3-3 *
3-7 *
3-8 *
3-9
3-11a*


4-la
4-lb
4-5


1
1.4
1.1
0.5
4.2
4
1.3
3
1.6
3
1.2
0.7

1.1
4
3
3.9
1.5
1.4
1.3

2.2
1.9
0.9


2
3c
4
5
6a
6b
7
9
10b
14
16
17


1
t_











Table 2-1--continued

BAY SIZE COGNATE NONCOGNATE NON-Y REPEATS Cot
CLONE Y Y 100
22a 4 H +
22b 1.8 H +
24 1.5 C H +
25a 3 +B 7 kbA H +
25b 1.2 C H +
26a 3.5 + H +
26b 1.6 + H +
29a 5 + H +
29b 1.4 4 kbt H +
30b 3 + 1.9kb H +
31a 1.7 + + C+N H +
31b 1 + C+N H +
33a 3 + H
33b 2.1 + -
33c 1.2 + 1.2kb -
34a 5 I H
35a 2.5 + 1.9kb 1.9kb H +
35b 1.8 + C H +
35c 1.4 + 3.5kb C+N H +
35d 1 + H +
35e 0.5 + N H +
36a 4 + C H +
41 1.7 + H

REPEATS: Indicates presence or absence of repeat elements in
probes
Cot 100: Indicates probes requiring preannealing of repeat
sequences
(*) Regionally mapped clones
(t) Bands analyzed if cognate bands undetected or non
Y-linked
(*) Probes containing internal Hind III sites
(I) Not determinable
(C) Cognate: probe detects genomic fragment of same size
(N) Noncognate: probe detects genomic fragment of different
size
(H) Highly repetitive: intense background smear
(M) Moderately repetitive: moderate background smear or
minisatellite sequence through which individual bands
may be detected
(A-Z) Indicates more than one Y-linked fragment detected by
a single probe
Band sizes reflect Hind III genomic digests









34

BAY31a, BAY31b, BAY33c, BAY35b, BAY35c, BAY36a). One of

these, BAY33c, detected Y and non-Y fragments of different

sizes when hybridized to Southern blots of Msp I-digested

genomic DNAs, and was thus included in the regional mapping

analysis. Twenty-nine of 64 recombinants produced one or

more male-specific bands that were also present in the

hybrid 3E7 and absent or of a different size in the female

lane (*, Table 2-1). These unambiguously Y-linked inserts

were included in the Y chromosome regional mapping studies.

Regional Mapping

Genomic DNA obtained from the Y translocation and

deletion cell lines and controls was digested with Hind III

and duplicate Southern blots were produced on charged nylon

membranes (Zetaprobe, Genescreen Plus, or Hybond N+).

Single blots were also produced using EcoR I and Msp I for

use with the probes obtained from other investigators

(EcoR I for MIAY, 4B-2, 1F5, Y3.4, and 49f; Msp I for

GMGY1). Probe BAY33c was also analyzed using Msp I as

mentioned previously. Southern blots on Genescreen Plus did

not produce signals as intense as those seen using Zetaprobe

and Hybond N+ (compare BAY3-7A on Genescreen Plus with

BAY2-5 on Hybond N+, Figure 2-3a, lanes 11-14, lanes F-P

were on Zetaprobe in both cases). Translocation and

deletion blots were annealed to multiple probes when

individual Y-linked fragments could be distinguished

unambiguously. Panel members were scored for either the








35

presence (+) or absence (-) of the Y-linked band(s) detected

on the initial Southern blots (Figures 2-3 and 2-4).

Results obtained from hybridizing 35 Y-linked sequences

to the ten cell lines in the mapping panel are summarized in

Figure 2-4. Seven intervals in the euchromatic region of

the human Y chromosome were defined. Twelve probes detected

sequences present in Yp or proximal Yq. BAY2-5, BAY2-10,

BAY3-7A, BAY4-lb, BAY4, BAY6a; BAY17, BAY25aB, BAY29a,

BAY29b, BAY33c, and BAY35a were localized to this first

interval. These probes detected sequences in DNA from the

deletion cell lines GM 2668, Pf, and Si but in none of the

translocation cell lines. Representative examples of the Y-

linked fragments mapping to this interval are shown in

Figure 2-3a (BAY2-5 and BAY3-7A). Probe BAY3-7 detected two

Y-linked fragments, A and B, that mapped to different

intervals. The higher molecular weight fragment, A, mapped

to the most proximal interval (Figure 2-3a), whereas the

lower molecular weight fragment, B, mapped more distally

(Figure 2-3b).

No probes isolated in this study detected sequences in

panel member GM 2730, which was reported by NIGMS to retain

some portion of Yqll (see Figure 2-2). GM 2730 was a mosaic

cell line with as few as 13% of its cells retaining a Y

chromosome fragment by cytogenetic analysis. However, the

single copy male testis-determining gene, SRY, was










Figure 2-3. Southern blot analyses of Y regional mapping panel. Genomic DNA was digested
with Hind III unless otherwise indicated to the right of the fragment size. Blots were
probed with the Y recombinants indicated in Table 2-1 (*). Data are presented for
representative sequences located in intervals 1-6 and all five sequences located in
interval 7. Probes are designated at the left of the Southern blots. Only the Y-linked
bands are shown and the sizes in kb are indicated to the right. Symbols below each data
set indicate the presence (+) or absence (-) of the Y-linked bands in the DNA of a panel
member, and the interval number is designated below. Lanes represent respectively: F,
normal female; M, normal male; 3, GM 0118; 4, GM 2103; 5, GM 2469; 6, GM 7970; 7, GM 8773;
8, GM 9403; Y, 3E7 (Y hybrid cell line); P, Rag (hybrid parent cell line); 11, Pf; 12, Si;
13, GM 2668; 14, GM 2730.

a) Representative Y recombinants located in the first three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal.

b) Representative Y recombinants located in the next three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal. Lane 4 (GM 2103)
is underloaded relative to the other lanes.

c) Two representative Southern blots hybridized with probe BAY3-8 from interval 7. Lanes
1-11 contain DNA from translocation-bearing cell lines; lanes 12-18 contain DNA from
deletion-bearing cell lines. Lanes represent 1, female; 2, male; 3, GM 0118; 4, GM 2103;
5, GM 2469; 6, marker; 7, GM 7970; 8, GM 8773; 9, GM 9403; 10, 3E7; 11, Rag; 12, female;
13, male; 14, Pf; 15, Si; 16, GM 2668; 17, GM 2730; 18, marker. Molecular weight markers
for the blot bearing lanes 1-11 are located to the left. Also at left are arrows numbered
1 7, and 5 indicating hybridization signals from probes BAY2-10, BAY3-8, and BAY3-lla
which map to intervals 1, 7, and 5 respectively. BAY3-8, BAY2-10 and BAY3-lla were
hybridized simultaneously to the blot at the left (lanes 1-11). Only BAY3-8 was
hybridized to the blot at the right (lanes 12-18). Molecular weight markers for the blot
bearing lanes 12-18 are located at the right. The arrow at the right indicates the
hybridization signal from BAY3-8 in lanes 12-18.







PROBE
BAY 2-5

BAY 3-7A


BAY 2-9a

BAY 4-1 a


BAY 2-4a
MIAY


FM 3 4 5






AdI
UI',
//o.

- i

jl 11

J I a I


7 8 Y P 11 121314
-i ~ klll,


i ll
Inberval 1
lil


_ + + -
Interval 2


+II -
1*11


11


1.4
S 3.6 (EcoRI)


+ -I---


Interval 3


Figure 2-3


Kb
4.2

3.9


3.1

2.2









PROBE
BAY 3-7B

BAY 5




BAY 3-1

BAY 3-11a


FM 34 5
, fill





1,1.,


678 Y





- + + +
In--rV 4


-- --+ + + +
WIONrv 5

4B-2 m----+

BAYb I -ft

-+- ++ +1 +
bItovf


P 11121314 Kb
2.5

4W ~ 1.4
- + + --



0*^ 1.1

1.3
+ + --



AN* 3.3 (EcoRI)
'M 1.9
-0.9
- + + --


Figure 2-3--continued







1 2 3 4 5
mm1m2


6 7 8 9 10 11
01- P.-


12 13 14 15 16 17 18

m -


.16


*5.5


3.5.
1


2.0.
1.9"



V .


I


fol 4


***t


C /

Figure 2-3--continued


*-2.7


l









40

detectable in all DNA samples of this cell line by Southern

analysis (not shown).

The first interval that was unambiguously localized to

Yqll was defined by two probes, BAY2-9a and BAY4-la

(Figure 2-3a). BAY2-9a also detected a non Y-linked

minisatellite repeated sequence in the human DNA on Southern

blots produced on Zetaprobe (Figure 2-3a, lanes 1-8) that

was not detectable on Genescreen Plus (lanes 11-14). The

BAY2-9a minisatellite was detectable on Southern blots

produced on Hybond N+ (not shown). Probes from this

interval detected sequences in the deletion cell lines

GM 2668, Pf, and Si which retain the entire Y short arm and

some portion of the proximal long arm (Ypter-Yqll)

(Figure 2-2). Sequences were detected in only one

translocation cell line, GM 9403; here, the Y fragment

extended from the terminus of the long arm to the proximal

long arm (Yqter-Yqll) (Figure 2-2).

BAY2-2, BAY2-4a, BAY16, BAY30b, BAY33b, and MIAY

defined the third interval, of which, MIAY had been

previously mapped to Yqll (Koenig et al., 1985). Two

representative sequences from this interval are shown in

Figure 2-3a (BAY2-4a and MIAY). The sequences were detected

in the translocation cell line GM 9403 and in the Yq

deletion cell lines Pf and Si, but not GM 2668. These data

indicated that the breakpoints in deletion samples Pf and Si

lay distal to the breakpoint in GM 2668.





























Figure 2-4. Hybridization of probes to DNA from regional
mapping panel. Genomic DNA from each cell line was digested
with the appropriate enzyme, separated by electrophoresis,
and transferred to nylon membranes. Southern blots were
sequentially hybridized with the Y chromosome recombinants
listed at the left. (+) indicates the presence of the
appropriate Y-linked fragment in the DNA from the panel
member, (-) indicates its absence. Cell line 3E7 is a
somatic cell hybrid bearing only the human Y chromosome, and
was included as a positive control for Y chromosomal
sequences. ND indicates the fragment was undetectable.





















11.3





11.2




1 1.1



1 1.1





11.2 1





1 1.22






11.23















12


Y3.4


I I


Translocations
Probes 3E7 9403 8773 2103 2469 118 7970 Pf
2-5 + +

2-10 + +
3-7A + +

4-lb + +
4 + +

6a + +
17 + +

25aB + +
29a + +
29b + +
33c + +

35a + +



2-9a + + +

4-la + + +
2-2 + + +

2-4a + + +
16 + + +

31b + + +

33b + + +
MIAY + + +

3-7B + + + +

5 + + + +

lOb + + + +
3-1 + + + + +

3-11a + + + + +

3c + + + + +

1-8b + + + + + +

2-7a + + + + + +
3-3 + + + + + +

25aA + + + + + +

4B-2 + + + + + +

GMGY1 + + + + + +

3-8 + + + + + +

2-11a + + + + + +
1F5 + + + + + +

p49f + + + ND + +


Deletions
Si 2668 2730
+ + -
+ + -
+ + -

+ + -
+ + -

+ + -
+ + -

+ + -
+ +
+ +
+ +

+ +









43

The fourth interval lay between the breakpoints in

GM 8773 and GM 2103, and contained three recombinant

fragments: BAY3-7B, BAY5, and BAY0lOb. These probes detected

sequences in two translocation cell lines, GM 9403 and

GM 8773, and two deletion cell lines, Pf and Si. BAY3-7B

and BAY5 represent two of the probes mapped to this interval

in Figure 2-3b. BAY3-7 had previously detected a higher

molecular weight fragment (A) in the most proximal Y

interval defined by the panel (Figure 2-3a).

The fifth interval was defined by BAY3-1, BAY3-lla, and

BAY3c which detected sequences in translocations GM 9403,

GM 8773, and GM 2103, and deletions Pf and Si. Two

representative probes from this interval are shown in Figure

2-3b. The fainter bands in GM 2103 (lane 4) were due to

underloading of the DNA.

The sixth interval contained 4B-2, BAY2-7a, BAY3-3,

BAY25aA and BAY1-8b, of which 4B-2 was described previously

(Burk et al., 1985). These probes detected sequences in

four translocation lines: GM 9403, GM 8773, GM 2103, and

GM 2469, and two deletion lines, Pf and Si. 4B-2 and

BAYl-8b represent probes defining the interval (Figure

2-3b). The faint band in GM 2103 (lane 4) detected by 4B-2

was due to incomplete digestion of the genomic DNA by EcoR I

as seen in the ethidium bromide stained gel prior to

transfer (not shown). The 0.9 and 1.2 kb bands detected by








44

the 2.1 kb BAYl-8b probe were due the previously mentioned

internal Hind III site.

The most distal interval, interval 7, in the

euchromatic region of Yq defined by this mapping panel also

contained five probes: BAY2-lla, BAY3-8, 1F5, 49f, and

GMGY1. Three of these sequences had been described in other

laboratories: 49f (Bishop et al., 1984), GMGY1 (Affara et

al., 1986), and 1F5 (Whisenant et al., 1991). The sequences

in this interval were detected in only one deletion cell

line, Pf, and were present in all the translocation cell

lines except GM 0118 and GM 7970. Figure 2-3c demonstrates

the two Southern blots hybridized with probe BAY3-8 which

maps to this interval.

The DYZ1 probe, Y3.4, was used as a positive control

for the presence of heterochromatin from Yql2 in the cell

lines (not shown). As seen in Figure 2-4, Y3.4 was present

in all the translocation cell lines, including GM 0118 and

GM 7970, as well as the deletion cell line, Pf. All these

cell lines had exhibited visible heterochromatin. As

expected from the cytogenetic analyses, Y3.4 was absent from

deletion cell lines GM 2668, GM 2730, and Si in which

heterochromatin was not visible.

The data from the regional mapping analyses are

summarized in Figure 2-5 and allow for the relative ordering

of the breakpoints. Distances between breakpoints, or the

physical sizes of the intervals those breakpoints bound, are









45

not addressed by the data. The order of individual

sequences within an interval has not been established except

for those sequences mapping to interval 7: cen--GMGYl,

BAY3-8, BAY2-lla, 1F5, 49f--tel. The experiments used to

achieve this ordering are described in Chapter 4. No

distinctions can be made between the breakpoints in the cell

lines GM 0118, GM 7970, and Pf by the sequences used in this

study. The data summarized in Figure 2-5 also indicate an

apparently unequal distribution of repeat-bearing probes

along the Y chromosome. Ten probes required a preannealing

step to a Cot of 100 prior to hybridization and mapped

within the three most proximal intervals (BAY4, BAY6a,

BAY17, BAY25aB, BAY29a, BAY29b, BAY35a, BAY2-2, BAY16, and

BAY30b). The repeat-bearing probes account for about 29 kb

out of the total 53 kb covered by all probes in the region.

Only four repeat-bearing probes (BAY5, BAY0lOb, BAY3c, and

BAY25aA) mapped to the three more distal intervals, while

none were present in interval 7. These four probes cover

only 6.5 kb in a total of 29 kb, or about 22% of the area

containing repeats as compared to 53% in the more proximal

intervals. Assuming that repeated elements are evenly

distributed along a segment of DNA, the probability that a

randomly isolated probe would contain such elements should

be independent of its regional location on a chromosome.

The area covered by repetitive elements in the first three

intervals was not significantly different from that expected




















GM 2730

GM 9403
GM 2668

GM 8773

GM 2103

GM 2469

Si


M2
4M3

AP 4
- 5

- 6


4. 6a. 17. 25aB. 29a. 29b. 35a.
2-5, 2-10, 3-7A, 4-1b, 33c
2-9a, 4-1a

2-4a, 33b, MIAY, 2-2. 16. 30b
3-7B, 5. 10b
3-1, 3-11a, 3c
1-8b, 2-7a, 3-3, 25aA. 4B-2


- 7 GMGY1, 3-8, 2-11a, 1F5, 49f


GM 118, Pf, GM 79704 8


Y3.4


Figure 2-5. Summary of Yq interval mapping. Breakpoints of
each cell line in the panel are ordered from proximal to
distal. The sets of sequences defining each interval and
the interval number are listed to the right of the arrows.
Distances between breakpoints are not implied.









47

by x2 analysis, however the next three intervals differed

significantly (0.001
distribution of repeats. This significant difference became

even greater (p<<0.001) if the analysis included sequences

from interval 7.

Submission of Sequences to the Genome Data Base

All the newly isolated Y chromosome probes that were

regionally mapped in Figure 2-4 were submitted to the Genome

Data Base at Johns Hopkins University. Anonymous DNA

segments are no longer being assigned D-numbers by GDB

without some evidence of function, but submitted information

is kept on file in the event that new data will provide that

evidence. Based upon their conservation in the higher

primate males as described in Chapter 3, nine sequences

listed in Table 2-2 were assigned D-numbers by GDB.

Discussion

Because of the lack of recombination in the region,

the development of a linkage map of the long arm of the

human Y chromosome depends upon physical analyses. Whereas

the majority of the physical mapping data obtained from the

Y chromosome was directed at the short arm, sequences

mapping to the long arm have been described; however, none

of the published mapping data have utilized common Y

chromosome translocations and deletions, making data

comparisons difficult. A standard panel of altered Y

































Table 2-2. Genome Data Base D-number assignments.

PROBE LOCUS
BAY2-10 DYS187
BAY4-la DYS188
BAY3-1 DYS189
BAY3-lla DYS190
BAY2-7a DYS191
BAYl-8b DYS192
BAY2-lla DYS193
BAY3-8 DYS194
BAY33b DYS195









49

chromosomes was needed to serve as a starting point for the

more detailed mapping of a specific region of the Y

chromosome that will follow.

In this chapter, I have introduced a reference panel of

Y chromosome translocations and deletions that others may

use for the generation of standardized maps. The data

presented here were used to produce a deletion map of the

euchromatic long arm of the human Y chromosome that is

divided into six intervals. This deletion map is consistent

with maps and in situ hybridization data that were generated

in other laboratories, although exact correlations are

difficult to make due to the different probes and cell lines

used in those studies. In the course of regionally mapping

29 newly isolated Y chromosomal sequences, an enrichment for

long arm sequences and an apparent paucity of GC-rich

islands was noted in the clones taken from a flow-sorted Y

chromosome library. The distribution of Y-linked sequences

obtained from the library was consistent with the

distributions noted in other laboratories.

The cell lines in the panel were chosen after meeting

certain criteria. Only unbalanced Y chromosomal

translocations were chosen so that the absence of a Y-linked

band would be indicative of an abnormal Y chromosome. Only

cell lines that were stable in culture were used to ensure

their suitability for molecular analyses. For example,

GM 3774 was rejected for use in the panel after cytogenetic









50

analyses revealed that the reported isochromosome Yq was

unstable, suggesting that the chromosome was dicentric

rather than an isochromosome. GM 3595 retained a stable

deleted Y chromosome, but the cells were intractable in

culture and could not be grown in quantities sufficient to

extract DNA, resulting in the exclusion of this cell line

from the panel.

The molecular data presented here indicate possible

discrepancies in the reported karyotypes of two cell lines

from NIGMS. GM 2730 reportedly contained a deleted Y

chromosome in which the deletion extended into Yqll. The

cytogenetic data do not appear to contradict this report

(see Figure 2-1). In the molecular data reported here,

twelve probes that map into the region reportedly present in

GM 2730 detected no sequences in DNA from this cell line.

Because GM 2730 was a mosaic cell line, the possibility

existed that the representation of the Y chromosome was

below the level of sensitivity for Southern blot analysis.

This was ruled out by the detection of the single copy SRY

gene in a Southern blot prepared with GM 2730 DNA samples.

One explanation for the lack of Yq sequences in GM 2730

would require all twelve probes to be in an interval just

distal to the Y centromere and proximal to the breakpoint in

GM 9403. This explanation would invoke the seemingly

unlikely event that the library screening procedure selected

against sequences mapping to the short arm of the Y









51

chromosome. Oosthuizen, however, has observed that 80% of

probes isolated from this library that identified

exclusively Y-linked sequences mapped to Yqll.23, indicating

a "nonrandom chromosomal distribution of randomly isolated

probes" (Oosthuizen et al., Human Gene Mapping 9, 1987).

An alternate explanation would suggest that the Y

chromosomal fragment in GM 2730 was formed by a complex

deletion/inversion event such that only distal short arm

sequences are retained, as evidenced by the retention of the

SRY gene. Complex cytogenetic events involving the Y have

been described, and most were determined only after

molecular analyses because of the inability to resolve these

events at the microscopic level (Zeuthen and Nielsen, 1973;

Affara et al., 1986; Page et al., 1990). This explanation

would allow for the regional localization of some of the

twelve most proximally mapping probes to the proximal Y

short arm.

The second discrepancy between a karyotype reported by

NIGMS and the molecular data from this study involves cell

line GM 0118. At the cytogenetic level, GM 0118 appears to

retain a large portion of the Y chromosome. Judging by the

size of the additional material on 15p, some portion of Yq

euchromatin should have been retained. However, none of the

probes used in the analyses detected any euchromatic

sequences. A closer look at the translocation chromosome

(Figure 2-1) reveals a banding pattern strongly similar to









52

an inversion of bands 15qll-ql2 at the top of the chromosome

15. The Yq material might, therefore, be only from the

heterochromatic region. The existence of sequences which

map more distally in Yqll cannot be ruled out, nor can the

possibility be excluded that more distally located probes

than those described here might detect euchromatic sequences

in GM 0118.

Regional mapping of 35 Y chromosomal probes, using the

ten member reference panel, indicated that seven euchromatic

intervals were distinguished along the Y chromosome

(Figures 2-4 and 2-5). Six of these intervals mapped

exclusively to the long arm of the Y. The majority of the

probes detected a single Y-linked fragment. Two, BAY3-7 and

BAY25a, detected multiple Y-linked fragments mapping to

different Y chromosome intervals (see Figures 2-3b, 2-4, and

2-5) and appeared to be similar to the multiple Y-linked

fragments that map to different intervals as previously

described for probes 50f2 (DYS7, Guellaen et al., 1984), 52d

(DYS3, Bishop et al., 1984), and 118 (DYS8, Guellaen et al.,

1984).

Vergnaud presented a deletion map of the human Y

chromosome in 1986 that divided the entire chromosome into

seven intervals. The majority of the DNAs in that study

were obtained from XX males, and the intervals were

concentrated on the short arm rather than the long arm.

Only two intervals were assigned to the euchromatic long








53

arm. Because none of the five cell lines containing Y long

arm deletions used in that study were available to us, it

was difficult to correlate these newly described intervals

with those previously postulated. Probe 49f (DYSl), which

localized to the most distal euchromatic interval defined by

the panel here, also localized to the most distal

euchromatic Yq interval in the panel used by Vergnaud

(1986). Because only two Yq euchromatic intervals were

defined at that time, the distal-most interval from that

study might correlate with several of the intervals defined

here. DYS1 has been demonstrated to be the most distal

locus in other studies using different cell lines or patient

samples (Johnson et al., 1989; Kotecki et al., 1991;

Nakahori et al., 1991). 49f was also mapped by in situ

hybridization to metaphase chromosomes to the Yqll.22-qll.23

region (Quack et al., 1988). These data would place

interval 7 into Yqll.22-qll.23, and indicate that DYS1 is

the most distal locus. The precise locations of the other

intervals with respect to cytogenetic subbands Yqll.21,

Yqll.22, and Yqll.23 were not determined.

One goal of this investigation was to survey the long

arm of the Y chromosome for the presence of sites for

infrequently cleaving restriction endonucleases. These

sites were of particular interest because of their usual

association with HTF islands, possibly indicating the

presence of genes in the region (Bird, 1986; Estivill and









54

Williamson, 1987; Lindsay and Bird, 1987). In an effort to

enrich for clones bearing GC-rich sequences associated with

infrequently cleaving restriction endonuclease sites, the Y

chromosome library was screened several times with various

oligonucleotides containing the Not I restriction site

sequence. Although no clone was shown to contain a Not I

restriction site, the technique has been successfully used

to enrich for clones bearing infrequently cleaved

restriction sites (Estivill and Williamson, 1987; Melmer and

Buchwald, 1989). Although approximately 40 kb of DNA were

surveyed for Not I sites, a potentially much larger area was

covered in the experiments to be described in Chapter 4.

This evidence indicated a possible dearth of Not I sites at

least in distal Yqll which might account in part for the

lack of success in screening for them. The overall

distribution of Not I sites in genomic DNA would be expected

to be about one per 65 kb for an eight base pair recognition

enzyme, but the actual distribution ranges from 50 kb to 2

Mb (H. Lehrach, personal communication), so the 40 kb

surveyed in this study may be underrepresentative.

Hybridizations of recombinant inserts to male, female,

3E7, and Rag genomic digests produced patterns consistent

with results reported in other laboratories. About 30% of

the inserts demonstrated exclusively Y-linked fragments

while some 36% produced Y-linked fragments that were shared

with the X or autosomes (Table 2-1). Most of the remainder









55

were laden with repetitive elements. Using a novel

subcloning vector, a Japanese group screened the EcoR I

library equivalent to the Hind III library used in this

study. They reported some 80% X, Y, or autosome shared

fragments and only 21% exclusively Y-linked sequences.

These results may be related to the library used and/or the

selection of subclones by the subcloning vector (Nakahori et

al., 1991). Randomly isolated single and low copy sequences

isolated from other flow sorted Y-DNA libraries have fallen

into similar categories to those reported here: 35% were

exclusively Y-linked, 46% were shared between the X and Y,

15% were shared between the Y and autosomes, and 4% were

shared with the X, Y, and autosomes (Bishop et al., 1984;

Affara et al., 1986; Oosthuizen et al., 1990).

Overall, the data obtained in this study indicate that

the regional mapping panel introduced here divides the

euchromatic Y chromosome long arm into six distinct

intervals. This amounts to more than twice the number of

intervals defined by Vergnaud (1986). Probes bearing

repetitive elements appear to be unequally distributed in

the long arm, with repeat-bearing sequences covering more

area in the proximal intervals than in the distal intervals.

The most distal euchromatic interval defined by the panel,

interval 7, probably lies within subband Yqll.23, the region

of the Y chromosome whose absence is associated with

spermatogenic failure (Tiepolo and Zuffardi, 1976; Bobrow,









56

1985; Hartung et al., 1988). The following chapters

describe experiments centered around this most distal

euchromatic interval. In Chapter 3, the evolutionary

conservation of these five sequences will be addressed,

while Chapter 4 will address the distances between the

probes, their order on the Y chromosome at the molecular

level, and the overall size of interval 7 itself.















CHAPTER 3

EVOLUTIONARY COMPARISONS OF SEQUENCES MAPPING
TO THE HUMAN Y CHROMOSOME

Introduction

The mammalian sex chromosomes differ structurally and

genetically from one another, but are thought to have

evolved from a homologous pair. Recombination between these

original homologous chromosomes is postulated to have become

suppressed after another gene became involved in sex

determination. The suppression of recombination then

allowed mutations to accumulate on the proto Y, and

eventually most of the chromosome became extinct (Ellis and

Goodfellow, 1989). The existence of a homologous pairing

region, termed the pseudoautosomal region, was postulated to

allow for correct meiotic segregation and to account for the

presence of X-Y bivalents seen in meiosis (Burgoyne, 1982).

The rest of the Y became a repository of 'junk' DNA

(Charlesworth, 1978) as evidenced by the preponderance of Y-

specific repeated sequences in the long arm that constitute

50-70% of the entire chromosome (Cooke et al., 1983; Burk et

al., 1985). These repeats contained Y-specific restriction

fragment classes that were species specific and might

indicate that Y chromosomal sequences had diverged rapidly

(Kunkel and Smith, 1982; Wolfe et al., 1985).

57









58

When anonymous unique-sequence elements began to be

cloned, many were found to recognize homologous sequences on

the X chromosome, but outside the putative pairing region

(Bishop et al., 1983, 1984; Cooke et al., 1984). The

presence of such X-Y homologous sequences was interpreted as

evidence supporting the primordial homology between the sex

chromosomes (Bishop et al., 1984) until the species

conservation of these X-Y homologous sequences was reported.

These studies indicated that the sequences had recently

arrived on the human Y chromosome from primate X

chromosomes, and therefore were not indicators of an ancient

homology between the X and Y (Page et al., 1984; Koenig et

al., 1984; Cooke et al., 1984). The cloning of separate X-

specific and Y-specific centromeric repeat sequences further

complicated the issue of the degree of X-Y homology (Yang et

al., 1982; Wolfe et al., 1985) and the very existence of the

pairing region predicted by Burgoyne was questioned (Ashley,

1984). The cloning of the predicted pseudoautosomal

sequences was soon reported in a series of papers (Cooke et

al., 1985; Simmler et al., 1985; Buckle et al., 1985) and

the sequences were later shown to be conserved on the sex

chromosomes of the hominid primates: chimpanzee, gorilla,

and orangutan (Weber et al., 1987; 1988). A candidate gene

for the testicular determining factor, ZFY, was shown to be

conserved on the Y chromosomes of all eutherian mammals

tested (Schneider-Gadicke, et al., 1989; Palmer et al.,









59

1990), as was the bona fide TDF gene, SRY (Sinclair, et al.

1990). These two genes and the pseudoautosomal sequences

were the only short arm sequences shown to have such an

evolutionary conservation on the Y chromosome.

The data on the repeated and unique sequences from

the human Y suggested a relatively recent origin of much of

the long arm of this chromosome. To date, only 4B-2, 69/6,

and 1F5 have been shown to retain their exclusive Y-linkage

in the great apes (Burk et al., 1985; Erickson, 1987;

Whisenant et al., 1991). This chapter describes the

analyses of a series of Y chromosome-specific clones

discussed in Chapter 2. These sequences were selected for

analyses of their conservation in the great apes: chimpanzee

(Pan troglodytes), gorilla (Gorilla gorilla), and orangutan

(Pongo pygmaeus). The five clones mapping to the most

distal euchromatic interval on the human Y long arm (see

Figure 2-5) were included because of the association of this

region with spermatogenesis in humans. Surprisingly, all

but one of the clones were exclusively Y-linked in the great

apes. Further analyses were performed on genomic DNA from

rhesus monkeys (Maccaca mulatta), cattle (Bos taurus), and

mice (Mus domesticus) with the five most distally mapping

clones. Only an autosomal allele of GMGY1 was found to be

conserved in those species as well as in the great apes.









60

Materials and Methods

Primate DNA Extraction

Male and female peripheral blood samples from the

following primate species were obtained from the Yerkes

Regional Primate Center in Atlanta, Georgia: gorilla,

chimpanzee, orangutan, and rhesus monkey. 10 to 20 ml of

peripheral blood were collected in vacuum tubes containing

0.1 ml of 15% EDTA and shipped overnight on ice. On

receipt, the tubes were centrifuged to separate plasma and

cells. The plasma was removed to just above the buffy coat.

The buffy coat and some of the underlying red cells were

transferred to 15 ml conical centrifuge tubes. DNA

extraction buffer (10 mM Tris, pH 7.8, 2 mM EDTA, 400 mM

NaCl) was added to 5 ml, SDS to 0.5%, and predigested

Pronase to 1 mg/ml. Tubes were incubated overnight at 37C

and extracted twice with 24:24:1 phenol:chloroform:isoamyl

alcohol and twice with 24:1 chloroform:isoamyl alcohol. DNA

was precipitated in ice cold ethanol. Precipitated DNA was

spooled onto a glass pipet tip and dipped into 70% ethanol

prior to a brief period of air drying. The DNA was

resuspended in 5004i TE, pH 8, with a drop of chloroform,

then rotated overnight at 4C. The concentrations of DNA

were calculated from their -absorbances at 260nm. Purity was

also calculated from the ratios of absorbances obtained at

260nm and 280nm (A260/280), with a ratio of 1.8-2.0

considered to be optimal.










Other DNA Sources

Peripheral blood samples from a yearling cow and bull

were obtained from the University of Florida College of

Veterinary Medicine and DNA was extracted as detailed above.

Male and female mouse tail DNA samples were kindly provided

by Karen Dukes in Dr. Philip Laipis' laboratory (Department

of Biochemistry and Molecular Biology).

Southern Blotting and Hybridizations

10 to 15 pg aliquots of DNA were digested with

restriction enzymes according to the manufacturer's

recommendations. Digestion products were separated by

electrophoresis in 0.75% agarose gels overnight. Following

electrophoresis, gels were stained with 0.25 gLg/ml ethidium

bromide, destined for 30 minutes to 2 hours in water,

photographed, then transferred to nylon membranes in 0.4 M

NaOH overnight. Great ape DNA digests were transferred to

either Genescreen Plus (NEN) or Hybond N+ (Amersham).

Monkey, bovine and mouse DNA digests were transferred to

Hybond N+, as were the Msp I digests used to analyze probe

GMGY1. After blotting, membranes were rinsed with 2X SSPE,

air dried for several hours or vacuum dried at 80C for 30

minutes to 2 hours.

Probe DNAs were radiolabeled with 32P dCTP (3000

Ci/mmol, ICN) by random primer extension (Feinberg and

Vogelstein, 1984) using a BRL kit. The unincorporated

nucleotides were removed using a NACS PREPAC column (BRL).









62

Hybridizations were performed for 18-24 hours in 5X SSPE, 1%

SDS, 5X Denhardt's (0.02% Ficoll, 0.02% polyvinyl

pyrrolidone, 0.02% bovine serum albumin), 50% formamide at

42C. Post-hybridization washes for the great ape blots

were in 2X SSPE, 0.1% SDS at room temperature for 20

minutes, and twice in 0.1X SSPE, 0.1% SDS at 55-60C for 30

minutes each. Final washes for the blots containing DNA

from the lower mammals and the GMGY1 Msp I blot were at

45C. Membranes were wrapped in plastic wrap and exposed to

Kodak XAR-5 film at -70C for 1-6 days. After

autoradiography, the probes were stripped from the Hybond N+

blots in a solution of 0.1X SSPE, 0.5% SDS at 95-100C for

20-30 minutes. Genescreen Plus blots proved to be

unsatisfactory for reuse despite numerous attempts to strip

probes from the blots. Wet blots were sealed in plastic

after stripping and refrigerated until the next use.

Results

Clones Mapping to the Six Most Proximal Y Intervals

BAY clones 2-10, 4-la, 33b, 3-1, 3-11a, 2-7a, and l-8b

were selected as probes for Southern blots generated from

male and female chimpanzee, gorilla, and orangutan genomic

DNA digested with Hind III. These clones represent all but

one of the six most proximal intervals (see Figure 2-5).

Except for BAY3-lla, all of these clones were used to probe

genomic Southern blots generated using EcoR I, BamH I, Taq

I, Pst I, Bgl II, and/or Sst II in order to estimate the









63

degree of sequence homology by conservation of restriction

sites (Table 3-1).

Several patterns were observed from the hybridizations,

the results of which are summarized in Table 3-1. All of

the probes recognized male-specific bands in all three great

ape species with at least one enzyme. No bands were seen in

female DNA from any species. BAY2-10 and BAY2-7a recognized

common male bands (10%) in chimpanzee, gorilla, and often

orangutan, using more than one enzyme. Five probes

(BAY2-10, BAY33b, BAY4-la, BAY2-7a, and BAYl-8b) detected

common male bands in all species with at least one enzyme.

Two probes, BAYl-8b and BAY2-7a, appeared to be single or

low-copy sequences by EcoR I and Hind III analyses, but in

hybridizations of Southern blots generated by BamH I and

Pst I they detected multiple bands, indicating that these

sequences may be low-level repeats (see Figure 3-1, BamH I

genomic digest probed with BAYl-8b). The regional mapping

analyses localized both of these sequences to the sixth

interval defined by the panel, indicating that the repeats

are located within a specific region. BAY3-1 detected

fragments of similar sizes for EcoR I in human, chimpanzee

and gorilla, but fragments of different sizes with Hind III.

Y-linked fragments were also detected in orangutan in both

cases. BAY3-lla detected only Y-linked fragments of

different sizes in all four species with one enzyme,

Hind III.














21.


HSA GGO PPY PTR
f m f m f m f m
a '.


16.



5.5.

4.7.

3.8.
3.2.

2.8.
2.6"



1.9. i


a
-,


Figure 3-1. Conservation of Y-linked sequences homologous
to BAYl-8b in great apes. BamH I-digested DNA was separated
by electrophoresis and transferred to Genescreen Plus. The
Southern blot was hybridized to probe BAYl-8b. DNA sources
are as follows: f female, m male. Human, HSA; gorilla, GGO;
orangutan, PPY; chimpanzee, PTR. Size markers in kb are
indicated at the right.


40











Table 3-1. Interspecies comparisons of Southern blot data.


PROBE &
INTERVAL
BAY2-10
#1


BAY4-la
#2
BAY33b
#2




BAY3-11a
#5

BAY3-1
#5

BAY2-7a
#6


ENZYME
HindIII
EcoRI
BamHI
TaqI


HindIII

HindIII





HindIII


HindIII
EcoRI

HindIII



EcoRI



BamHI


PstI



TaqI


HUMAN
2.65
2.55
21
16
1.65


1.65


CHIMP
2.65
2.55
21

1.65


1.65


GORILLA
2.65
2.55
21


ORANGUTAN
10

21


1.65

1.65


4.6


1.9


1.3


0.9
1.6

4.4


0.8
6.1

3.0

>21
20


2.2
0.73
15
6.6
4.7
<1.0


1.9


12.8
1.9

1.3
1.6

4.3


0.8
6.0

3.0

>21


2.3

15


2.8
2.5
1.8


1.65

1.65

13
5.5

1.8
1.5

2.2


0.9
1.15


1.3
1.6

4.5
2.65
2.0
0.8
6.0
5.0
3.0


>21
20


4.3
1.9
1.8
0.8


2.95
2.7
21
11
7.2
5.5
3.3


2.1
0.76
21
18
15
<1.0


3.9
3.3
2.2
0.74
18
15
10


3.6











PROBE &
INTERVAL
BAY1-8b
#6


BAY3-8
#7


BAY2-11a
#7

1F5
#7











49f
#7


ENZYME
HindIII

EcoRI


BamHI
PstI


HindIII
EcoRI


HindIII


HindIII

BamHI

PstI


BglII

SstII


HindIII


HUMAN
1.1
0.95
17.5
12
rpt x13
rpt x9

1.6
18


5.0

4.6
2.2
2.0
1.4
1.25
2.1

2.4
1.5

1.6


CHIMP
1.1
0.95
18.5
8.6
rpt x7
rpt x4

3.5
18
13


5.0

4.6
2.2
2.0
1.4
1.25
2.1
0.7
NA


1.6


GORILLA
1.1
0.95
20
9.2
rpt x13
rpt x9


1.6
8.6
7.4


ORANGUTAN

0.95
20
9.9
rpt x12
rpt xlO


3.5
8.6
7.4


5.0


2.5
1.8
5.0


4.6
2.2
2.0
1.4
1.25
2.1

2.4
1.5

1.6


0.9
20
1.3
3.0


2.8
2.5
2.1


Band sizes are in kb. Bands shared in 3 or more species are
underlined. Numbers of BamH I and Pst I repeats detected by
BAY1-8b are indicated by "rpt times number of repeats". NA
indicates Not analyzed. Interval numbers beneath probe
designations refer to the intervals defined in Chapter 2,
Figure 2-5.









67

Analysis of Clones from the Distal Yqcr Euchromatic Region

The five clones (GMGY1, BAY3-8, BAY2-lla, 1F5, and 49f)

located in interval 7 of the long arm were used to probe

Southern blots generated from DNA from the great apes as

well as from rhesus monkeys, cattle, and mice. All except

GMGY1 detected male-specific sequences in all the great apes

(Figures 3-2 and 3-3), but not in rhesus monkeys, cattle, or

mice (not shown). BAY3-8, BAY2-lla, 1F5, and 49f detected

common fragments in man, chimpanzee and gorilla. BAY2-lla

also detected a common fragment in orangutan. 1F5 and 49f

both have been shown to detect non-Y-linked fragments at

reduced stringency in humans. These probes detected

fragments in gorilla females, but these migrated differently

from the common Y-linked fragments seen in the males and

were assumed to be non-Y-linked.

GMGY1 required analysis on a Southern blot generated by

the enzyme Msp I, because a Hind Ill-generated genomic

Southern blot did not differentiate a Y-linked fragment in

any of the great apes tested (not shown). Genomic DNA from

male and female humans, chimpanzees, gorillas, orangutans,

rhesus monkeys, cattle, and mice was digested with Msp I and

used to generate a Southern blot. This blot was washed at

reduced stringency (0.1X SSPE, 45C). A male-specific band

was detected only in human, but an autosomal sequence from

males and females was detected in all lanes except male

gorilla (Figure 3-4). The DNA sample from the male gorilla













HSA
f m


GGO PPY
fm f m


PTR
f m


Figure 3-2. Conservation of Y-linked sequences homologous
to BAY3-8 in great apes. EcoR I-digested DNA was
electrophoretically separated and transferred to Genescreen
Plus. The Southern blot was hybridized to probe BAY3-8.
DNA sources are as follows: f female, m male. Human, HSA;
gorilla, GGO; orangutan, PPY; chimpanzee, PTR. Band sizes
in kb are indicated at the right.


.18

013

o8.6
.7.4











1


a .,


i


i
f!


2 3 4 5 6 7 8


.7

' -7

95





.2.5
.1.8


03.4

.2.8
*2.5
-2.1


*1.6


P
C


Figure 3-3. Conservation of Y-linked sequences homologous
to probes BAY2-lla, 1F5, and 49f in great apes. Panels are:
a) BAY2-lla; b) 1F5; and c) 49f. Hind Ill-digested genomic
DNA was electrophoretically separated and transferred to
Hybond N+. The Southern blot was sequentially hybridized
with the probes. Lanes are as follows: 1 human female, 2
chimpanzee female, 3 gorilla female, 4 orangutan female, 5
human male, 6 chimpanzee male, 7 gorilla male, 8 orangutan
male. Band sizes in kb are indicated at the right.














HSA GGO PTR PPY MMU BTA MDO
f m fm f m F f m fm fm
ma~ _


21




-5.5
.4.7

-3.8
t .3.2

.2.8
-2.6

.1.9







Figure 3-4. Conservation of GMGYl-like sequences. Msp I-
digested genomic DNA was electrophoretically separated and
transferred to Hybond N+. The Southern blot was hybridized
with GMGY1. DNA sources are as follows: f female, m male.
Human, HSA; gorilla, GGO; chimpanzee, PTR; orangutan, PPY;
rhesus monkey, MMU; cow, BTA; mouse, MDO. Band sizes in kb
are indicated at the right.









71

was incompletely digested as indicated in the ethidium

bromide stained gel prior to transfer (not shown). It would

seem unlikely that the sequence is not conserved in male

gorillas since it is detected in female gorilla and is known

to be autosomal in humans (Affara et al., 1986).

The Hind III data for all probes are summarized in

Figure 3-5. The order of the probes on the human Y is from

proximal to distal. Common fragments in male chimpanzee and

gorilla or male chimpanzee, gorilla and orangutan are

underlined.

Discussion

Several reports have been published regarding the

conservation of Y chromosome-linked restriction fragments in

the great apes. Whereas many of these fragments are shared

among humans, chimpanzees, and gorillas, some divergence has

been noted in orangutan (Page et al., 1984; Koenig et al.,

1985; Erickson, 1987; Whisenant et al., 1991). An even

greater degree of divergence was observed in gibbons and

macaques (Page et al., 1984; Whisenant et al., 1991).

Whereas many human Y-linked fragments have been found to be

conserved, especially among the great apes, few have been Y-

linked in lower primates and other mammals. It has been

argued that the human Y is of recent evolutionary origin and

therefore any sequences that are Y-specific in closely

related species may be functionally significant (Burk et

al., 1985).












Human Chimpanzee Gorilla Orangutan


2-10 2.65
4-1a 1.65

33b


3-1


3-11a
2-7a


1


1.9
0.9
1.3
4.4


0.8
-8b 1.1
0.95
2-11a 7
3-8 1.5


1F5
49f


5.0
1.6


2.65
1.65
4.6, 3.2
1.9
1.3


12.8


4.3

0.8
1.1
0.95
7
3.5
5.0
1.6


2.65
1.65
11.5, 3.8
1.8
1.3
1.85, 1.2
4.5
2.65, 2.0
0.8
1.1
0.95
7
3.5
5.0
1.6


10
1.65
13, 5.5
1.8, 1.5
0.9
2.2
4.3
1.9, 1.8
0.8

0.95
7
1.5
2.5, 1.8
2.8, 2.5


2.1






Figure 3-5. Comparative evolutionary analysis of Hind III
fragments in great ape males. Hind III fragment sizes
detected in male but not female DNA by the probes listed at
the left are indicated for each of the great ape species.
Probes are ordered from proximal to distal corresponding to
the orders of the intervals into which each falls on the
human Y. Common fragments in male chimpanzee and gorilla or
male chimpanzee, gorilla and orangutan are underlined.


Probe









73

When viewed cytogenetically, great ape Y chromosomes

appear markedly similar. To date, only three anonymous Y

chromosome DNA sequences outside the pseudoautosomal region

have been demonstrated to be conserved on the Y chromosomes

of chimpanzees, gorillas, and orangutans: 4B-2 (Burk et al.,

1985), 69/6 (Erickson, 1987), and 1F5 (Whisenant et al.,

1991). The results reported here are unprecedented in that

all of the eleven Y chromosome-specific sequences studied

detected similar sequences on the Y chromosomes of the great

apes. These same sequences were not detected in male or

female DNA from rhesus monkeys, cattle, or mice. A twelfth

sequence, GMGY1, which detects autosomal and Y alleles in

human DNA, demonstrated conservation of one autosomal

homologue in all six mammalian species studied.

Transcription from the GMGY1 locus has not been reported,

but its high degree of conservation argues for some type of

functional role for the autosomal sequence. The human Y-

linked homologue might represent a newly arrived pseudogene,

as was described for the argininosuccinate synthetase and

steroid sulfatase Y-linked fragments (Daiger et al., 1982;

Yen et al., 1988). The absence of data from the male

gorilla prevents a definitive statement about the Y-linked

allele, although data gathered to date indicate that Y-

linked sequences shared by humans and gorillas are also

shared by chimpanzees.









74

The results of this study contrast with many earlier

findings that human Y-linked sequences, while evolutionarily

conserved, were not Y-linked in other higher primate genomes

(Page et al., 1984; Koenig et al., 1984, 1985; Erickson,

1987; Bickmore and Cooke, 1987). This conservation of

sequences is similar to what has been observed on the

autosomes of higher primates. The interpreters of the early

data suggested that a substantial portion of the single- and

multi-copy sequences on the human Y chromosome were acquired

during human evolution (Kunkel and Smith, 1982; Burk et al.,

1985). The proposal that the human Y chromosome is composed

primarily of newly arrived sequences is refuted by the

conservation of the exclusively Y-linked sequences described

in this chapter.

A model explaining the different Y chromosome sequence

elements might be thus. The pseudoautosomal region,

conserved on the sex chromosomes of the higher primates, and

some other X- and Y-linked genes may be the last remnants of

an ancient homologous pair of chromosomes. The sequences

detecting homologues on both the human X and Y chromosomes

outside of this pairing region and found only on the X

chromosomes of chimpanzees, gorillas, and sometimes

orangutans, may represent the sequences transposed to the

human Y after the divergence from the chimpanzee/gorilla

ancestor. Tandem duplication of repeated sequences on the

human Y also occurred after this divergence. By contrast,








75

many of the sequences on the long arm of the Y arose during

or after the divergence of higher primates from their lower

primate ancestors. These sequences have accumulated new

mutations and in some cases duplicated over the past 10

million years.

The human Y chromosome appears to be a mosaic of

sequences that have arrived at different times during its

evolution. The retention of Y-specificity among related

species has been suggested to be functionally significant

(Burk et al., 1985). Some of these sequences may represent

genes that affect the fertility of male higher primates, for

example, TSPY (Arnemann et al., 1987). Because very few

genetic loci have been assigned to the Y (Goodfellow et al.,

1985), it seems unlikely that all of these evolutionarily

conserved, anonymous DNA sequences encode functional

proteins. Some other selective pressure may be exerted on

these exclusively Y-linked sequences to cause them to remain

Y-associated over a period of approximately 10 million

years. This block of evolutionarily conserved, exclusively

Y-linked sequences may be related to the structure of the

region, assuring proper replication and condensation.

Alternatively, they may play a role in pairing or in

blocking recombination with the X or other chromosomes.

These experiments represent a first step in

understanding the extent of conservation of the euchromatic

long arm of the Y chromosome among higher primates. Genes








76

that are located within this region may be difficult to

identify because of a paucity of HTF islands (presented in

Chapter 4) as well as absence of conservation in other

species. The development of maps for this region in all of

these organisms will provide greater insight into the

molecular mechanisms of evolution. Because this region is

not subject to recombination, DNA sequence analysis will

provide a molecular clock to determine more precisely the

phylogenetic relationships among these organisms. Unlike

more global models, these genetic events represent fine

tuning in the evolution of sex chromosomes.















CHAPTER 4

LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME


Introduction

The long arm of the human Y chromosome is hemizygous in

males and therefore does not normally participate in

recombination during meiosis. The only technique currently

available for long-range mapping of such a non-recombinant

region is pulsed field gel electrophoresis, PFGE. This

technique used in conjunction with enzymes that cleave

infrequently in mammalian genomes, allows the analysis of

DNA fragments up to 9,000 kb (Schwartz and Cantor, 1984;

Barlow and Lehrach, 1987). The restriction endonucleases

used for this technique have recognition sites that tend to

be clustered in HTF islands. The sites are G + C rich and

contain one or more CpG dinucleotides. When HTF islands

were first described (Cooper et al., 1983; Bird et al.,

1985), they were hypothesized to be associated with genes.

Almost all cleavage by CG enzymes occurs in HTF islands

(Brown and Bird, 1986) which are now known to mark many

genes in mammalian genomes (Bird et al., 1987; Abe et al.,

1988; Pontarotti et al., 1988; Goodfellow et al., 1988;

Sargent et al., 1989). The most useful enzymes contain only

77








78

G and C in their recognition sequences, with two CpGs per

site. Double CpGs are rare between islands and common

within them, such that three out of every four genomic sites

occur in islands (Lindsay and Bird, 1987). The presence of

island-related genes on distal Yqll has not yet been

addressed.

Several genes in and near the pseudoautosomal region

have been localized by constructing restriction maps

covering long stretches of genomic DNA. These genes include

MIC2, ZFY, SRY, and a gene possibly involved in linear

growth (Pritchard et al., 1987; Page et al., 1987; Sinclair

et al., 1990; Henke et al., 1991). Several of these genes

were ascertained in genomic DNA by the production of

similarly sized fragments by different infrequently cleaving

restriction endonucleases, an observation often associated

with HTF islands (Brown and Bird, 1986; Pritchard et al.,

1987; Henke et al., 1991).

The distal portion of Yqll has been of interest because

of its association with spermatogenesis (Tiepolo and

Zuffardi, 1976), and the physical characterization of the

area might aid in the isolation of the responsible gene(s).

Deletion of the distal euchromatic long arm of the human Y

chromosome has been associated with azoospermia, or

spermatogenic failure, in otherwise normal men (Tiepolo and

Zuffardi, 1976; Bobrow, 1985; BUhler, 1985; Andersson et

al., 1988; Hartung et al., 1988). A complete long-range map









79

of the Y will be useful because cytogenetic landmarks are

difficult to visualize. The critical subband of Yqll to

which the azoospermia factor, AZF, has been assigned is

Yqll.23 (Human Gene Mapping, 1989). This subband would

therefore reside in "interval 6" as described by Vergnaud

which corresponds to distal Yqll (Vergnaud et al., 1986;

Andersson et al., 1988; Johnson et al., 1989). Sequences

such as pDP105/B (DYZ4, Andersson et al., 1988), 50f2/C,E

(DYS7, Guellaen et al., 1984) and 49f (DYS1, Bishop et al.

1984) have been shown to be deleted from some infertile

azoospermic men (Andersson et al., 1988; Nakahori et al.,

1991) as well as from other individuals with abnormalities

in sexual development (Kotecki et al., 1991). Probe 4B-2

(DYS15, Burk et al., 1985) was included in one study to

screen for the integrity of sequences proximal to the AZF

region (Johnson et al., 1989), and will be shown in this

study to anchor the proximal end of the restriction map

described.

The experiments in Chapter 2 allowed the separation of

the Y chromosome into eight intervals, six of which lie in

Yqll. The most distal euchromatic interval described in

that study, interval 7, contains locus DYS1 (49f) and would

therefore correspond roughly to "interval 6" as described by

Vergnaud (Vergnaud et al., 1986). The experiments to be

described here were used to develop a long-range restriction

map of interval 7, and should be near the location of the








80

azoospermia factor. The five sequences mapping to this

interval were used to develop the map, and probe 4B-2

(DYS15) was included because it detected significant

restriction fragment overlap with distal sequences, and was

therefore useful in distinguishing the centromeric and

telomeric ends of the map.

Materials and Methods

High Molecular Weight DNA Preparation

Lymphoblastoid cell cultures or separated peripheral

blood lymphocytes (PEL) were counted using a Neubauer

hemocytometer after washing one to two times with SE (75 mM

NaCl, 25 mM EDTA, pH 7.4). Lymphoblastoid cultures were

derived from a normal male and Oxen, a cell line derived

from a male with four Y chromosomes, 49,XYYYY (Sirota et

al., 1981). Female control cells were obtained from

peripheral blood lymphocytes from normal female donors.

Single cell suspensions were prepared at 1-2 x 106 cells per

50 4i SE at room temperature, which yielded about 10 gg of

DNA per agarose block. A 1.2% solution of nucleic acid

grade agarose (Pharmacia) in SE was melted and held at about

50C and equal volumes of agarose and cell suspension

(prewarmed to 37C) were mixed and immediately dispensed

into a mold with a capacity of 100 gi per slot. The blocks

were allowed to solidify at 4C and then were removed from

the mold to a sterile mixture of 0.5 M EDTA, pH 9/1% (v/v)

Sarkosyl (Sigma)/2 mg per ml predigested Pronase (Boehringer









81

Mannheim Biochemicals) at 0.5 ml per block in a 50 ml

conical centrifuge tube (Falcon) for 48 hours at 55C. A

second aliquot of Pronase was added to 2 mg/ml after 24

hours to ensure complete proteolysis. The blocks were

rinsed three times with sterile TE, pH 8, by filling the

tube, allowing the blocks to settle to the bottom, and

pouring the buffer away. The tube was then filled with TE

plus 0.04 mg/ml PMSF (Sigma) dissolved in DMSO (Fisher) and

incubated at 55C for 30 minutes. This step was repeated

once and was included to inactivate the Pronase and remove

the Sarkosyl. The blocks were rinsed with TE to remove the

PMSF and stored in TE or 0.5 M EDTA, pH 8 at 4C. All steps

were performed with gloves, sterile solutions, and sterile

supplies in order to avoid nuclease contamination.

Restriction Enzyme Digestion

Blocks from the Oxen and normal male cell lines and

female PBL were decanted from centrifuge tubes and

manipulated with an alcohol-flamed spatula into 1.5 ml

microfuge tubes. All solutions and supplies were sterile,

and gloves were worn throughout the procedure to reduce

nuclease contamination. Each block was immersed in two 1 ml

changes of TE, pH 8 and refrigerated for 30 minutes to

several hours each. The TE was replaced with two changes of

1 ml of the appropriate IX restriction buffer at 4C and

preequilibrated for 1-2 hours each, after which the

restriction buffer was removed. A typical restriction









82

digest was set up in a total volume of 300 .l: 100 Al

agarose block, 30 gl 10X restriction buffer, 0.1 mg/ml BSA,

0.5 mM DTT, and 20 units of enzyme. Spermidine was added to

10 mM in digests performed in buffers 50 mM or higher in

salt. Incubations were performed overnight at the

appropriate temperature and a second aliquot of enzyme was

added after 1-2 hours of digestion. The reaction was

terminated by the addition of 1/10 volume of 0.5 M EDTA,

pH 8, and the blocks refrigerated prior to loading the gel.

Double digests were set up such that an enzyme

requiring low salt buffer or 37C incubation was used before

an enzyme requiring high salt or 50C incubation. After

digestion with the first enzyme, 1/10 volume of 0.5 M EDTA,

pH 8 was added to the tube to inactivate the enzyme. The

tube was then rinsed twice with 1 ml of TE, pH 8 before

preequilibrating the block for several hours with two

changes of the next IX buffer at 4C. The second digest was

performed in the same manner as the first. Agarose-embedded

cells from a single harvest of male and Oxen lymphoblastoids

were used to produce the blots shown in Figures 4-1 and 4-2.

Gel Running Conditions

PFGE was performed in a BioRad CHEF DRII apparatus.

Digested DNA samples in agarose blocks were manipulated into

the wells of 1% agarose gels formed in 0.5X TBE (IX TBE is

89 mM Tris, 89 mM boric acid, 1 mM EDTA, pH 8). Sample and

marker blocks were overlaid with molten 1% agarose to fix








83

the blocks into the wells. Gels were run in recirculating

0.5X TBE at 200 V at 14C for 24 hours using a ramped switch

time of 60-90 seconds. Some gel runs were performed in a

cold room at 4C with buffer recirculation and all other

conditions remaining the same. These conditions were used

to separate fragments from about 20 kb to 1600 kb. To

separate fragments from 200 kb to 5 Mb, 0.8% gels were run

at 150 V, 4C with buffer recirculation, using ramped switch

times of 90-3600 seconds for 30-36 hours.

Large genomic fragments were sized by comparison with

yeast chromosomes (Saccharomyces cerevisiae AB972, 240 kb-

1600 kb; Schizosaccharomyces pombe, 3.6 Mb-5.7 Mb [BioRad])

and lambda concatamers (FMC Bioproducts), 50 kb-400 kb. The

error involved in sizing genomic fragments above 400 kb may

be as large as 50 kb (Kenwrick et al., 1987).

Preparation of Blocks Containing Yeast Chromosomes

Haploid Saccharomyces cerevisiae strain AB972, a gift

from Dr. Maynard V. Olson, Washington University, was grown

in YEPD (1% yeast extract, 2% peptone, 2% glucose) to mid-

logarithmic phase at 30C and harvested by centrifugation.

The cells were washed twice in 50 mM EDTA, pH 8 and

resuspended to a final concentration of 5 X 109 cells per

ml. The cells were mixed volume to volume with 1% agarose

in 50 mM EDTA, pH 8 maintained at 55C, dispensed into a

mold, and allowed to solidify at 4C. Spheroplasts were

obtained from the embedded cells using the protocol of








84

Bellis et al. (1987) in 0.5 M NaCl, 0.25 M EDTA, 0.125 M

Tris, pH 7.5, and 0.5 M beta-mercaptoethanol. The

suspension was incubated at 37C for 6 hours and then made

1% in SDS and 2 mg/ml in Pronase and incubated at 42C for

24 hours. The blocks were rinsed three times in TE, pH 8 at

50C and stored at 4C in 0.5 M EDTA, pH 8. The chromosome

sizes for this strain provided by Dr. Olson are 240, 280,

350, 440, 590, 680, 755, 810, 840, 950, 985, 1095, 1120,

1130, and 1640 kb.

Transfer and Hybridization

Ethidium bromide stained gels were destined in water

for 30 minutes to 2 hours, photographed, and acid hydrolyzed

in 0.25 M HCl for 20 minutes. The DNA was transferred

according to the manufacturer's instructions to Hybond N+ in

0.4 M NaOH. Before the first use with an unknown probe,

each blot was test hybridized with the pseudoautosomal probe

19b (MIC2 genomic clone), and autoradiography performed.

Each lane was examined for the known pattern of bands

detected by 19b with a particular enzyme to test for

completion of digestion and efficiency of DNA transfer.

None of the blots indicated incomplete digestion of DNA at

the MIC2 locus in any lane.

DNA probes were labeled with 32P dCTP (3000 Ci/mmol,

ICN) by random primer extension (Feinberg and Vogelstein,

1984) using a BRL kit to a specific activity of at least

1 X 109 cpm/gg. Membranes were hybridized in 5X SSPE, 1%








85

SDS, 5X Denhardt's, 10% dextran sulfate at 65C. Total

human genomic DNA derived from placenta and sheared to 300-

500 bp, was denatured and added to 30 jg/ml in both

prehybridization and hybridization solutions to block

background smears in the lanes. Post-hybridization washes

were performed at high stringency (0.1X SSPE, 0.1% SDS,

65C) and autoradiography was performed at -70C for 4-6

days with intensifying screens (Lightening Plus, DuPont).

Blots were washed in 0.1X SSPE, 0.5% SDS at 95C after

autoradiography, exposed for 48-72 hours to ensure that

residual probe was removed, sealed in plastic and reused.

Assumptions Affecting Long-Range Map Development

The map to be presented here was composed on the basis

of several assumptions. Restriction sites from single

enzyme digests were placed in the individual maps such that

the fragments would nest, following the routine

interpretation that these fragments represent partial

digestion of methylated sites (Nicholls et al., 1989; Li et

al., 1990; Kirkilionis et al., 1991). Wolf and Migeon

demonstrated that multiple restriction fragments detected by

single copy probes hybridized to Southern blots of human

genomic DNA generated by methylation-sensitive enzymes

represent fixed methylation patterns that differ from cell

to cell (Wolf and Migeon, 1982). The differences in band

intensities in a lane were assumed to be related to the

percentages of cells present in the sample that bore a









86

particular fixed methylation pattern (Wolf and Migeon, 1982;

Goodfellow et al., 1987; Nicholls et al., 1989).

The extremely complex patterns detected at all loci

when digested with two enzymes were assumed to be composed

primarily of single digestion products, because most of the

bands in those double digests were present when either of

the enzymes was used singly (see Table 4-1). The sequence

in which the digestions were performed did not appear to

influence which single digestion products were produced.

For example, most of the Mlu I restriction fragments were

present in both Mlu I/BssH II and Ksp I/Mlu I digestions, in

which Mlu I was used first and last, respectively. For the

double digests, only those bands which were not seen in

either single digest were considered to be unambiguous

double digestion products.

Results

A Restriction Map of the Distal Euchromatic Long Arm of the

Y Chromosome

DNA was digested with Not I, BssH II, Ksp I, and Mlu I.

Double digests were performed using combinations of BssH II,

Ksp I, and Mlu I. Blots containing Oxen, normal male, and

normal female DNA digested with BssH II, Ksp I, and Mlu I

singly and in combination were sequentially hybridized with

the five probes from interval 7, and 4B-2 from interval 6.

Before the first use with a Yqll probe, all blots were test

hybridized with 19b, the MIC2 genomic clone mapping to the









87

pseudoautosomal region. The blots were analyzed for the

presence of bands known to be produced at that locus by the

various enzymes used in this study and to ascertain the

completion of digestion and efficiency of DNA transfer. The

Y chromosome restriction fragment data from single and

double digests of Oxen DNA for these six loci are compiled

in Table 4-1. The Southern blot data from the cell line

Oxen, containing four copies of the Y, are shown in Figures

4-1 and 4-2. Female control lanes were included on the

blots to visualize non Y-linked bands detected by 49f, 1F5,

and GMGY1 (not shown). In Figures 4-1 and 4-2 the non Y-

linked bands detected in female lanes are marked for probes

GMGY1, 1F5, and 49f.

Not I restriction fragments were not detected at any of

the loci when probes were hybridized to PFGE-generated

Southern blots containing fragments from about 200 kb to

5 Mb (not shown). No Ksp I fragments were detected by 1F5

and 49f, whereas a minor fragment of 480 kb was detected by

BAY3-8 and BAY2-11a. GMGY1 and 4B-2 detected common Ksp I

fragments of 250, 320, and 370 kb; and an additional six

fragments of 150, 190, 420, 490, 520, and 590 kb were

detected by 4B-2. A major 900 kb BssH II fragment was

detected by BAY3-8, BAY2-lla, 1F5 and 49f. These probes all

detected a minor 780 kb fragment; this fragment was weak

using the blot in Figure 4-1, but was stronger on a blot

produced from a different harvest of the Oxen cells (not











Table 4-1. High molecular weight restriction fragments
detected by distal Yqll probes.


Bss KsD Mlu B/K


420


590
520
490
420
370
320
250
190
150


820 (650)
780 (620)
760 (420)
550 360
510 190
300 150


PROBE
4B-2









GMGY1






BAY3-8


BAY2-lla 900 (480) 820
780 780
(300) 630
(200)


900
120


B/M K/M

(1Mb) (960)
(820) 820
(780) 780
630 (630)
550 (550)
510 (510)
420 (200)
300 (190)
(40)

(1Mb) (960)
(820) (820)
(780) (780)
(590) 550
(550) 420


(820) (960)
(780) 820
630 780
590 630
(550)
470
440
300
120

(820) (960)
(780) 820
630 780
590 710
(550) 630
470 550
440 400
300
120


(360) (370) 820
(320) 780
(250) 760
550
510
420

900 (480) 820 900
(780) 780 (580)
(300) 630 (480)
120 120











Table 4-1--continued


Bss KSP Mlu B/K


900
(780)


820
780
(550)
200


820
(550)
200
60
(20)


900
(780)


900


(900)


B/M K/M


(820)
(780)
630
(590)
(550)
470
440
200

820
630
(590)
(550)
470
200
60
(20)


(960)
(820)
(780)
710
630
200


820
(550)
200
60
(20)


Band sizes are in kb. Enzymes are Bss: BssH II, Ksp: Ksp I,
Mlu: Mlu I. B/K: BssH II and Ksp I, B/M: BssH II and Mlu I,
K/M: Ksp I and Mlu I. Weakly hybridizing fragments are
indicated in parentheses. Restriction enzyme fragments
recognized by multiple probes are underlined for the single
digests.


PROBE
1F5


49f





























Figure 4-1. Hybridization of distal Yqll probes to PFGE
Southern blots. High molecular weight DNA from a 49,XYYYY
individual was digested with various enzymes, separated by
PFGE, and transferred to Hybond N+. The blot was
sequentially hybridized with sequences mapping to the
distal-most interval defined by the mapping panel in Chapter
2 as well as probe 4B-2 from the next more proximal
interval. Lanes: 1 BssH II, 2 Ksp I, 3 Mlu I. Probes: a
4B-2 (DYS15), b GMGY1 (DYS12), c BAY3-8 (DYS194), d BAY2-lla
(DYS193), e 1F5 (DYS128), f 49f (DYS1). Molecular weight
markers are in kb. Dots (.) indicate fragments detected in
female DNA and which are not Y-linked.




Full Text
81
Mannheim Biochemicals) at 0.5 ml per block in a 50 ml
conical centrifuge tube (Falcon) for 48 hours at 55C. A
second aliquot of Pronase was added to 2 mg/ml after 24
hours to ensure complete proteolysis. The blocks were
rinsed three times with sterile TE, pH 8, by filling the
tube, allowing the blocks to settle to the bottom, and
pouring the buffer away. The tube was then filled with TE
plus 0.04 mg/ml PMSF (Sigma) dissolved in DMSO (Fisher) and
incubated at 55C for 30 minutes. This step was repeated
once and was included to inactivate the Pronase and remove
the Sarkosyl. The blocks were rinsed with TE to remove the
PMSF and stored in TE or 0.5 M EDTA, pH 8 at 4C. All steps
were performed with gloves, sterile solutions, and sterile
supplies in order to avoid nuclease contamination.
Restriction Enzyme Digestion
Blocks from the Oxen and normal male cell lines and
female PBL were decanted from centrifuge tubes and
manipulated with an alcohol-flamed spatula into 1.5 ml
microfuge tubes. All solutions and supplies were sterile,
and gloves were worn throughout the procedure to reduce
nuclease contamination. Each block was immersed in two 1 ml
changes of TE, pH 8 and refrigerated for 30 minutes to
several hours each. The TE was replaced with two changes of
1 ml of the appropriate IX restriction buffer at 4C and
preequilibrated for 1-2 hours each, after which the
restriction buffer was removed. A typical restriction


Figure 2-3. Southern blot analyses of Y regional mapping panel. Genomic DNA was digested
with Hind III unless otherwise indicated to the right of the fragment size. Blots were
probed with the Y recombinants indicated in Table 2-1 (*). Data are presented for
representative sequences located in intervals 1-6 and all five sequences located in
interval 7. Probes are designated at the left of the Southern blots. Only the Y-linked
bands are shown and the sizes in kb are indicated to the right. Symbols below each data
set indicate the presence (+) or absence (-) of the Y-linked bands in the DNA of a panel
member, and the interval number is designated below. Lanes represent respectively: F,
normal female; M, normal male; 3, GM 0118; 4, GM 2103; 5, GM 2469; 6, GM 7970; 7, GM 8773;
8, GM 9403; Y, 3E7 (Y hybrid cell line); P, Rag (hybrid parent cell line); 11, Pf; 12, Si;
13, GM 2668; 14, GM 2730.
a) Representative Y recombinants located in the first three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal.
b) Representative Y recombinants located in the next three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal. Lane 4 (GM 2103)
is underloaded relative to the other lanes.
c) Two representative Southern blots hybridized with probe BAY3-8 from interval 7. Lanes
1-11 contain DNA from translocation-bearing cell lines; lanes 12-18 contain DNA from
deletion-bearing cell lines. Lanes represent 1, female; 2, male; 3, GM 0118; 4, GM 2103;
5, GM 2469; 6, marker; 7, GM 7970; 8, GM 8773; 9, GM 9403; 10, 3E7; 11, Rag; 12, female;
13, male; 14, Pf; 15, Si; 16, GM 2668; 17, GM 2730; 18, marker. Molecular weight markers
for the blot bearing lanes 1-11 are located to the left. Also at left are arrows numbered
1,7, and 5 indicating hybridization signals from probes BAY2-10, BAY3-8, and BAY3-lla
which map to intervals 1, 7, and 5 respectively. BAY3-8, BAY2-10 and BAY3-lla were
hybridized simultaneously to the blot at the left (lanes 1-11). Only BAY3-8 was
hybridized to the blot at the right (lanes 12-18). Molecular weight markers for the blot
bearing lanes 12-18 are located at the right. The arrow at the right indicates the
hybridization signal from BAY3-8 in lanes 12-18.


50
analyses revealed that the reported isochromosome Yq was
unstable, suggesting that the chromosome was dicentric
rather than an isochromosome. GM 3595 retained a stable
deleted Y chromosome, but the cells were intractable in
culture and could not be grown in quantities sufficient to
extract DNA, resulting in the exclusion of this cell line
from the panel.
The molecular data presented here indicate possible
discrepancies in the reported karyotypes of two cell lines
from NIGMS. GM 2730 reportedly contained a deleted Y
chromosome in which the deletion extended into Yqll. The
cytogenetic data do not appear to contradict this report
(see Figure 2-1). In the molecular data reported here,
twelve probes that map into the region reportedly present in
GM 2730 detected no sequences in DNA from this cell line.
Because GM 2730 was a mosaic cell line, the possibility
existed that the representation of the Y chromosome was
below the level of sensitivity for Southern blot analysis.
This was ruled out by the detection of the single copy SRY
gene in a Southern blot prepared with GM 2730 DNA samples.
One explanation for the lack of Yq sequences in GM 2730
would require all twelve probes to be in an interval just
distal to the Y centromere and proximal to the breakpoint in
GM 9403. This explanation would invoke the seemingly
unlikely event that the library screening procedure selected
against sequences mapping to the short arm of the Y


6
PROBE
F M 3 4 5
BAY 3-7B
-mm
BAY 5
1*
- + - -
BAY 3-1
# Ci
BAY 3-11a
t
- + + -
Figure 2
7 8 Y P 11 1213 14
+ + + + +- -
Interval 4
+ + +- + +- -
Interval 5
* 3.3 (EcoRI)
+ + +- + +
Interval 6
03
00
3continued


47
by x2 analysis, however the next three intervals differed
significantly (0.001 distribution of repeats. This significant difference became
even greater (p<<0.001) if the analysis included sequences
from interval 7.
Submission of Sequences to the Genome Data Base
All the newly isolated Y chromosome probes that were
regionally mapped in Figure 2-4 were submitted to the Genome
Data Base at Johns Hopkins University. Anonymous DNA
segments are no longer being assigned D-numbers by GDB
without some evidence of function, but submitted information
is kept on file in the event that new data will provide that
evidence. Based upon their conservation in the higher
primate males as described in Chapter 3, nine sequences
listed in Table 2-2 were assigned D-numbers by GDB.
Discussion
Because of the lack of recombination in the region,
the development of a linkage map of the long arm of the
human Y chromosome depends upon physical analyses. Whereas
the majority of the physical mapping data obtained from the
Y chromosome was directed at the short arm, sequences
mapping to the long arm have been described; however, none
of the published mapping data have utilized common Y
chromosome translocations and deletions, making data
comparisons difficult. A standard panel of altered Y


102
considered to be due to variable methylation in different
clonal populations of cells.
Discussion
Pulsed field gel electrophoresis allows for the
correlation of genetic and physical distances. In the case
of the euchromatic long arm of the Y chromosome, genetic
distances cannot be ascertained because of the absence of
recombination. PFGE was used to establish the order and
physical positions of five loci mapping to the most distal
euchromatic region, interval 7, defined by the deletion
mapping panel in Chapter 2. A sixth locus, mapping to
interval 6, was included in the proposed restriction map to
establish the orientation of the map with regard to the Y
chromosome centromere and telomere. The purpose of this
study was to provide physical landmarks required to further
characterize this genetically interesting region of the Y
chromosome.
The AZF locus lies in the region distal to 4B-2
(Johnson et al., 1989) which anchors the proximal border of
the map presented here, while 49f localizes to the distal
end of the map. 49f and other more proximal loci have been
shown to be deleted in a number of individuals with
azoospermia (Vergnaud et al., 1986; Johnson et al., 1989;
Nakahori et al., 1991). In additional hybridization
analyses of individuals with deletions of distal Yqll, 49f
has been reported as the most distal locus in a series of


117
Nakahori, Y., Tamura, T., Nagafuchi, S., Fujieda, K.,
Minowada, S., Fukutani, K., Fuse, H., Hayashi, K.,
Kuroki, Y. Fukushima, Y., Agematsu, K., Kuno, T.,
Kaneko, S., Yamada, K., Kitagawa, T., Nonomura, M.,
Fukuda, S., Kusano, M., Onigata, S. Hibi, I.,
Nakagome, Y. 1991. Molecular cloning and mapping of 10
new probes on the human Y chromosome. Genomics 9:765-
769.
Ngo, K., Vergnaud, G., Johnsson, C., Lucotte, G.,
Weissenbach, J. 1986. A DNA probe detecting multiple
haplotypes of the human Y chromosome. Am J Hum Genet
38:407-418.
Nicholls, R. Knoll, J., Butler, M., Karam, S., Lalande, M.
1989. Genetic imprinting suggested by maternal
heterodisomy in non-deletion Prader-Willi syndrome.
Nature 342:281-285.
Oosthuizen, C., Herbert, J., Vermaak, L., Brusnicky, J.,
Fricke, J., du Plessis, L., Reteif, A. 1990. Deletion
mapping of 39 random isolated Y-chromosome DNA
fragments. Hum Genet 85:205-210.
Page, D., de Martinville, B., Barker, D., Wyman, A., White,
R., Francke, U., Botstein, D. 1982. Single-copy
sequence hybridizes to polymorphic and homologous loci
on human X and Y chromosomes. Proc Natl Acad Sci USA
79:5352-5356.
Page, D., Fisher, E., McGillivray, B., Brown, L. 1990.
Additional deletion in sex-determining region of human
Y chromosome resolves paradox of X,t(Y;22) female.
Nature 346:279-281.
Page, D., Harper, M., Love, J., Botstein, D. 1984.
Occurrence of a transposition from the X-chromosome
long arm to the Y-chromosome short arm during human
evolution. Nature 311:119-123.
Page, D., Mosher, R., Simpson, E., Fisher, E., Mardon, G.,
Pollack, J., McGillivray, B., de la Chapelle, A.,
Brown, L. 1987. The sex-determining region of the human
Y chromosome encodes a finger protein. Cell 51:1091-
1104.
Palmer, M., Berta, P., Sinclair, A., Pym, B., Goodfellow, P.
N. 1990. Comparison of human ZFY and ZFX transcripts.
Proc Natl Acad Sci USA 87:1681-1685.


71
was incompletely digested as indicated in the ethidium
bromide stained gel prior to transfer (not shown). It would
seem unlikely that the sequence is not conserved in male
gorillas since it is detected in female gorilla and is known
to be autosomal in humans (Affara et al., 1986).
The Hind III data for all probes are summarized in
Figure 3-5. The order of the probes on the human Y is from
proximal to distal. Common fragments in male chimpanzee and
gorilla or male chimpanzee, gorilla and orangutan are
underlined.
Discussion
Several reports have been published regarding the
conservation of Y chromosome-linked restriction fragments in
the great apes. Whereas many of these fragments are shared
among humans, chimpanzees, and gorillas, some divergence has
been noted in orangutan (Page et al., 1984; Koenig et al.,
1985; Erickson, 1987; Whisenant et al., 1991). An even
greater degree of divergence was observed in gibbons and
macaques (Page et al., 1984; Whisenant et al., 1991).
Whereas many human Y-linked fragments have been found to be
conserved, especially among the great apes, few have been Y-
linked in lower primates and other mammals. It has been
argued that the human Y is of recent evolutionary origin and
therefore any sequences that are Y-specific in closely
related species may be functionally significant (Burk et
al.. 1985).


108
previously localized to Yqll can now be sublocalized with
these cell lines to facilitate the production of a common
deletion map of the long arm. A cooperative mapping effort
would greatly facilitate the production of a deletion map of
the human Y chromosome that is composed from common probes
and common cell lines.
To further address the origins of the human Y
chromosome, a greater number of Y-linked probes need to be
mapped comparatively and categorized as to their X- or
autosomal-linkage in humans and their subsequent association
or non-association with primate Y chromosomes. The two
postulates addressing the origin of the chromosome need not
be mutually exclusive; certain portions of the human Y
chromosome may indeed be composed of recently transposed
sequences, while others have a more ancient association with
the Y. It will be interesting to investigate whether the
different classes of sequences have intermixed or have
remained together in large blocks on the human Y, or
conversely on other great ape Y chromosomes. The only
evidence touching on the long-range distribution of
sequences relates to the probe 2:13 (Cooke et al., 1984), an
X-Y homologous sequence that is only found on the X
chromosomes of chimpanzees and gorillas. The X and Y
homology was shown to extend for at least 50 kb in humans
(Bickmore and Cooke, 1987). The continuity of this block
with other sequences such as DXYS1 and DYS22 that show a


4
may represent the TSY locus (Salido et al.. 1992).
Pseudogenes for gamma actin (Heilig et al., 1984), steroid
sulfatase (Yen et al., 1988), argininosuccinate synthetase
(Daiger et al., 1982), and several retroviral sequences
(Silver et al., 1987) also have been found to reside on the
human Y.
The molecular model of the human Y chromosome
necessarily derives from analysis of anonymous cloned
sequences rather than from classical genetic studies that
depend upon recombination between linked loci. Despite the
dearth of genes, the chromosome is well suited to molecular
genetic mapping. Most sequences have only a single Y-linked
allele because of the hemizygous nature of the chromosome.
Three methods have been used to isolate unique sequence
probes for the Y chromosome. Some Y-linked probes have been
fortuitously isolated from human cDNA or genomic DNA
libraries. While not well suited to generating large
numbers of Y-linked probes, the first unique sequence probe
shared by the sex chromosomes was isolated in this manner
(Page et al., 1982). Genomic libraries constructed from DNA
from Y chromosomes separated from the other chromosomes by a
fluorescence activated cell sorter have proven to be a more
efficient means of obtaining Y chromosome-specific probes
(Affara et al., 1986; Oosthuizen et al., 1990; Nakahori et
al., 1991). The third technique involves screening genomic
libraries constructed from human-rodent somatic cell hybrids


40
detectable in all DNA samples of this cell line by Southern
analysis (not shown).
The first interval that was unambiguously localized to
Yqll was defined by two probes, BAY2-9a and BAY4-la
(Figure 2-3a). BAY2-9a also detected a non Y-linked
minisatellite repeated sequence in the human DNA on Southern
blots produced on Zetaprobe (Figure 2-3a, lanes 1-8) that
was not detectable on Genescreen Plus (lanes 11-14). The
BAY2-9a minisatellite was detectable on Southern blots
produced on Hybond N+ (not shown). Probes from this
interval detected sequences in the deletion cell lines
GM 2668, Pf, and Si which retain the entire Y short arm and
some portion of the proximal long arm (Ypter-Yqll)
(Figure 2-2). Sequences were detected in only one
translocation cell line, GM 9403; here, the Y fragment
extended from the terminus of the long arm to the proximal
long arm (Yqter-Yqll) (Figure 2-2).
BAY2-2, BAY2-4a, BAY16, BAY3 0b, BAY3 3b, and MIAY
defined the third interval, of which, MIAY had been
previously mapped to Yqll (Koenig et al., 1985). Two
representative sequences from this interval are shown in
Figure 2-3a (BAY2-4a and MIAY). The sequences were detected
in the translocation cell line GM 9403 and in the Yq
deletion cell lines Pf and Si, but not GM 2668. These data
indicated that the breakpoints in deletion samples Pf and Si
lay distal to the breakpoint in GM 2668.


4B-2 GMGY1
J L
BAY3-8,2-lla

J I IJ
1F5 49f

l l l ll
telomere>
r
420
I
J 300 1
J 550
I
360
120
J 420 L
I
J 510
J 550
-* 760
T~
i 300
J 780
J 820
J 630
J 200
1 200
u 20
60
I 900
i 780
CD
00
370
320
250
190
480
150
-100 kb
BssH II
HKsP 1
1I Mlu I


60
Materials and Methods
Primate DNA Extraction
Male and female peripheral blood samples from the
following primate species were obtained from the Yerkes
Regional Primate Center in Atlanta, Georgia: gorilla,
chimpanzee, orangutan, and rhesus monkey. 10 to 20 ml of
peripheral blood were collected in vacuum tubes containing
0.1 ml of 15% EDTA and shipped overnight on ice. On
receipt, the tubes were centrifuged to separate plasma and
cells. The plasma was removed to just above the buffy coat.
The buffy coat and some of the underlying red cells were
transferred to 15 ml conical centrifuge tubes. DNA
extraction buffer (10 mM Tris, pH 7.8, 2 mM EDTA, 400 mM
NaCl) was added to 5 ml, SDS to 0.5%, and predigested
Pronase to 1 mg/ml. Tubes were incubated overnight at 37C
and extracted twice with 24:24:1 phenol:chloroform:isoamyl
alcohol and twice with 24:1 chloroform:isoamyl alcohol. DNA
was precipitated in ice cold ethanol. Precipitated DNA was
spooled onto a glass pipet tip and dipped into 70% ethanol
prior to a brief period of air drying. The DNA was
resuspended in 500/xl TE, pH 8, with a drop of chloroform,
then rotated overnight at 4C. The concentrations of DNA
were calculated from their -absorbances at 260nm. Purity was
also calculated from the ratios of absorbances obtained at
260nm and 280nm (A260/280), with a ratio of 1.8-2.0
considered to be optimal.


14
phage plaques to nitrocellulose filters (Schleicher and
Schull). Marked filters were placed on plates until
thoroughly wet and plate bottoms were correspondingly
marked. A duplicate filter was laid on the plate after the
first was removed and allowed to remain for 30 seconds to 1
minute longer than the previous filter to equalize the
quantity of phage adhering to the two filters.
Corresponding marks were also made on the plate bottoms for
the second filters. Filters were dipped into denaturing and
neutralizing washes as described in Maniatis et al. (1982),
then allowed to air dry. The filters were placed between
sheets of Whatman 3MM paper and baked at 80C in a vacuum
oven for 2 hours. Duplicate filter pairs were sequentially
hybridized as described below.
Phage Selection
Duplicate filters were first screened for the presence
of clones containing GC-rich sequences. A series of
oligonucleotides containing the recognition sequence of the
restriction enzyme Not I were synthesized by the Health
Science Center DNA Core Facility. The oligonucleotides were
radiolabeled using T4 kinase (Maniatis et al., 1982) and
hybridized to duplicate filters. The first oligonucleotide
used for screening was 8 nt in length and consisted of only
the recognition sequence for the restriction endonuclease,
Not I (GCGGCCGC). Hybridization for this oligonucleotide
was performed in 6X SSC (IX SSC is 0.15 M NaCl, 0.015 M


53
arm. Because none of the five cell lines containing Y long
arm deletions used in that study were available to us, it
was difficult to correlate these newly described intervals
with those previously postulated. Probe 49f (DYS1), which
localized to the most distal euchromatic interval defined by
the panel here, also localized to the most distal
euchromatic Yq interval in the panel used by Vergnaud
(1986). Because only two Yq euchromatic intervals were
defined at that time, the distal-most interval from that
study might correlate with several of the intervals defined
here. DYS1 has been demonstrated to be the most distal
locus in other studies using different cell lines or patient
samples (Johnson et al. 1989; Kotecki et a_l. 1991;
Nakahori et al., 1991). 49f was also mapped by in situ
hybridization to metaphase chromosomes to the Yqll.22-qll.23
region (Quack et al., 1988). These data would place
interval 7 into Yqll.22-qll. 23, and indicate that DYS1 is
the most distal locus. The precise locations of the other
intervals with respect to cytogenetic subbands Yqll.21,
Yqll.22, and Yqll.23 were not determined.
One goal of this investigation was to survey the long
arm of the Y chromosome for the presence of sites for
infrequently cleaving restriction endonucleases. These
sites were of particular interest because of their usual
association with HTF islands, possibly indicating the
presence of genes in the region (Bird, 1986; Estivill and


91
1 2 3
2 3 12 3
*
b


78
G and C in their recognition sequences, with two CpGs per
site. Double CpGs are rare between islands and common
within them, such that three out of every four genomic sites
occur in islands (Lindsay and Bird, 1987). The presence of
island-related genes on distal Yqll has not yet been
addressed.
Several genes in and near the pseudoautosomal region
have been localized by constructing restriction maps
covering long stretches of genomic DNA. These genes include
MIC2, ZFY, SRY, and a gene possibly involved in linear
growth (Pritchard et al.. 1987; Page et al., 1987; Sinclair
et al.. 1990; Henke et al., 1991). Several of these genes
were ascertained in genomic DNA by the production of
similarly sized fragments by different infrequently cleaving
restriction endonucleases, an observation often associated
with HTF islands (Brown and Bird, 1986; Pritchard et al..
1987; Henke et al., 1991).
The distal portion of Yqll has been of interest because
of its association with spermatogenesis (Tiepolo and
Zuffardi, 1976), and the physical characterization of the
area might aid in the isolation of the responsible gene(s).
Deletion of the distal euchromatic long arm of the human Y
chromosome has been associated with azoospermia, or
spermatogenic failure, in otherwise normal men (Tiepolo and
Zuffardi, 1976; Bobrow, 1985; Bhler, 1985; Andersson et
al., 1988; Hartung et al., 1988). A complete long-range map


12
cells or trypsinized fibroblasts were pelleted and
resuspended in 3-5 ml of DNA extraction buffer (10 mM Tris,
pH 7.8, 2 mM EDTA, 400 mM NaCl). After addition of SDS to
0.5% and predigested Pronase (Boehringer Mannheim) to a
final concentration of 1 mg/ml, the solution was incubated
overnight at 37C. The solution was extracted with 24:24:1
phenol:chloroform:isoamyl alcohol, pH 8.0, followed by one
to two extractions with 24:1 chloroform:isoamyl alcohol.
DNA was precipitated with ice cold 95% ethanol. The DNA was
spooled onto a glass pipette and rinsed briefly with 70%
ethanol before air drying. The DNA was resuspended in 3 00-
500 /I TE, pH 8, with a drop of chloroform, then rotated
overnight at 4C. DNA concentration was calculated from the
absorbance at 260nm. Purity was also calculated from the
ratios of absorbances obtained at 260nm and 280nm
(A260/280), with a ratio of 1.8-2.0 considered to be
optimal.
Normal male and female donors provided blood samples
from which DNA was extracted. These provided controls for
the presence and absence of a Y chromosome. Blood obtained
by venipuncture into 10 ml Vacutainer tubes (Becton
Dickinson) containing 0.1 ml of 15% EDTA was centrifuged to
separate plasma and cells. The plasma was removed to just
above the buffy coat. The buffy coat and some of the
underlying red cells were transferred to 15 ml conical
centrifuge tubes. DNA extraction buffer was added to 5 ml,


118
Petit, C., Levilliers, J., Weissenbach, J. 1988. Physical
mapping of the human pseudoautosomal region; comparison
with genetic linkage map. EMBO 7:2369-2376.
Petit, C., Levilliers, J., Weissenbach, J. 1990. Long-range
restriction map of the terminal part of the short arm
of the human X chromosome. Proc Natl Acad Sci USA
87:3680-3684.
Polani, P. 1982. Pairing of X and Y chromosomes, non
inactivation of X-linked genes, and the maleness
factor. Hum Genet 60:207-211.
Pontarotti, P., Chimini, G., Nguyen, C., Boretto, J.,
Jordan, B. 1988. CpG islands and HTF islands in the HLA
class I region: investigation of the methylation status
of class I genes leads to precise physical mapping of
the HLA-B and -C genes. Nuc Acids Res 16:6767-6778.
Pritchard, C., Goodfellow, P. J., Goodfellow, P. N. 1987.
Mapping the limits of the human pseudoautosomal region
and a candidate sequence of the male-determining gene.
Nature 328:273-275.
Quack, B., Guerin, P., Ruffie, J., Lucotte, G. 1988. Mapping
of probe 49f to the proximal part of the human Y
chromosome long arm. Cytogenet Cell Genet 47:232.
Rappold, G., Lehrach, H. 1988. A long range map of the
pseudoautosomal region by partial digest PFGE analysis
from the telomere. Nuc Acids Res 16:5361-5377.
Rouyer, F., de la Chapelle, Andersson, M., Weissenbach, J.
1990. An interspersed repeated sequence specific for
human subtelomeric regions. EMBO 9:505-514.
Salido, E., Yen, P., Koprivnikar, K., Yu, L.-C., Shapiro, L.
1992. The human enamel protein gene amelogenin is
expressed from both the X and Y chromosomes. Am J Hum
Genet 50:303-316.
Sargent, C., Dunham, I., Campbell, R. D. 1989.
Identification of multiple HTF-island associated genes
in the human major histocompatibility complex class III
region. EMBO 8:2305-2312.
Schneider-Gadicke, A., Beer-Romero, P., Brown, L., Nussbaum,
R., Page, D. 1989. ZFX has a gene structure similar to
ZFY, the putative human sex determinant, and escapes X
inactivation. Cell 57:1247-1258.


65
Table 3-1. Interspecies comparisons of Southern blot data
PROBE &
INTERVAL
BAY 2-10
#1
ENZYME
Hindlll
EcoRI
BamHI
TaqI
HUMAN
2.65
2.55
21
16
1.65
BAY4-la
#2
BAY 33b
#2
Hindlll
Hindlll
1.65
1.9
BAY3-lla
#5
Hindlll
1.3
BAY 3-1
#5
Hindlll
EcoRI
0.9
1.6
BAY2-7a
#6
Hindlll
4.4
EcoRI
0.8
6.1
3.0
BamHI
>21
20
PstI
2.2
0.73
TaqI 15
6.6
4.7
<1.0
CHIMP
GORILLA
ORANGUTAN
2.65
2.65
10
2.55
2.55
21
21
21
1.65
1.65
1.65
1.65
1.65
1.65
4.6
13
2.8
5.5
2.5
1.9
1.8
1.8
1.5
12.8
NA
2.2
1.9
1.3
1.3
0.9
1.6
1.6
1.15
4.3
4.5
4.3
2.65
1.9
2.0
1.8
0.8
0.8
0.8
6.0
6.0
5.0
3.0
3.0
2.95
2.7
>21
>21
21
20
11
7.2
5.5
3.3
3.9
3.3
2.3
2.2
2.1
0.74
0.76
15
18
21
15
18
10
15
<1.0
3.6


67
Analysis of Clones from the Distal Ya Euchromatic Region
The five clones (GMGY1, BAY3-8, BAY2-lla, 1F5, and 49f)
located in interval 7 of the long arm were used to probe
Southern blots generated from DNA from the great apes as
well as from rhesus monkeys, cattle, and mice. All except
GMGY1 detected male-specific sequences in all the great apes
(Figures 3-2 and 3-3), but not in rhesus monkeys, cattle, or
mice (not shown). BAY3-8, BAY2-lla, 1F5, and 49f detected
common fragments in man, chimpanzee and gorilla. BAY2-lla
also detected a common fragment in orangutan. 1F5 and 49f
both have been shown to detect non-Y-linked fragments at
reduced stringency in humans. These probes detected
fragments in gorilla females, but these migrated differently
from the common Y-linked fragments seen in the males and
were assumed to be non-Y-linked.
GMGY1 required analysis on a Southern blot generated by
the enzyme Msp I, because a Hind Ill-generated genomic
Southern blot did not differentiate a Y-linked fragment in
any of the great apes tested (not shown). Genomic DNA from
male and female humans, chimpanzees, gorillas, orangutans,
rhesus monkeys, cattle, and mice was digested with Msp I and
used to generate a Southern blot. This blot was washed at
reduced stringency (0.1X SSPE, 45C). A male-specific band
was detected only in human, but an autosomal sequence from
males and females was detected in all lanes except male
gorilla (Figure 3-4). The DNA sample from the male gorilla


GM 0118
GM 2103
GM 2469
GM 2668
k*GM 2730
GM 7970
GM 8773
^)GM 9403
SZ


33
Table 2-1continued
BAY SIZE COGNATE NONCOGNATE NON-Y REPEATS Cot
CLONE Y Y 100
22a
4
-
-
-
H
+
22b
1.8
-
-
-
H
+
24
1.5
-
-
C
H
+
25a
*
3
+B
7 kbA
-
H
+
25b
1.2
-
-
c
H
+
26a
3.5
-
-
+
H
+
26b
1.6
-
-
+
H
+
29a
*
5
+
-
-
H
+
29b
*
1.4
-
4 kb*
-
H
+
30b
*
3
+
-
1.9kb
H
+
31a
1.7
+
+
C+N
H
+
31b
1
+
-
C+N
H
+
33a
3
+
-
-
H
-
33b
*
2.1
+
-
-
-
-
33c
*
1.2
+
-
1.2kb
-
-
34a
5
-
-
I
H
-
35a
*
2.5
+
1.9kb
1.9kb
H
+
35b
1.8
+
-
C
H
+
35c
1.4
+
3.5kb
C+N
H
+
35d
1
+
-
-
H
+
35e
0.5
-
+
N
H
+
36a
4
+
-
C
H
+
41
1.7
+


H

REPEATS:
Indicates presence or absence of
repeat
el erne:
probes
Cot 100: Indicates probes requiring preannealing of repeat
sequences
Regionally mapped clones
Bands analyzed if cognate bands undetected or non
Y-linked
Probes containing internal Hind III sites
Not determinable
Cognate: probe detects genomic fragment of same size
Noncognate: probe detects genomic fragment of different
size
(H) Highly repetitive: intense background smear
(M) Moderately repetitive: moderate background smear or
minisatellite sequence through which individual bands
may be detected
) Indicates more than one Y-linked fragment detected by
a single probe
Band sizes reflect Hind III genomic digests
(*)
(+)
(*)
(I)
(C)
(N)
(
A-Z <


husband, Bert, during this time. His intercession and
spiritual leadership were also greatly appreciated. I thank
our families for their love and encouragement over the
years. I also thank our sisters and brothers in Christ, the
Lees, Ahns, Barnards and many others for their prayers,
love, and understanding. My thanks go also to Dr. Catherine
Ketcham for her friendship and long-distance encouragement,
and to my shootin' buddy Kathy Mercer for fun times on the
range.
iii


13
SDS to 0.5%, and predigested Pronase to 1 mg/ml. Tubes were
incubated overnight at 37C and extracted twice with
phenol:chloroform:isoamyl alcohol and twice with
chloroform:isoamyl alcohol before ethanol precipitation as
above.
Library Screening
The methods used for phage library screening were
generally as described in Maniatis et al. (1982). Phage
were isolated from a Y chromosome-enriched library that was
obtained from the Lawrence Livermore National Laboratory
(LLOYNSOl). The library was prepared from flow-sorted human
Y chromosomes that were cut to completion with the
restriction endonuclease, Hind III. The fragments were
inserted into the Hind III cloning site of the cloning
vector, lambda Charon 21A. The insert sizes ranged up to
9 kb. The yield of independent recombinants was 2.5 x 105,
and the amplified library contained 27 genome equivalents.
The library was plated at approximately 20,000 pfu/plate on
150 mm petri dishes in three separate experiments. Host
bacteria, E. coli LE392, were cultured in NZY medium
supplemented with 0.2% maltose. Appropriate numbers of
phage were mixed with host cells and incubated at 37C for
20 minutes. Infected cells were mixed with molten (47C)
0.7% agarose, poured over the surfaces of 1.5% agar plates
and incubated for 12 to 16 hours at 37C. Plates were
allowed to cool to room temperature prior to transferring


22
detects a 3.6 kb Y-specific fragment in EcoR I-digested DNA
and defines locus DYS22. MIAY was kindly provided by Dr.
Michael Koenig.
Probe Y3.4 is a 3.4 kb Pst I insert in pBR322. It was
derived from the 3.4 kb Hae III repeat seen in male, but not
female DNA. It defines the Y-specific repeat family DYZ1
(Cooke, 1976). Y3.4 was kindly provided by Dr. Kirby Smith.
These plasmids were transformed into appropriate host
bacteria, amplified, purified, and restricted with the
appropriate enzyme(s), separated in low melt agarose, and
extracted by standard procedures (Maniatis et al., 1982).
Results
Mapping Panel Selection
Ten cell lines were initially obtained from the Human
Genetic Mutant Cell Repository (NIGMS) in Camden, NJ. The
karyotype of each line was verified prior to inclusion of
the cell line into the mapping panel. The Y chromosomal
fragments present in each NIGMS cell line used in the panel
are shown in Figure 2-1. Two of the ten cell lines
originally obtained from NIGMS, GM 3774 and GM 3595, were
excluded from the panel. GM 3774 reportedly contained an
isochromosome Yq, but this was not verified after receipt of
the culture. Two separate cultures of GM 3595 exhibited
extremely poor growth, resulting in its exclusion from the
panel even though its reported karyotype was confirmed.
GM 7970, reported by NIGMS to contribute only band Yql2, was


105
frequently than the other enzymes because of its recognition
sequence, and did produce more numerous restriction
fragments than either Ksp I or BssH II in most cases.
The proposed long-range restriction map presented
represents the most parsimonious arrangement of restriction
sites accounting for all of the fragments produced by the
single enzyme digestions, and at the same time confining all
six loci to an area of no more than 820 kb as indicated by
the Mlu I data. The map represents the first description of
mammalian genomic DNA that is devoid of HTF islands in an
area covering more than 1500 kb. Although these results
were not expected, the nonrecombinant nature of Yqll
represents a unique situation in the genome as well.
Because all genes are not linked to HTF islands, this map
indicates that genes in distal Yq may not be associated with
clusters of unmethylated CpG dinucleotides. The absence of
this segment of the Y chromosome is associated with abnormal
spermatogenesis, and implies the presence of at least one
gene. Evidence gathered from several laboratories,
including that from this study, indicates that the
spermatogenesis locus, AZF, may reside within the region for
which a map is now presented. This map provides a starting
point from which a detailed characterization of this
important area may begin.


31
BAY6b, BAY9, BAY14, and BAY22a and BAY22b), whereas six
inserts produced background signals of such intensity that
the presence of non Y-linked bands could not be ascertained
(BAYl-7a, BAYl-7b, BAYl-8a, BAY3-2, BAY3-9, BAY34a). Two
recombinants, BAYl-8b and BAY2-7a, contained internal
Hind III sites and therefore detected two genomic fragments
that mapped to the same interval. The sizes of the genomic
fragments added up to the sizes of their respective cloned
genomic inserts. In the case of BAY2-2, an intense
background smear was observed in the male and female lanes
while a clear 1 kb signal was detected in the 3E7 hybrid
lane (not shown). After the repetitive elements in the
probe were preannealed to total genomic DNA to a Cot of 100
the background smear was eliminated from the human lanes,
allowing for the unambiguous detection of the 1 kb band in
both the male and 3E7 lanes. This band was absent in the
female and Rag lanes. With the exceptions of BAY33a,
BAY33b, BAY33c, BAY34a, and BAY41 all clones numbered BAY1
to BAY50 were preannealed to a Cot of 100 prior to
hybridization to compete out the repetitive elements that
had appeared as faint gray signals in the library screening
Eleven inserts detected Y and non-Y fragments of the same
sizes in Hind Ill-digested genomic DNAs of males, females,
and 3E7 (BAYl-lla, BAY2-7b, BAY2-9b, BAY2-lla, BAY 2-llb,


43
The fourth interval lay between the breakpoints in
GM 8773 and GM 2103, and contained three recombinant
fragments: BAY3-7B, BAY5, and BAYlOb. These probes detected
sequences in two translocation cell lines, GM 9403 and
GM 8773, and two deletion cell lines, Pf and Si. BAY3-7B
and BAY5 represent two of the probes mapped to this interval
in Figure 2-3b. BAY3-7 had previously detected a higher
molecular weight fragment (A) in the most proximal Y
interval defined by the panel (Figure 2-3a).
The fifth interval was defined by BAY3-1, BAY3-lla, and
BAY3c which detected sequences in translocations GM 9403,
GM 8773, and GM 2103, and deletions Pf and Si. Two
representative probes from this interval are shown in Figure
2-3b. The fainter bands in GM 2103 (lane 4) were due to
underloading of the DNA.
The sixth interval contained 4B-2, BAY2-7a, BAY3-3,
BAY25aA and BAYl-8b, of which 4B-2 was described previously
(Burk et al., 1985). These probes detected sequences in
four translocation lines: GM 9403, GM 8773, GM 2103, and
GM 2469, and two deletion lines, Pf and Si. 4B-2 and
BAYl-8b represent probes defining the interval (Figure
2-3b). The faint band in GM 2103 (lane 4) detected by 4B-2
was due to incomplete digestion of the genomic DNA by EcoR I
as seen in the ethidium bromide stained gel prior to
transfer (not shown). The 0.9 and 1.2 kb bands detected by


66
GORILLA ORANGUTAN
PROBE &
INTERVAL
BAYl-8b
#6
BAY3-8
#7
BAY2-lla
#7
1F5
#7
49f
#7
ENZYME
Hindlll
EcoRI
BamHI
PstI
Hindlll
EcoRI
Hindlll
Hindlll
BamHI
PstI
Bglll
Sstll
Hindlll
HUMAN
1.1
0.95
17.5
12
rpt xl3
rpt x9
1.6
18
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
2.4
1.5
1.6
CHIMP
1.1
0.95
18.5
8.6
rpt x7
rpt x4
3.5
18
13
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
0.7
NA
1.6
1.1
0.95
20
9.2
rpt xl3
rpt x9
1.6
8.6
7.4
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
2.4
1.5
1.6
0.95
20
9.9
rpt xl2
rpt xlO
3.5
8.6
7.4
7
2.5
1.8
5.0
0.9
20
1.3
3.0
2.8
2.5
2.1
Band sizes are in kb. Bands shared in 3 or more species are
underlined. Numbers of BamH I and Pst I repeats detected by
BAYl-8b are indicated by "rpt times number of repeats". NA
indicates Not analyzed. Interval numbers beneath probe
designations refer to the intervals defined in Chapter 2,
Figure 2-5.


20
filtrate was kept on ice throughout the processing. The
filtrate was poured into 50 ml conical centrifuge tubes and
the nuclei pelleted at 4C, 1500 rpm. Nuclei were washed
once in homogenization buffer without Triton and pooled.
Pellets were resuspended in DNA extraction buffer. SDS was
added to 0.5%, and Pronase to 1 mg/ml. This solution was
incubated overnight at 37C. The solution was extracted
once with phenol:chloroform:isoamyl alcohol and twice with
chloroform:isoamyl alcohol. RNase was added to a final
concentration of 50 /xg/ml. The solution was incubated at
37C for 2 hours. The solution was reextracted, ethanol
precipitated, and spooled onto a glass pipette tip, dipped
into 70% ethanol, and air dried briefly. The DNA was
resuspended in TE, pH 8, sonicated to about 300-500 bp, then
ethanol precipitated and resuspended to 10 or 20 mg/ml.
Y Chromosome Probes Provided by Other Investigators
In order to correlate the data produced in this study
with published information about the region, probes that
mapped to the euchromatic long arm of the human Y chromosome
were obtained from other investigators. All probes
described below are subclones into plasmids.
Probe 49f, a 2.8 kb EcoR I insert in pBR322, was
derived from a Y-enriched cosmid library (Bishop et al.,
1983, 1984). It detects a 2.8 kb Y-specific band in EcoR I-
digested DNA at high stringency ("cognate band"). At low
stringency, it detects two autosomal bands as well as


87
pseudoautosomal region. The blots were analyzed for the
presence of bands known to be produced at that locus by the
various enzymes used in this study and to ascertain the
completion of digestion and efficiency of DNA transfer. The
Y chromosome restriction fragment data from single and
double digests of Oxen DNA for these six loci are compiled
in Table 4-1. The Southern blot data from the cell line
Oxen, containing four copies of the Y, are shown in Figures
4-1 and 4-2. Female control lanes were included on the
blots to visualize non Y-linked bands detected by 49f, 1F5,
and GMGY1 (not shown). In Figures 4-1 and 4-2 the non Y-
linked bands detected in female lanes are marked for probes
GMGY1, 1F5, and 49f.
Not I restriction fragments were not detected at any of
the loci when probes were hybridized to PFGE-generated
Southern blots containing fragments from about 200 kb to
5 Mb (not shown). No Ksp I fragments were detected by 1F5
and 49f, whereas a minor fragment of 480 kb was detected by
BAY3-8 and BAY2-lla. GMGY1 and 4B-2 detected common Ksp I
fragments of 250, 320, and 370 kb; and an additional six
fragments of 150, 190, 420, 490, 520, and 590 kb were
detected by 4B-2. A major 900 kb BssH II fragment was
detected by BAY3-8, BAY2-lla, 1F5 and 49f. These probes all
detected a minor 780 kb fragment; this fragment was weak
using the blot in Figure 4-1, but was stronger on a blot
produced from a different harvest of the Oxen cells (not


79
of the Y will be useful because cytogenetic landmarks are
difficult to visualize. The critical subband of Yqll to
which the azoospermia factor, AZF, has been assigned is
Yqll.23 (Human Gene Mapping, 1989). This subband would
therefore reside in "interval 6" as described by Vergnaud
which corresponds to distal Yqll (Vergnaud et al. 1986;
Andersson et al., 1988; Johnson et al., 1989). Sequences
such as pDP105/B (DYZ4, Andersson et al., 1988), 50f2/C,E
(DYS7, Guellaen et al.. 1984) and 49f (DYS1, Bishop et al.,
1984) have been shown to be deleted from some infertile
azoospermic men (Andersson et al., 1988; Nakahori et al. ,
1991) as well as from other individuals with abnormalities
in sexual development (Kotecki et a_l. 1991). Probe 4B-2
(DYS15, Burk et al., 1985) was included in one study to
screen for the integrity of sequences proximal to the AZF
region (Johnson et al., 1989), and will be shown in this
study to anchor the proximal end of the restriction map
described.
The experiments in Chapter 2 allowed the separation of
the Y chromosome into eight intervals, six of which lie in
Yqll. The most distal euchromatic interval described in
that study, interval 7, contains locus DYS1 (49f) and would
therefore correspond roughly to "interval 6" as described by
Vergnaud (Vergnaud et al., 1986). The experiments to be
described here were used to develop a long-range restriction
map of interval 7, and should be near the location of the


75
many of the sequences on the long arm of the Y arose during
or after the divergence of higher primates from their lower
primate ancestors. These sequences have accumulated new
mutations and in some cases duplicated over the past 10
million years.
The human Y chromosome appears to be a mosaic of
sequences that have arrived at different times during its
evolution. The retention of Y-specificity among related
species has been suggested to be functionally significant
(Burk et al., 1985). Some of these sequences may represent
genes that affect the fertility of male higher primates, for
example, TSPY (Arnemann et al., 1987). Because very few
genetic loci have been assigned to the Y (Goodfellow et al.,
1985), it seems unlikely that all of these evolutionarily
conserved, anonymous DNA sequences encode functional
proteins. Some other selective pressure may be exerted on
these exclusively Y-linked sequences to cause them to remain
Y-associated over a period of approximately 10 million
years. This block of evolutionarily conserved, exclusively
Y-linked sequences may be related to the structure of the
region, assuring proper replication and condensation.
Alternatively, they may play a role in pairing or in
blocking recombination with the X or other chromosomes.
These experiments represent a first step in
understanding the extent of conservation of the euchromatic
long arm of the Y chromosome among higher primates. Genes


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor oi^Philosophy.
Thomas P. Yarn
Assistant Pro
Biochemistry'a
Biology
I certify that I have read this study and tfta£_An my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Harry bstrer
Associate Professor of
Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Philip J. Atai£is C
Professor^of'Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Harry £J. Nick
Associate Professor of
Biochemistry and Molecular
Biology


82
digest was set up in a total volume of 300 /zl: 100 /I
agarose block, 30 /z 1 10X restriction buffer, 0.1 mg/ml BSA,
0.5 mM DTT, and 20 units of enzyme. Spermidine was added to
10 mM in digests performed in buffers 50 mM or higher in
salt. Incubations were performed overnight at the
appropriate temperature and a second aliquot of enzyme was
added after 1-2 hours of digestion. The reaction was
terminated by the addition of 1/10 volume of 0.5 M EDTA,
pH 8, and the blocks refrigerated prior to loading the gel.
Double digests were set up such that an enzyme
requiring low salt buffer or 37C incubation was used before
an enzyme requiring high salt or 50C incubation. After
digestion with the first enzyme, 1/10 volume of 0.5 M EDTA,
pH 8 was added to the tube to inactivate the enzyme. The
tube was then rinsed twice with 1 ml of TE, pH 8 before
preequilibrating the block for several hours with two
changes of the next IX buffer at 4C. The second digest was
performed in the same manner as the first. Agarose-embedded
cells from a single harvest of male and Oxen lymphoblastoids
were used to produce the blots shown in Figures 4-1 and 4-2.
Gel Running Conditions
PFGE was performed in a BioRad CHEF DRII apparatus.
Digested DNA samples in agarose blocks were manipulated into
the wells of 1% agarose gels formed in 0.5X TBE (IX TBE is
89 mM Tris, 89 mM boric acid, 1 mM EDTA, pH 8). Sample and
marker blocks were overlaid with molten 1% agarose to fix


94
shown). BAY3-8 and BAY2-lla each detected an additional
weak 300 kb fragment and a major 120 kb fragment with
BssH II. GMGY1 and 4B-2 detected single BssH II fragments
of 360 and 420 kb, respectively. All probes detected a
common 820 kb Mlu I fragment, and all except 49f detected a
780 kb Mlu I fragment as well. GMGY1 and 4B-2 detected
common Mlu I fragments of 760, 550, and 510 kb; and unique
fragments of 420 and 300 kb, respectively. BAY3-8 and
BAY2-lla detected a common 630 kb Mlu I fragment, whereas
BAY2-lla detected a weak Mlu I fragment of 200 kb. 1F5 and
49f detected common fragments of 550 and 200 kb, while 49f
detected unique Mlu I fragments of approximately 60 and
20 kb. The 200 kb fragment detected by BAY2-lla was
considered to be different from the 200 kb fragment shared
by 1F5 and 49f for several reasons. First, its
hybridization signal was much weaker than that detected by
1F5 and 49f (see Figure 4-1, d-f). Second, the weak 200 kb
fragment detected by BAY2-lla was absent from double digests
involving Mlu I, while the strong 200 kb fragment seen with
1F5 and 49f remained visible in Mlu I double digests with
both BssH II and Ksp I (Figure 4-2, d-f). Third, the same
set of double digest blots was sequentially hybridized with
BAY2-lla and 1F5. The absence on these blots of the 200 kb
band in the BAY2-lla hybridization, followed by the
detection of a strong 200 kb fragment by 1F5 indicated that
the different intensities of the fragments detected on the


CHAPTER 2
DELETION MAPPING OF THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME
Introduction
A physical map has been developed for the distal short
arm of the human Y chromosome. This area includes the
pseudoautosomal region, where sequences are exchanged
actively with the X chromosome (Cooke et al.. 1985; Simmler
et al., 1985; Goodfellow et ad., 1986), and the next most
distal interval where the testis-determining gene lies
(Sinclair et al., 1990). Other genes including GM-CSF, ZFY,
RPS4Y, TSPY, and amelogenin have been cloned from the short
arm region (Gough et al., 1990; Page et al.. 1987; Fisher et
al., 1990; Arnemann et al., 1987; Salido et al.. 1992). At
the time that these studies were undertaken, a restriction
map had not yet been constructed for the rest of the Y
chromosome. Deletion maps have been constructed with novel
Y probes, but different reference translocations and
deletions were used in each case, making comparisons
difficult (Affara et ad., 1986, Vergnaud et al., 1986,
Oosthuizen et al., 1990, Nakahori et al., 1991). Other
studies have addressed the presence or absence of Y
chromosomal sequences in various patients exhibiting
abnormalities in fertility or sexual development (Andersson
8


70
HSA GGO PIR PPY MMU BTA MPO
f m f m f m f m f m f m f m
Figure 3-4. Conservation of GMGYl-like sequences. Msp in
digested genomic DNA was electrophoretically separated and
transferred to Hybond N+. The Southern blot was hybridized
with GMGY1. DNA sources are as follows: f female, m male.
Human, HSA; gorilla, GGO; chimpanzee, PTR; orangutan, PPY;
rhesus monkey, MMU; cow, BTA; mouse, MDO. Band sizes in kb
are indicated at the right.


9
et al.. 1988; Johnson et al., 1989; Skare et al., 1990;
Kotecki et al.. 1991; Nakahori et al., 1991), again
providing no composite picture for comparative purposes.
HTF islands are located in the pseudoautosomal region
(Goodfellow et al.. 1988; Henke et al.. 1991) as well as in
the proximally located Y-specific region near the ZFY gene
(Page et ad., 1987). At the time this study was undertaken,
no information existed regarding either the presence or
distribution of these gene-associated islands on the long
arm of the Y. Identifying HTF islands represented one means
to locate genes on the long arm of the Y chromosome.
These experiments were undertaken to map the
euchromatic long arm of the human Y chromosome, Yqll. The
goals were to characterize new Y chromosomal probes, derive
a regional map for these new and previously described Y
chromosomal sequences, and to address the distribution of
HTF islands on the long arm. The anticipated result of
these mapping studies was the generation of a long-range
restriction map of the most distal interval defined by the
mapping panel. Such a restriction map might facilitate the
localization of the spermatogenesis-related gene(s) that
were provisionally mapped to the region (Tiepolo and
Zuffardi, 1976; Bobrow, 1985; Hartung et al., 1988). An
additional aim of this project was to test a newly assembled
mapping panel for the Y chromosome long arm.


119
Schwartz, C. and Cantor, C. 1984. Separation of yeast
chromosome sized DNAs by pulsed field gradient gel
electrophoresis. Cell 37:67-75.
Sealy, P., Whittaker, P., Southern, E. 1985. Removal of
repeated sequences from hybridisation probes. Nuc Acids
Res 13:1905-1922.
Silver, J., Rabson, A., Bryan, T., Willey, R., Martin, M.
1987. Human retroviral sequences on the Y chromosome.
Mol Cell Biol 7:1559-1562.
Simmler, M.-C., Rouyer, F., Vergnaud, G., Nystrom-Lahti, M.,
Ngo, K., de la Chapelle, A., Weissenbach, J. 1985.
Pseudoautosomal DNA sequences in the pairing region of
the human sex chromosomes. Nature 317:692-697.
Simpson, E., Chandler, P., Goulmy, E., Disteche, C.,
Ferguson-Smith, M., Page, D. 1987. Separation of the
genetic loci for the H-Y antigen and for testis
determination on the human Y chromosome. Nature
326:876-878.
Sinclair, A., Berta, P., Palmer, M., Hawkins, J., Griffiths,
B., Smith, M., Foster, J., Frischauf, A.-M., Lovell-
Badge, R., Goodfellow, P. 1990. A gene from the human
sex-determining region encodes a protein with homology
to a conserved DNA-binding motif. Nature 346:240-244.
Sirota, L., Zlotogora, Y., Shabtai, F., Halbrecht, I.,
Elian, E. 1981. 49,XYYYY. A case report. Clinical Genet
19:87-93.
Skare, J., Drwinga, H., Wyandt, H., vanderSpek, J., Troxler,
R., Milunsky, A. 1990. Interstitial deletion involving
most of Yq. Am J Med Genet 36:394-397.
Smith, K., Young, K., Talbot, C., Schmeckpeper, B. 1987.
Repeated DNA of the human Y chromosome. Development
101:77-92.
Tiepolo, L., Zuffardi, 0. 1976. Localization of factors
controlling spermatogenesis in the nonfluorescent
portion of the human Y chromosome long arm. Hum Genet
34:119-124.
Vergnaud, G., Page, D., Simmler, M.-C., Brown, L., Rouyer,
F., Noel, B., Botstein, D., de la Chapelle, A.,
Weissenbach, J. 1986. A deletion map of the human Y
chromosome based on DNA hybridization. Am J Hum Genet
38:109-124.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Susan A. Moyer
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Edward E. Wakeland
Professor of Pathology and
Laboratory Medicine
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy^
May, 1992
Dean, College of Medicine
' 'r,~ s* *
Dean, Graduate School


Figure 2-2. Y Chromosome Mapping Panel. Representations of the Y chromosomal fragments
present in each cell line as judged by hybridization mapping are indicated by the
horizontal lines. The positions of the breakpoints are relative and do not represent the
resolution used in cytogenetic analyses. Karyotypes are indicated above the lines.


110
This project represents a starting point from which to
generate a composite molecular map of the human Y chromosome
long arm. A panel of cell lines useful for producing a
common Yq deletion map has been introduced in Chapter 2.
The feasibility of linking intervals in which the order and
physical positions of individual probes was established, has
been demonstrated in Chapter 4. Continuing on in that
manner, the remaining intervals may be connected and the
resultant molecular map correlated with the cytogenetic map.
The conservation of sequences on the Y chromosomes of
closely related species seen in Chapter 3, coupled with the
unique arrangement of infrequently cleaving restriction
enzyme sites observed in Chapter 4, suggests the intriguing
possibility that these observations are interconnected. A
restriction map of the entire long arm of the Y chromosome
may be of use in elucidating the extent of that
relationship, as well as in ultimately detecting other genes
that are thought to reside there.


Figure 2-4. Hybridization of probes to DNA from regional
mapping panel. Genomic DNA from each cell line was digested
with the appropriate enzyme, separated by electrophoresis,
and transferred to nylon membranes. Southern blots were
sequentially hybridized with the Y chromosome recombinants
listed at the left. (+) indicates the presence of the
appropriate Y-linked fragment in the DNA from the panel
member, (-) indicates its absence. Cell line 3E7 is a
somatic cell hybrid bearing only the human Y chromosome, and
was included as a positive control for Y chromosomal
sequences. ND indicates the fragment was undetectable.


54
Williamson, 1987; Lindsay and Bird, 1987). In an effort to
enrich for clones bearing GC-rich sequences associated with
infrequently cleaving restriction endonuclease sites, the Y
chromosome library was screened several times with various
oligonucleotides containing the Not I restriction site
sequence. Although no clone was shown to contain a Not I
restriction site, the technique has been successfully used
to enrich for clones bearing infrequently cleaved
restriction sites (Estivill and Williamson, 1987; Melmer and
Buchwald, 1989). Although approximately 40 kb of DNA were
surveyed for Not I sites, a potentially much larger area was
covered in the experiments to be described in Chapter 4.
This evidence indicated a possible dearth of Not I sites at
least in distal Yqll which might account in part for the
lack of success in screening for them. The overall
distribution of Not I sites in genomic DNA would be expected
to be about one per 65 kb for an eight base pair recognition
enzyme, but the actual distribution ranges from 50 kb to 2
Mb (H. Lehrach, personal communication), so the 40 kb
surveyed in this study may be underrepresentative.
Hybridizations of recombinant inserts to male, female,
3E7, and Rag genomic digests produced patterns consistent
with results reported in other laboratories. About 30% of
the inserts demonstrated exclusively Y-linked fragments
while some 36% produced Y-linked fragments that were shared
with the X or autosomes (Table 2-1). Most of the remainder


PROBE
BAY 2-5
BAY 3-7A
BAY 2-9a
BAY 4-1 a
BAY 2-4a
Ml AY
F M 3 4 5 6 7 8 Y P 11 121314
ill '* m
m in
- + - ___ + + + +-
Interval 1
iljj lie
! '* til-
- - + + ~ + + + -
Interval 2
- 1-4
3.6 (EcoRI)
+ -- --+ + + 4- -
Interval 3
Figure 2-3


72
Probe
Human
ChimDanzee Gorilla
Oranautan
2-10
2.65
2.65
2.65
10
4-1a
1.65
1.65
1.65
1.65
33b
4.6, 3.2
11.5, 3.8
13, 5.5
1.9
1.9
1.8
1.8, 1.5
3-1
0.9
1.3
1.3
0.9
3-11a
1.3
12.8
1.85, 1.2
2.2
2-7a
4.4
4.3
4.5
4.3
2.65, 2.0
1.9, 1.8
0.8
0.8
0.8
0.8
1-8b
1.1
1.1
1.1
0.95
0.95
0.95
0.95
2-11a
7
7
7
z
3-8
1.5
3.5
3.5
1.5
1F5
5.0
5.0
5.0
2.5, 1.8
49f
1.6
1.6
1.6
2.8, 2.5
2.1
Figure 3-5. Comparative evolutionary analysis of Hind III
fragments in great ape males. Hind III fragment sizes
detected in male but not female DNA by the probes listed at
the left are indicated for each of the great ape species.
Probes are ordered from proximal to distal corresponding to
the orders of the intervals into which each falls on the
human Y. Common fragments in male chimpanzee and gorilla or
male chimpanzee, gorilla and orangutan are underlined.


2
14 kilobases proximal to the pseudoautosomal boundary
(Sinclair et al. 1990). Other Y chromosome-specific
sequences extend to the centromere then distally to the
heterochromatic region. DNA sequences are interspersed
among the Y-linked sequences that exhibit varying degrees of
homology with the X chromosome and/or the autosomes (Bishop
et al.. 1984; Cooke et al., 1984; Page et al., 1984; Wolfe
et al., 1984; Geldwerth et al.. 1985; Koenig et al., 1985;
Bickmore and Cooke, 1987). DXYS1 and related single-copy
sequences have been demonstrated to share blocks of homology
with the human X chromosome (Page et al., 1982; 1984; Koenig
et al.. 1984; 1985; Cooke et al.. 1984) that in one case
extend for 50 kb (Bickmore and Cooke, 1987). Alu- and Kpn-
related repetitive elements are also found along the Y
chromosome (Smith et al., 1987). The heterochromatic region
of the human Y chromosome lies distal to these predominantly
low-copy number sequences and extends to the telomere of the
long arm. The heterochromatic region, constituting 50-70%
of the total length of the human Y chromosome (Golomb and
Bahr, 1971), contains two sets of Y-specific repeats. These
repeats appear in agarose gels as 3.4 and 2.1 kb bands in
male DNA digested with the restriction endonuclease Hae III
and are absent from similarly prepared female DNA (Cooke,
1976). As a consequence of varying numbers of copies of
these Y-specific repeat families, the heterochromatic region


interval were included in the analyses. These were used as
probes for Southern blots of genomic DNA generated from
rhesus monkeys, cattle, and mice. Only GMGY1 was conserved
in all species studied, and may represent a newly arrived
pseudogene, as it was Y-linked only in humans. All of the
other sequences were conserved on the Y chromosomes of the
great apes, but were absent from rhesus monkey genomes. The
association of these sequences with the Y chromosomes of the
great apes is hypothesized to be related to their marked
cytogenetic similarities.
A long-range molecular map of the most distal
euchromatic interval was produced that covers approximately
1600 kb, of which some 1000 kb are from that interval. The
map was oriented by mapping a probe from the next most
proximal interval such that the centromeric and telomeric
ends were distinguished. No HTF islands were observed over
the entire length of the map. Instead, sites for
infrequently cleaving restriction endonucleases were
randomly distributed throughout the region. No sites for
the enzyme Not I were noted. The long-range mapping
analyses indicated that this region of the Y chromosome is
heavily methylated.
x


15
sodium citrate, pH 7.0), 0.1% SDS at 27C overnight,
followed by washing at 4C in 4X SSC, 0.1% SDS, and 2X SSC,
0.1% SDS, and finally at 27C in 2X SSC, 0.1% SDS (Melmer
and Buchwald, 1989). Two additional screenings were
performed with 12 nt degenerate oligonucleotides:
TNGCGGCCGCNN; and a mixture of oligonucleotides
ANGCGGCCGCNN, TNGCGGCCGCNN, GNCGCGGCCGCNN, CNGCGGCCGCNN.
Hybridization conditions were the same except that the
hybridization and final washes were at 37C, room
temperature, and 37C, respectively (Melmer and Buchwald,
1989). Filters were wrapped in plastic wrap and exposed to
Kodak XAR-5 film at -70C. After autoradiography, plates
were aligned with films and positively hybridizing plaques
were removed and placed in 1 ml of SM (0.1 M NaCl, 0.01 M
MgS04, 0.05 M Tris, 0.01% gelatin, pH 7.5) as described in
Maniatis et al. (1982). The filters were stripped of
oligonucleotide probe in a solution of 0.IX SSPE (0.15 M
NaCl, 0.01 M NaH2P04, 1 mM EDTA, pH 7.4) and 0.5% SDS at
90C, and were rehybridized with radiolabeled total human
genomic DNA in 6X SSPE, 0.2% SDS, 2% Blotto (10% (w/v)
Carnation Non-Fat Dry Milk in water, plus 1% (w/v) sodium
azide as a preservative) at 60C overnight. Final washes
were at 60C in 2X SSPE, 0.1% SDS, followed by
autoradiography at -70C. Duplicate filters were aligned
again with their plates and only those plaques with no
hybridization signal (designated BAY1-1 to BAY4-12) or with


120
Verma, R. and Babu, A. 1989. "Human Chromosomes Manual of
Basic Techniques". Pergamon Press, Inc., New York.
Weber, B., Weissenbach, J., Schempp, W. 1987. Conservation
of human-derived pseudoautosomal sequences on the sex
chromosomes of the great apes. Cytogenet Cell Genet
45:26-29.
Weber, B., Weissenbach, J., Schempp, W. 1988. X-Y crossing
over in the chimpanzee. Hum Genet 80:301-303.
Whisenant, E., Rasheed, B. K. A., Ostrer, H., Bhatnagar, Y.
M. 1991. Evolution and sequence analysis of a human Y-
chromosomal DNA fragment. J Mol Evol 33:133-141.
Wolf, S., Migeon, B. 1982. Studies of X chromosome DNA
methylation in normal human cells. Nature 295:667-671.
Wolfe, J., Darling, S., Erickson, R., Craig, I., Buckle, V.
Rigby, P., Willard, H., Goodfellow, P. N. 1985.
Isolation and characterization of an alphoid
centromeric repeat family from the human Y chromosome.
J Mol Biol 182:477-485.
Wolfe, J., Erickson, R., Rigby, P., Goodfellow, P. N. 1984.
Regional localization of 3 Y-derived sequences on the
human X and Y chromosomes. Ann Hum Genet 48:253-259.
Yang, T., Hansen, S., Oishi, K., Ryder, O., Hamkalo, B.
1982. Characterization of a cloned repetitive DNA
sequence concentrated on the human X chromosome. Proc
Natl Acad Sci USA 79:6593-6597.
Yen, P., Marsh, B., Allen, E., Tsai, S. P., Ellison, J.,
Connolly, L., Neiswanger, K., Shapiro, L. 1988. The
human X-linked steroid sulfatase gene and a Y-encoded
pseudogene: evidence for an inversion of the Y
chromosome during primate evolution. Cell 55:1123-1135
Yunis, J., Prakash, 0. 1982. The origin of man: a
chromosomal pictorial legacy. Science 215:1525-1529.
Zeuthen, E., Nielsen, J. 1973. Pericentric Y inversion in
the general population. Humangenetik 19:265-270.


63
degree of sequence homology by conservation of restriction
sites (Table 3-1).
Several patterns were observed from the hybridizations,
the results of which are summarized in Table 3-1. All of
the probes recognized male-specific bands in all three great
ape species with at least one enzyme. No bands were seen in
female DNA from any species. BAY2-10 and BAY2-7a recognized
common male bands (10%) in chimpanzee, gorilla, and often
orangutan, using more than one enzyme. Five probes
(BAY2-10, BAY33b, BAY4-la, BAY2-7a, and BAYl-8b) detected
common male bands in all species with at least one enzyme.
Two probes, BAYl-8b and BAY2-7a, appeared to be single or
low-copy sequences by EcoR I and Hind III analyses, but in
hybridizations of Southern blots generated by BamH I and
Pst I they detected multiple bands, indicating that these
sequences may be low-level repeats (see Figure 3-1, BamH I
genomic digest probed with BAYl-8b). The regional mapping
analyses localized both of these sequences to the sixth
interval defined by the panel, indicating that the repeats
are located within a specific region. BAY3-1 detected
fragments of similar sizes for EcoR I in human, chimpanzee
and gorilla, but fragments of different sizes with Hind III.
Y-linked fragments were also detected in orangutan in both
cases. BAY3-lla detected only Y-linked fragments of
different sizes in all four species with one enzyme,
Hind III.


112
Bickmore, W. Cooke, H. 1987. Evolution of homologous
sequences on the human X and Y chromosomes, outside of
the meiotic pairing segment. Nuc Acids Res 15:6261-
6271.
Bird, A. 1986. CpG-rich islands and the function of DNA
methylation. Nature 321:209-213.
Bird, A. 1987. CpG islands as gene markers in the vertebrate
nucleus. Trends Genet 3:342-347.
Bird, A., Taggart, M., Frommer, M., Miller, O., Macleod, D.
1985. A fraction of the mouse genome that is derived
from islands of nonmethylated, CpG-rich DNA. Cell
40:91-99.
Bird, A., Taggart, M., Nicholls, R., Higgs, D. 1987. Non
methylated CpG-rich islands at the human a-globin
locus: implications for evolution of the a-globin
pseudogene. EMBO 6:999-1004.
Bishop, C., Guellaen, G., Geldwerth, D., Fellous, M.,
Weissenbach, J. 1984. Extensive sequence homologies
between Y and other human chromosomes. J Mol Biol
173:403-417.
Bishop, C., Guellaen, G., Geldwerth, D., Voss, R., Fellous,
M., Weissenbach, J. 1983. Single-copy DNA sequences for
the human Y chromosome. Nature 303:831-832.
Bobrow, M. 1985. Heterochromatic chromosome variation and
reproductive failure. Expl Clin Immunogenet 2:97-105.
Brown, W. R. A. 1988. A physical map of the human
pseudoautosomal region. EMBO 7:2377-2385.
Brown, W. R. A., Bird, A., 1986. Long-range restriction site
mapping of mammalian genomic DNA. Nature 322:477-481.
Buckle, V., Mondello, C., Darling, S., Craig, I.,
Goodfellow, P. N. 1985. Homologous expressed genes in
the human sex chromosome pairing region. Nature
317:739-741.
Bhler, E. 1985. Clinical and cytologic impact of Y-
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Clinical Aspects of Y Chromosome Abnormalities", pp.
61-93. Alan R. Liss, Inc, New York.
Burgoyne, P., 1982. Genetic homology and crossing over in
the X and Y chromosomes of mammals. Hum Genet 61:85-90.


48
Table 2-2. Genome
Data Base D-number assignments
PROBE
BAY2-10
BAY4-la
BAY 3-1
BAY3-lla
BAY2-7a
BAYl-8b
BAY2-lla
BAY3-8
BAY 33b
LOCUS
DYS187
DYS188
DYS189
DYS190
DYS191
DYS192
DYS193
DYS194
DYS195


80
azoospermia factor. The five sequences mapping to this
interval were used to develop the map, and probe 4B-2
(DYS15) was included because it detected significant
restriction fragment overlap with distal sequences, and was
therefore useful in distinguishing the centromeric and
telomeric ends of the map.
Materials and Methods
High Molecular Weight DNA Preparation
Lymphoblastoid cell cultures or separated peripheral
blood lymphocytes (PBL) were counted using a Neubauer
hemocytometer after washing one to two times with SE (75 mM
NaCl, 25 mM EDTA, pH 7.4). Lymphoblastoid cultures were
derived from a normal male and Oxen, a cell line derived
from a male with four Y chromosomes, 49,XYYYY (Sirota et
al., 1981). Female control cells were obtained from
peripheral blood lymphocytes from normal female donors.
Single cell suspensions were prepared at 1-2 x 106 cells per
50 jul SE at room temperature, which yielded about 10 /g of
DNA per agarose block. A 1.2% solution of nucleic acid
grade agarose (Pharmacia) in SE was melted and held at about
50C and equal volumes of agarose and cell suspension
(prewarmed to 37C) were mixed and immediately dispensed
into a mold with a capacity of 100 ¡il per slot. The blocks
were allowed to solidify at 4C and then were removed from
the mold to a sterile mixture of 0.5 M EDTA, pH 9/1% (v/v)
Sarkosyl (Sigma)/2 mg per ml predigested Pronase (Boehringer


76
that are located within this region may be difficult to
identify because of a paucity of HTF islands (presented in
Chapter 4) as well as absence of conservation in other
species. The development of maps for this region in all of
these organisms will provide greater insight into the
molecular mechanisms of evolution. Because this region is
not subject to recombination, DNA sequence analysis will
provide a molecular clock to determine more precisely the
phylogenetic relationships among these organisms. Unlike
more global models, these genetic events represent fine
tuning in the evolution of sex chromosomes.


85
SDS, 5X Denhardt's, 10% dextran sulfate at 65C. Total
human genomic DNA derived from placenta and sheared to 3 00-
500 bp, was denatured and added to 30 nq/ral in both
prehybridization and hybridization solutions to block
background smears in the lanes. Post-hybridization washes
were performed at high stringency (0.1X SSPE, 0.1% SDS,
65C) and autoradiography was performed at -70C for 4-6
days with intensifying screens (Lightening Plus, DuPont).
Blots were washed in 0.1X SSPE, 0.5% SDS at 95C after
autoradiography, exposed for 48-72 hours to ensure that
residual probe was removed, sealed in plastic and reused.
Assumptions Affecting Long-Range Map Development
The map to be presented here was composed on the basis
of several assumptions. Restriction sites from single
enzyme digests were placed in the individual maps such that
the fragments would nest, following the routine
interpretation that these fragments represent partial
digestion of methylated sites (Nicholls et al.. 1989; Li et
al.. 1990; Kirkilionis et al.. 1991). Wolf and Migeon
demonstrated that multiple restriction fragments detected by
single copy probes hybridized to Southern blots of human
genomic DNA generated by methylation-sensitive enzymes
represent fixed methylation patterns that differ from cell
to cell (Wolf and Migeon, 1982). The differences in band
intensities in a lane were assumed to be related to the
percentages of cells present in the sample that bore a


CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
The studies in this dissertation present a model for
the long arm of the human Y chromosome. In Chapter 2, a
mapping panel is described that was assembled from the Human
Genetic Mutant Cell Repository (NIGMS). Using this panel, a
series of novel and previously described Y chromosomal
sequences were mapped. The cell lines in the panel divided
the Y chromosome into eight intervals, six of which are in
the euchromatic long arm. Where comparisons can be made
between this deletion map and those from other laboratories,
they are consistent with one another. These results
indicate that the panel can be used for more precise
deletion mapping of the long arm of the Y.
The results of the comparative evolutionary analyses
described in Chapter 3 were unexpected in that eleven
sequences mapping to various intervals of the human Y
chromosome were found to be retained on the Y chromosomes of
the hominid primates: chimpanzees, gorillas, and orangutans.
Although unexpected relative to sequences previously
studied, this sequence conservation appears to parallel the
cytogenetic similarities seen among higher primate Y
chromosomes. The sequences conserved on the Y chromosomes
106


45
not addressed by the data. The order of individual
sequences within an interval has not been established except
for those sequences mapping to interval 7: cenGMGY1,
BAY3-8, BAY2-lla, 1F5, 49ftel. The experiments used to
achieve this ordering are described in Chapter 4. No
distinctions can be made between the breakpoints in the cell
lines GM 0118, GM 7970, and Pf by the sequences used in this
study. The data summarized in Figure 2-5 also indicate an
apparently unequal distribution of repeat-bearing probes
along the Y chromosome. Ten probes required a preannealing
step to a Cot of 100 prior to hybridization and mapped
within the three most proximal intervals (BAY4, BAY6a,
BAY17, BAY25aB, BAY29a, BAY29b, BAY35a, BAY2-2, BAY16, and
BAY30b). The repeat-bearing probes account for about 29 kb
out of the total 53 kb covered by all probes in the region.
Only four repeat-bearing probes (BAY5, BAYlOb, BAY3c, and
BAY25aA) mapped to the three more distal intervals, while
none were present in interval 7. These four probes cover
only 6.5 kb in a total of 29 kb, or about 22% of the area
containing repeats as compared to 53% in the more proximal
intervals. Assuming that repeated elements are evenly
distributed along a segment of DNA, the probability that a
randomly isolated probe would contain such elements should
be independent of its regional location on a chromosome.
The area covered by repetitive elements in the first three
intervals was not significantly different from that expected


61
Other DNA Sources
Peripheral blood samples from a yearling cow and bull
were obtained from the University of Florida College of
Veterinary Medicine and DNA was extracted as detailed above.
Male and female mouse tail DNA samples were kindly provided
by Karen Dukes in Dr. Philip Laipis' laboratory (Department
of Biochemistry and Molecular Biology).
Southern Blotting and Hybridizations
10 to 15 nq aliguots of DNA were digested with
restriction enzymes according to the manufacturer's
recommendations. Digestion products were separated by
electrophoresis in 0.75% agarose gels overnight. Following
electrophoresis, gels were stained with 0.25 jug/ml ethidium
bromide, destained for 30 minutes to 2 hours in water,
photographed, then transferred to nylon membranes in 0.4 M
NaOH overnight. Great ape DNA digests were transferred to
either Genescreen Plus (NEN) or Hybond N+ (Amersham).
Monkey, bovine and mouse DNA digests were transferred to
Hybond N+, as were the Msp I digests used to analyze probe
GMGY1. After blotting, membranes were rinsed with 2X SSPE,
air dried for several hours or vacuum dried at 80C for 30
minutes to 2 hours.
Probe DNAs were radiolabeled with 32P dCTP (3000
Ci/mmol, ICN) by random primer extension (Feinberg and
Vogelstein, 1984) using a BRL kit. The unincorporated
nucleotides were removed using a NACS PREPAC column (BRL).


Figure 4-3. Proposed long-range map of interval 7 on the long arm of the Y chromosome.
The composite map at the top indicates the relative positions of the probes (not to
scale). Fragments detected by each probe in Mlu I and BssH II digests are arranged below
the composite map in their proposed order and relative degree of overlap. The Ksp I
fragments indicated in brackets below do not indicate their arrangements relative to one
another. Ksp I sites are not included on this composite proposed map due to an inability
to establish the relationship among individual fragments. B: BssH II, K: Ksp I, M: Mlu I.


59
1990), as was the bona fide TDF gene, SRY (Sinclair, et al.,
1990). These two genes and the pseudoautosomal sequences
were the only short arm sequences shown to have such an
evolutionary conservation on the Y chromosome.
The data on the repeated and unique sequences from
the human Y suggested a relatively recent origin of much of
the long arm of this chromosome. To date, only 4B-2, 69/6,
and 1F5 have been shown to retain their exclusive Y-linkage
in the great apes (Burk et ad., 1985; Erickson, 1987;
Whisenant et al., 1991). This chapter describes the
analyses of a series of Y chromosome-specific clones
discussed in Chapter 2. These sequences were selected for
analyses of their conservation in the great apes: chimpanzee
(Pan troglodytes), gorilla (Gorilla gorilla), and orangutan
(Pongo pvqmaeus). The five clones mapping to the most
distal euchromatic interval on the human Y long arm (see
Figure 2-5) were included because of the association of this
region with spermatogenesis in humans. Surprisingly, all
but one of the clones were exclusively Y-linked in the great
apes. Further analyses were performed on genomic DNA from
rhesus monkeys (Maccaca mulatta), cattle (Bos taurus), and
mice (Mus domesticus) with the five most distally mapping
clones. Only an autosomal allele of GMGY1 was found to be
conserved in those species as well as in the great apes.


44
the 2.1 kb BAYl-8b probe were due the previously mentioned
internal Hind III site.
The most distal interval, interval 7, in the
euchromatic region of Yq defined by this mapping panel also
contained five probes: BAY2-lla, BAY3-8, 1F5, 49f, and
GMGY1. Three of these sequences had been described in other
laboratories: 49f (Bishop et al.. 1984), GMGY1 (Affara et
al.. 1986), and 1F5 (Whisenant et al., 1991). The sequences
in this interval were detected in only one deletion cell
line, Pf, and were present in all the translocation cell
lines except GM 0118 and GM 7970. Figure 2-3c demonstrates
the two Southern blots hybridized with probe BAY3-8 which
maps to this interval.
The DYZ1 probe, Y3.4, was used as a positive control
for the presence of heterochromatin from Yql2 in the cell
lines (not shown). As seen in Figure 2-4, Y3.4 was present
in all the translocation cell lines, including GM 0118 and
GM 7970, as well as the deletion cell line, Pf. All these
cell lines had exhibited visible heterochromatin. As
expected from the cytogenetic analyses, Y3.4 was absent from
deletion cell lines GM 2668, GM 2730, and Si in which
heterochromatin was not visible.
The data from the regional mapping analyses are
summarized in Figure 2-5 and allow for the relative ordering
of the breakpoints. Distances between breakpoints, or the
physical sizes of the intervals those breakpoints bound, are


104
for islands. The test hybridizations of pulsed-field blots
with 19b also demonstrated the existence of the MIC2-
associated islands. The absence of HTF islands should not
be construed to mean that genes are likewise absent from
distal Yqll, but only that the region lacks island-
associated genes.
In addition to the unexpectedly random distribution of
double CG enzyme restriction sites, sites for Not I were
undetected. The Not I fragment recognized by probe 19b from
the pseudoautosomal region was detected using these blots,
indicating that the Not I digests and DNA transfers were
successful. The apparent absence of Not I sites might be
due either to methylation of sites in the region or to a
lack of sites.
Methylation was expected to be encountered in Yqll, so
the restriction endonuclease Ksp I was included in the
analyses because it was reported by the manufacturer,
Boehringer Mannheim Biochemicals, to be a methylation-
insensitive isoschizomer of Sac II. Southern blot analyses
of male, Oxen, and female DNA restricted with either of
these enzymes and separated in parallel on the same gel
demonstrated identical banding patterns (not shown). Mlu I,
with a recognition sequence of ACGCGT, was chosen for the
analyses in case the CG-only enzymes Not I, Ksp I, and
BssH II should detect restriction fragments of the same
sizes. Mlu I was expected to cleave somewhat more


86
particular fixed methylation pattern (Wolf and Migeon, 1982;
Goodfellow et ad., 1987; Nicholls et al.. 1989).
The extremely complex patterns detected at all loci
when digested with two enzymes were assumed to be composed
primarily of single digestion products, because most of the
bands in those double digests were present when either of
the enzymes was used singly (see Table 4-1). The sequence
in which the digestions were performed did not appear to
influence which single digestion products were produced.
For example, most of the Mlu I restriction fragments were
present in both Mlu I/BssH II and Ksp I/Mlu I digestions, in
which Mlu I was used first and last, respectively. For the
double digests, only those bands which were not seen in
either single digest were considered to be unambiguous
double digestion products.
Results
A Restriction Map of the Distal Euchromatic Long Arm of the
Y Chromosome
DNA was digested with Not I, BssH II, Ksp I, and Mlu I.
Double digests were performed using combinations of BssH II,
Ksp I, and Mlu I. Blots containing Oxen, normal male, and
normal female DNA digested with BssH II, Ksp I, and Mlu I
singly and in combination were sequentially hybridized with
the five probes from interval 7, and 4B-2 from interval 6.
Before the first use with a Yqll probe, all blots were test
hybridized with 19b, the MIC2 genomic clone mapping to the


23
chosen for the panel for two reasons. First, if GM 7970
truly retained only Yql2, it would serve as a control for Y
heterochromatin. Conversely, should GM 7970 retain a
cytogenetically indistinguishable portion of band Yqll, the
cell line would be a source of distal Yq euchromatin. Two
deletion cell lines already available in our laboratory (Pf
and Si) were added to the panel and will be submitted to
NIGMS. The final mapping panel consisted of ten cell lines:
six unbalanced translocations and four terminal deletions.
All of the translocations and one deletion, Pf, exhibited
cytogenetically detectable heterochromatin. The Y
chromosomal fragments represented in each line are depicted
in Figure 2-2. Breakpoint locations are approximations
based on the molecular data obtained in this study and do
not represent the cytogenetic resolution used to verify the
karyotypes in the various cell lines (compare Figure 2-1).
Isolation of Probes
Most of the Y chromosomal fragments utilized in this
project were novel isolates from a Y chromosome-specific
library, and six were obtained from other investigators.
Approximately 100,000 pfu were screened initially from
plates with well-isolated plaques. Duplicate filters were
hybridized first with the 8 bp oligonucleotide consisting of
the recognition sequence for the restriction endonuclease,
Not I. Fewer than ten positive signals were obtained, and
only one appeared as a duplicate signal on the second


116
Kenwrick, S., Patterson, M., Speer, A., Fischbeck, K.,
Davies, K. 1987. Molecular analysis of the Duchenne
muscular dystrophy region using pulsed field gel
electrophoresis. Cell 48:351-357.
Kirkilionis, A., Gregory, C., Hamerton, J. 1991. Long-range
restriction mapping and linkage analysis of the Prader-
Willi chromosome region (PWCR). Genomics 9:524-535.
Klebe, R., Chen, T., Ruddle, F. 1970. Controlled production
of proliferating somatic cell hybrids. J Cell Biol
45:74-82.
Koenig, M., Camerino, G., Heilig, R., Mandel, J.-L. 1984. A
DNA fragment from the human X chromosome short arm
which detects a partially homologous sequence on the Y
chromosome long arm. Nuc Acids Res 12:4097-4109.
Koenig, M., Moisan, J., Heilig, R., Mandel, J.-L. 1985.
Homologies between X and Y chromosomes detected by DNA
probes: localisation and evolution. Nuc Acids Res
13:5485-5501.
Kotecki, M., Jaruzelska, J., Skowronska, J., Fichna, P.
1991. Deletion mapping of interval 6 of the human Y
chromosome. Hum Genet 87:234-236.
Kunkel, L., Smith, K. 1982. Evolution of human Y-chromosome
DNA. Chromosoma 86:209-228.
Li, X-M., Yen, P., Mohandas, T., Shapiro, L. 1990. A long
range restriction map of the distal human X chromosome
short arm around the steroid sulfatase locus. Nuc Acids
Res 18:2783-2788.
Lindsay, S., Bird, A. 1987. Use of restriction enzymes to
detect potential gene sequences in mammalian DNA.
Nature 327:336-338.
Maniatis, T., Fritsch, E., Sambrook, J. 1982. "Molecular
Cloning: A Laboratory Manual". Cold Spring Harbor
Publishers, New York.
Melmer, G. and Buchwald, M. 1989. Identification of G/C-
containing genomic sequences by screening with
oligonucleotides. Am J Human Genet. 45:A151.
Mondello, C., Ropers, H.-H., Craig, I., Tolley, E.,
Goodfellow, P. 1987. Physical mapping of genes and
sequences at the end of the human X chromosome short
arm. Ann Human Genet 51:137-143.


107
of the hominid primates differed from most sequences for
which comparative mapping data were gathered, in that most
of the sequences used in this study were exclusively Y-
linked in humans. Whereas all of these sequences are
unlikely to be protein-encoding genes, their exclusive Y-
linkage in humans and conservation on the Y chromosomes of
related species suggests some functional significance.
The long-range map presented in Chapter 4 demonstrates
that sequences within different intervals may be ordered and
that the different intervals defined by the mapping panel
may be linked to one another by long-range mapping
techniques. The random distribution of infrequently
cleaving restriction endonuclease sites seen in distal Yq
was unexpected, as was the apparent lack of sites in the
region for the enzyme, Not I. The probes from the most
distal euchromatic interval were chosen for study because of
their proximity to the region associated with the AZF
gene(s). If preliminary indications about the location of
AZF gathered from several laboratories are correct, this
locus may be contained on the Mlu I fragment bounded by
probes 4B-2 and 49f.
In future studies, the mapping panel presented in
Chapter 2 may serve two purposes. First, on the basis of
its demonstrated suitability for mapping sequences to
different intervals on Yq, the cell lines may be
incorporated into future mapping analyses. Second, probes


CHAPTER 1
INTRODUCTION
The Y chromosome is the heterogametic sex chromosome of
mammalian males. Normally, the Y chromosome is present in
the haploid state. The human Y consists of two
cytogenetically distinct regions. The genetically active
euchromatic region comprises the short and proximal long
arms. The adjacent heterochromatic region comprises the
distal long arm and is considered to be genetically inert.
Based upon electron microscopic measurements (Golomb and
Bahr, 1971), the length of the human Y chromosome is
estimated to be approximately 30 Mb (Goodfellow et ad.,
1985; Smith et al.. 1987). The pseudoautosoma1 region,
consisting of X-Y homologous sequences that pair and
exchange during meiosis (Burgoyne, 1982; Buckle et al.,
1985; Cooke et al., 1985; Simmler et al., 1985), begins at
the telomere of the short arm and extends about 2.5 Mb
toward the centromere (Pritchard et al., 1987; Brown, 1988;
Petit et al.. 1988; Rappold and Lehrach, 1988; Petit et al..
1990). The border between the pseudoautosomal region and
sequences specific to the Y chromosome contains Alu repeated
sequences (Ellis et al., 1989). The testis determining
gene, SRY, resides within this Y-specific area approximately
1


30
human Y chromosome (Goodfellow et al.. 1983); and Rag, the
mouse parental cell line from which 3E7 was produced (Klebe
et al., 1970). The optimal pattern indicating Y-linkage
consisted of one or more identical bands in the male and 3E7
lanes that were absent in the female and Rag negative
control lanes. Another pattern seen with some Y-linked
sequences demonstrated bands in female, male and 3E7 lanes,
indicative of X chromosome or autosomal linkage. Such
probes were usable for regional mapping only if at least one
band of a different molecular weight was seen in the male
and 3E7 lanes than was present in the female lane (for
example, BAY4 detected a 2.3 kb male and 3E7 Y-linked band,
as well as 3 and 1.6 kb male and female X-/autosome-linked
bands, Table 2-1). In some cases, identically sized bands
were seen in male, female and 3E7 lanes, indicating Y- and
X-/autosome-linkage. This pattern could sometimes be
resolved into different sized Y and X/autosome bands by
hybridizing the probe to genomic Southern blots generated
from other restriction enzymes such as Taq I, Msp I, or
EcoR I. One probe from this study, BAY33c, was analyzed in
this manner, as was GMGY1 (Affara et al., 1986).
Inserts from 64 recombinants produced different
patterns when hybridized to these Southern blots. Table 2-1
summarizes the observations made about the recombinants from
these preliminary analyses. Nine inserts detected no bands
in the genomic digests (BAYl-llc, BAY2-4C, BAY4-5, BAY2,


29
plates, designated BAY1-1 to BAY4-12. A third screening for
clones with infrequently cleaving restriction sites was
performed on another 80,000 pfu and filters were hybridized
with the mixed degenerate 12 bp Not I oligonucleotides.
Again, Not I sites were not detected in six putatively
positive plaques.
Human inserts in the recombinant phage were amplified
directly by PCR thereby avoiding difficulties associated
with growing and purifying large quantities of phage.
Because there were no commercially available primers
specific to the cloning site of lambda Charon 21A, primers
were designed that annealed on either side of the Hind III
site and not elsewhere in the vector. Plaques did not
necessarily contain a single population of recombinant
phage. PCR products amplified from a single plaque might
demonstrate as many as five separate bands (for example,
BAY35a-e, Table 2-1).
Characterization of Probes
Y chromosome linkage of sequences was ascertained by
hybridization of radiolabeled probes to Southern blots of
Hind Ill-digested genomic DNAs. Since the library was
produced from a complete Hind III digest, amplified inserts
should recognize genomic Hind III fragments of the same
sizes as the probes ("cognate" bands). Genomic DNA was
obtained from human male and female donors; 3E7, a mouse
hybrid cell line containing from one to four copies of the


114
Estivill, X., Farrall, M., Scambler, P., Bell, G., Hawley,
K., Lench, N., Bates, G., Kruyer, H., Frederick, P.,
Stanier, P., Watson, E., Williamson, R., Wainwright, B.
1987. A candidate for the cystic fibrosis locus
isolated by selection for methylation-free islands.
Nature 326:840-845.
Estivill, X., Williamson, R. 1987. A rapid method to
identify cosmids containing rare restriction sites. Nuc
Acids Res 15:1415-1425.
Feinberg, A., Vogelstein, B. 1984. A technique for
radiolabeling DNA restriction endonuclease fragments to
high specific activity. Analyt Biochem 137:266-267.
Fisher, E., Beer-Romero, P., Brown, L., Ridley, A., McNeil,
J., Lawrence, J., Willard, H., Bieber, F., Page, D.
1990. Homologous ribosomal protein genes on the human X
and Y chromosomes: escape from X inactivation and
possible implications for Turner syndrome. Cell
63:1205-1218.
Fitch, N., Richer, C.-L., Pinsky, L., Kahn, A. 1985.
Deletion of the long arm of the Y chromosome and review
of Y chromosome abnormalities. Am J Med Genet 20:31-42.
Geldwerth, D., Bishop, C., Guellaen, G., Koenig, M.,
Vergnaud, G., Mandel, J.-L., Weissenbach, J. 1985.
Extensive DNA sequence homologies between the human Y
and the long arm of the X chromosome. EMBO 4:1739-1743.
Golomb, H., Bahr, G. 1971. Analysis of an isolated metaphase
plate by quantitative electron microscopy. Exp Cell Res
68:65-74.
Goodfellow, P. J., Banting, G., Sheer, D., Ropers, H-H.,
Caine, A., Ferguson-Smith, M., Povey, S., Voss, R.
1983. Genetic evidence that a Y-linked gene in man is
homologous to a gene on the X chromosome. Nature
302:346-349.
Goodfellow, P. J., Darling, S., Thomas, N., Goodfellow, P.
N. 1986. A pseudoautosomal gene in man. Science
234:740-743.
Goodfellow, P. J., Darling, S., Wolfe, J. 1985. The human Y
chromosome. J Med Genet 22:329-344.


62
Hybridizations were performed for 18-24 hours in 5X SSPE, 1%
SDS, 5X Denhardt's (0.02% Ficoll, 0.02% polyvinyl
pyrrolidone, 0.02% bovine serum albumin), 50% formamide at
42C. Post-hybridization washes for the great ape blots
were in 2X SSPE, 0.1% SDS at room temperature for 20
minutes, and twice in 0.1X SSPE, 0.1% SDS at 55-60C for 30
minutes each. Final washes for the blots containing DNA
from the lower mammals and the GMGY1 Msp I blot were at
45C. Membranes were wrapped in plastic wrap and exposed to
Kodak XAR-5 film at -70C for 1-6 days. After
autoradiography, the probes were stripped from the Hybond N+
blots in a solution of 0.1X SSPE, 0.5% SDS at 95-100C for
20-30 minutes. Genescreen Plus blots proved to be
unsatisfactory for reuse despite numerous attempts to strip
probes from the blots. Wet blots were sealed in plastic
after stripping and refrigerated until the next use.
Results
Clones Mapping to the Six Most Proximal Y Intervals
BAY clones 2-10, 4-la, 33b, 3-1, 3-lla, 2-7a, and l-8b
were selected as probes for Southern blots generated from
male and female chimpanzee, gorilla, and orangutan genomic
DNA digested with Hind III. These clones represent all but
one of the six most proximal intervals (see Figure 2-5).
Except for BAY3-lla, all of these clones were used to probe
genomic Southern blots generated using EcoR I, BamH I, Taq
I, Pst I, Bgl II, and/or Sst II in order to estimate the


5
containing the human Y as the only detectable human
chromosome (Bishop et al.. 1983; Wolfe et al., 1984).
Y-linked sequences isolated from these libraries have fallen
into several categories: some are exclusively Y-linked, and
some have homologues on both the X and Y chromosomes. Some
have homologues on the Y and autosomes, whereas a small
minority have homologues on the X, Y, and autosomes (Bishop
et al.. 1984; Affara et al., 1986; Oosthuizen et al.. 1990;
Nakahori et al.. 1991).
The mammalian X and Y chromosomes are thought to have
originated from a homologous pair of chromosomes (Polani,
1982) The terminal short arms of the X and Y chromosomes
pair and exchange sequences during meiosis, possibly a
remnant of that homology. These pseudoautosomal sequences
in humans are conserved on the sex chromosomes of the great
apes (chimpanzees, gorillas, and orangutans) (Weber et al.,
1987; 1988). Nearly half of randomly isolated single-copy
Y-linked sequences share homologies with the X chromosome
outside the pairing region. A number of human Y-linked
sequences from both the short and long arms have been
studied and most are conserved in the great apes, but are
not Y-linked (Page et al.. 1984; Bickmore and Cooke, 1987;
Koenig et al., 1985; Erickson, 1987; Burk et al., 1985).
The human male-specific repetitive elements from the
heterochromatic region of the long arm are conserved in
these hominid primates, but are autosomally-linked rather


16
only a faint gray signal (designated from BAY1 to BAY50)
were removed and placed in 1 ml SM at 4C. Plate lysates
were prepared from 10 /l of the SM solution as described in
Maniatis (1982). Following overnight incubation, 5 ml of SM
was poured on to the surfaces of the plates. These were
incubated overnight at 4C, then tilted and the phage-
containing SM was removed. After centrifugation to remove
bacterial particulates, the supernatants were treated with
0.3% chloroform and stored at 4C prior to insert
amplification. Aliquots of the supernatants were stored at
-70C with DMSO added to 7%.
Insert Amplification from Phage
Human DNA inserts in recombinant phages were directly
amplified using the polymerase chain reaction (PCR). Single
stranded DNA primers 24 nt in length were constructed that
would anneal specifically to the immediate right and left
sides of the single Hind III cloning site in lambda Charon
21A; thus the PCR reaction would amplify the human DNA
insert flanked on either side by a short stretch of lambda
DNA. The primer seguences were synthesized by the DNA Core
Facility at the University of Florida Health Science Center.
The sequences were: 5'AGA AGA GTT AGT TGA CTA TAC AGC-31
(right side of Hind III site), and 5'ATG TTT GAA TGT GAT AAC
CGT CCT-31 (left side of Hind III site). PCR reactions were
performed in a Perkin Elmer Cetus Thermal Cycler in 0.5 ml
microcentrifuge tubes in volumes of 100 ¡il under mineral


CHAPTER 4
LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME
Introduction
The long arm of the human Y chromosome is hemizygous in
males and therefore does not normally participate in
recombination during meiosis. The only technique currently
available for long-range mapping of such a non-recombinant
region is pulsed field gel electrophoresis, PFGE. This
technique used in conjunction with enzymes that cleave
infrequently in mammalian genomes, allows the analysis of
DNA fragments up to 9,000 kb (Schwartz and Cantor, 1984;
Barlow and Lehrach, 1987). The restriction endonucleases
used for this technique have recognition sites that tend to
be clustered in HTF islands. The sites are G + C rich and
contain one or more CpG dinucleotides. When HTF islands
were first described (Cooper et al., 1983; Bird et al..
1985), they were hypothesized to be associated with genes.
Almost all cleavage by CG enzymes occurs in HTF islands
(Brown and Bird, 1986) which are now known to mark many
genes in mammalian genomes (Bird et al., 1987; Abe et al.,
1988; Pontarotti et al.. 1988; Goodfellow et al., 1988;
Sargent et al., 1989). The most useful enzymes contain only
77


58
When anonymous unique-sequence elements began to be
cloned, many were found to recognize homologous sequences on
the X chromosome, but outside the putative pairing region
(Bishop et al., 1983, 1984; Cooke et al. 1984). The
presence of such X-Y homologous sequences was interpreted as
evidence supporting the primordial homology between the sex
chromosomes (Bishop et al., 1984) until the species
conservation of these X-Y homologous sequences was reported.
These studies indicated that the sequences had recently
arrived on the human Y chromosome from primate X
chromosomes, and therefore were not indicators of an ancient
homology between the X and Y (Page et al., 1984; Koenig et
al.. 1984; Cooke et al., 1984). The cloning of separate X-
specific and Y-specific centromeric repeat sequences further
complicated the issue of the degree of X-Y homology (Yang et
al., 1982; Wolfe et al., 1985) and the very existence of the
pairing region predicted by Burgoyne was questioned (Ashley,
1984). The cloning of the predicted pseudoautosomal
sequences was soon reported in a series of papers (Cooke et
al. 1985; Simmler et al. 1985; Buckle et al.. 1985) and
the sequences were later shown to be conserved on the sex
chromosomes of the hominid primates: chimpanzee, gorilla,
and orangutan (Weber et al., 1987; 1988). A candidate gene
for the testicular determining factor, ZFY, was shown to be
conserved on the Y chromosomes of all eutherian mammals
tested (Schneider-Gadicke, et al., 1989; Palmer et al.,


99
The distance between the endmost loci of the map
presented in Figure 4-3 is approximately 820 kb, with the
entire map covering some 1600 kb of distal Yqll. Because of
the complexity of the Southern blot data compiled in Table
4-1, the single digest products are included below the
composite map in Figure 4-3. The fragments produced by each
enzyme are drawn beneath the probes which detect them.
Several trial maps were generated for each of the
single enzyme digestion products. The ordering of the loci
relative to one another was dictated by the Mlu I and
BssH II fragments. The Ksp I fragments were not useful for
ordering the loci because of the absence of shared fragments
detected at different loci (see Table 4-1). Several
orientations for some loci were suggested by the BssH II
data, but only one was consistent with the linkage required
by the Mlu I data. Once the probe order was established,
the individual maps were aligned. Initially the BssH II
fragments appeared to be explainable by at least two maps
which differed in overall length, but scale drawings of each
could not be aligned in such a manner that all probes would
coexist on an 820 kb Mlu I fragment. An early version of
the BssH II map placed 4B-2 and GMGY1 approximately 180 kb
closer to BAY3-8 and BAY2-lla. This placement was
inconsistent with the presence of the 630 kb Mlu I fragment
detected by BAY3-8 and BAY2-lla that was not detected by any
of the other probes. The presence of this fragment placed


68
HSA GGO PPY PTR
fmfmfmfm
Figure 3-2. Conservation of Y-linked sequences homologous
to BAY3-8 in great apes. EcoR I-digested DNA was
electrophoretically separated and transferred to Genescreen
Plus. The Southern blot was hybridized to probe BAY3-8.
DNA sources are as follows: f female, m male. Human, HSA;
gorilla, GGO; orangutan, PPY; chimpanzee, PTR. Band sizes
in kb are indicated at the right.


ABBREVIATIONS
bp
Base pair(s)
BSA
Bovine serum albumin
dCTP
Deoxycytidine triphosphate
DMSO
Dimethylsulfoxide
dNTPs
Deoxynucleotide triphosphates
DTT
Dithiothreitol
EDTA
Ethylenediaminetetraacetate
Kb
Kilobases
Mb
Megabases
nt
Nucleotide(s)
PBS
Phosphate-buffered saline
PMSF
Phenylmethylsulfonylfluoride
rpm
Revolutions per minute
SDS
Sodium dodecyl sulfate
SSC
Saline sodium citrate
SSPE
Saline sodium phosphate EDTA
TE
Tris-EDTA buffer
viii


103
loci studied (Kotecki et al. 1991; Nakahori et al., 1991).
To date, euchromatic loci have not been detected distal to
49f, which may place this locus at the distal end of the
region associated with the AZF gene. If 49f is truly distal
to AZF, then 4B-2 and 49f would bound that locus, indicating
that the gene may lie on the 820 kb Mlu I fragment common to
49f and 4B-2.
Clusters of infrequently cleaving restriction sites
associated with HTF islands are not evident in the map in
Figure 4-3. Restriction sites for the endonucleases used in
this study appear to be distributed randomly throughout the
region. None of the infrequently cleaving restriction
enzymes detected distributions of fragments similar to those
produced by the others; thus indicating that HTF islands
were not in the vicinity. This finding is unlike the
prediction by Brown and Bird (1986) that most sites for
infrequently cleaving endonucleases should be concentrated
into HTF islands and not be distributed randomly. Long-
range restriction maps constructed from the short arm of the
Y and from other chromosomes bear out their prediction of
the island association of these sites (Pritchard et al.,
1987; Sargent et al., 1989; Li et al., 1990; Henke et al.,
1991; Kirkilionis et al., 1991). As expected for sequences
associated with islands, both 1F5 and 49f detected common
non-Y-linked fragments in Ksp I and BssH II digests (see
Figure 4-1, e and f) and thereby served as internal controls


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS
By
Beverly Steele Allen
May, 1992
Chairman: Dr. Thomas P. Yang
Major Department: Biochemistry and Molecular Biology
A deletion map of the human Y chromosome was produced
from a novel panel of Y translocation-and deletion-bearing
cell lines assembled for the purpose of mapping the
euchromatic long arm. Southern blot data from 35 cloned
sequences indicate an ordered arrangement of eight
intervals, six of which are located in the euchromatic long
arm. Twenty-nine of the cloned sequences were novel
isolates from a Y chromosome-specific library. The most
distal interval was chosen for comparative evolutionary and
pulsed-field gel electrophoretic analyses because of its
likely proximity to the region whose deletion is associated
with abnormal spermatogenesis.
Twelve regionally mapped probes were chosen for studies
of their conservation in the genomes of the great apes. All
of the sequences that mapped to the most distal euchromatic
IX


93


7
physically mapping a chromosomal region using PFGE may
provisionally locate genes (Brown and Bird, 1986; Bird,
1987). These islands are rich in the dinucleotide CpG, non-
methylated and usually associated with the 5' ends of
housekeeping genes (Bird, 1986; Bird, 1987; Bird et al.,
1987; Pontarotti et al., 1988; Sargent et al.. 1989). Long-
range restriction mapping of other areas in the human genome
has been used to locate a number of genes (Hardy et al.,
1986; Bird, 1987; Bird et al. 1987; Estivill et 1987;
Sargent et al., 1989; Henke et al., 1991) and has not yet
been applied to the distal euchromatic long arm of the Y
where it may facilitate the localization of genes in the
region, especially the AZF gene(s) associated with
spermatogenesis (Human Gene Mapping 10, 1989).
In the experiments described in Chapter 2, anonymous
sequences isolated from a flow-sorted Y chromosome library
were characterized for linkage to the Y or other
chromosomes, and regionally mapped on a panel of cell lines
containing deleted or translocated Y chromosomes. A number
of probes, including those mapping to the most distal region
of the euchromatic long arm, were analyzed for their
conservation in several mammalian species. The results of
these experiments are presented in Chapter 3. Chapter 4
presents a description of the experiments used to prepare a
long-range restriction map of sequences mapping to the
distal euchromatic long arm of the Y chromosome.


Figure 4-2. Hybridization of distal Yqll probes to PFGE
Southern blots produced from digests with two restriction
enzymes. High molecular weight DNA from a 49,XYYYY
individual was digested with various combinations of
enzymes, separated by PFGE, and transferred to Hybond N+.
The blots were seguentially hybridized with seguences
mapping to the distal-most interval defined by the mapping
panel in Chapter 2 as well as probe 4B-2 from the next more
proximal interval. Lanes: 1 BssH II/Ksp I, 2 BssH II/Mlu I,
3 Ksp I/Mlu I. Probes: a 4B-2 (DYS15), b GMGY1 (DYS12), c
BAY3-8 (DYS194), d BAY2-lla (DYS193), e 1F5 (DYS128), f 49f
(DYS1). Molecular weight markers are in kb. Dots (.)
indicate fragments detected in female DNA and which are not
Y-linked.


35
presence (+) or absence (-) of the Y-linked band(s) detected
on the initial Southern blots (Figures 2-3 and 2-4).
Results obtained from hybridizing 35 Y-linked sequences
to the ten cell lines in the mapping panel are summarized in
Figure 2-4. Seven intervals in the euchromatic region of
the human Y chromosome were defined. Twelve probes detected
sequences present in Yp or proximal Yq. BAY2-5, BAY2-10,
BAY3-7A, BAY4-lb, BAY4, BAY6a, BAY17, BAY25aB, BAY29a,
BAY29b, BAY33c, and BAY35a were localized to this first
interval. These probes detected sequences in DNA from the
deletion cell lines GM 2668, Pf, and Si but in none of the
translocation cell lines. Representative examples of the Y-
linked fragments mapping to this interval are shown in
Figure 2-3a (BAY2-5 and BAY3-7A). Probe BAY3-7 detected two
Y-linked fragments, A and B, that mapped to different
intervals. The higher molecular weight fragment, A, mapped
to the most proximal interval (Figure 2-3a), whereas the
lower molecular weight fragment, B, mapped more distally
(Figure 2-3b).
No probes isolated in this study detected sequences in
panel member GM 2730, which was reported by NIGMS to retain
some portion of Yqll (see Figure 2-2). GM 2730 was a mosaic
cell line with as few as 13% of its cells retaining a Y
chromosome fragment by cytogenetic analysis. However, the
single copy male testis-determining gene, SRY, was


LIST OF FIGURES
Figure 2-1. NIGMS Y chromosome translocations and
deletions 25
Figure 2-2. Y Chromosome Mapping Panel 28
Figure 2-3. Southern blot analyses of Y regional
mapping panel 37
Figure 2-4. Hybridization of probes to DNA from
regional mapping panel 42
Figure 2-5. Summary of Yq interval mapping 46
Figure 3-1. Conservation of Y-linked sequences
homologous to BAYl-8b in great apes 64
Figure 3-2. Conservation of Y-linked sequences
homologous to BAY3-8 in great apes 68
Figure 3-3. Conservation of Y-linked sequences
homologous to BAY2-lla, 1F5, and 49f in great
apes 69
Figure 3-4. Conservation of GMGYl-like sequences ... 70
Figure 3-5. Comparative evolutionary analysis of Hind
III fragments in great ape males 72
Figure 4-1. Hybridization of distal Yqll probes to
PFGE Southern blots 91
Figure 4-2. Hybridization of distal Yqll probes to
PFGE Southern blots produced from digests with two
restriction enzymes 93
Figure 4-3. Proposed long range map of interval 7 on
the long arm of the Y chromosome 98
vii


83
the blocks into the wells. Gels were run in recirculating
0.5X TBE at 200 V at 14C for 24 hours using a ramped switch
time of 60-90 seconds. Some gel runs were performed in a
cold room at 4C with buffer recirculation and all other
conditions remaining the same. These conditions were used
to separate fragments from about 20 kb to 1600 kb. To
separate fragments from 200 kb to 5 Mb, 0.8% gels were run
at 150 V, 4C with buffer recirculation, using ramped switch
times of 90-3600 seconds for 30-36 hours.
Large genomic fragments were sized by comparison with
yeast chromosomes (Saccharomvces cerevisiae AB972, 240 kb-
1600 kb; Schizosaccharomvces pombe. 3.6 Mb-5.7 Mb [BioRad])
and lambda concatamers (FMC Bioproducts), 50 kb-400 kb. The
error involved in sizing genomic fragments above 400 kb may
be as large as 50 kb (Kenwrick et al., 1987).
Preparation of Blocks Containing Yeast Chromosomes
Haploid Saccharomvces cerevisiae strain AB972, a gift
from Dr. Maynard V. Olson, Washington University, was grown
in YEPD (1% yeast extract, 2% peptone, 2% glucose) to mid-
logarithmic phase at 30C and harvested by centrifugation.
The cells were washed twice in 50 mM EDTA, pH 8 and
resuspended to a final concentration of 5 X 109 cells per
ml. The cells were mixed volume to volume with 1% agarose
in 50 mM EDTA, pH 8 maintained at 55C, dispensed into a
mold, and allowed to solidify at 4C. Spheroplasts were
obtained from the embedded cells using the protocol of


17
oil. Reaction mixtures contained 500 ng of each primer,
Triton X-100 to 0.01%, 1/10 volume of Perkin Elmer Cetus 10X
Reaction Buffer, MgCl2 added to 3 mM, 200 /M dNTPs, 5 ¡jl 1 of
phage lysate solution and 2.5 units Taq DNA polymerase
(Perkin Elmer Cetus). Mixtures were heated to 95C for 5
minutes to break open the phage heads prior to thermocycling
at 94C for 30 seconds, 52C for 2 minutes annealing time,
and 72C for 5 minutes extension time for 35 cycles. A
final 10 minute extension at 72C was performed to extend
any incomplete reaction products. The reaction mixture was
chilled at 4C until collected. Reaction products were
extracted once with chloroform to eliminate mineral oil
overlays. The contents of each tube were separated by
electrophoresis in 0.8% low melting temperature (LMT)
agarose gels. Individual inserts were extracted from the
agarose or used directly in LMT agarose for radiolabeling.
Multiple bands amplified from a single lysate were labeled
from highest (a) to lowest molecular weight (b, c, etc) with
lower case letters (see Table 2-1).
Southern Blotting and Hybridizations
10 to 15 jLtg aliquots of DNA were digested with Hind III
or other appropriate enzymes according to the manufacturer's
recommendations. Digestion products were separated by
electrophoresis in 0.75% agarose gels overnight. Following
electrophoresis, gels were stained with 0.25 jug/ml ethidium
bromide, destained for 30 minutes to 2 hours in water, then


109
similar pattern and date of arrival (Page et al., 1984;
Koenig et al., 1985) has not been addressed. Additional
important experiments indicated by the strong conservation
of GMGYl-related sequences in mammalian species involves
investigating whether or not the autosomal allele of GMGY1
represents a functional gene.
A long-range map that includes the more proximal
intervals may demonstrate the presence of HTF islands or
continue to demonstrate the pattern of randomly distributed
sites for infrequently cleaving enzymes seen in the most
distal interval. It may be important to demonstrate a point
where these randomly distributed sites give way to an
arrangement of sites into islands as seen on the short arm
of the Y and on other chromosomes. Although such a long-
range genomic restriction map is subject to methylation,
maps from other chromosomes have proved useful for
comparisons to maps generated from yeast artificial
chromosomes (YACs). A YAC-derived map of the human Y
chromosome would be unaffected by the methylation
encountered on the Y chromosome at the genomic level. YACs
may indicate whether the randomly distributed infrequently
cleaving restriction sites mapped in interval 7 are present
on very few chromosomes, or that the sites are present on
all the Y chromosomes, but are not detectable at the genomic
level because of methylation.


21
numerous Y-specific bands in Taq I-digested DNA. It defines
locus DYS1. 49f was kindly provided by Dr. Jean
Weissenbach.
Probe GMGY1, a 0.8 kb EcoR I insert in pUC19, was
derived from a library constructed from a hamster-human
hybrid cell line containing the Y chromosome as the only
identifiable human chromosome (Affara et al.. 1986). In
Mspl-digested DNA, it detects a 2.4 kb Y-specific band and 2
autosomal bands of 3 kb and 1.7 kb. It defines locus DYS12.
GMGY1 was kindly provided by Dr. N. A. Affara.
Probe 1F5, a 4.1 kb EcoR I insert in pBR325, was
derived from a ACharon 21A human Y chromosome library
(LAOYNSOl, ATCC) (Whisenant, et al.. 1991). It detects a
cognate 4.1 kb Y-specific band in EcoR I-digested DNA. It
defines locus DYS128. 1F5 was kindly provided by Dr. Mohan
Bhatnagar.
Probe 4B-2, a 3.3 kb EcoR I insert in pBR322, was
derived from a library constructed from a hamster-human
hybrid cell line containing the Y chromosome as the only
identifiable human chromosome (Burk et al., 1985). It
detects a cognate 3.3 kb Y-specific band in EcoR I-digested
DNA and defines locus DYS15. 4B-2 was kindly provided by
Dr. Kirby Smith.
Probe MIAY, a 2.9 kb EcoR I-Hind III insert in pBR322,
was derived from a genomic library constructed from purified
human X chromosomes (Koenig, et al., 1984, 1985). It


73
When viewed cytogenetically, great ape Y chromosomes
appear markedly similar. To date, only three anonymous Y
chromosome DNA sequences outside the pseudoautosomal region
have been demonstrated to be conserved on the Y chromosomes
of chimpanzees, gorillas, and orangutans: 4B-2 (Burk et al.,
1985), 69/6 (Erickson, 1987), and 1F5 (Whisenant et al..
1991). The results reported here are unprecedented in that
all of the eleven Y chromosome-specific sequences studied
detected similar sequences on the Y chromosomes of the great
apes. These same sequences were not detected in male or
female DNA from rhesus monkeys, cattle, or mice. A twelfth
sequence, GMGY1, which detects autosomal and Y alleles in
human DNA, demonstrated conservation of one autosomal
homologue in all six mammalian species studied.
Transcription from the GMGY1 locus has not been reported,
but its high degree of conservation argues for some type of
functional role for the autosomal sequence. The human Y-
1inked homologue might represent a newly arrived pseudogene,
as was described for the argininosuccinate synthetase and
steroid sulfatase Y-linked fragments (Daiger et al., 1982;
Yen et al., 1988). The absence of data from the male
gorilla prevents a definitive statement about the Y-linked
allele, although data gathered to date indicate that Y-
linked sequences shared by humans and gorillas are also
shared by chimpanzees.


Figure 4-1. Hybridization of distal Yqll probes to PFGE
Southern blots. High molecular weight DNA from a 49,XYYYY
individual was digested with various enzymes, separated by
PFGE, and transferred to Hybond N+. The blot was
sequentially hybridized with sequences mapping to the
distal-most interval defined by the mapping panel in Chapter
2 as well as probe 4B-2 from the next more proximal
interval. Lanes: 1 BssH II, 2 Ksp I, 3 Mlu I. Probes: a
4B-2 (DYS15), b GMGY1 (DYS12), C BAY3-8 (DYS194), d BAY2-lla
(DYS193), e 1F5 (DYS128), f 49f (DYS1). Molecular weight
markers are in kb. Dots (.) indicate fragments detected in
female DNA and which are not Y-linked.


42
Translocations Deletions
Probes
3 E 7
9403
8 773
2 103
2 469
1 1 8
7970
Pt
Si
2668
2 730
2 5
+
-
-
-
-
-
-
+
+
+
-
/ A
2-10
+
-
-
-
-
-
-
+
+
+
-
11.3

3 7 A
+
-
-
-
-
-
+
+
+

4 -1b
+
-
.

-
+
+
+
4
+
-
-
-
-
-
-
+
+
+
-
11.2
6a
+



_
_
_
+
+
+

1 7
+
-
-
-
-
-
-
+
+
+
-
2 5a B
+
-
-
-
-
-
-
+
+
+
-
11.1
put
29a
+
-
-
-
-
-
-
+
+
+
29b
+
-
-
-
-
-
-
+
+
+

11.1
Jill
33c
+
-
-
-
-
-
-
+
+
+
-
-
35a
+
-
-
-
-
-
-
+
+
+
-
11.21
2 -9a
+
+
_
_
_
+
+
+
_
4 -1a
+
+
-
-
-
-
-
+
+
+
-

2 2
+
+
-
-
-
-
-
+
+
-
-
11.22
2 4 a
+
+
-
-
-
-
-
+
+
-
-
in
\
1 6
+
+
-
-
-
-
-
+
+
-
-
31b
33b
+
+
+
+
_
\
+
+
_
_
_
_
_
+
+


11.23
Ml AY
+
+
-
-
-
-
-
+
+
-
_
3 7 B
+
+
+
-
-
-
-
+
+

up
5
+
+
+
-
-
-
-
+
+
-

10b
+
+
+
-
-
-
-
+
+
-
-
3 1
+
+
+
+
-
-
-
+
+
-
-
3-Ha
+
+
+
+
-
-
-
+
+
-
-
3 c
+
+
+
+
-
-
-
+
+
-
-
1 -8b
+
+
+
+
+
-
-
+
+
-
-
1
2 7 a
+
+
+
+
+
-
-
+
+
-
-
1 2
3-3
+
+
+
+
+
-
-
+
+
-
-
25a A
+
+
+
+
+
-
-
+
+
-
-
4 B 2
+
+
+
+
+
-
-
+
+
-
-
||§¡|
GMGY1
+
+
+
+
+
-
-
+
-
-
-
§§§§
3-8
+
+
+
+
+
-
-
+
-
-
-
|¡¡¡¡
2 -11a
+
+
+
+
+
-
-
+
-
-
-
m¡¡|
1F5
+
+
+
+
+
-
-
+
-
-
-
f§§
0 4 9 f
+
+
+
ND
+
-
-
+
-
-
-
ill
Y 3.4
+
+
+
+
+
+
+
+
_
-j


REFERENCES
Abe, K., Wei, J.-F., Fu-Sheng, W., Hsu, Y.-C., Uehara, H.,
Artzt, K., Bennet, D. 1988. Searching for coding
sequences in the mammalian genome: the H-2K region of
the mouse MHC is replete with genes expressed in
embryos. EMBO 7:3441-3449.
Affara, N., Florentin, L., Morrison, N., Kwok, K., Mitchell,
M., Cook, A., Jamieson, D., Glasgow, L., Meredith, L.,
Boyd, E., Ferguson-Smith, M. 1986. Regional assignment
of Y-linked DNA probes by deletion mapping and their
homology with X-chromosome and autosomal sequences. Nuc
Acids Res 14:5353-5373.
Alvesalo, L., de la Chapelle, A. 1981. Tooth sizes in two
males with deletions of the long arm of the Y-
chromosome. Ann Human Genet 45:49-54.
Andersson, M., Page, D., Pettay, D., Subrt, I., Turleau, C.,
deGrouchy, J., de la Chapelle, A. 1988. Y;autosome
translocations and mosaicism in the aetiology of 45,X
maleness: assignment of fertility factor to distal
Yqll. Hum Genet 79:2-7.
Arnemann, J., Epplen, J., Cooke, H., Sauermann, U., Engel,
W., Schmidtke, J. 1987. A human Y-chromosomal DNA
sequence expressed in testicular tissue. Nuc Acids Res
15:8713-8724.
Ashley, T. 1984. A re-examination of the case for homology
between the X and Y chromosomes of mouse and man. Hum
Genet 67:372-377.
Barlow, D., Lehrach, H. 1987. Genetics by gel
electrophoresis: the impact of pulsed field gel
electrophoresis on mammalian genetics. Trends Genet
3:157-171.
Beilis, M., Pages, M., Roizes, G. 1987. A simple and rapid
method for preparing yeast chromosomes for pulse field
gel electrophoresis. Nuc Acids Res 15:6749.
Ill


96
BAY3-8) and molecular weights estimated from different
autoradiograms were comparable.
Physical Linkage of 4B-2. GMGY1. BAY3-8. BAY2-lla. 1F5. and
49f.
All probes apparently shared a common 820 kb Mlu I
fragment, simplifying the positioning of the BssH II and
Mlu I maps relative to one another. A proposed long-range
restriction map covering interval 7 and some of interval 6
on the Y chromosome long arm is presented in Figure 4-3.
This preliminary map is based upon the assumption that all
six probes detecting an 820 kb Mlu I restriction fragment
were recognizing a single fragment. Probes 4B-2, BAY2-7a,
BAY25a, and BAYl-8b, from interval 6, were hybridized to a
different set of PFGE Southern blots than that shown in
Figure 4-1 in the effort to establish physical linkage of
intervals 6 and 7 (not shown). Hybridizations of those
initial blots indicated that various Ksp I and BssH II
fragments were shared between these loci and the loci in
interval 7. 4B-2 appeared to overlap the most with loci in
interval 7 using the blots analyzed in Figures 4-1 and 4-2
and was therefore included in the mapping analyses to
indicate the directions of the centromere and telomere. In
the map presented here, the order of loci is: cenDYS15
(4B-2), DYS12 (GMGY1), DYS194 (BAY3-8), DYS193 (BAY2-lla),
DYS128 (1F5), DYS1 (49f)tel.


11
previously prepared in the laboratory were added to the
panel. Each cell line was cultured, and chromosomes were
prepared by standard techniques (Verma and Babu, 1989).
Air-dried slides with mitotic chromosomes were aged several
days before staining. For G-banding, slides were treated
with 1 ml of 0.025% trypsin-EDTA (GIBCO) in 100 ml Tyrodes
buffer (8 g/L NaCl, 1 g/L glucose, 1 g/L sodium bicarbonate,
0.05 g/L sodium phosphate monobasic, 0.2 g/L KC1) for 1-2
minutes prior to staining for 1-3 minutes in 2% Giemsa
(Harleco). Using another method for producing G-bands, some
slides were pretreated by immersion for 20 minutes in 0.1 N
HC1, and rinsed in water. The slides were then immersed for
90 minutes in 2X SSC, 50% formamide at 37C. After rinsing
in distilled water, the slides were dehydrated in 95%
ethanol. Air-dried slides were stained with Wrights stain
(Verma and Babu, 1989) mixed 1:3 with 0.6 M phosphate
buffer, pH 6.8 for 1-4 minutes. Y chromosomal
translocations and deletions were verified for each cell
line and compared to the documentation provided by NIGMS.
Only those cell lines that retained the reported Y
chromosomal translocations or deletions were used for the
mapping panel.
DNA Extraction
At least ten confluent T25 monolayer cultures or
approximately 20 ml of actively dividing lymphoblastoid
cells were harvested for DNA extraction. Lymphoblastoid


115
Goodfellow, P. J., Mondello, C., Darling, S., Pym, B.,
Little, P., Goodfellow, P. N. 1988. Absence of
methylation of a CpG-rich region at the 51 end of the
MIC2 gene on the active X, the inactive X, and the Y
chromosome. Proc Natl Acad Sci USA 85:5605-5609.
Gough, N., Gearing, D., Nicola, N., Baker, E., Pritchard,
M., Callen, D., Sutherland, G. 1990. Localization of
the human GM-CSF receptor gene to the X-Y
pseudoautosomal region. Nature 345:734-736.
Guellaen, G., Casanova, M., Bishop, C., Geldwerth, D.,
Andre, G., Fellous, M., Weissenbach, J. 1984. Human XX
males with Y single-copy DNA fragments. Nature 307:172-
173.
Hartung, M., Devictor, M., Codaccioni, J., Stahl, A. 1988.
Yq deletion and failure of spermatogenesis. Ann Genet
31:21-26.
Heilig, R., Hanauer, A., Grzeschik, K., Hors-Cayla, M.,
Mandel, J.-L. 1984. Actin like sequences are present on
the X and Y chromosomes. EMBO 3:1803-1807.
Henke, A., Wapenaar, M., van Ommen, G., Maraschio, P.,
Camerino, G., Rappold, G. 1991. Deletions within the
pseudoautosomal region help map three new markers and
indicate a possible role of this region in linear
growth. Am J Human Genet 49:811-819.
Human Gene Mapping 9. 1987. Cytogenet Cell Genet 46:1-762.
Human Gene Mapping 10. 1989. Cytogenet Cell Genet 51:1-1147.
Johnson, M., Tho, S., Behzadian, A., McDonough, P. 1989.
Molecular scanning of Yqll (interval 6) in men with
Sertoli-cell-only syndrome. Am J Obstet Gynecol
161:1732-1737.
Jones, K. 1977. Repetitive DNA and primate evolution, in
"Molecular Structure of Human Chromosomes", pp. 295-
326. Academic Press, New York.
Keitges, E., Rivest, M., Siniscalco, M., Gartler, S. 1985.
X-linkage of steroid sulphatase in the mouse is
evidence for a functional Y-linked allele. Nature
315:226-227.


3
may vary greatly in size without phenotypic effects (Bhler,
1985).
Few genes have been assigned to the human Y chromosome
on the basis of their phenotypic effects, and the existence
of most of the Y-assigned genes remains contested. SRY, the
candidate gene for testis determination, and ZFY, the
earlier candidate gene for that phenotype, are the only Y-
linked genes that have been cloned on the basis of their
chromosomal location (Sinclair et al., 1990; Page et al..
1987). A few genes that encode proteins have been cloned
without previous suspicion of their Y-linkage. MIC2, the
first such gene cloned, encodes a cell surface antigen, 12E7
(Goodfellow et al., 1986), and represents the first cloned
pseudoautosomal gene. Other genes include TSPY, a testis-
expressed sequence (Arnemann et al.. 1987), RPS4Y, a
ribosomal protein gene that has been proposed to be involved
in the etiology of Turner syndrome (Fisher et al., 1990),
and GM-CSF, the granulocyte-macrophage colony stimulating
factor gene (Gough et al., 1990). Genes provisionally
mapped to the Y on the bases of imparted phenotypes include
growth control Y (Human Gene Mapping 10, 1989; Henke et al.,
1991), tooth size (TSY) (Alvesalo and de la Chapelle, 1981),
H-Y antigen (Simpson et al., 1987), and the azoospermia
factor, AZF (Tiepolo and Zuffardi, 1976; Fitch et al., 1985;
Hartung et al., 1988). The amelogenin gene, recently
reported to be expressed from both the X and Y chromosomes,


101
probes could be explained on the map by a variety of sites.
These variations could involve the choice of left and right
restriction sites (1,2 or 2,1) and/or the placement of the
fragment along the map. The extremely intense 550 kb band
detected in BssH II/Mlu I double digests by BAY3-8 and
BAY2-lla on different blots may represent a number of
overlapping 550 kb fragments that vary with respect to their
right and left restriction sites and their relative map
positions. Observations such as these served to further
strengthen the relative placements of the three individual
maps that went to form the composite map.
The map does not address the question of why the
numerous other fragments presumably possible were not
detected. The double digest data suggest that many more
fragments than those listed in Table 4-1 were indeed
produced. In Figure 4-2, faint smears in many of the lanes
appeared to be bands. They were not included in the
analyses because their lack of intensity and resolution was
considered to be inadequate for positive identification as a
restriction fragment. Even the 780 kb BssH II fragment
detected by BAY3-8, BAY2-lla, 1F5, and 49f was barely
adequate for positive identification in the blot shown in
Figure 4-1. This band was included in the analysis because
it had produced a stronger signal in blots prepared from
Oxen cells harvested at a different time, and was therefore


100
severe restrictions on the placement of loci on the Mlu I
map which required that all loci share the previously
mentioned 820 kb fragment. This fragment was considered to
be common to all the probes because of its relatively
invariant intensity on autoradiograms of all blots analyzed
during the study. Another consideration was that the
calculated size of the area in question (6 euchromatic
intervals covering 6-10 Mb would each be approximately 1-1.6
Mb in length) would be unlikely to support two such
fragments. The proposed map also would not support another
820 kb Mlu I fragment in an overlapping orientation. As
can be seen from the products of single enzyme digestion
below the composite map, very little shifting of the single
maps relative to one another is allowed. Probably less than
a 20 kb shift in either direction would be tolerated, well
within the 10% resolution of a restriction map covering
over 1 Mb. The strength of this map lies in the consistency
of probe order and site placement imposed upon it by the
combined single digest data.
Because of the constraints imposed upon the production
of a composite map from the three single digest maps by the
Mlu I data, the double digests were needed only to
corroborate the placements of the maps relative to one
another. The double digests served to strengthen the map
again by confirming the probe order already deduced. Often,
a fragment of a particular size detected by one or more


113
Burk, R., Ma, P., Smith, K. 1985. Characterization and
evolution of a single-copy sequence from the human Y
chromosome. Mol Cell Biol 5:576-581.
Charlesworth, B. 1978. Model for evolution of Y chromosomes
and dosage compensation. Proc Natl Acad Sci USA
75:5618-5622.
Church, G. and Gilbert, W. 1984. Genomic sequencing. Proc
Natl Acad Sci USA 81:1991-1995.
Cooke, H. 1976. Repeated sequence specific to human males.
Nature 262:182-186.
Cooke, H., Brown, W. R. A., Rappold, G. 1984. Closely
related sequences on human X and Y chromosomes outside
the pairing region. Nature 311:259-261.
Cooke, H., Brown, W. R. A., Rappold, G. 1985. Hypervariable
telomeric sequences from the human sex chromosomes are
pseudoautosomal. Nature 317:687-692.
Cooke, H., Fantes, J., Green, D. 1983. Structure and
evolution of human Y chromosome DNA. Differentiation
2 3:S48-S55.
Cooke, H., Schmidtke, J., Gosden, J. 1982. Characterization
of a human Y chromosome repeated sequence and related
sequences in higher primates. Chromosoma 87:491-502.
Cooper, D., Taggart, M., Bird, A. 1983. Unmethylated domains
in vertebrate DNA. Nuc Acids Res 11:647-658.
Daiger, S., Wildin, R., Su, T.-S. 1982. Sequences on the
human Y chromosome homologous to the autosomal gene for
argininosuccinate synthetase. Nature 298:682-684.
Ellis, N. Goodfellow, P. J., Pym, B., Smith, M., Palmer,
M., Frischauf, A.-M., Goodfellow, P. N. 1989. The
pseudoautosomal boundary in man is defined by an Alu
repeat sequence inserted on the Y chromosome. Nature
337:81-84.
Ellis, N., Goodfellow, P. N. 1989. The mammalian
pseudoautosomal region. Trends Genet 5:406-410.
Erickson, R. 1987. Evolution of four human Y chromosomal
unique sequences. J Mol Evol 25:300-307.


19
Probes containing repeated sequences were preannealed
to total genomic human DNA by the method of Sealy et al.
(1983). NACS-purified probes were ethanol precipitated at
-70C for 20 minutes, pelleted, and briefly air dried.
Pellets were resuspended in TE, pH 8 and heated to 65C for
2 minutes. 25 jitl of 2OX SSC and 0.4 mg of human placental
DNA sheared to 300-500 bp were added in a final volume of
0.1 ml. The mixture was boiled for 10 minutes, chilled one
minute, and incubated for 25 minutes at 65C to a Cot of
100. This mixture was added directly to the
prehybridization solution and hybridization was performed
for 16-24 hours. Washes were performed as already
described.
Placental DNA Extraction
Freshly delivered placenta was placed on ice and
transported to the laboratory where it was washed several
times in cold PBS. One inch pieces were cut off and washed
in PBS until somewhat clear and cut to about 1 cm2.
Approximately 3-4 grams of tissue pieces were placed in a
blender with 200 ml of homogenization buffer (0.25 M
sucrose, 3 mM CaCl2, 10 mM Tris, pH 7.9, 1% Triton X-100
(v/v)) and blended on lowest speed for about 5 minutes.
Blender contents were filtered through six pieces of gauze
and the foamy solids on top were added back to the blender
along with the next batch of tissue. Blending and filtering
were repeated until the entire placenta was processed. The


10
This chapter describes the isolation and regional
mapping of 29 novel Y-linked sequences randomly isolated
from a Y chromosome-specific library. Most of the probes
chosen from the library exhibited no highly repetitive
sequences; any sequences containing such repeats were
preannealed to total human genomic DNA to facilitate their
use in the mapping analyses. Several attempts were made to
isolate CG-rich clones that might contain HTF islands
(Lindsay and Bird, 1987). Six probes that were previously
mapped to the long arm of the Y were included to correlate
the mapping data with existing information about the region.
A regional mapping panel was assembled from the Human
Genetic Mutant Cell Repository (NIGMS), in Camden, NJ. Two
cell lines that were already available in the laboratory
were included in the panel. Mapping of new and existing Y
chromosomal probes against a standard panel, such as that
introduced here, presented a means to facilitate the
production of a common physical map of the long arm of the
human Y chromosome and help in localizing genes that are
postulated to reside on Yq.
Materials and Methods
Mapping Panel Cytogenetics
A group of 10 fibroblast or lymphoblast cell lines was
purchased from the National Institute of General Medical
Science (NIGMS) Human Genetic Mutant Cell Repository,
Camden, NJ. Two additional lymphoblastoid cell lines


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INGEST IEID EFWKSUTN3_H7N84U INGEST_TIME 2014-05-23T23:28:55Z PACKAGE AA00020013_00001
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FILES


MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS
By
BEVERLY STEELE ALLEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1992


56
1985; Hartung et al., 1988). The following chapters
describe experiments centered around this most distal
euchromatic interval. In Chapter 3, the evolutionary
conservation of these five sequences will be addressed,
while Chapter 4 will address the distances between the
probes, their order on the Y chromosome at the molecular
level, and the overall size of interval 7 itself.


69
1 2
3 4
5 6
7 8
a
!
It
111
|
7
5
2.5
1.8
Figure 3-3. Conservation of Y-linked sequences homologous
to probes BAY2-lla, 1F5, and 49f in great apes. Panels are
a) BAY2-lla; b) 1F5; and c) 49f. Hind Ill-digested genomic
DNA was electrophoretically separated and transferred to
Hybond N+. The Southern blot was sequentially hybridized
with the probes. Lanes are as follows: 1 human female, 2
chimpanzee female, 3 gorilla female, 4 orangutan female, 5
human male, 6 chimpanzee male, 7 gorilla male, 8 orangutan
male. Band sizes in kb are indicated at the right.


88
Table 4-1. High molecular weight restriction fragments
detected by distal Yqll probes.
Bss
KSP
Mlu
B/K
B/M
K/M
PROBE
4B-2
420
590
820
(650)
(1Mb)
(960)
520
780
(620)
(820)
820
490
760
(420)
(780)
780
420
550
360
630
(630)
370
510
190
550
(550)
320
300
150
510
(510)
250
420
(200)
190
300
(190)
150
(40)
GMGY1
(360)
370)
820
(1Mb)
(960)
(320)
780
(820)
(820)
(250)
760
(780)
(780)
550
(590)
550
510
(550)
420
420
BAY3-8
900
(480)
820
900
(820)
(960)
(780)
780
(580)
(780)
820
f300)
630
(480)
630
780
120
120
590
630
(550)
470
440
300
120
BAY211a
900
(480)
820
900
(820)
(960)
780
780
120
(780)
820
3001
630
630
780
(200)
590
710
(550) 630
470 550
440 400
300
120


55
were laden with repetitive elements. Using a novel
subcloning vector, a Japanese group screened the EcoR I
library equivalent to the Hind III library used in this
study. They reported some 80% X, Y, or autosome shared
fragments and only 21% exclusively Y-linked sequences.
These results may be related to the library used and/or the
selection of subclones by the subcloning vector (Nakahori et
al., 1991). Randomly isolated single and low copy sequences
isolated from other flow sorted Y-DNA libraries have fallen
into similar categories to those reported here: 35% were
exclusively Y-linked, 46% were shared between the X and Y,
15% were shared between the Y and autosomes, and 4% were
shared with the X, Y, and autosomes (Bishop et al., 1984;
Affara et al., 1986; Oosthuizen et al., 1990).
Overall, the data obtained in this study indicate that
the regional mapping panel introduced here divides the
euchromatic Y chromosome long arm into six distinct
intervals. This amounts to more than twice the number of
intervals defined by Vergnaud (1986). Probes bearing
repetitive elements appear to be unequally distributed in
the long arm, with repeat-bearing sequences covering more
area in the proximal intervals than in the distal intervals.
The most distal euchromatic interval defined by the panel,
interval 7, probably lies within subband Yqll.23, the region
of the Y chromosome whose absence is associated with
spermatogenic failure (Tiepolo and Zuffardi, 1976; Bobrow,


FiSrure 2~o
cntinued
7


64
HSA GGO PPY PTR
fmfm fmfm
Figure 3-1. Conservation of Y-linked sequences homologous
to BAYl-8b in great apes. BamH I-digested DNA was separated
by electrophoresis and transferred to Genescreen Plus. The
Southern blot was hybridized to probe BAYl-8b. DNA sources
are as follows: f female, m male. Human, HSA; gorilla, GGO;
orangutan, PPY; chimpanzee, PTR. Size markers in kb are
indicated at the right.


18
photographed. The DNA was acid hydrolyzed with 0.25 M HC1
for 20 minutes, then transferred to nylon membranes
(Zetaprobe [BioRad], Genescreen Plus [NEN], or Hybond N+
[Amersham]) in 0.4 M NaOH overnight. After blotting,
membranes were rinsed with 2X SSPE, air dried for several
hours or vacuum dried at 80C for 30 minutes to 2 hours.
Zetaprobe membranes were prewashed at 65C for one hour in
0.1X SSPE, 0.5% SDS prior to the first use.
Probe DNAs were radiolabeled with 32P dCTP (3000
Ci/mmol, ICN) by random primer extension (Feinberg and
Vogelstein, 1984) using a BRL kit. The unincorporated
nucleotides were removed using a NACS PREPAC column (BRL).
Hybridizations were performed for 16-24 hours using
conditions recommended by the manufacturers or in Church
buffer (1% BSA, fraction V [Sigma], 1 mM EDTA, 0.5 M sodium
phosphate, pH 7.2, 7% SDS) (Church and Gilbert, 1984).
Post-hybridization washes were in 2X SSPE, 0.1% SDS at room
temperature for 20 minutes, and twice in 0.1X SSPE, 0.1% SDS
at 65C for 10-30 minutes each. Membranes were blotted to
remove excess fluid, wrapped in plastic wrap then exposed to
Kodak XAR-5 film at for 1-6 days at -70C. After
autoradiography, the probes were stripped from the blots
without allowing membranes to dry in a solution of 0.1X
SSPE, 0.5% SDS at 95-100C for 20-30 minutes. Wet blots
were sealed in plastic and refrigerated until the next use.


6
than Y-linked (Cooke et al.. 1982; Kunkel and Smith, 1982;
Cooke et al., 1983). These findings suggest a recent origin
for a significant portion of the long arm of the human Y
chromosome and some part of the proximal short arm as well
(Page et al., 1984; Burk et al., 1985; Bickmore and Cooke,
1987) The observation that some of the randomly isolated
human Y chromosomal sequences share homology with the X
chromosome and are located on the primate X but not Y
chromosomes, suggests that the human Y is the result of
recent sequence rearrangements (Page et al., 1984; Bickmore
and Cooke, 1987). The possibility of functional
significance has been suggested for 4B-2, the first human Y-
linked sequence found to be Y-linked in the higher primates
(Burk et al., 1985). Since then, only two other anonymous
sequences from the human Y, p69/6 and 1F5, have been shown
to be conserved on the Y chromosomes of the great apes
(Erickson, 1987; Whisenant et al., 1991).
The small size of the human Y chromosome makes it
amenable to the newly developed long-range molecular mapping
techniques. Pulsed-field gel electrophoresis (PFGE) used in
conjunction with enzymes that cleave infrequently in
mammalian genomes, allows the analysis of DNA fragments up
to 9,000 kb (Schwartz and Cantor, 1984; Barlow and Lehrach,
1987) Because of the concentration of sites for the
infrequently cleaving restriction endonucleases in regions
termed HTF (Hpa II tiny fragment) islands, the process of


Figure 2-1. NIGMS Y chromosome translocations and deletions. Metaphase chromosomes from
cultures of each NIGMS cell line were G-banded. Translocations and deletions are
indicated by t or d, respectively, below the chromosome. The arrowheads indicate
translocated or deleted Y chromosomal material.


34
BAY3la, BAY3lb, BAY3 3C, BAY3 5b, BAY35C, BAY36a) One of
these, BAY3 3c, detected Y and non-Y fragments of different
sizes when hybridized to Southern blots of Msp I-digested
genomic DNAs, and was thus included in the regional mapping
analysis. Twenty-nine of 64 recombinants produced one or
more male-specific bands that were also present in the
hybrid 3E7 and absent or of a different size in the female
lane (*, Table 2-1). These unambiguously Y-linked inserts
were included in the Y chromosome regional mapping studies.
Regional Mapping
Genomic DNA obtained from the Y translocation and
deletion cell lines and controls was digested with Hind III
and duplicate Southern blots were produced on charged nylon
membranes (Zetaprobe, Genescreen Plus, or Hybond N+).
Single blots were also produced using EcoR I and Msp I for
use with the probes obtained from other investigators
(EcoR I for MIAY, 4B-2, 1F5, Y3.4, and 49f; Msp I for
GMGY1). Probe BAY33c was also analyzed using Msp I as
mentioned previously. Southern blots on Genescreen Plus did
not produce signals as intense as those seen using Zetaprobe
and Hybond N+ (compare BAY3-7A on Genescreen Plus with
BAY2-5 on Hybond N+, Figure 2-3a, lanes 11-14, lanes F-P
were on Zetaprobe in both cases). Translocation and
deletion blots were annealed to multiple probes when
individual Y-linked fragments could be distinguished
unambiguously. Panel members were scored for either the


95
single digest blot were not the result of differing
conditions of hybridization, but that the fragments
themselves were different.
Restriction maps covering hundreds of kilobase pairs
were constructed for each enzyme in a manner analogous to
conventional restriction mapping. To establish a composite
long-range restriction map of interval 7, double digests
were performed with combinations of BssH II, Ksp I, and
Mlu I (see Table 4-1). Two blots were required to include
all enzyme combinations and controls, and two sets of these
blots were produced and sequentially hybridized with probes
from interval 7. Multiple fragments were detected by each
probe in most double digestions, however, most of these
fragments were the same sizes as fragments produced by one
or the other of the enzymes when used singly (see Table 4-1)
and were therefore assumed to be single digestion products.
Because of the complexity of the banding patterns produced
at each of the loci, the double digest data were only used
to corroborate the relative positions of the maps produced
from the single digests. Southern blot data from double
digests of the Oxen cell line are presented in Figure 4-2.
The rates of migration on the gels from which the multiple
blots were produced varied, even under standardized
conditions. Banding patterns from different autoradiograms
could be superimposed upon one another (for example, 1F5 and


52
an inversion of bands 15qll-ql2 at the top of the chromosome
15. The Yq material might, therefore, be only from the
heterochromatic region. The existence of sequences which
map more distally in Yqll cannot be ruled out, nor can the
possibility be excluded that more distally located probes
than those described here might detect euchromatic sequences
in GM 0118.
Regional mapping of 35 Y chromosomal probes, using the
ten member reference panel, indicated that seven euchromatic
intervals were distinguished along the Y chromosome
(Figures 2-4 and 2-5). Six of these intervals mapped
exclusively to the long arm of the Y. The majority of the
probes detected a single Y-linked fragment. Two, BAY3-7 and
BAY25a, detected multiple Y-linked fragments mapping to
different Y chromosome intervals (see Figures 2-3b, 2-4, and
2-5) and appeared to be similar to the multiple Y-linked
fragments that map to different intervals as previously
described for probes 50f2 (DYS7, Guellaen et al. 1984), 52d
(DYS3, Bishop et al.. 1984), and 118 (DYS8, Guellaen et al.,
1984) .
Vergnaud presented a deletion map of the human Y
chromosome in 1986 that divided the entire chromosome into
seven intervals. The majority of the DNAs in that study
were obtained from XX males, and the intervals were
concentrated on the short arm rather than the long arm.
Only two intervals were assigned to the euchromatic long


32
Table 2-1. Y chromosome linkage of BAY clones.
BAY SIZE COGNATE NONCOGNATE NON-Y REPEATS Cot
CLONE Y Y 100
l-7a
3.5
-
I
H
-
l-7b
1.2
-
I
H
-
l-8a
4
-
I
H
-
l-8b
*
2.1
-
1.2, 0.9 kb* -
-
-
1-lla
2.0
+
C
M
-
1-llb
0.8
-
+ C
M
-
1-llc
0.5




2-2
*
1
+

H
+
2-4a
*
1.4
+
-
-
-
2-4b
1.1
-
+ c
-
-
2-4c
0.5
-
-
-
-
2-5
*
4.2
+
- -

-
2-1 a.
*
4
-
3, 1 kb*
M
-
2-lb
1.3
+
C
M
-
2-9a
*
3
+
+
M
-
2-9b
1.6
+
+ C
-
-
2-10
*
3
+
- -
-
-
2-lla*
1.2
+
"7 kb* C
M
-
2-llb
0.7
+
C
M

3-1
*
1.1
+


-
3-2
4
-
I
H
-
3-3
*
3
+
-
-
-
3-7
*
3.9
+A
2.5 kbB
-
-
3-8
*
1.5
+
-
-
-
3-9
1.4
-
-
M
-
3-lla*
1.3
+



4-la
*
2.2
+



4-lb
*
1.9
+
-
-
4-5
0.9




2
1.3


H
+
3c
*
1.5
+
-
H
+
4
*
2.3
+
(3, 1.
6kb)
H
+
5
*
1.4
+
(1.2kb)
H
+
6a
*
5
+
2.3kb (3, 1.
6kb)
H
+
6b
0.6
-
-
H
+
7
3
+
+ +
H
+
9
5
-
-
H
+
10b
*
0.6
+
N
M
+
14
3
-
-
H
+
16
*
3
+
-
H
+
17
*
3
+
5kb
H
+


26
filter. All putative Not I-containing phages were subjected
to PCR to amplify the inserts, and aliquots of the inserts
were restricted with Not I and electrophoretically separated
next to uncut controls. None of the inserts was cleaved by
Not I, indicating a relatively high production of false
positives by the screening procedure.
Rehybridization of the filters with radiolabeled total
human genomic DNA was intended to distinguish between
plaques containing highly repeated sequences and those with
few or no repetitive elements. In order to visualize
signals from repeated elements on the Y chromosome, the
stringency of washing conditions was reduced by raising the
salt content to 2X SSPE and decreasing the hybridization and
wash temperatures to 60C. In the initial screening,
plaques producing a faint gray signal on autoradiographs
were removed. These clones were designated BAY1 to BAY50.
In the second screening, 80,000 pfu were plated and screened
with the single degenerate 12 bp Not I oligonucleotide in
another attempt to detect clones with infrequently cleaving
restriction sites. As in the initial screening with the
shorter oligonucleotide, fewer than ten putatively positive
clones were detected, and none was determined to contain
Not I sites after digestion of the purified inserts with
that enzyme. Selecting only those plaques producing no
signals, twelve clones were removed from each of four


84
Beilis et al. (1987) in 0.5 M NaCl, 0.25 M EDTA, 0.125 M
Tris, pH 7.5, and 0.5 M beta-mercaptoethanol. The
suspension was incubated at 37C for 6 hours and then made
1% in SDS and 2 mg/ml in Pronase and incubated at 42C for
24 hours. The blocks were rinsed three times in TE, pH 8 at
50C and stored at 4C in 0.5 M EDTA, pH 8. The chromosome
sizes for this strain provided by Dr. Olson are 240, 280,
350, 440, 590, 680, 755, 810, 840, 950, 985, 1095, 1120,
1130, and 1640 kb.
Transfer and Hybridization
Ethidium bromide stained gels were destained in water
for 30 minutes to 2 hours, photographed, and acid hydrolyzed
in 0.25 M HC1 for 20 minutes. The DNA was transferred
according to the manufacturer's instructions to Hybond N+ in
0.4 M NaOH. Before the first use with an unknown probe,
each blot was test hybridized with the pseudoautosomal probe
19b (MIC2 genomic clone), and autoradiography performed.
Each lane was examined for the known pattern of bands
detected by 19b with a particular enzyme to test for
completion of digestion and efficiency of DNA transfer.
None of the blots indicated incomplete digestion of DNA at
the MIC2 locus in any lane.
DNA probes were labeled with 32P dCTP (3000 Ci/mmol,
ICN) by random primer extension (Feinberg and Vogelstein,
1984) using a BRL kit to a specific activity of at least
1 X 109 cpm/jiig. Membranes were hybridized in 5X SSPE, 1%


ACKNOWLEDGEMENTS
I have been privileged to know a number of individuals
during my graduate education, and would like to acknowledge
them here. First, I thank my mentor, Dr. Harry Ostrer, for
his guidance, support, enthusiasm, and encouragement. My
education and training under his direction have been a
valuable and rewarding experience. Thanks also go to my co-
chairman, Dr. Tom Yang, and the members of his lab, for
helpful discussions and guidance. Special thanks go to Dr.
Harry Nick for his efforts on my behalf in coordinating my
position in the Division of Genetics with that in the
Department of Biochemistry and Molecular Biology. I would
like to thank my other committee members Dr. Philip Laipis,
Dr. Sue Moyer, and Dr. Ward Wakeland for helpful discussions
and encouragement.
In addition, I would like to acknowledge the financial
support of the Division of Genetics in the Department of
Pediatrics, and the R.C. Phillips Research and Education
Unit. I would like to thank Dr. Charles Williams, Chief of
Genetics, for his support and for the use of laboratory
space after Dr. Ostrer's departure to New York University.
Finally, I would like to acknowledge the unfaltering
love, support, encouragement, and occasional bullying of my
11


45,X/46.X,del(Y)(pter>q11:) GM 2730
Deletions
45,X/46,Xldel(Y)(per>q11.2:) GM 2668
46,X,del(Y)(pter>q11:) S
46,X,del(Y)(pter>q12:) Pf
GM
GM
GM
GM
GM
9403 46,X,-X, + der(X;Y)(Xqter>Xp22.3::Yq11.2>Yqter)
8773 46>X.t(X;Y)(Xqter>Xp22.1::Yq11.2>Yqter)
2103 4 6, X, t (X ¡ Y) (X p t e r > Xq 11:: Yq 11 > Yq te r)
2469 4 6,X,t(X;Y)(Xpter>Xq2 2::Yq11>Yqter)
118 4 6lXXl-15,*der(15),t(Y;15)(15qter>15p1::Yq11>Yqter)
Translocati
ons
to
00
GM 7970 47,XX, + der(9),t(Y¡9)(9pter>9q13::Yq12>Yqter)


74
The results of this study contrast with many earlier
findings that human Y-linked sequences, while evolutionarily
conserved, were not Y-linked in other higher primate genomes
(Page et al.. 1984; Koenig et al.. 1984, 1985; Erickson,
1987; Bickmore and Cooke, 1987). This conservation of
sequences is similar to what has been observed on the
autosomes of higher primates. The interpreters of the early
data suggested that a substantial portion of the single- and
multi-copy sequences on the human Y chromosome were acquired
during human evolution (Kunkel and Smith, 1982; Burk et al.,
1985) The proposal that the human Y chromosome is composed
primarily of newly arrived sequences is refuted by the
conservation of the exclusively Y-linked sequences described
in this chapter.
A model explaining the different Y chromosome sequence
elements might be thus. The pseudoautosomal region,
conserved on the sex chromosomes of the higher primates, and
some other X- and Y-linked genes may be the last remnants of
an ancient homologous pair of chromosomes. The sequences
detecting homologues on both the human X and Y chromosomes
outside of this pairing region and found only on the X
chromosomes of chimpanzees, gorillas, and sometimes
orangutans, may represent the sequences transposed to the
human Y after the divergence from the chimpanzee/gorilla
ancestor. Tandem duplication of repeated sequences on the
human Y also occurred after this divergence. By contrast,


TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES V
ABBREVIATIONS viii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
2 DELETION MAPPING OF THE EUCHROMATIC LONG
ARM OF THE HUMAN Y CHROMOSOME 8
Introduction 8
Materials and Methods 10
Results 22
Discussion 47
3 EVOLUTIONARY COMPARISONS OF SEQUENCES
MAPPING TO THE HUMAN Y CHROMOSOME 57
Introduction 57
Materials and Methods 60
Results 62
Discussion 71
4 LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM OF
THE HUMAN Y CHROMOSOME 77
Introduction 77
Materials and Methods 80
Results 86
Discussion 102
5 CONCLUSIONS AND FUTURE DIRECTIONS 106
iv


46
GM 2730
GM 9403
GM 2668
GM 8773
GM 2103
GM 2469
Si
1
2
3
4
5
6
7
4, 6a. 17. 25aB. 29a, 29b. 35a.
2-5, 2-10, 3-7A, 4-1b, 33c
2-9a, 4-1a
2-4a, 33b, MI AY, 2-2. 16. 30b
3-7B, 5. 10b
3-1, 311a, 3c
1-8b, 2-7a, 3-3, 25aA. 4B-2
GMGY1, 3-8, 2-11a, 1F5, 49f
GM 118, Pf, GM 7970^ 8 Y3-4
Figure 2-5. Summary of Yq interval mapping. Breakpoints of
each cell line in the panel are ordered from proximal to
distal. The sets of sequences defining each interval and
the interval number are listed to the right of the arrows.
Distances between breakpoints are not implied.


CHAPTER 3
EVOLUTIONARY COMPARISONS OF SEQUENCES MAPPING
TO THE HUMAN Y CHROMOSOME
Introduction
The mammalian sex chromosomes differ structurally and
genetically from one another, but are thought to have
evolved from a homologous pair. Recombination between these
original homologous chromosomes is postulated to have become
suppressed after another gene became involved in sex
determination. The suppression of recombination then
allowed mutations to accumulate on the proto Y, and
eventually most of the chromosome became extinct (Ellis and
Goodfellow, 1989). The existence of a homologous pairing
region, termed the pseudoautosomal region, was postulated to
allow for correct meiotic segregation and to account for the
presence of X-Y bivalents seen in meiosis (Burgoyne, 1982).
The rest of the Y became a repository of 'junk' DNA
(Charlesworth, 1978) as evidenced by the preponderance of Y-
specific repeated sequences in the long arm that constitute
50-70% of the entire chromosome (Cooke et al.. 1983; Burk et
al., 1985). These repeats contained Y-specific restriction
fragment classes that were species specific and might
indicate that Y chromosomal sequences had diverged rapidly
(Kunkel and Smith, 1982; Wolfe et al., 1985).
57


MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS
By
BEVERLY STEELE ALLEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1992

ACKNOWLEDGEMENTS
I have been privileged to know a number of individuals
during my graduate education, and would like to acknowledge
them here. First, I thank my mentor, Dr. Harry Ostrer, for
his guidance, support, enthusiasm, and encouragement. My
education and training under his direction have been a
valuable and rewarding experience. Thanks also go to my co-
chairman, Dr. Tom Yang, and the members of his lab, for
helpful discussions and guidance. Special thanks go to Dr.
Harry Nick for his efforts on my behalf in coordinating my
position in the Division of Genetics with that in the
Department of Biochemistry and Molecular Biology. I would
like to thank my other committee members Dr. Philip Laipis,
Dr. Sue Moyer, and Dr. Ward Wakeland for helpful discussions
and encouragement.
In addition, I would like to acknowledge the financial
support of the Division of Genetics in the Department of
Pediatrics, and the R.C. Phillips Research and Education
Unit. I would like to thank Dr. Charles Williams, Chief of
Genetics, for his support and for the use of laboratory
space after Dr. Ostrer's departure to New York University.
Finally, I would like to acknowledge the unfaltering
love, support, encouragement, and occasional bullying of my
11

husband, Bert, during this time. His intercession and
spiritual leadership were also greatly appreciated. I thank
our families for their love and encouragement over the
years. I also thank our sisters and brothers in Christ, the
Lees, Ahns, Barnards and many others for their prayers,
love, and understanding. My thanks go also to Dr. Catherine
Ketcham for her friendship and long-distance encouragement,
and to my shootin' buddy Kathy Mercer for fun times on the
range.
iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES V
ABBREVIATIONS viii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
2 DELETION MAPPING OF THE EUCHROMATIC LONG
ARM OF THE HUMAN Y CHROMOSOME 8
Introduction 8
Materials and Methods 10
Results 22
Discussion 47
3 EVOLUTIONARY COMPARISONS OF SEQUENCES
MAPPING TO THE HUMAN Y CHROMOSOME 57
Introduction 57
Materials and Methods 60
Results 62
Discussion 71
4 LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM OF
THE HUMAN Y CHROMOSOME 77
Introduction 77
Materials and Methods 80
Results 86
Discussion 102
5 CONCLUSIONS AND FUTURE DIRECTIONS 106
iv

REFERENCES Ill
BIOGRAPHICAL SKETCH 121
V

LIST OF TABLES
Table 2-1. Y chromosome linkage of BAY clones .... 32
Table 2-2. Genome Data Base D-number assignments ... 48
Table 3-1. Interspecies comparisons of Southern blot
data 65
Table 4-1. High molecular weight restriction fragments
detected by distal Ygll probes 88

LIST OF FIGURES
Figure 2-1. NIGMS Y chromosome translocations and
deletions 25
Figure 2-2. Y Chromosome Mapping Panel 28
Figure 2-3. Southern blot analyses of Y regional
mapping panel 37
Figure 2-4. Hybridization of probes to DNA from
regional mapping panel 42
Figure 2-5. Summary of Yq interval mapping 46
Figure 3-1. Conservation of Y-linked sequences
homologous to BAYl-8b in great apes 64
Figure 3-2. Conservation of Y-linked sequences
homologous to BAY3-8 in great apes 68
Figure 3-3. Conservation of Y-linked sequences
homologous to BAY2-lla, 1F5, and 49f in great
apes 69
Figure 3-4. Conservation of GMGYl-like sequences ... 70
Figure 3-5. Comparative evolutionary analysis of Hind
III fragments in great ape males 72
Figure 4-1. Hybridization of distal Yqll probes to
PFGE Southern blots 91
Figure 4-2. Hybridization of distal Yqll probes to
PFGE Southern blots produced from digests with two
restriction enzymes 93
Figure 4-3. Proposed long range map of interval 7 on
the long arm of the Y chromosome 98
vii

ABBREVIATIONS
bp
Base pair(s)
BSA
Bovine serum albumin
dCTP
Deoxycytidine triphosphate
DMSO
Dimethylsulfoxide
dNTPs
Deoxynucleotide triphosphates
DTT
Dithiothreitol
EDTA
Ethylenediaminetetraacetate
Kb
Kilobases
Mb
Megabases
nt
Nucleotide(s)
PBS
Phosphate-buffered saline
PMSF
Phenylmethylsulfonylfluoride
rpm
Revolutions per minute
SDS
Sodium dodecyl sulfate
SSC
Saline sodium citrate
SSPE
Saline sodium phosphate EDTA
TE
Tris-EDTA buffer
viii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MAPPING THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME:
CYTOGENETIC AND MOLECULAR CORRELATIONS
By
Beverly Steele Allen
May, 1992
Chairman: Dr. Thomas P. Yang
Major Department: Biochemistry and Molecular Biology
A deletion map of the human Y chromosome was produced
from a novel panel of Y translocation-and deletion-bearing
cell lines assembled for the purpose of mapping the
euchromatic long arm. Southern blot data from 35 cloned
sequences indicate an ordered arrangement of eight
intervals, six of which are located in the euchromatic long
arm. Twenty-nine of the cloned sequences were novel
isolates from a Y chromosome-specific library. The most
distal interval was chosen for comparative evolutionary and
pulsed-field gel electrophoretic analyses because of its
likely proximity to the region whose deletion is associated
with abnormal spermatogenesis.
Twelve regionally mapped probes were chosen for studies
of their conservation in the genomes of the great apes. All
of the sequences that mapped to the most distal euchromatic
IX

interval were included in the analyses. These were used as
probes for Southern blots of genomic DNA generated from
rhesus monkeys, cattle, and mice. Only GMGY1 was conserved
in all species studied, and may represent a newly arrived
pseudogene, as it was Y-linked only in humans. All of the
other sequences were conserved on the Y chromosomes of the
great apes, but were absent from rhesus monkey genomes. The
association of these sequences with the Y chromosomes of the
great apes is hypothesized to be related to their marked
cytogenetic similarities.
A long-range molecular map of the most distal
euchromatic interval was produced that covers approximately
1600 kb, of which some 1000 kb are from that interval. The
map was oriented by mapping a probe from the next most
proximal interval such that the centromeric and telomeric
ends were distinguished. No HTF islands were observed over
the entire length of the map. Instead, sites for
infrequently cleaving restriction endonucleases were
randomly distributed throughout the region. No sites for
the enzyme Not I were noted. The long-range mapping
analyses indicated that this region of the Y chromosome is
heavily methylated.
x

CHAPTER 1
INTRODUCTION
The Y chromosome is the heterogametic sex chromosome of
mammalian males. Normally, the Y chromosome is present in
the haploid state. The human Y consists of two
cytogenetically distinct regions. The genetically active
euchromatic region comprises the short and proximal long
arms. The adjacent heterochromatic region comprises the
distal long arm and is considered to be genetically inert.
Based upon electron microscopic measurements (Golomb and
Bahr, 1971), the length of the human Y chromosome is
estimated to be approximately 30 Mb (Goodfellow et ad.,
1985; Smith et al.. 1987). The pseudoautosoma1 region,
consisting of X-Y homologous sequences that pair and
exchange during meiosis (Burgoyne, 1982; Buckle et al.,
1985; Cooke et al., 1985; Simmler et al., 1985), begins at
the telomere of the short arm and extends about 2.5 Mb
toward the centromere (Pritchard et al., 1987; Brown, 1988;
Petit et al.. 1988; Rappold and Lehrach, 1988; Petit et al..
1990). The border between the pseudoautosomal region and
sequences specific to the Y chromosome contains Alu repeated
sequences (Ellis et al., 1989). The testis determining
gene, SRY, resides within this Y-specific area approximately
1

2
14 kilobases proximal to the pseudoautosomal boundary
(Sinclair et al. 1990). Other Y chromosome-specific
sequences extend to the centromere then distally to the
heterochromatic region. DNA sequences are interspersed
among the Y-linked sequences that exhibit varying degrees of
homology with the X chromosome and/or the autosomes (Bishop
et al.. 1984; Cooke et al., 1984; Page et al., 1984; Wolfe
et al., 1984; Geldwerth et al.. 1985; Koenig et al., 1985;
Bickmore and Cooke, 1987). DXYS1 and related single-copy
sequences have been demonstrated to share blocks of homology
with the human X chromosome (Page et al., 1982; 1984; Koenig
et al.. 1984; 1985; Cooke et al.. 1984) that in one case
extend for 50 kb (Bickmore and Cooke, 1987). Alu- and Kpn-
related repetitive elements are also found along the Y
chromosome (Smith et al., 1987). The heterochromatic region
of the human Y chromosome lies distal to these predominantly
low-copy number sequences and extends to the telomere of the
long arm. The heterochromatic region, constituting 50-70%
of the total length of the human Y chromosome (Golomb and
Bahr, 1971), contains two sets of Y-specific repeats. These
repeats appear in agarose gels as 3.4 and 2.1 kb bands in
male DNA digested with the restriction endonuclease Hae III
and are absent from similarly prepared female DNA (Cooke,
1976). As a consequence of varying numbers of copies of
these Y-specific repeat families, the heterochromatic region

3
may vary greatly in size without phenotypic effects (Bhler,
1985).
Few genes have been assigned to the human Y chromosome
on the basis of their phenotypic effects, and the existence
of most of the Y-assigned genes remains contested. SRY, the
candidate gene for testis determination, and ZFY, the
earlier candidate gene for that phenotype, are the only Y-
linked genes that have been cloned on the basis of their
chromosomal location (Sinclair et al., 1990; Page et al..
1987). A few genes that encode proteins have been cloned
without previous suspicion of their Y-linkage. MIC2, the
first such gene cloned, encodes a cell surface antigen, 12E7
(Goodfellow et al., 1986), and represents the first cloned
pseudoautosomal gene. Other genes include TSPY, a testis-
expressed sequence (Arnemann et al.. 1987), RPS4Y, a
ribosomal protein gene that has been proposed to be involved
in the etiology of Turner syndrome (Fisher et al., 1990),
and GM-CSF, the granulocyte-macrophage colony stimulating
factor gene (Gough et al., 1990). Genes provisionally
mapped to the Y on the bases of imparted phenotypes include
growth control Y (Human Gene Mapping 10, 1989; Henke et al.,
1991), tooth size (TSY) (Alvesalo and de la Chapelle, 1981),
H-Y antigen (Simpson et al., 1987), and the azoospermia
factor, AZF (Tiepolo and Zuffardi, 1976; Fitch et al., 1985;
Hartung et al., 1988). The amelogenin gene, recently
reported to be expressed from both the X and Y chromosomes,

4
may represent the TSY locus (Salido et al.. 1992).
Pseudogenes for gamma actin (Heilig et al., 1984), steroid
sulfatase (Yen et al., 1988), argininosuccinate synthetase
(Daiger et al., 1982), and several retroviral sequences
(Silver et al., 1987) also have been found to reside on the
human Y.
The molecular model of the human Y chromosome
necessarily derives from analysis of anonymous cloned
sequences rather than from classical genetic studies that
depend upon recombination between linked loci. Despite the
dearth of genes, the chromosome is well suited to molecular
genetic mapping. Most sequences have only a single Y-linked
allele because of the hemizygous nature of the chromosome.
Three methods have been used to isolate unique sequence
probes for the Y chromosome. Some Y-linked probes have been
fortuitously isolated from human cDNA or genomic DNA
libraries. While not well suited to generating large
numbers of Y-linked probes, the first unique sequence probe
shared by the sex chromosomes was isolated in this manner
(Page et al., 1982). Genomic libraries constructed from DNA
from Y chromosomes separated from the other chromosomes by a
fluorescence activated cell sorter have proven to be a more
efficient means of obtaining Y chromosome-specific probes
(Affara et al., 1986; Oosthuizen et al., 1990; Nakahori et
al., 1991). The third technique involves screening genomic
libraries constructed from human-rodent somatic cell hybrids

5
containing the human Y as the only detectable human
chromosome (Bishop et al.. 1983; Wolfe et al., 1984).
Y-linked sequences isolated from these libraries have fallen
into several categories: some are exclusively Y-linked, and
some have homologues on both the X and Y chromosomes. Some
have homologues on the Y and autosomes, whereas a small
minority have homologues on the X, Y, and autosomes (Bishop
et al.. 1984; Affara et al., 1986; Oosthuizen et al.. 1990;
Nakahori et al.. 1991).
The mammalian X and Y chromosomes are thought to have
originated from a homologous pair of chromosomes (Polani,
1982) The terminal short arms of the X and Y chromosomes
pair and exchange sequences during meiosis, possibly a
remnant of that homology. These pseudoautosomal sequences
in humans are conserved on the sex chromosomes of the great
apes (chimpanzees, gorillas, and orangutans) (Weber et al.,
1987; 1988). Nearly half of randomly isolated single-copy
Y-linked sequences share homologies with the X chromosome
outside the pairing region. A number of human Y-linked
sequences from both the short and long arms have been
studied and most are conserved in the great apes, but are
not Y-linked (Page et al.. 1984; Bickmore and Cooke, 1987;
Koenig et al., 1985; Erickson, 1987; Burk et al., 1985).
The human male-specific repetitive elements from the
heterochromatic region of the long arm are conserved in
these hominid primates, but are autosomally-linked rather

6
than Y-linked (Cooke et al.. 1982; Kunkel and Smith, 1982;
Cooke et al., 1983). These findings suggest a recent origin
for a significant portion of the long arm of the human Y
chromosome and some part of the proximal short arm as well
(Page et al., 1984; Burk et al., 1985; Bickmore and Cooke,
1987) The observation that some of the randomly isolated
human Y chromosomal sequences share homology with the X
chromosome and are located on the primate X but not Y
chromosomes, suggests that the human Y is the result of
recent sequence rearrangements (Page et al., 1984; Bickmore
and Cooke, 1987). The possibility of functional
significance has been suggested for 4B-2, the first human Y-
linked sequence found to be Y-linked in the higher primates
(Burk et al., 1985). Since then, only two other anonymous
sequences from the human Y, p69/6 and 1F5, have been shown
to be conserved on the Y chromosomes of the great apes
(Erickson, 1987; Whisenant et al., 1991).
The small size of the human Y chromosome makes it
amenable to the newly developed long-range molecular mapping
techniques. Pulsed-field gel electrophoresis (PFGE) used in
conjunction with enzymes that cleave infrequently in
mammalian genomes, allows the analysis of DNA fragments up
to 9,000 kb (Schwartz and Cantor, 1984; Barlow and Lehrach,
1987) Because of the concentration of sites for the
infrequently cleaving restriction endonucleases in regions
termed HTF (Hpa II tiny fragment) islands, the process of

7
physically mapping a chromosomal region using PFGE may
provisionally locate genes (Brown and Bird, 1986; Bird,
1987). These islands are rich in the dinucleotide CpG, non-
methylated and usually associated with the 5' ends of
housekeeping genes (Bird, 1986; Bird, 1987; Bird et al.,
1987; Pontarotti et al., 1988; Sargent et al.. 1989). Long-
range restriction mapping of other areas in the human genome
has been used to locate a number of genes (Hardy et al.,
1986; Bird, 1987; Bird et al. 1987; Estivill et 1987;
Sargent et al., 1989; Henke et al., 1991) and has not yet
been applied to the distal euchromatic long arm of the Y
where it may facilitate the localization of genes in the
region, especially the AZF gene(s) associated with
spermatogenesis (Human Gene Mapping 10, 1989).
In the experiments described in Chapter 2, anonymous
sequences isolated from a flow-sorted Y chromosome library
were characterized for linkage to the Y or other
chromosomes, and regionally mapped on a panel of cell lines
containing deleted or translocated Y chromosomes. A number
of probes, including those mapping to the most distal region
of the euchromatic long arm, were analyzed for their
conservation in several mammalian species. The results of
these experiments are presented in Chapter 3. Chapter 4
presents a description of the experiments used to prepare a
long-range restriction map of sequences mapping to the
distal euchromatic long arm of the Y chromosome.

CHAPTER 2
DELETION MAPPING OF THE EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME
Introduction
A physical map has been developed for the distal short
arm of the human Y chromosome. This area includes the
pseudoautosomal region, where sequences are exchanged
actively with the X chromosome (Cooke et al.. 1985; Simmler
et al., 1985; Goodfellow et ad., 1986), and the next most
distal interval where the testis-determining gene lies
(Sinclair et al., 1990). Other genes including GM-CSF, ZFY,
RPS4Y, TSPY, and amelogenin have been cloned from the short
arm region (Gough et al., 1990; Page et al.. 1987; Fisher et
al., 1990; Arnemann et al., 1987; Salido et al.. 1992). At
the time that these studies were undertaken, a restriction
map had not yet been constructed for the rest of the Y
chromosome. Deletion maps have been constructed with novel
Y probes, but different reference translocations and
deletions were used in each case, making comparisons
difficult (Affara et ad., 1986, Vergnaud et al., 1986,
Oosthuizen et al., 1990, Nakahori et al., 1991). Other
studies have addressed the presence or absence of Y
chromosomal sequences in various patients exhibiting
abnormalities in fertility or sexual development (Andersson
8

9
et al.. 1988; Johnson et al., 1989; Skare et al., 1990;
Kotecki et al.. 1991; Nakahori et al., 1991), again
providing no composite picture for comparative purposes.
HTF islands are located in the pseudoautosomal region
(Goodfellow et al.. 1988; Henke et al.. 1991) as well as in
the proximally located Y-specific region near the ZFY gene
(Page et ad., 1987). At the time this study was undertaken,
no information existed regarding either the presence or
distribution of these gene-associated islands on the long
arm of the Y. Identifying HTF islands represented one means
to locate genes on the long arm of the Y chromosome.
These experiments were undertaken to map the
euchromatic long arm of the human Y chromosome, Yqll. The
goals were to characterize new Y chromosomal probes, derive
a regional map for these new and previously described Y
chromosomal sequences, and to address the distribution of
HTF islands on the long arm. The anticipated result of
these mapping studies was the generation of a long-range
restriction map of the most distal interval defined by the
mapping panel. Such a restriction map might facilitate the
localization of the spermatogenesis-related gene(s) that
were provisionally mapped to the region (Tiepolo and
Zuffardi, 1976; Bobrow, 1985; Hartung et al., 1988). An
additional aim of this project was to test a newly assembled
mapping panel for the Y chromosome long arm.

10
This chapter describes the isolation and regional
mapping of 29 novel Y-linked sequences randomly isolated
from a Y chromosome-specific library. Most of the probes
chosen from the library exhibited no highly repetitive
sequences; any sequences containing such repeats were
preannealed to total human genomic DNA to facilitate their
use in the mapping analyses. Several attempts were made to
isolate CG-rich clones that might contain HTF islands
(Lindsay and Bird, 1987). Six probes that were previously
mapped to the long arm of the Y were included to correlate
the mapping data with existing information about the region.
A regional mapping panel was assembled from the Human
Genetic Mutant Cell Repository (NIGMS), in Camden, NJ. Two
cell lines that were already available in the laboratory
were included in the panel. Mapping of new and existing Y
chromosomal probes against a standard panel, such as that
introduced here, presented a means to facilitate the
production of a common physical map of the long arm of the
human Y chromosome and help in localizing genes that are
postulated to reside on Yq.
Materials and Methods
Mapping Panel Cytogenetics
A group of 10 fibroblast or lymphoblast cell lines was
purchased from the National Institute of General Medical
Science (NIGMS) Human Genetic Mutant Cell Repository,
Camden, NJ. Two additional lymphoblastoid cell lines

11
previously prepared in the laboratory were added to the
panel. Each cell line was cultured, and chromosomes were
prepared by standard techniques (Verma and Babu, 1989).
Air-dried slides with mitotic chromosomes were aged several
days before staining. For G-banding, slides were treated
with 1 ml of 0.025% trypsin-EDTA (GIBCO) in 100 ml Tyrodes
buffer (8 g/L NaCl, 1 g/L glucose, 1 g/L sodium bicarbonate,
0.05 g/L sodium phosphate monobasic, 0.2 g/L KC1) for 1-2
minutes prior to staining for 1-3 minutes in 2% Giemsa
(Harleco). Using another method for producing G-bands, some
slides were pretreated by immersion for 20 minutes in 0.1 N
HC1, and rinsed in water. The slides were then immersed for
90 minutes in 2X SSC, 50% formamide at 37C. After rinsing
in distilled water, the slides were dehydrated in 95%
ethanol. Air-dried slides were stained with Wrights stain
(Verma and Babu, 1989) mixed 1:3 with 0.6 M phosphate
buffer, pH 6.8 for 1-4 minutes. Y chromosomal
translocations and deletions were verified for each cell
line and compared to the documentation provided by NIGMS.
Only those cell lines that retained the reported Y
chromosomal translocations or deletions were used for the
mapping panel.
DNA Extraction
At least ten confluent T25 monolayer cultures or
approximately 20 ml of actively dividing lymphoblastoid
cells were harvested for DNA extraction. Lymphoblastoid

12
cells or trypsinized fibroblasts were pelleted and
resuspended in 3-5 ml of DNA extraction buffer (10 mM Tris,
pH 7.8, 2 mM EDTA, 400 mM NaCl). After addition of SDS to
0.5% and predigested Pronase (Boehringer Mannheim) to a
final concentration of 1 mg/ml, the solution was incubated
overnight at 37C. The solution was extracted with 24:24:1
phenol:chloroform:isoamyl alcohol, pH 8.0, followed by one
to two extractions with 24:1 chloroform:isoamyl alcohol.
DNA was precipitated with ice cold 95% ethanol. The DNA was
spooled onto a glass pipette and rinsed briefly with 70%
ethanol before air drying. The DNA was resuspended in 3 00-
500 /I TE, pH 8, with a drop of chloroform, then rotated
overnight at 4C. DNA concentration was calculated from the
absorbance at 260nm. Purity was also calculated from the
ratios of absorbances obtained at 260nm and 280nm
(A260/280), with a ratio of 1.8-2.0 considered to be
optimal.
Normal male and female donors provided blood samples
from which DNA was extracted. These provided controls for
the presence and absence of a Y chromosome. Blood obtained
by venipuncture into 10 ml Vacutainer tubes (Becton
Dickinson) containing 0.1 ml of 15% EDTA was centrifuged to
separate plasma and cells. The plasma was removed to just
above the buffy coat. The buffy coat and some of the
underlying red cells were transferred to 15 ml conical
centrifuge tubes. DNA extraction buffer was added to 5 ml,

13
SDS to 0.5%, and predigested Pronase to 1 mg/ml. Tubes were
incubated overnight at 37C and extracted twice with
phenol:chloroform:isoamyl alcohol and twice with
chloroform:isoamyl alcohol before ethanol precipitation as
above.
Library Screening
The methods used for phage library screening were
generally as described in Maniatis et al. (1982). Phage
were isolated from a Y chromosome-enriched library that was
obtained from the Lawrence Livermore National Laboratory
(LLOYNSOl). The library was prepared from flow-sorted human
Y chromosomes that were cut to completion with the
restriction endonuclease, Hind III. The fragments were
inserted into the Hind III cloning site of the cloning
vector, lambda Charon 21A. The insert sizes ranged up to
9 kb. The yield of independent recombinants was 2.5 x 105,
and the amplified library contained 27 genome equivalents.
The library was plated at approximately 20,000 pfu/plate on
150 mm petri dishes in three separate experiments. Host
bacteria, E. coli LE392, were cultured in NZY medium
supplemented with 0.2% maltose. Appropriate numbers of
phage were mixed with host cells and incubated at 37C for
20 minutes. Infected cells were mixed with molten (47C)
0.7% agarose, poured over the surfaces of 1.5% agar plates
and incubated for 12 to 16 hours at 37C. Plates were
allowed to cool to room temperature prior to transferring

14
phage plaques to nitrocellulose filters (Schleicher and
Schull). Marked filters were placed on plates until
thoroughly wet and plate bottoms were correspondingly
marked. A duplicate filter was laid on the plate after the
first was removed and allowed to remain for 30 seconds to 1
minute longer than the previous filter to equalize the
quantity of phage adhering to the two filters.
Corresponding marks were also made on the plate bottoms for
the second filters. Filters were dipped into denaturing and
neutralizing washes as described in Maniatis et al. (1982),
then allowed to air dry. The filters were placed between
sheets of Whatman 3MM paper and baked at 80C in a vacuum
oven for 2 hours. Duplicate filter pairs were sequentially
hybridized as described below.
Phage Selection
Duplicate filters were first screened for the presence
of clones containing GC-rich sequences. A series of
oligonucleotides containing the recognition sequence of the
restriction enzyme Not I were synthesized by the Health
Science Center DNA Core Facility. The oligonucleotides were
radiolabeled using T4 kinase (Maniatis et al., 1982) and
hybridized to duplicate filters. The first oligonucleotide
used for screening was 8 nt in length and consisted of only
the recognition sequence for the restriction endonuclease,
Not I (GCGGCCGC). Hybridization for this oligonucleotide
was performed in 6X SSC (IX SSC is 0.15 M NaCl, 0.015 M

15
sodium citrate, pH 7.0), 0.1% SDS at 27C overnight,
followed by washing at 4C in 4X SSC, 0.1% SDS, and 2X SSC,
0.1% SDS, and finally at 27C in 2X SSC, 0.1% SDS (Melmer
and Buchwald, 1989). Two additional screenings were
performed with 12 nt degenerate oligonucleotides:
TNGCGGCCGCNN; and a mixture of oligonucleotides
ANGCGGCCGCNN, TNGCGGCCGCNN, GNCGCGGCCGCNN, CNGCGGCCGCNN.
Hybridization conditions were the same except that the
hybridization and final washes were at 37C, room
temperature, and 37C, respectively (Melmer and Buchwald,
1989). Filters were wrapped in plastic wrap and exposed to
Kodak XAR-5 film at -70C. After autoradiography, plates
were aligned with films and positively hybridizing plaques
were removed and placed in 1 ml of SM (0.1 M NaCl, 0.01 M
MgS04, 0.05 M Tris, 0.01% gelatin, pH 7.5) as described in
Maniatis et al. (1982). The filters were stripped of
oligonucleotide probe in a solution of 0.IX SSPE (0.15 M
NaCl, 0.01 M NaH2P04, 1 mM EDTA, pH 7.4) and 0.5% SDS at
90C, and were rehybridized with radiolabeled total human
genomic DNA in 6X SSPE, 0.2% SDS, 2% Blotto (10% (w/v)
Carnation Non-Fat Dry Milk in water, plus 1% (w/v) sodium
azide as a preservative) at 60C overnight. Final washes
were at 60C in 2X SSPE, 0.1% SDS, followed by
autoradiography at -70C. Duplicate filters were aligned
again with their plates and only those plaques with no
hybridization signal (designated BAY1-1 to BAY4-12) or with

16
only a faint gray signal (designated from BAY1 to BAY50)
were removed and placed in 1 ml SM at 4C. Plate lysates
were prepared from 10 /l of the SM solution as described in
Maniatis (1982). Following overnight incubation, 5 ml of SM
was poured on to the surfaces of the plates. These were
incubated overnight at 4C, then tilted and the phage-
containing SM was removed. After centrifugation to remove
bacterial particulates, the supernatants were treated with
0.3% chloroform and stored at 4C prior to insert
amplification. Aliquots of the supernatants were stored at
-70C with DMSO added to 7%.
Insert Amplification from Phage
Human DNA inserts in recombinant phages were directly
amplified using the polymerase chain reaction (PCR). Single
stranded DNA primers 24 nt in length were constructed that
would anneal specifically to the immediate right and left
sides of the single Hind III cloning site in lambda Charon
21A; thus the PCR reaction would amplify the human DNA
insert flanked on either side by a short stretch of lambda
DNA. The primer seguences were synthesized by the DNA Core
Facility at the University of Florida Health Science Center.
The sequences were: 5'AGA AGA GTT AGT TGA CTA TAC AGC-31
(right side of Hind III site), and 5'ATG TTT GAA TGT GAT AAC
CGT CCT-31 (left side of Hind III site). PCR reactions were
performed in a Perkin Elmer Cetus Thermal Cycler in 0.5 ml
microcentrifuge tubes in volumes of 100 ¡il under mineral

17
oil. Reaction mixtures contained 500 ng of each primer,
Triton X-100 to 0.01%, 1/10 volume of Perkin Elmer Cetus 10X
Reaction Buffer, MgCl2 added to 3 mM, 200 /M dNTPs, 5 ¡jl 1 of
phage lysate solution and 2.5 units Taq DNA polymerase
(Perkin Elmer Cetus). Mixtures were heated to 95C for 5
minutes to break open the phage heads prior to thermocycling
at 94C for 30 seconds, 52C for 2 minutes annealing time,
and 72C for 5 minutes extension time for 35 cycles. A
final 10 minute extension at 72C was performed to extend
any incomplete reaction products. The reaction mixture was
chilled at 4C until collected. Reaction products were
extracted once with chloroform to eliminate mineral oil
overlays. The contents of each tube were separated by
electrophoresis in 0.8% low melting temperature (LMT)
agarose gels. Individual inserts were extracted from the
agarose or used directly in LMT agarose for radiolabeling.
Multiple bands amplified from a single lysate were labeled
from highest (a) to lowest molecular weight (b, c, etc) with
lower case letters (see Table 2-1).
Southern Blotting and Hybridizations
10 to 15 jLtg aliquots of DNA were digested with Hind III
or other appropriate enzymes according to the manufacturer's
recommendations. Digestion products were separated by
electrophoresis in 0.75% agarose gels overnight. Following
electrophoresis, gels were stained with 0.25 jug/ml ethidium
bromide, destained for 30 minutes to 2 hours in water, then

18
photographed. The DNA was acid hydrolyzed with 0.25 M HC1
for 20 minutes, then transferred to nylon membranes
(Zetaprobe [BioRad], Genescreen Plus [NEN], or Hybond N+
[Amersham]) in 0.4 M NaOH overnight. After blotting,
membranes were rinsed with 2X SSPE, air dried for several
hours or vacuum dried at 80C for 30 minutes to 2 hours.
Zetaprobe membranes were prewashed at 65C for one hour in
0.1X SSPE, 0.5% SDS prior to the first use.
Probe DNAs were radiolabeled with 32P dCTP (3000
Ci/mmol, ICN) by random primer extension (Feinberg and
Vogelstein, 1984) using a BRL kit. The unincorporated
nucleotides were removed using a NACS PREPAC column (BRL).
Hybridizations were performed for 16-24 hours using
conditions recommended by the manufacturers or in Church
buffer (1% BSA, fraction V [Sigma], 1 mM EDTA, 0.5 M sodium
phosphate, pH 7.2, 7% SDS) (Church and Gilbert, 1984).
Post-hybridization washes were in 2X SSPE, 0.1% SDS at room
temperature for 20 minutes, and twice in 0.1X SSPE, 0.1% SDS
at 65C for 10-30 minutes each. Membranes were blotted to
remove excess fluid, wrapped in plastic wrap then exposed to
Kodak XAR-5 film at for 1-6 days at -70C. After
autoradiography, the probes were stripped from the blots
without allowing membranes to dry in a solution of 0.1X
SSPE, 0.5% SDS at 95-100C for 20-30 minutes. Wet blots
were sealed in plastic and refrigerated until the next use.

19
Probes containing repeated sequences were preannealed
to total genomic human DNA by the method of Sealy et al.
(1983). NACS-purified probes were ethanol precipitated at
-70C for 20 minutes, pelleted, and briefly air dried.
Pellets were resuspended in TE, pH 8 and heated to 65C for
2 minutes. 25 jitl of 2OX SSC and 0.4 mg of human placental
DNA sheared to 300-500 bp were added in a final volume of
0.1 ml. The mixture was boiled for 10 minutes, chilled one
minute, and incubated for 25 minutes at 65C to a Cot of
100. This mixture was added directly to the
prehybridization solution and hybridization was performed
for 16-24 hours. Washes were performed as already
described.
Placental DNA Extraction
Freshly delivered placenta was placed on ice and
transported to the laboratory where it was washed several
times in cold PBS. One inch pieces were cut off and washed
in PBS until somewhat clear and cut to about 1 cm2.
Approximately 3-4 grams of tissue pieces were placed in a
blender with 200 ml of homogenization buffer (0.25 M
sucrose, 3 mM CaCl2, 10 mM Tris, pH 7.9, 1% Triton X-100
(v/v)) and blended on lowest speed for about 5 minutes.
Blender contents were filtered through six pieces of gauze
and the foamy solids on top were added back to the blender
along with the next batch of tissue. Blending and filtering
were repeated until the entire placenta was processed. The

20
filtrate was kept on ice throughout the processing. The
filtrate was poured into 50 ml conical centrifuge tubes and
the nuclei pelleted at 4C, 1500 rpm. Nuclei were washed
once in homogenization buffer without Triton and pooled.
Pellets were resuspended in DNA extraction buffer. SDS was
added to 0.5%, and Pronase to 1 mg/ml. This solution was
incubated overnight at 37C. The solution was extracted
once with phenol:chloroform:isoamyl alcohol and twice with
chloroform:isoamyl alcohol. RNase was added to a final
concentration of 50 /xg/ml. The solution was incubated at
37C for 2 hours. The solution was reextracted, ethanol
precipitated, and spooled onto a glass pipette tip, dipped
into 70% ethanol, and air dried briefly. The DNA was
resuspended in TE, pH 8, sonicated to about 300-500 bp, then
ethanol precipitated and resuspended to 10 or 20 mg/ml.
Y Chromosome Probes Provided by Other Investigators
In order to correlate the data produced in this study
with published information about the region, probes that
mapped to the euchromatic long arm of the human Y chromosome
were obtained from other investigators. All probes
described below are subclones into plasmids.
Probe 49f, a 2.8 kb EcoR I insert in pBR322, was
derived from a Y-enriched cosmid library (Bishop et al.,
1983, 1984). It detects a 2.8 kb Y-specific band in EcoR I-
digested DNA at high stringency ("cognate band"). At low
stringency, it detects two autosomal bands as well as

21
numerous Y-specific bands in Taq I-digested DNA. It defines
locus DYS1. 49f was kindly provided by Dr. Jean
Weissenbach.
Probe GMGY1, a 0.8 kb EcoR I insert in pUC19, was
derived from a library constructed from a hamster-human
hybrid cell line containing the Y chromosome as the only
identifiable human chromosome (Affara et al.. 1986). In
Mspl-digested DNA, it detects a 2.4 kb Y-specific band and 2
autosomal bands of 3 kb and 1.7 kb. It defines locus DYS12.
GMGY1 was kindly provided by Dr. N. A. Affara.
Probe 1F5, a 4.1 kb EcoR I insert in pBR325, was
derived from a ACharon 21A human Y chromosome library
(LAOYNSOl, ATCC) (Whisenant, et al.. 1991). It detects a
cognate 4.1 kb Y-specific band in EcoR I-digested DNA. It
defines locus DYS128. 1F5 was kindly provided by Dr. Mohan
Bhatnagar.
Probe 4B-2, a 3.3 kb EcoR I insert in pBR322, was
derived from a library constructed from a hamster-human
hybrid cell line containing the Y chromosome as the only
identifiable human chromosome (Burk et al., 1985). It
detects a cognate 3.3 kb Y-specific band in EcoR I-digested
DNA and defines locus DYS15. 4B-2 was kindly provided by
Dr. Kirby Smith.
Probe MIAY, a 2.9 kb EcoR I-Hind III insert in pBR322,
was derived from a genomic library constructed from purified
human X chromosomes (Koenig, et al., 1984, 1985). It

22
detects a 3.6 kb Y-specific fragment in EcoR I-digested DNA
and defines locus DYS22. MIAY was kindly provided by Dr.
Michael Koenig.
Probe Y3.4 is a 3.4 kb Pst I insert in pBR322. It was
derived from the 3.4 kb Hae III repeat seen in male, but not
female DNA. It defines the Y-specific repeat family DYZ1
(Cooke, 1976). Y3.4 was kindly provided by Dr. Kirby Smith.
These plasmids were transformed into appropriate host
bacteria, amplified, purified, and restricted with the
appropriate enzyme(s), separated in low melt agarose, and
extracted by standard procedures (Maniatis et al., 1982).
Results
Mapping Panel Selection
Ten cell lines were initially obtained from the Human
Genetic Mutant Cell Repository (NIGMS) in Camden, NJ. The
karyotype of each line was verified prior to inclusion of
the cell line into the mapping panel. The Y chromosomal
fragments present in each NIGMS cell line used in the panel
are shown in Figure 2-1. Two of the ten cell lines
originally obtained from NIGMS, GM 3774 and GM 3595, were
excluded from the panel. GM 3774 reportedly contained an
isochromosome Yq, but this was not verified after receipt of
the culture. Two separate cultures of GM 3595 exhibited
extremely poor growth, resulting in its exclusion from the
panel even though its reported karyotype was confirmed.
GM 7970, reported by NIGMS to contribute only band Yql2, was

23
chosen for the panel for two reasons. First, if GM 7970
truly retained only Yql2, it would serve as a control for Y
heterochromatin. Conversely, should GM 7970 retain a
cytogenetically indistinguishable portion of band Yqll, the
cell line would be a source of distal Yq euchromatin. Two
deletion cell lines already available in our laboratory (Pf
and Si) were added to the panel and will be submitted to
NIGMS. The final mapping panel consisted of ten cell lines:
six unbalanced translocations and four terminal deletions.
All of the translocations and one deletion, Pf, exhibited
cytogenetically detectable heterochromatin. The Y
chromosomal fragments represented in each line are depicted
in Figure 2-2. Breakpoint locations are approximations
based on the molecular data obtained in this study and do
not represent the cytogenetic resolution used to verify the
karyotypes in the various cell lines (compare Figure 2-1).
Isolation of Probes
Most of the Y chromosomal fragments utilized in this
project were novel isolates from a Y chromosome-specific
library, and six were obtained from other investigators.
Approximately 100,000 pfu were screened initially from
plates with well-isolated plaques. Duplicate filters were
hybridized first with the 8 bp oligonucleotide consisting of
the recognition sequence for the restriction endonuclease,
Not I. Fewer than ten positive signals were obtained, and
only one appeared as a duplicate signal on the second

Figure 2-1. NIGMS Y chromosome translocations and deletions. Metaphase chromosomes from
cultures of each NIGMS cell line were G-banded. Translocations and deletions are
indicated by t or d, respectively, below the chromosome. The arrowheads indicate
translocated or deleted Y chromosomal material.

GM 0118
GM 2103
GM 2469
GM 2668
k*GM 2730
GM 7970
GM 8773
^)GM 9403
SZ

26
filter. All putative Not I-containing phages were subjected
to PCR to amplify the inserts, and aliquots of the inserts
were restricted with Not I and electrophoretically separated
next to uncut controls. None of the inserts was cleaved by
Not I, indicating a relatively high production of false
positives by the screening procedure.
Rehybridization of the filters with radiolabeled total
human genomic DNA was intended to distinguish between
plaques containing highly repeated sequences and those with
few or no repetitive elements. In order to visualize
signals from repeated elements on the Y chromosome, the
stringency of washing conditions was reduced by raising the
salt content to 2X SSPE and decreasing the hybridization and
wash temperatures to 60C. In the initial screening,
plaques producing a faint gray signal on autoradiographs
were removed. These clones were designated BAY1 to BAY50.
In the second screening, 80,000 pfu were plated and screened
with the single degenerate 12 bp Not I oligonucleotide in
another attempt to detect clones with infrequently cleaving
restriction sites. As in the initial screening with the
shorter oligonucleotide, fewer than ten putatively positive
clones were detected, and none was determined to contain
Not I sites after digestion of the purified inserts with
that enzyme. Selecting only those plaques producing no
signals, twelve clones were removed from each of four

Figure 2-2. Y Chromosome Mapping Panel. Representations of the Y chromosomal fragments
present in each cell line as judged by hybridization mapping are indicated by the
horizontal lines. The positions of the breakpoints are relative and do not represent the
resolution used in cytogenetic analyses. Karyotypes are indicated above the lines.

45,X/46.X,del(Y)(pter>q11:) GM 2730
Deletions
45,X/46,Xldel(Y)(per>q11.2:) GM 2668
46,X,del(Y)(pter>q11:) S
46,X,del(Y)(pter>q12:) Pf
GM
GM
GM
GM
GM
9403 46,X,-X, + der(X;Y)(Xqter>Xp22.3::Yq11.2>Yqter)
8773 46>X.t(X;Y)(Xqter>Xp22.1::Yq11.2>Yqter)
2103 4 6, X, t (X ¡ Y) (X p t e r > Xq 11:: Yq 11 > Yq te r)
2469 4 6,X,t(X;Y)(Xpter>Xq2 2::Yq11>Yqter)
118 4 6lXXl-15,*der(15),t(Y;15)(15qter>15p1::Yq11>Yqter)
Translocati
ons
to
00
GM 7970 47,XX, + der(9),t(Y¡9)(9pter>9q13::Yq12>Yqter)

29
plates, designated BAY1-1 to BAY4-12. A third screening for
clones with infrequently cleaving restriction sites was
performed on another 80,000 pfu and filters were hybridized
with the mixed degenerate 12 bp Not I oligonucleotides.
Again, Not I sites were not detected in six putatively
positive plaques.
Human inserts in the recombinant phage were amplified
directly by PCR thereby avoiding difficulties associated
with growing and purifying large quantities of phage.
Because there were no commercially available primers
specific to the cloning site of lambda Charon 21A, primers
were designed that annealed on either side of the Hind III
site and not elsewhere in the vector. Plaques did not
necessarily contain a single population of recombinant
phage. PCR products amplified from a single plaque might
demonstrate as many as five separate bands (for example,
BAY35a-e, Table 2-1).
Characterization of Probes
Y chromosome linkage of sequences was ascertained by
hybridization of radiolabeled probes to Southern blots of
Hind Ill-digested genomic DNAs. Since the library was
produced from a complete Hind III digest, amplified inserts
should recognize genomic Hind III fragments of the same
sizes as the probes ("cognate" bands). Genomic DNA was
obtained from human male and female donors; 3E7, a mouse
hybrid cell line containing from one to four copies of the

30
human Y chromosome (Goodfellow et al.. 1983); and Rag, the
mouse parental cell line from which 3E7 was produced (Klebe
et al., 1970). The optimal pattern indicating Y-linkage
consisted of one or more identical bands in the male and 3E7
lanes that were absent in the female and Rag negative
control lanes. Another pattern seen with some Y-linked
sequences demonstrated bands in female, male and 3E7 lanes,
indicative of X chromosome or autosomal linkage. Such
probes were usable for regional mapping only if at least one
band of a different molecular weight was seen in the male
and 3E7 lanes than was present in the female lane (for
example, BAY4 detected a 2.3 kb male and 3E7 Y-linked band,
as well as 3 and 1.6 kb male and female X-/autosome-linked
bands, Table 2-1). In some cases, identically sized bands
were seen in male, female and 3E7 lanes, indicating Y- and
X-/autosome-linkage. This pattern could sometimes be
resolved into different sized Y and X/autosome bands by
hybridizing the probe to genomic Southern blots generated
from other restriction enzymes such as Taq I, Msp I, or
EcoR I. One probe from this study, BAY33c, was analyzed in
this manner, as was GMGY1 (Affara et al., 1986).
Inserts from 64 recombinants produced different
patterns when hybridized to these Southern blots. Table 2-1
summarizes the observations made about the recombinants from
these preliminary analyses. Nine inserts detected no bands
in the genomic digests (BAYl-llc, BAY2-4C, BAY4-5, BAY2,

31
BAY6b, BAY9, BAY14, and BAY22a and BAY22b), whereas six
inserts produced background signals of such intensity that
the presence of non Y-linked bands could not be ascertained
(BAYl-7a, BAYl-7b, BAYl-8a, BAY3-2, BAY3-9, BAY34a). Two
recombinants, BAYl-8b and BAY2-7a, contained internal
Hind III sites and therefore detected two genomic fragments
that mapped to the same interval. The sizes of the genomic
fragments added up to the sizes of their respective cloned
genomic inserts. In the case of BAY2-2, an intense
background smear was observed in the male and female lanes
while a clear 1 kb signal was detected in the 3E7 hybrid
lane (not shown). After the repetitive elements in the
probe were preannealed to total genomic DNA to a Cot of 100
the background smear was eliminated from the human lanes,
allowing for the unambiguous detection of the 1 kb band in
both the male and 3E7 lanes. This band was absent in the
female and Rag lanes. With the exceptions of BAY33a,
BAY33b, BAY33c, BAY34a, and BAY41 all clones numbered BAY1
to BAY50 were preannealed to a Cot of 100 prior to
hybridization to compete out the repetitive elements that
had appeared as faint gray signals in the library screening
Eleven inserts detected Y and non-Y fragments of the same
sizes in Hind Ill-digested genomic DNAs of males, females,
and 3E7 (BAYl-lla, BAY2-7b, BAY2-9b, BAY2-lla, BAY 2-llb,

32
Table 2-1. Y chromosome linkage of BAY clones.
BAY SIZE COGNATE NONCOGNATE NON-Y REPEATS Cot
CLONE Y Y 100
l-7a
3.5
-
I
H
-
l-7b
1.2
-
I
H
-
l-8a
4
-
I
H
-
l-8b
*
2.1
-
1.2, 0.9 kb* -
-
-
1-lla
2.0
+
C
M
-
1-llb
0.8
-
+ C
M
-
1-llc
0.5




2-2
*
1
+

H
+
2-4a
*
1.4
+
-
-
-
2-4b
1.1
-
+ c
-
-
2-4c
0.5
-
-
-
-
2-5
*
4.2
+
- -

-
2-1 a.
*
4
-
3, 1 kb*
M
-
2-lb
1.3
+
C
M
-
2-9a
*
3
+
+
M
-
2-9b
1.6
+
+ C
-
-
2-10
*
3
+
- -
-
-
2-lla*
1.2
+
"7 kb* C
M
-
2-llb
0.7
+
C
M

3-1
*
1.1
+


-
3-2
4
-
I
H
-
3-3
*
3
+
-
-
-
3-7
*
3.9
+A
2.5 kbB
-
-
3-8
*
1.5
+
-
-
-
3-9
1.4
-
-
M
-
3-lla*
1.3
+



4-la
*
2.2
+



4-lb
*
1.9
+
-
-
4-5
0.9




2
1.3


H
+
3c
*
1.5
+
-
H
+
4
*
2.3
+
(3, 1.
6kb)
H
+
5
*
1.4
+
(1.2kb)
H
+
6a
*
5
+
2.3kb (3, 1.
6kb)
H
+
6b
0.6
-
-
H
+
7
3
+
+ +
H
+
9
5
-
-
H
+
10b
*
0.6
+
N
M
+
14
3
-
-
H
+
16
*
3
+
-
H
+
17
*
3
+
5kb
H
+

33
Table 2-1continued
BAY SIZE COGNATE NONCOGNATE NON-Y REPEATS Cot
CLONE Y Y 100
22a
4
-
-
-
H
+
22b
1.8
-
-
-
H
+
24
1.5
-
-
C
H
+
25a
*
3
+B
7 kbA
-
H
+
25b
1.2
-
-
c
H
+
26a
3.5
-
-
+
H
+
26b
1.6
-
-
+
H
+
29a
*
5
+
-
-
H
+
29b
*
1.4
-
4 kb*
-
H
+
30b
*
3
+
-
1.9kb
H
+
31a
1.7
+
+
C+N
H
+
31b
1
+
-
C+N
H
+
33a
3
+
-
-
H
-
33b
*
2.1
+
-
-
-
-
33c
*
1.2
+
-
1.2kb
-
-
34a
5
-
-
I
H
-
35a
*
2.5
+
1.9kb
1.9kb
H
+
35b
1.8
+
-
C
H
+
35c
1.4
+
3.5kb
C+N
H
+
35d
1
+
-
-
H
+
35e
0.5
-
+
N
H
+
36a
4
+
-
C
H
+
41
1.7
+


H

REPEATS:
Indicates presence or absence of
repeat
el erne:
probes
Cot 100: Indicates probes requiring preannealing of repeat
sequences
Regionally mapped clones
Bands analyzed if cognate bands undetected or non
Y-linked
Probes containing internal Hind III sites
Not determinable
Cognate: probe detects genomic fragment of same size
Noncognate: probe detects genomic fragment of different
size
(H) Highly repetitive: intense background smear
(M) Moderately repetitive: moderate background smear or
minisatellite sequence through which individual bands
may be detected
) Indicates more than one Y-linked fragment detected by
a single probe
Band sizes reflect Hind III genomic digests
(*)
(+)
(*)
(I)
(C)
(N)
(
A-Z <

34
BAY3la, BAY3lb, BAY3 3C, BAY3 5b, BAY35C, BAY36a) One of
these, BAY3 3c, detected Y and non-Y fragments of different
sizes when hybridized to Southern blots of Msp I-digested
genomic DNAs, and was thus included in the regional mapping
analysis. Twenty-nine of 64 recombinants produced one or
more male-specific bands that were also present in the
hybrid 3E7 and absent or of a different size in the female
lane (*, Table 2-1). These unambiguously Y-linked inserts
were included in the Y chromosome regional mapping studies.
Regional Mapping
Genomic DNA obtained from the Y translocation and
deletion cell lines and controls was digested with Hind III
and duplicate Southern blots were produced on charged nylon
membranes (Zetaprobe, Genescreen Plus, or Hybond N+).
Single blots were also produced using EcoR I and Msp I for
use with the probes obtained from other investigators
(EcoR I for MIAY, 4B-2, 1F5, Y3.4, and 49f; Msp I for
GMGY1). Probe BAY33c was also analyzed using Msp I as
mentioned previously. Southern blots on Genescreen Plus did
not produce signals as intense as those seen using Zetaprobe
and Hybond N+ (compare BAY3-7A on Genescreen Plus with
BAY2-5 on Hybond N+, Figure 2-3a, lanes 11-14, lanes F-P
were on Zetaprobe in both cases). Translocation and
deletion blots were annealed to multiple probes when
individual Y-linked fragments could be distinguished
unambiguously. Panel members were scored for either the

35
presence (+) or absence (-) of the Y-linked band(s) detected
on the initial Southern blots (Figures 2-3 and 2-4).
Results obtained from hybridizing 35 Y-linked sequences
to the ten cell lines in the mapping panel are summarized in
Figure 2-4. Seven intervals in the euchromatic region of
the human Y chromosome were defined. Twelve probes detected
sequences present in Yp or proximal Yq. BAY2-5, BAY2-10,
BAY3-7A, BAY4-lb, BAY4, BAY6a, BAY17, BAY25aB, BAY29a,
BAY29b, BAY33c, and BAY35a were localized to this first
interval. These probes detected sequences in DNA from the
deletion cell lines GM 2668, Pf, and Si but in none of the
translocation cell lines. Representative examples of the Y-
linked fragments mapping to this interval are shown in
Figure 2-3a (BAY2-5 and BAY3-7A). Probe BAY3-7 detected two
Y-linked fragments, A and B, that mapped to different
intervals. The higher molecular weight fragment, A, mapped
to the most proximal interval (Figure 2-3a), whereas the
lower molecular weight fragment, B, mapped more distally
(Figure 2-3b).
No probes isolated in this study detected sequences in
panel member GM 2730, which was reported by NIGMS to retain
some portion of Yqll (see Figure 2-2). GM 2730 was a mosaic
cell line with as few as 13% of its cells retaining a Y
chromosome fragment by cytogenetic analysis. However, the
single copy male testis-determining gene, SRY, was

Figure 2-3. Southern blot analyses of Y regional mapping panel. Genomic DNA was digested
with Hind III unless otherwise indicated to the right of the fragment size. Blots were
probed with the Y recombinants indicated in Table 2-1 (*). Data are presented for
representative sequences located in intervals 1-6 and all five sequences located in
interval 7. Probes are designated at the left of the Southern blots. Only the Y-linked
bands are shown and the sizes in kb are indicated to the right. Symbols below each data
set indicate the presence (+) or absence (-) of the Y-linked bands in the DNA of a panel
member, and the interval number is designated below. Lanes represent respectively: F,
normal female; M, normal male; 3, GM 0118; 4, GM 2103; 5, GM 2469; 6, GM 7970; 7, GM 8773;
8, GM 9403; Y, 3E7 (Y hybrid cell line); P, Rag (hybrid parent cell line); 11, Pf; 12, Si;
13, GM 2668; 14, GM 2730.
a) Representative Y recombinants located in the first three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal.
b) Representative Y recombinants located in the next three Y chromosome intervals
defined by the panel. Intervals are in order from proximal to distal. Lane 4 (GM 2103)
is underloaded relative to the other lanes.
c) Two representative Southern blots hybridized with probe BAY3-8 from interval 7. Lanes
1-11 contain DNA from translocation-bearing cell lines; lanes 12-18 contain DNA from
deletion-bearing cell lines. Lanes represent 1, female; 2, male; 3, GM 0118; 4, GM 2103;
5, GM 2469; 6, marker; 7, GM 7970; 8, GM 8773; 9, GM 9403; 10, 3E7; 11, Rag; 12, female;
13, male; 14, Pf; 15, Si; 16, GM 2668; 17, GM 2730; 18, marker. Molecular weight markers
for the blot bearing lanes 1-11 are located to the left. Also at left are arrows numbered
1,7, and 5 indicating hybridization signals from probes BAY2-10, BAY3-8, and BAY3-lla
which map to intervals 1, 7, and 5 respectively. BAY3-8, BAY2-10 and BAY3-lla were
hybridized simultaneously to the blot at the left (lanes 1-11). Only BAY3-8 was
hybridized to the blot at the right (lanes 12-18). Molecular weight markers for the blot
bearing lanes 12-18 are located at the right. The arrow at the right indicates the
hybridization signal from BAY3-8 in lanes 12-18.

PROBE
BAY 2-5
BAY 3-7A
BAY 2-9a
BAY 4-1 a
BAY 2-4a
Ml AY
F M 3 4 5 6 7 8 Y P 11 121314
ill '* m
m in
- + - ___ + + + +-
Interval 1
iljj lie
! '* til-
- - + + ~ + + + -
Interval 2
- 1-4
3.6 (EcoRI)
+ -- --+ + + 4- -
Interval 3
Figure 2-3

6
PROBE
F M 3 4 5
BAY 3-7B
-mm
BAY 5
1*
- + - -
BAY 3-1
# Ci
BAY 3-11a
t
- + + -
Figure 2
7 8 Y P 11 1213 14
+ + + + +- -
Interval 4
+ + +- + +- -
Interval 5
* 3.3 (EcoRI)
+ + +- + +
Interval 6
03
00
3continued

FiSrure 2~o
cntinued
7

40
detectable in all DNA samples of this cell line by Southern
analysis (not shown).
The first interval that was unambiguously localized to
Yqll was defined by two probes, BAY2-9a and BAY4-la
(Figure 2-3a). BAY2-9a also detected a non Y-linked
minisatellite repeated sequence in the human DNA on Southern
blots produced on Zetaprobe (Figure 2-3a, lanes 1-8) that
was not detectable on Genescreen Plus (lanes 11-14). The
BAY2-9a minisatellite was detectable on Southern blots
produced on Hybond N+ (not shown). Probes from this
interval detected sequences in the deletion cell lines
GM 2668, Pf, and Si which retain the entire Y short arm and
some portion of the proximal long arm (Ypter-Yqll)
(Figure 2-2). Sequences were detected in only one
translocation cell line, GM 9403; here, the Y fragment
extended from the terminus of the long arm to the proximal
long arm (Yqter-Yqll) (Figure 2-2).
BAY2-2, BAY2-4a, BAY16, BAY3 0b, BAY3 3b, and MIAY
defined the third interval, of which, MIAY had been
previously mapped to Yqll (Koenig et al., 1985). Two
representative sequences from this interval are shown in
Figure 2-3a (BAY2-4a and MIAY). The sequences were detected
in the translocation cell line GM 9403 and in the Yq
deletion cell lines Pf and Si, but not GM 2668. These data
indicated that the breakpoints in deletion samples Pf and Si
lay distal to the breakpoint in GM 2668.

Figure 2-4. Hybridization of probes to DNA from regional
mapping panel. Genomic DNA from each cell line was digested
with the appropriate enzyme, separated by electrophoresis,
and transferred to nylon membranes. Southern blots were
sequentially hybridized with the Y chromosome recombinants
listed at the left. (+) indicates the presence of the
appropriate Y-linked fragment in the DNA from the panel
member, (-) indicates its absence. Cell line 3E7 is a
somatic cell hybrid bearing only the human Y chromosome, and
was included as a positive control for Y chromosomal
sequences. ND indicates the fragment was undetectable.

42
Translocations Deletions
Probes
3 E 7
9403
8 773
2 103
2 469
1 1 8
7970
Pt
Si
2668
2 730
2 5
+
-
-
-
-
-
-
+
+
+
-
/ A
2-10
+
-
-
-
-
-
-
+
+
+
-
11.3

3 7 A
+
-
-
-
-
-
+
+
+

4 -1b
+
-
.

-
+
+
+
4
+
-
-
-
-
-
-
+
+
+
-
11.2
6a
+



_
_
_
+
+
+

1 7
+
-
-
-
-
-
-
+
+
+
-
2 5a B
+
-
-
-
-
-
-
+
+
+
-
11.1
put
29a
+
-
-
-
-
-
-
+
+
+
29b
+
-
-
-
-
-
-
+
+
+

11.1
Jill
33c
+
-
-
-
-
-
-
+
+
+
-
-
35a
+
-
-
-
-
-
-
+
+
+
-
11.21
2 -9a
+
+
_
_
_
+
+
+
_
4 -1a
+
+
-
-
-
-
-
+
+
+
-

2 2
+
+
-
-
-
-
-
+
+
-
-
11.22
2 4 a
+
+
-
-
-
-
-
+
+
-
-
in
\
1 6
+
+
-
-
-
-
-
+
+
-
-
31b
33b
+
+
+
+
_
\
+
+
_
_
_
_
_
+
+


11.23
Ml AY
+
+
-
-
-
-
-
+
+
-
_
3 7 B
+
+
+
-
-
-
-
+
+

up
5
+
+
+
-
-
-
-
+
+
-

10b
+
+
+
-
-
-
-
+
+
-
-
3 1
+
+
+
+
-
-
-
+
+
-
-
3-Ha
+
+
+
+
-
-
-
+
+
-
-
3 c
+
+
+
+
-
-
-
+
+
-
-
1 -8b
+
+
+
+
+
-
-
+
+
-
-
1
2 7 a
+
+
+
+
+
-
-
+
+
-
-
1 2
3-3
+
+
+
+
+
-
-
+
+
-
-
25a A
+
+
+
+
+
-
-
+
+
-
-
4 B 2
+
+
+
+
+
-
-
+
+
-
-
||§¡|
GMGY1
+
+
+
+
+
-
-
+
-
-
-
§§§§
3-8
+
+
+
+
+
-
-
+
-
-
-
|¡¡¡¡
2 -11a
+
+
+
+
+
-
-
+
-
-
-
m¡¡|
1F5
+
+
+
+
+
-
-
+
-
-
-
f§§
0 4 9 f
+
+
+
ND
+
-
-
+
-
-
-
ill
Y 3.4
+
+
+
+
+
+
+
+
_
-j

43
The fourth interval lay between the breakpoints in
GM 8773 and GM 2103, and contained three recombinant
fragments: BAY3-7B, BAY5, and BAYlOb. These probes detected
sequences in two translocation cell lines, GM 9403 and
GM 8773, and two deletion cell lines, Pf and Si. BAY3-7B
and BAY5 represent two of the probes mapped to this interval
in Figure 2-3b. BAY3-7 had previously detected a higher
molecular weight fragment (A) in the most proximal Y
interval defined by the panel (Figure 2-3a).
The fifth interval was defined by BAY3-1, BAY3-lla, and
BAY3c which detected sequences in translocations GM 9403,
GM 8773, and GM 2103, and deletions Pf and Si. Two
representative probes from this interval are shown in Figure
2-3b. The fainter bands in GM 2103 (lane 4) were due to
underloading of the DNA.
The sixth interval contained 4B-2, BAY2-7a, BAY3-3,
BAY25aA and BAYl-8b, of which 4B-2 was described previously
(Burk et al., 1985). These probes detected sequences in
four translocation lines: GM 9403, GM 8773, GM 2103, and
GM 2469, and two deletion lines, Pf and Si. 4B-2 and
BAYl-8b represent probes defining the interval (Figure
2-3b). The faint band in GM 2103 (lane 4) detected by 4B-2
was due to incomplete digestion of the genomic DNA by EcoR I
as seen in the ethidium bromide stained gel prior to
transfer (not shown). The 0.9 and 1.2 kb bands detected by

44
the 2.1 kb BAYl-8b probe were due the previously mentioned
internal Hind III site.
The most distal interval, interval 7, in the
euchromatic region of Yq defined by this mapping panel also
contained five probes: BAY2-lla, BAY3-8, 1F5, 49f, and
GMGY1. Three of these sequences had been described in other
laboratories: 49f (Bishop et al.. 1984), GMGY1 (Affara et
al.. 1986), and 1F5 (Whisenant et al., 1991). The sequences
in this interval were detected in only one deletion cell
line, Pf, and were present in all the translocation cell
lines except GM 0118 and GM 7970. Figure 2-3c demonstrates
the two Southern blots hybridized with probe BAY3-8 which
maps to this interval.
The DYZ1 probe, Y3.4, was used as a positive control
for the presence of heterochromatin from Yql2 in the cell
lines (not shown). As seen in Figure 2-4, Y3.4 was present
in all the translocation cell lines, including GM 0118 and
GM 7970, as well as the deletion cell line, Pf. All these
cell lines had exhibited visible heterochromatin. As
expected from the cytogenetic analyses, Y3.4 was absent from
deletion cell lines GM 2668, GM 2730, and Si in which
heterochromatin was not visible.
The data from the regional mapping analyses are
summarized in Figure 2-5 and allow for the relative ordering
of the breakpoints. Distances between breakpoints, or the
physical sizes of the intervals those breakpoints bound, are

45
not addressed by the data. The order of individual
sequences within an interval has not been established except
for those sequences mapping to interval 7: cenGMGY1,
BAY3-8, BAY2-lla, 1F5, 49ftel. The experiments used to
achieve this ordering are described in Chapter 4. No
distinctions can be made between the breakpoints in the cell
lines GM 0118, GM 7970, and Pf by the sequences used in this
study. The data summarized in Figure 2-5 also indicate an
apparently unequal distribution of repeat-bearing probes
along the Y chromosome. Ten probes required a preannealing
step to a Cot of 100 prior to hybridization and mapped
within the three most proximal intervals (BAY4, BAY6a,
BAY17, BAY25aB, BAY29a, BAY29b, BAY35a, BAY2-2, BAY16, and
BAY30b). The repeat-bearing probes account for about 29 kb
out of the total 53 kb covered by all probes in the region.
Only four repeat-bearing probes (BAY5, BAYlOb, BAY3c, and
BAY25aA) mapped to the three more distal intervals, while
none were present in interval 7. These four probes cover
only 6.5 kb in a total of 29 kb, or about 22% of the area
containing repeats as compared to 53% in the more proximal
intervals. Assuming that repeated elements are evenly
distributed along a segment of DNA, the probability that a
randomly isolated probe would contain such elements should
be independent of its regional location on a chromosome.
The area covered by repetitive elements in the first three
intervals was not significantly different from that expected

46
GM 2730
GM 9403
GM 2668
GM 8773
GM 2103
GM 2469
Si
1
2
3
4
5
6
7
4, 6a. 17. 25aB. 29a, 29b. 35a.
2-5, 2-10, 3-7A, 4-1b, 33c
2-9a, 4-1a
2-4a, 33b, MI AY, 2-2. 16. 30b
3-7B, 5. 10b
3-1, 311a, 3c
1-8b, 2-7a, 3-3, 25aA. 4B-2
GMGY1, 3-8, 2-11a, 1F5, 49f
GM 118, Pf, GM 7970^ 8 Y3-4
Figure 2-5. Summary of Yq interval mapping. Breakpoints of
each cell line in the panel are ordered from proximal to
distal. The sets of sequences defining each interval and
the interval number are listed to the right of the arrows.
Distances between breakpoints are not implied.

47
by x2 analysis, however the next three intervals differed
significantly (0.001 distribution of repeats. This significant difference became
even greater (p<<0.001) if the analysis included sequences
from interval 7.
Submission of Sequences to the Genome Data Base
All the newly isolated Y chromosome probes that were
regionally mapped in Figure 2-4 were submitted to the Genome
Data Base at Johns Hopkins University. Anonymous DNA
segments are no longer being assigned D-numbers by GDB
without some evidence of function, but submitted information
is kept on file in the event that new data will provide that
evidence. Based upon their conservation in the higher
primate males as described in Chapter 3, nine sequences
listed in Table 2-2 were assigned D-numbers by GDB.
Discussion
Because of the lack of recombination in the region,
the development of a linkage map of the long arm of the
human Y chromosome depends upon physical analyses. Whereas
the majority of the physical mapping data obtained from the
Y chromosome was directed at the short arm, sequences
mapping to the long arm have been described; however, none
of the published mapping data have utilized common Y
chromosome translocations and deletions, making data
comparisons difficult. A standard panel of altered Y

48
Table 2-2. Genome
Data Base D-number assignments
PROBE
BAY2-10
BAY4-la
BAY 3-1
BAY3-lla
BAY2-7a
BAYl-8b
BAY2-lla
BAY3-8
BAY 33b
LOCUS
DYS187
DYS188
DYS189
DYS190
DYS191
DYS192
DYS193
DYS194
DYS195

49
chromosomes was needed to serve as a starting point for the
more detailed mapping of a specific region of the Y
chromosome that will follow.
In this chapter, I have introduced a reference panel of
Y chromosome translocations and deletions that others may
use for the generation of standardized maps. The data
presented here were used to produce a deletion map of the
euchromatic long arm of the human Y chromosome that is
divided into six intervals. This deletion map is consistent
with maps and in situ hybridization data that were generated
in other laboratories, although exact correlations are
difficult to make due to the different probes and cell lines
used in those studies. In the course of regionally mapping
29 newly isolated Y chromosomal sequences, an enrichment for
long arm sequences and an apparent paucity of GC-rich
islands was noted in the clones taken from a flow-sorted Y
chromosome library. The distribution of Y-linked sequences
obtained from the library was consistent with the
distributions noted in other laboratories.
The cell lines in the panel were chosen after meeting
certain criteria. Only unbalanced Y chromosomal
translocations were chosen so that the absence of a Y-linked
band would be indicative of an abnormal Y chromosome. Only
cell lines that were stable in culture were used to ensure
their suitability for molecular analyses. For example,
GM 3774 was rejected for use in the panel after cytogenetic

50
analyses revealed that the reported isochromosome Yq was
unstable, suggesting that the chromosome was dicentric
rather than an isochromosome. GM 3595 retained a stable
deleted Y chromosome, but the cells were intractable in
culture and could not be grown in quantities sufficient to
extract DNA, resulting in the exclusion of this cell line
from the panel.
The molecular data presented here indicate possible
discrepancies in the reported karyotypes of two cell lines
from NIGMS. GM 2730 reportedly contained a deleted Y
chromosome in which the deletion extended into Yqll. The
cytogenetic data do not appear to contradict this report
(see Figure 2-1). In the molecular data reported here,
twelve probes that map into the region reportedly present in
GM 2730 detected no sequences in DNA from this cell line.
Because GM 2730 was a mosaic cell line, the possibility
existed that the representation of the Y chromosome was
below the level of sensitivity for Southern blot analysis.
This was ruled out by the detection of the single copy SRY
gene in a Southern blot prepared with GM 2730 DNA samples.
One explanation for the lack of Yq sequences in GM 2730
would require all twelve probes to be in an interval just
distal to the Y centromere and proximal to the breakpoint in
GM 9403. This explanation would invoke the seemingly
unlikely event that the library screening procedure selected
against sequences mapping to the short arm of the Y

51
chromosome. Oosthuizen, however, has observed that 80% of
probes isolated from this library that identified
exclusively Y-linked sequences mapped to Yqll.23, indicating
a "nonrandom chromosomal distribution of randomly isolated
probes" (Oosthuizen et al.. Human Gene Mapping 9, 1987).
An alternate explanation would suggest that the Y
chromosomal fragment in GM 2730 was formed by a complex
deletion/inversion event such that only distal short arm
sequences are retained, as evidenced by the retention of the
SRY gene. Complex cytogenetic events involving the Y have
been described, and most were determined only after
molecular analyses because of the inability to resolve these
events at the microscopic level (Zeuthen and Nielsen, 1973;
Affara et al., 1986; Page et al., 1990). This explanation
would allow for the regional localization of some of the
twelve most proximally mapping probes to the proximal Y
short arm.
The second discrepancy between a karyotype reported by
NIGMS and the molecular data from this study involves cell
line GM 0118. At the cytogenetic level, GM 0118 appears to
retain a large portion of the Y chromosome. Judging by the
size of the additional material on 15p, some portion of Yq
euchromatin should have been retained. However, none of the
probes used in the analyses detected any euchromatic
sequences. A closer look at the translocation chromosome
(Figure 2-1) reveals a banding pattern strongly similar to

52
an inversion of bands 15qll-ql2 at the top of the chromosome
15. The Yq material might, therefore, be only from the
heterochromatic region. The existence of sequences which
map more distally in Yqll cannot be ruled out, nor can the
possibility be excluded that more distally located probes
than those described here might detect euchromatic sequences
in GM 0118.
Regional mapping of 35 Y chromosomal probes, using the
ten member reference panel, indicated that seven euchromatic
intervals were distinguished along the Y chromosome
(Figures 2-4 and 2-5). Six of these intervals mapped
exclusively to the long arm of the Y. The majority of the
probes detected a single Y-linked fragment. Two, BAY3-7 and
BAY25a, detected multiple Y-linked fragments mapping to
different Y chromosome intervals (see Figures 2-3b, 2-4, and
2-5) and appeared to be similar to the multiple Y-linked
fragments that map to different intervals as previously
described for probes 50f2 (DYS7, Guellaen et al. 1984), 52d
(DYS3, Bishop et al.. 1984), and 118 (DYS8, Guellaen et al.,
1984) .
Vergnaud presented a deletion map of the human Y
chromosome in 1986 that divided the entire chromosome into
seven intervals. The majority of the DNAs in that study
were obtained from XX males, and the intervals were
concentrated on the short arm rather than the long arm.
Only two intervals were assigned to the euchromatic long

53
arm. Because none of the five cell lines containing Y long
arm deletions used in that study were available to us, it
was difficult to correlate these newly described intervals
with those previously postulated. Probe 49f (DYS1), which
localized to the most distal euchromatic interval defined by
the panel here, also localized to the most distal
euchromatic Yq interval in the panel used by Vergnaud
(1986). Because only two Yq euchromatic intervals were
defined at that time, the distal-most interval from that
study might correlate with several of the intervals defined
here. DYS1 has been demonstrated to be the most distal
locus in other studies using different cell lines or patient
samples (Johnson et al. 1989; Kotecki et a_l. 1991;
Nakahori et al., 1991). 49f was also mapped by in situ
hybridization to metaphase chromosomes to the Yqll.22-qll.23
region (Quack et al., 1988). These data would place
interval 7 into Yqll.22-qll. 23, and indicate that DYS1 is
the most distal locus. The precise locations of the other
intervals with respect to cytogenetic subbands Yqll.21,
Yqll.22, and Yqll.23 were not determined.
One goal of this investigation was to survey the long
arm of the Y chromosome for the presence of sites for
infrequently cleaving restriction endonucleases. These
sites were of particular interest because of their usual
association with HTF islands, possibly indicating the
presence of genes in the region (Bird, 1986; Estivill and

54
Williamson, 1987; Lindsay and Bird, 1987). In an effort to
enrich for clones bearing GC-rich sequences associated with
infrequently cleaving restriction endonuclease sites, the Y
chromosome library was screened several times with various
oligonucleotides containing the Not I restriction site
sequence. Although no clone was shown to contain a Not I
restriction site, the technique has been successfully used
to enrich for clones bearing infrequently cleaved
restriction sites (Estivill and Williamson, 1987; Melmer and
Buchwald, 1989). Although approximately 40 kb of DNA were
surveyed for Not I sites, a potentially much larger area was
covered in the experiments to be described in Chapter 4.
This evidence indicated a possible dearth of Not I sites at
least in distal Yqll which might account in part for the
lack of success in screening for them. The overall
distribution of Not I sites in genomic DNA would be expected
to be about one per 65 kb for an eight base pair recognition
enzyme, but the actual distribution ranges from 50 kb to 2
Mb (H. Lehrach, personal communication), so the 40 kb
surveyed in this study may be underrepresentative.
Hybridizations of recombinant inserts to male, female,
3E7, and Rag genomic digests produced patterns consistent
with results reported in other laboratories. About 30% of
the inserts demonstrated exclusively Y-linked fragments
while some 36% produced Y-linked fragments that were shared
with the X or autosomes (Table 2-1). Most of the remainder

55
were laden with repetitive elements. Using a novel
subcloning vector, a Japanese group screened the EcoR I
library equivalent to the Hind III library used in this
study. They reported some 80% X, Y, or autosome shared
fragments and only 21% exclusively Y-linked sequences.
These results may be related to the library used and/or the
selection of subclones by the subcloning vector (Nakahori et
al., 1991). Randomly isolated single and low copy sequences
isolated from other flow sorted Y-DNA libraries have fallen
into similar categories to those reported here: 35% were
exclusively Y-linked, 46% were shared between the X and Y,
15% were shared between the Y and autosomes, and 4% were
shared with the X, Y, and autosomes (Bishop et al., 1984;
Affara et al., 1986; Oosthuizen et al., 1990).
Overall, the data obtained in this study indicate that
the regional mapping panel introduced here divides the
euchromatic Y chromosome long arm into six distinct
intervals. This amounts to more than twice the number of
intervals defined by Vergnaud (1986). Probes bearing
repetitive elements appear to be unequally distributed in
the long arm, with repeat-bearing sequences covering more
area in the proximal intervals than in the distal intervals.
The most distal euchromatic interval defined by the panel,
interval 7, probably lies within subband Yqll.23, the region
of the Y chromosome whose absence is associated with
spermatogenic failure (Tiepolo and Zuffardi, 1976; Bobrow,

56
1985; Hartung et al., 1988). The following chapters
describe experiments centered around this most distal
euchromatic interval. In Chapter 3, the evolutionary
conservation of these five sequences will be addressed,
while Chapter 4 will address the distances between the
probes, their order on the Y chromosome at the molecular
level, and the overall size of interval 7 itself.

CHAPTER 3
EVOLUTIONARY COMPARISONS OF SEQUENCES MAPPING
TO THE HUMAN Y CHROMOSOME
Introduction
The mammalian sex chromosomes differ structurally and
genetically from one another, but are thought to have
evolved from a homologous pair. Recombination between these
original homologous chromosomes is postulated to have become
suppressed after another gene became involved in sex
determination. The suppression of recombination then
allowed mutations to accumulate on the proto Y, and
eventually most of the chromosome became extinct (Ellis and
Goodfellow, 1989). The existence of a homologous pairing
region, termed the pseudoautosomal region, was postulated to
allow for correct meiotic segregation and to account for the
presence of X-Y bivalents seen in meiosis (Burgoyne, 1982).
The rest of the Y became a repository of 'junk' DNA
(Charlesworth, 1978) as evidenced by the preponderance of Y-
specific repeated sequences in the long arm that constitute
50-70% of the entire chromosome (Cooke et al.. 1983; Burk et
al., 1985). These repeats contained Y-specific restriction
fragment classes that were species specific and might
indicate that Y chromosomal sequences had diverged rapidly
(Kunkel and Smith, 1982; Wolfe et al., 1985).
57

58
When anonymous unique-sequence elements began to be
cloned, many were found to recognize homologous sequences on
the X chromosome, but outside the putative pairing region
(Bishop et al., 1983, 1984; Cooke et al. 1984). The
presence of such X-Y homologous sequences was interpreted as
evidence supporting the primordial homology between the sex
chromosomes (Bishop et al., 1984) until the species
conservation of these X-Y homologous sequences was reported.
These studies indicated that the sequences had recently
arrived on the human Y chromosome from primate X
chromosomes, and therefore were not indicators of an ancient
homology between the X and Y (Page et al., 1984; Koenig et
al.. 1984; Cooke et al., 1984). The cloning of separate X-
specific and Y-specific centromeric repeat sequences further
complicated the issue of the degree of X-Y homology (Yang et
al., 1982; Wolfe et al., 1985) and the very existence of the
pairing region predicted by Burgoyne was questioned (Ashley,
1984). The cloning of the predicted pseudoautosomal
sequences was soon reported in a series of papers (Cooke et
al. 1985; Simmler et al. 1985; Buckle et al.. 1985) and
the sequences were later shown to be conserved on the sex
chromosomes of the hominid primates: chimpanzee, gorilla,
and orangutan (Weber et al., 1987; 1988). A candidate gene
for the testicular determining factor, ZFY, was shown to be
conserved on the Y chromosomes of all eutherian mammals
tested (Schneider-Gadicke, et al., 1989; Palmer et al.,

59
1990), as was the bona fide TDF gene, SRY (Sinclair, et al.,
1990). These two genes and the pseudoautosomal sequences
were the only short arm sequences shown to have such an
evolutionary conservation on the Y chromosome.
The data on the repeated and unique sequences from
the human Y suggested a relatively recent origin of much of
the long arm of this chromosome. To date, only 4B-2, 69/6,
and 1F5 have been shown to retain their exclusive Y-linkage
in the great apes (Burk et ad., 1985; Erickson, 1987;
Whisenant et al., 1991). This chapter describes the
analyses of a series of Y chromosome-specific clones
discussed in Chapter 2. These sequences were selected for
analyses of their conservation in the great apes: chimpanzee
(Pan troglodytes), gorilla (Gorilla gorilla), and orangutan
(Pongo pvqmaeus). The five clones mapping to the most
distal euchromatic interval on the human Y long arm (see
Figure 2-5) were included because of the association of this
region with spermatogenesis in humans. Surprisingly, all
but one of the clones were exclusively Y-linked in the great
apes. Further analyses were performed on genomic DNA from
rhesus monkeys (Maccaca mulatta), cattle (Bos taurus), and
mice (Mus domesticus) with the five most distally mapping
clones. Only an autosomal allele of GMGY1 was found to be
conserved in those species as well as in the great apes.

60
Materials and Methods
Primate DNA Extraction
Male and female peripheral blood samples from the
following primate species were obtained from the Yerkes
Regional Primate Center in Atlanta, Georgia: gorilla,
chimpanzee, orangutan, and rhesus monkey. 10 to 20 ml of
peripheral blood were collected in vacuum tubes containing
0.1 ml of 15% EDTA and shipped overnight on ice. On
receipt, the tubes were centrifuged to separate plasma and
cells. The plasma was removed to just above the buffy coat.
The buffy coat and some of the underlying red cells were
transferred to 15 ml conical centrifuge tubes. DNA
extraction buffer (10 mM Tris, pH 7.8, 2 mM EDTA, 400 mM
NaCl) was added to 5 ml, SDS to 0.5%, and predigested
Pronase to 1 mg/ml. Tubes were incubated overnight at 37C
and extracted twice with 24:24:1 phenol:chloroform:isoamyl
alcohol and twice with 24:1 chloroform:isoamyl alcohol. DNA
was precipitated in ice cold ethanol. Precipitated DNA was
spooled onto a glass pipet tip and dipped into 70% ethanol
prior to a brief period of air drying. The DNA was
resuspended in 500/xl TE, pH 8, with a drop of chloroform,
then rotated overnight at 4C. The concentrations of DNA
were calculated from their -absorbances at 260nm. Purity was
also calculated from the ratios of absorbances obtained at
260nm and 280nm (A260/280), with a ratio of 1.8-2.0
considered to be optimal.

61
Other DNA Sources
Peripheral blood samples from a yearling cow and bull
were obtained from the University of Florida College of
Veterinary Medicine and DNA was extracted as detailed above.
Male and female mouse tail DNA samples were kindly provided
by Karen Dukes in Dr. Philip Laipis' laboratory (Department
of Biochemistry and Molecular Biology).
Southern Blotting and Hybridizations
10 to 15 nq aliguots of DNA were digested with
restriction enzymes according to the manufacturer's
recommendations. Digestion products were separated by
electrophoresis in 0.75% agarose gels overnight. Following
electrophoresis, gels were stained with 0.25 jug/ml ethidium
bromide, destained for 30 minutes to 2 hours in water,
photographed, then transferred to nylon membranes in 0.4 M
NaOH overnight. Great ape DNA digests were transferred to
either Genescreen Plus (NEN) or Hybond N+ (Amersham).
Monkey, bovine and mouse DNA digests were transferred to
Hybond N+, as were the Msp I digests used to analyze probe
GMGY1. After blotting, membranes were rinsed with 2X SSPE,
air dried for several hours or vacuum dried at 80C for 30
minutes to 2 hours.
Probe DNAs were radiolabeled with 32P dCTP (3000
Ci/mmol, ICN) by random primer extension (Feinberg and
Vogelstein, 1984) using a BRL kit. The unincorporated
nucleotides were removed using a NACS PREPAC column (BRL).

62
Hybridizations were performed for 18-24 hours in 5X SSPE, 1%
SDS, 5X Denhardt's (0.02% Ficoll, 0.02% polyvinyl
pyrrolidone, 0.02% bovine serum albumin), 50% formamide at
42C. Post-hybridization washes for the great ape blots
were in 2X SSPE, 0.1% SDS at room temperature for 20
minutes, and twice in 0.1X SSPE, 0.1% SDS at 55-60C for 30
minutes each. Final washes for the blots containing DNA
from the lower mammals and the GMGY1 Msp I blot were at
45C. Membranes were wrapped in plastic wrap and exposed to
Kodak XAR-5 film at -70C for 1-6 days. After
autoradiography, the probes were stripped from the Hybond N+
blots in a solution of 0.1X SSPE, 0.5% SDS at 95-100C for
20-30 minutes. Genescreen Plus blots proved to be
unsatisfactory for reuse despite numerous attempts to strip
probes from the blots. Wet blots were sealed in plastic
after stripping and refrigerated until the next use.
Results
Clones Mapping to the Six Most Proximal Y Intervals
BAY clones 2-10, 4-la, 33b, 3-1, 3-lla, 2-7a, and l-8b
were selected as probes for Southern blots generated from
male and female chimpanzee, gorilla, and orangutan genomic
DNA digested with Hind III. These clones represent all but
one of the six most proximal intervals (see Figure 2-5).
Except for BAY3-lla, all of these clones were used to probe
genomic Southern blots generated using EcoR I, BamH I, Taq
I, Pst I, Bgl II, and/or Sst II in order to estimate the

63
degree of sequence homology by conservation of restriction
sites (Table 3-1).
Several patterns were observed from the hybridizations,
the results of which are summarized in Table 3-1. All of
the probes recognized male-specific bands in all three great
ape species with at least one enzyme. No bands were seen in
female DNA from any species. BAY2-10 and BAY2-7a recognized
common male bands (10%) in chimpanzee, gorilla, and often
orangutan, using more than one enzyme. Five probes
(BAY2-10, BAY33b, BAY4-la, BAY2-7a, and BAYl-8b) detected
common male bands in all species with at least one enzyme.
Two probes, BAYl-8b and BAY2-7a, appeared to be single or
low-copy sequences by EcoR I and Hind III analyses, but in
hybridizations of Southern blots generated by BamH I and
Pst I they detected multiple bands, indicating that these
sequences may be low-level repeats (see Figure 3-1, BamH I
genomic digest probed with BAYl-8b). The regional mapping
analyses localized both of these sequences to the sixth
interval defined by the panel, indicating that the repeats
are located within a specific region. BAY3-1 detected
fragments of similar sizes for EcoR I in human, chimpanzee
and gorilla, but fragments of different sizes with Hind III.
Y-linked fragments were also detected in orangutan in both
cases. BAY3-lla detected only Y-linked fragments of
different sizes in all four species with one enzyme,
Hind III.

64
HSA GGO PPY PTR
fmfm fmfm
Figure 3-1. Conservation of Y-linked sequences homologous
to BAYl-8b in great apes. BamH I-digested DNA was separated
by electrophoresis and transferred to Genescreen Plus. The
Southern blot was hybridized to probe BAYl-8b. DNA sources
are as follows: f female, m male. Human, HSA; gorilla, GGO;
orangutan, PPY; chimpanzee, PTR. Size markers in kb are
indicated at the right.

65
Table 3-1. Interspecies comparisons of Southern blot data
PROBE &
INTERVAL
BAY 2-10
#1
ENZYME
Hindlll
EcoRI
BamHI
TaqI
HUMAN
2.65
2.55
21
16
1.65
BAY4-la
#2
BAY 33b
#2
Hindlll
Hindlll
1.65
1.9
BAY3-lla
#5
Hindlll
1.3
BAY 3-1
#5
Hindlll
EcoRI
0.9
1.6
BAY2-7a
#6
Hindlll
4.4
EcoRI
0.8
6.1
3.0
BamHI
>21
20
PstI
2.2
0.73
TaqI 15
6.6
4.7
<1.0
CHIMP
GORILLA
ORANGUTAN
2.65
2.65
10
2.55
2.55
21
21
21
1.65
1.65
1.65
1.65
1.65
1.65
4.6
13
2.8
5.5
2.5
1.9
1.8
1.8
1.5
12.8
NA
2.2
1.9
1.3
1.3
0.9
1.6
1.6
1.15
4.3
4.5
4.3
2.65
1.9
2.0
1.8
0.8
0.8
0.8
6.0
6.0
5.0
3.0
3.0
2.95
2.7
>21
>21
21
20
11
7.2
5.5
3.3
3.9
3.3
2.3
2.2
2.1
0.74
0.76
15
18
21
15
18
10
15
<1.0
3.6

66
GORILLA ORANGUTAN
PROBE &
INTERVAL
BAYl-8b
#6
BAY3-8
#7
BAY2-lla
#7
1F5
#7
49f
#7
ENZYME
Hindlll
EcoRI
BamHI
PstI
Hindlll
EcoRI
Hindlll
Hindlll
BamHI
PstI
Bglll
Sstll
Hindlll
HUMAN
1.1
0.95
17.5
12
rpt xl3
rpt x9
1.6
18
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
2.4
1.5
1.6
CHIMP
1.1
0.95
18.5
8.6
rpt x7
rpt x4
3.5
18
13
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
0.7
NA
1.6
1.1
0.95
20
9.2
rpt xl3
rpt x9
1.6
8.6
7.4
7
5.0
4.6
2.2
2.0
1.4
1.25
2.1
2.4
1.5
1.6
0.95
20
9.9
rpt xl2
rpt xlO
3.5
8.6
7.4
7
2.5
1.8
5.0
0.9
20
1.3
3.0
2.8
2.5
2.1
Band sizes are in kb. Bands shared in 3 or more species are
underlined. Numbers of BamH I and Pst I repeats detected by
BAYl-8b are indicated by "rpt times number of repeats". NA
indicates Not analyzed. Interval numbers beneath probe
designations refer to the intervals defined in Chapter 2,
Figure 2-5.

67
Analysis of Clones from the Distal Ya Euchromatic Region
The five clones (GMGY1, BAY3-8, BAY2-lla, 1F5, and 49f)
located in interval 7 of the long arm were used to probe
Southern blots generated from DNA from the great apes as
well as from rhesus monkeys, cattle, and mice. All except
GMGY1 detected male-specific sequences in all the great apes
(Figures 3-2 and 3-3), but not in rhesus monkeys, cattle, or
mice (not shown). BAY3-8, BAY2-lla, 1F5, and 49f detected
common fragments in man, chimpanzee and gorilla. BAY2-lla
also detected a common fragment in orangutan. 1F5 and 49f
both have been shown to detect non-Y-linked fragments at
reduced stringency in humans. These probes detected
fragments in gorilla females, but these migrated differently
from the common Y-linked fragments seen in the males and
were assumed to be non-Y-linked.
GMGY1 required analysis on a Southern blot generated by
the enzyme Msp I, because a Hind Ill-generated genomic
Southern blot did not differentiate a Y-linked fragment in
any of the great apes tested (not shown). Genomic DNA from
male and female humans, chimpanzees, gorillas, orangutans,
rhesus monkeys, cattle, and mice was digested with Msp I and
used to generate a Southern blot. This blot was washed at
reduced stringency (0.1X SSPE, 45C). A male-specific band
was detected only in human, but an autosomal sequence from
males and females was detected in all lanes except male
gorilla (Figure 3-4). The DNA sample from the male gorilla

68
HSA GGO PPY PTR
fmfmfmfm
Figure 3-2. Conservation of Y-linked sequences homologous
to BAY3-8 in great apes. EcoR I-digested DNA was
electrophoretically separated and transferred to Genescreen
Plus. The Southern blot was hybridized to probe BAY3-8.
DNA sources are as follows: f female, m male. Human, HSA;
gorilla, GGO; orangutan, PPY; chimpanzee, PTR. Band sizes
in kb are indicated at the right.

69
1 2
3 4
5 6
7 8
a
!
It
111
|
7
5
2.5
1.8
Figure 3-3. Conservation of Y-linked sequences homologous
to probes BAY2-lla, 1F5, and 49f in great apes. Panels are
a) BAY2-lla; b) 1F5; and c) 49f. Hind Ill-digested genomic
DNA was electrophoretically separated and transferred to
Hybond N+. The Southern blot was sequentially hybridized
with the probes. Lanes are as follows: 1 human female, 2
chimpanzee female, 3 gorilla female, 4 orangutan female, 5
human male, 6 chimpanzee male, 7 gorilla male, 8 orangutan
male. Band sizes in kb are indicated at the right.

70
HSA GGO PIR PPY MMU BTA MPO
f m f m f m f m f m f m f m
Figure 3-4. Conservation of GMGYl-like sequences. Msp in
digested genomic DNA was electrophoretically separated and
transferred to Hybond N+. The Southern blot was hybridized
with GMGY1. DNA sources are as follows: f female, m male.
Human, HSA; gorilla, GGO; chimpanzee, PTR; orangutan, PPY;
rhesus monkey, MMU; cow, BTA; mouse, MDO. Band sizes in kb
are indicated at the right.

71
was incompletely digested as indicated in the ethidium
bromide stained gel prior to transfer (not shown). It would
seem unlikely that the sequence is not conserved in male
gorillas since it is detected in female gorilla and is known
to be autosomal in humans (Affara et al., 1986).
The Hind III data for all probes are summarized in
Figure 3-5. The order of the probes on the human Y is from
proximal to distal. Common fragments in male chimpanzee and
gorilla or male chimpanzee, gorilla and orangutan are
underlined.
Discussion
Several reports have been published regarding the
conservation of Y chromosome-linked restriction fragments in
the great apes. Whereas many of these fragments are shared
among humans, chimpanzees, and gorillas, some divergence has
been noted in orangutan (Page et al., 1984; Koenig et al.,
1985; Erickson, 1987; Whisenant et al., 1991). An even
greater degree of divergence was observed in gibbons and
macaques (Page et al., 1984; Whisenant et al., 1991).
Whereas many human Y-linked fragments have been found to be
conserved, especially among the great apes, few have been Y-
linked in lower primates and other mammals. It has been
argued that the human Y is of recent evolutionary origin and
therefore any sequences that are Y-specific in closely
related species may be functionally significant (Burk et
al.. 1985).

72
Probe
Human
ChimDanzee Gorilla
Oranautan
2-10
2.65
2.65
2.65
10
4-1a
1.65
1.65
1.65
1.65
33b
4.6, 3.2
11.5, 3.8
13, 5.5
1.9
1.9
1.8
1.8, 1.5
3-1
0.9
1.3
1.3
0.9
3-11a
1.3
12.8
1.85, 1.2
2.2
2-7a
4.4
4.3
4.5
4.3
2.65, 2.0
1.9, 1.8
0.8
0.8
0.8
0.8
1-8b
1.1
1.1
1.1
0.95
0.95
0.95
0.95
2-11a
7
7
7
z
3-8
1.5
3.5
3.5
1.5
1F5
5.0
5.0
5.0
2.5, 1.8
49f
1.6
1.6
1.6
2.8, 2.5
2.1
Figure 3-5. Comparative evolutionary analysis of Hind III
fragments in great ape males. Hind III fragment sizes
detected in male but not female DNA by the probes listed at
the left are indicated for each of the great ape species.
Probes are ordered from proximal to distal corresponding to
the orders of the intervals into which each falls on the
human Y. Common fragments in male chimpanzee and gorilla or
male chimpanzee, gorilla and orangutan are underlined.

73
When viewed cytogenetically, great ape Y chromosomes
appear markedly similar. To date, only three anonymous Y
chromosome DNA sequences outside the pseudoautosomal region
have been demonstrated to be conserved on the Y chromosomes
of chimpanzees, gorillas, and orangutans: 4B-2 (Burk et al.,
1985), 69/6 (Erickson, 1987), and 1F5 (Whisenant et al..
1991). The results reported here are unprecedented in that
all of the eleven Y chromosome-specific sequences studied
detected similar sequences on the Y chromosomes of the great
apes. These same sequences were not detected in male or
female DNA from rhesus monkeys, cattle, or mice. A twelfth
sequence, GMGY1, which detects autosomal and Y alleles in
human DNA, demonstrated conservation of one autosomal
homologue in all six mammalian species studied.
Transcription from the GMGY1 locus has not been reported,
but its high degree of conservation argues for some type of
functional role for the autosomal sequence. The human Y-
1inked homologue might represent a newly arrived pseudogene,
as was described for the argininosuccinate synthetase and
steroid sulfatase Y-linked fragments (Daiger et al., 1982;
Yen et al., 1988). The absence of data from the male
gorilla prevents a definitive statement about the Y-linked
allele, although data gathered to date indicate that Y-
linked sequences shared by humans and gorillas are also
shared by chimpanzees.

74
The results of this study contrast with many earlier
findings that human Y-linked sequences, while evolutionarily
conserved, were not Y-linked in other higher primate genomes
(Page et al.. 1984; Koenig et al.. 1984, 1985; Erickson,
1987; Bickmore and Cooke, 1987). This conservation of
sequences is similar to what has been observed on the
autosomes of higher primates. The interpreters of the early
data suggested that a substantial portion of the single- and
multi-copy sequences on the human Y chromosome were acquired
during human evolution (Kunkel and Smith, 1982; Burk et al.,
1985) The proposal that the human Y chromosome is composed
primarily of newly arrived sequences is refuted by the
conservation of the exclusively Y-linked sequences described
in this chapter.
A model explaining the different Y chromosome sequence
elements might be thus. The pseudoautosomal region,
conserved on the sex chromosomes of the higher primates, and
some other X- and Y-linked genes may be the last remnants of
an ancient homologous pair of chromosomes. The sequences
detecting homologues on both the human X and Y chromosomes
outside of this pairing region and found only on the X
chromosomes of chimpanzees, gorillas, and sometimes
orangutans, may represent the sequences transposed to the
human Y after the divergence from the chimpanzee/gorilla
ancestor. Tandem duplication of repeated sequences on the
human Y also occurred after this divergence. By contrast,

75
many of the sequences on the long arm of the Y arose during
or after the divergence of higher primates from their lower
primate ancestors. These sequences have accumulated new
mutations and in some cases duplicated over the past 10
million years.
The human Y chromosome appears to be a mosaic of
sequences that have arrived at different times during its
evolution. The retention of Y-specificity among related
species has been suggested to be functionally significant
(Burk et al., 1985). Some of these sequences may represent
genes that affect the fertility of male higher primates, for
example, TSPY (Arnemann et al., 1987). Because very few
genetic loci have been assigned to the Y (Goodfellow et al.,
1985), it seems unlikely that all of these evolutionarily
conserved, anonymous DNA sequences encode functional
proteins. Some other selective pressure may be exerted on
these exclusively Y-linked sequences to cause them to remain
Y-associated over a period of approximately 10 million
years. This block of evolutionarily conserved, exclusively
Y-linked sequences may be related to the structure of the
region, assuring proper replication and condensation.
Alternatively, they may play a role in pairing or in
blocking recombination with the X or other chromosomes.
These experiments represent a first step in
understanding the extent of conservation of the euchromatic
long arm of the Y chromosome among higher primates. Genes

76
that are located within this region may be difficult to
identify because of a paucity of HTF islands (presented in
Chapter 4) as well as absence of conservation in other
species. The development of maps for this region in all of
these organisms will provide greater insight into the
molecular mechanisms of evolution. Because this region is
not subject to recombination, DNA sequence analysis will
provide a molecular clock to determine more precisely the
phylogenetic relationships among these organisms. Unlike
more global models, these genetic events represent fine
tuning in the evolution of sex chromosomes.

CHAPTER 4
LONG-RANGE ANALYSIS OF SEQUENCES MAPPING
TO THE DISTAL EUCHROMATIC LONG ARM
OF THE HUMAN Y CHROMOSOME
Introduction
The long arm of the human Y chromosome is hemizygous in
males and therefore does not normally participate in
recombination during meiosis. The only technique currently
available for long-range mapping of such a non-recombinant
region is pulsed field gel electrophoresis, PFGE. This
technique used in conjunction with enzymes that cleave
infrequently in mammalian genomes, allows the analysis of
DNA fragments up to 9,000 kb (Schwartz and Cantor, 1984;
Barlow and Lehrach, 1987). The restriction endonucleases
used for this technique have recognition sites that tend to
be clustered in HTF islands. The sites are G + C rich and
contain one or more CpG dinucleotides. When HTF islands
were first described (Cooper et al., 1983; Bird et al..
1985), they were hypothesized to be associated with genes.
Almost all cleavage by CG enzymes occurs in HTF islands
(Brown and Bird, 1986) which are now known to mark many
genes in mammalian genomes (Bird et al., 1987; Abe et al.,
1988; Pontarotti et al.. 1988; Goodfellow et al., 1988;
Sargent et al., 1989). The most useful enzymes contain only
77

78
G and C in their recognition sequences, with two CpGs per
site. Double CpGs are rare between islands and common
within them, such that three out of every four genomic sites
occur in islands (Lindsay and Bird, 1987). The presence of
island-related genes on distal Yqll has not yet been
addressed.
Several genes in and near the pseudoautosomal region
have been localized by constructing restriction maps
covering long stretches of genomic DNA. These genes include
MIC2, ZFY, SRY, and a gene possibly involved in linear
growth (Pritchard et al.. 1987; Page et al., 1987; Sinclair
et al.. 1990; Henke et al., 1991). Several of these genes
were ascertained in genomic DNA by the production of
similarly sized fragments by different infrequently cleaving
restriction endonucleases, an observation often associated
with HTF islands (Brown and Bird, 1986; Pritchard et al..
1987; Henke et al., 1991).
The distal portion of Yqll has been of interest because
of its association with spermatogenesis (Tiepolo and
Zuffardi, 1976), and the physical characterization of the
area might aid in the isolation of the responsible gene(s).
Deletion of the distal euchromatic long arm of the human Y
chromosome has been associated with azoospermia, or
spermatogenic failure, in otherwise normal men (Tiepolo and
Zuffardi, 1976; Bobrow, 1985; Bhler, 1985; Andersson et
al., 1988; Hartung et al., 1988). A complete long-range map

79
of the Y will be useful because cytogenetic landmarks are
difficult to visualize. The critical subband of Yqll to
which the azoospermia factor, AZF, has been assigned is
Yqll.23 (Human Gene Mapping, 1989). This subband would
therefore reside in "interval 6" as described by Vergnaud
which corresponds to distal Yqll (Vergnaud et al. 1986;
Andersson et al., 1988; Johnson et al., 1989). Sequences
such as pDP105/B (DYZ4, Andersson et al., 1988), 50f2/C,E
(DYS7, Guellaen et al.. 1984) and 49f (DYS1, Bishop et al.,
1984) have been shown to be deleted from some infertile
azoospermic men (Andersson et al., 1988; Nakahori et al. ,
1991) as well as from other individuals with abnormalities
in sexual development (Kotecki et a_l. 1991). Probe 4B-2
(DYS15, Burk et al., 1985) was included in one study to
screen for the integrity of sequences proximal to the AZF
region (Johnson et al., 1989), and will be shown in this
study to anchor the proximal end of the restriction map
described.
The experiments in Chapter 2 allowed the separation of
the Y chromosome into eight intervals, six of which lie in
Yqll. The most distal euchromatic interval described in
that study, interval 7, contains locus DYS1 (49f) and would
therefore correspond roughly to "interval 6" as described by
Vergnaud (Vergnaud et al., 1986). The experiments to be
described here were used to develop a long-range restriction
map of interval 7, and should be near the location of the

80
azoospermia factor. The five sequences mapping to this
interval were used to develop the map, and probe 4B-2
(DYS15) was included because it detected significant
restriction fragment overlap with distal sequences, and was
therefore useful in distinguishing the centromeric and
telomeric ends of the map.
Materials and Methods
High Molecular Weight DNA Preparation
Lymphoblastoid cell cultures or separated peripheral
blood lymphocytes (PBL) were counted using a Neubauer
hemocytometer after washing one to two times with SE (75 mM
NaCl, 25 mM EDTA, pH 7.4). Lymphoblastoid cultures were
derived from a normal male and Oxen, a cell line derived
from a male with four Y chromosomes, 49,XYYYY (Sirota et
al., 1981). Female control cells were obtained from
peripheral blood lymphocytes from normal female donors.
Single cell suspensions were prepared at 1-2 x 106 cells per
50 jul SE at room temperature, which yielded about 10 /g of
DNA per agarose block. A 1.2% solution of nucleic acid
grade agarose (Pharmacia) in SE was melted and held at about
50C and equal volumes of agarose and cell suspension
(prewarmed to 37C) were mixed and immediately dispensed
into a mold with a capacity of 100 ¡il per slot. The blocks
were allowed to solidify at 4C and then were removed from
the mold to a sterile mixture of 0.5 M EDTA, pH 9/1% (v/v)
Sarkosyl (Sigma)/2 mg per ml predigested Pronase (Boehringer

81
Mannheim Biochemicals) at 0.5 ml per block in a 50 ml
conical centrifuge tube (Falcon) for 48 hours at 55C. A
second aliquot of Pronase was added to 2 mg/ml after 24
hours to ensure complete proteolysis. The blocks were
rinsed three times with sterile TE, pH 8, by filling the
tube, allowing the blocks to settle to the bottom, and
pouring the buffer away. The tube was then filled with TE
plus 0.04 mg/ml PMSF (Sigma) dissolved in DMSO (Fisher) and
incubated at 55C for 30 minutes. This step was repeated
once and was included to inactivate the Pronase and remove
the Sarkosyl. The blocks were rinsed with TE to remove the
PMSF and stored in TE or 0.5 M EDTA, pH 8 at 4C. All steps
were performed with gloves, sterile solutions, and sterile
supplies in order to avoid nuclease contamination.
Restriction Enzyme Digestion
Blocks from the Oxen and normal male cell lines and
female PBL were decanted from centrifuge tubes and
manipulated with an alcohol-flamed spatula into 1.5 ml
microfuge tubes. All solutions and supplies were sterile,
and gloves were worn throughout the procedure to reduce
nuclease contamination. Each block was immersed in two 1 ml
changes of TE, pH 8 and refrigerated for 30 minutes to
several hours each. The TE was replaced with two changes of
1 ml of the appropriate IX restriction buffer at 4C and
preequilibrated for 1-2 hours each, after which the
restriction buffer was removed. A typical restriction

82
digest was set up in a total volume of 300 /zl: 100 /I
agarose block, 30 /z 1 10X restriction buffer, 0.1 mg/ml BSA,
0.5 mM DTT, and 20 units of enzyme. Spermidine was added to
10 mM in digests performed in buffers 50 mM or higher in
salt. Incubations were performed overnight at the
appropriate temperature and a second aliquot of enzyme was
added after 1-2 hours of digestion. The reaction was
terminated by the addition of 1/10 volume of 0.5 M EDTA,
pH 8, and the blocks refrigerated prior to loading the gel.
Double digests were set up such that an enzyme
requiring low salt buffer or 37C incubation was used before
an enzyme requiring high salt or 50C incubation. After
digestion with the first enzyme, 1/10 volume of 0.5 M EDTA,
pH 8 was added to the tube to inactivate the enzyme. The
tube was then rinsed twice with 1 ml of TE, pH 8 before
preequilibrating the block for several hours with two
changes of the next IX buffer at 4C. The second digest was
performed in the same manner as the first. Agarose-embedded
cells from a single harvest of male and Oxen lymphoblastoids
were used to produce the blots shown in Figures 4-1 and 4-2.
Gel Running Conditions
PFGE was performed in a BioRad CHEF DRII apparatus.
Digested DNA samples in agarose blocks were manipulated into
the wells of 1% agarose gels formed in 0.5X TBE (IX TBE is
89 mM Tris, 89 mM boric acid, 1 mM EDTA, pH 8). Sample and
marker blocks were overlaid with molten 1% agarose to fix

83
the blocks into the wells. Gels were run in recirculating
0.5X TBE at 200 V at 14C for 24 hours using a ramped switch
time of 60-90 seconds. Some gel runs were performed in a
cold room at 4C with buffer recirculation and all other
conditions remaining the same. These conditions were used
to separate fragments from about 20 kb to 1600 kb. To
separate fragments from 200 kb to 5 Mb, 0.8% gels were run
at 150 V, 4C with buffer recirculation, using ramped switch
times of 90-3600 seconds for 30-36 hours.
Large genomic fragments were sized by comparison with
yeast chromosomes (Saccharomvces cerevisiae AB972, 240 kb-
1600 kb; Schizosaccharomvces pombe. 3.6 Mb-5.7 Mb [BioRad])
and lambda concatamers (FMC Bioproducts), 50 kb-400 kb. The
error involved in sizing genomic fragments above 400 kb may
be as large as 50 kb (Kenwrick et al., 1987).
Preparation of Blocks Containing Yeast Chromosomes
Haploid Saccharomvces cerevisiae strain AB972, a gift
from Dr. Maynard V. Olson, Washington University, was grown
in YEPD (1% yeast extract, 2% peptone, 2% glucose) to mid-
logarithmic phase at 30C and harvested by centrifugation.
The cells were washed twice in 50 mM EDTA, pH 8 and
resuspended to a final concentration of 5 X 109 cells per
ml. The cells were mixed volume to volume with 1% agarose
in 50 mM EDTA, pH 8 maintained at 55C, dispensed into a
mold, and allowed to solidify at 4C. Spheroplasts were
obtained from the embedded cells using the protocol of

84
Beilis et al. (1987) in 0.5 M NaCl, 0.25 M EDTA, 0.125 M
Tris, pH 7.5, and 0.5 M beta-mercaptoethanol. The
suspension was incubated at 37C for 6 hours and then made
1% in SDS and 2 mg/ml in Pronase and incubated at 42C for
24 hours. The blocks were rinsed three times in TE, pH 8 at
50C and stored at 4C in 0.5 M EDTA, pH 8. The chromosome
sizes for this strain provided by Dr. Olson are 240, 280,
350, 440, 590, 680, 755, 810, 840, 950, 985, 1095, 1120,
1130, and 1640 kb.
Transfer and Hybridization
Ethidium bromide stained gels were destained in water
for 30 minutes to 2 hours, photographed, and acid hydrolyzed
in 0.25 M HC1 for 20 minutes. The DNA was transferred
according to the manufacturer's instructions to Hybond N+ in
0.4 M NaOH. Before the first use with an unknown probe,
each blot was test hybridized with the pseudoautosomal probe
19b (MIC2 genomic clone), and autoradiography performed.
Each lane was examined for the known pattern of bands
detected by 19b with a particular enzyme to test for
completion of digestion and efficiency of DNA transfer.
None of the blots indicated incomplete digestion of DNA at
the MIC2 locus in any lane.
DNA probes were labeled with 32P dCTP (3000 Ci/mmol,
ICN) by random primer extension (Feinberg and Vogelstein,
1984) using a BRL kit to a specific activity of at least
1 X 109 cpm/jiig. Membranes were hybridized in 5X SSPE, 1%

85
SDS, 5X Denhardt's, 10% dextran sulfate at 65C. Total
human genomic DNA derived from placenta and sheared to 3 00-
500 bp, was denatured and added to 30 nq/ral in both
prehybridization and hybridization solutions to block
background smears in the lanes. Post-hybridization washes
were performed at high stringency (0.1X SSPE, 0.1% SDS,
65C) and autoradiography was performed at -70C for 4-6
days with intensifying screens (Lightening Plus, DuPont).
Blots were washed in 0.1X SSPE, 0.5% SDS at 95C after
autoradiography, exposed for 48-72 hours to ensure that
residual probe was removed, sealed in plastic and reused.
Assumptions Affecting Long-Range Map Development
The map to be presented here was composed on the basis
of several assumptions. Restriction sites from single
enzyme digests were placed in the individual maps such that
the fragments would nest, following the routine
interpretation that these fragments represent partial
digestion of methylated sites (Nicholls et al.. 1989; Li et
al.. 1990; Kirkilionis et al.. 1991). Wolf and Migeon
demonstrated that multiple restriction fragments detected by
single copy probes hybridized to Southern blots of human
genomic DNA generated by methylation-sensitive enzymes
represent fixed methylation patterns that differ from cell
to cell (Wolf and Migeon, 1982). The differences in band
intensities in a lane were assumed to be related to the
percentages of cells present in the sample that bore a

86
particular fixed methylation pattern (Wolf and Migeon, 1982;
Goodfellow et ad., 1987; Nicholls et al.. 1989).
The extremely complex patterns detected at all loci
when digested with two enzymes were assumed to be composed
primarily of single digestion products, because most of the
bands in those double digests were present when either of
the enzymes was used singly (see Table 4-1). The sequence
in which the digestions were performed did not appear to
influence which single digestion products were produced.
For example, most of the Mlu I restriction fragments were
present in both Mlu I/BssH II and Ksp I/Mlu I digestions, in
which Mlu I was used first and last, respectively. For the
double digests, only those bands which were not seen in
either single digest were considered to be unambiguous
double digestion products.
Results
A Restriction Map of the Distal Euchromatic Long Arm of the
Y Chromosome
DNA was digested with Not I, BssH II, Ksp I, and Mlu I.
Double digests were performed using combinations of BssH II,
Ksp I, and Mlu I. Blots containing Oxen, normal male, and
normal female DNA digested with BssH II, Ksp I, and Mlu I
singly and in combination were sequentially hybridized with
the five probes from interval 7, and 4B-2 from interval 6.
Before the first use with a Yqll probe, all blots were test
hybridized with 19b, the MIC2 genomic clone mapping to the

87
pseudoautosomal region. The blots were analyzed for the
presence of bands known to be produced at that locus by the
various enzymes used in this study and to ascertain the
completion of digestion and efficiency of DNA transfer. The
Y chromosome restriction fragment data from single and
double digests of Oxen DNA for these six loci are compiled
in Table 4-1. The Southern blot data from the cell line
Oxen, containing four copies of the Y, are shown in Figures
4-1 and 4-2. Female control lanes were included on the
blots to visualize non Y-linked bands detected by 49f, 1F5,
and GMGY1 (not shown). In Figures 4-1 and 4-2 the non Y-
linked bands detected in female lanes are marked for probes
GMGY1, 1F5, and 49f.
Not I restriction fragments were not detected at any of
the loci when probes were hybridized to PFGE-generated
Southern blots containing fragments from about 200 kb to
5 Mb (not shown). No Ksp I fragments were detected by 1F5
and 49f, whereas a minor fragment of 480 kb was detected by
BAY3-8 and BAY2-lla. GMGY1 and 4B-2 detected common Ksp I
fragments of 250, 320, and 370 kb; and an additional six
fragments of 150, 190, 420, 490, 520, and 590 kb were
detected by 4B-2. A major 900 kb BssH II fragment was
detected by BAY3-8, BAY2-lla, 1F5 and 49f. These probes all
detected a minor 780 kb fragment; this fragment was weak
using the blot in Figure 4-1, but was stronger on a blot
produced from a different harvest of the Oxen cells (not

88
Table 4-1. High molecular weight restriction fragments
detected by distal Yqll probes.
Bss
KSP
Mlu
B/K
B/M
K/M
PROBE
4B-2
420
590
820
(650)
(1Mb)
(960)
520
780
(620)
(820)
820
490
760
(420)
(780)
780
420
550
360
630
(630)
370
510
190
550
(550)
320
300
150
510
(510)
250
420
(200)
190
300
(190)
150
(40)
GMGY1
(360)
370)
820
(1Mb)
(960)
(320)
780
(820)
(820)
(250)
760
(780)
(780)
550
(590)
550
510
(550)
420
420
BAY3-8
900
(480)
820
900
(820)
(960)
(780)
780
(580)
(780)
820
f300)
630
(480)
630
780
120
120
590
630
(550)
470
440
300
120
BAY211a
900
(480)
820
900
(820)
(960)
780
780
120
(780)
820
3001
630
630
780
(200)
590
710
(550) 630
470 550
440 400
300
120

89
Table 4-1continued
Bss
Ksp
Mlu
B/K B/M
K/M
PROBE
1F5 900
820
900 (820)
(960)
(780}
780
(780)
(820)
(550}
630
(780)
200
(590)
710
(550)
630
470
200
440
200
49f 900
820
(900) 820
820
(780}
(550}
630
(550)
200
(590)
200
60
(550)
60
(20)
470
(20)
200
60
(20)
Band sizes are in
kb.
Enzymes
are Bss: BssH II
, Ksp
Mlu: Mlu I. B/K:
BssH
II and
Ksp I, B/M: BssH
II anc
Ksp I,
Mlu I,
K/M: Ksp I and Mlu I. Weakly hybridizing fragments are
indicated in parentheses. Restriction enzyme fragments
recognized by multiple probes are underlined for the single
digests.

Figure 4-1. Hybridization of distal Yqll probes to PFGE
Southern blots. High molecular weight DNA from a 49,XYYYY
individual was digested with various enzymes, separated by
PFGE, and transferred to Hybond N+. The blot was
sequentially hybridized with sequences mapping to the
distal-most interval defined by the mapping panel in Chapter
2 as well as probe 4B-2 from the next more proximal
interval. Lanes: 1 BssH II, 2 Ksp I, 3 Mlu I. Probes: a
4B-2 (DYS15), b GMGY1 (DYS12), C BAY3-8 (DYS194), d BAY2-lla
(DYS193), e 1F5 (DYS128), f 49f (DYS1). Molecular weight
markers are in kb. Dots (.) indicate fragments detected in
female DNA and which are not Y-linked.

91
1 2 3
2 3 12 3
*
b

Figure 4-2. Hybridization of distal Yqll probes to PFGE
Southern blots produced from digests with two restriction
enzymes. High molecular weight DNA from a 49,XYYYY
individual was digested with various combinations of
enzymes, separated by PFGE, and transferred to Hybond N+.
The blots were seguentially hybridized with seguences
mapping to the distal-most interval defined by the mapping
panel in Chapter 2 as well as probe 4B-2 from the next more
proximal interval. Lanes: 1 BssH II/Ksp I, 2 BssH II/Mlu I,
3 Ksp I/Mlu I. Probes: a 4B-2 (DYS15), b GMGY1 (DYS12), c
BAY3-8 (DYS194), d BAY2-lla (DYS193), e 1F5 (DYS128), f 49f
(DYS1). Molecular weight markers are in kb. Dots (.)
indicate fragments detected in female DNA and which are not
Y-linked.

93

94
shown). BAY3-8 and BAY2-lla each detected an additional
weak 300 kb fragment and a major 120 kb fragment with
BssH II. GMGY1 and 4B-2 detected single BssH II fragments
of 360 and 420 kb, respectively. All probes detected a
common 820 kb Mlu I fragment, and all except 49f detected a
780 kb Mlu I fragment as well. GMGY1 and 4B-2 detected
common Mlu I fragments of 760, 550, and 510 kb; and unique
fragments of 420 and 300 kb, respectively. BAY3-8 and
BAY2-lla detected a common 630 kb Mlu I fragment, whereas
BAY2-lla detected a weak Mlu I fragment of 200 kb. 1F5 and
49f detected common fragments of 550 and 200 kb, while 49f
detected unique Mlu I fragments of approximately 60 and
20 kb. The 200 kb fragment detected by BAY2-lla was
considered to be different from the 200 kb fragment shared
by 1F5 and 49f for several reasons. First, its
hybridization signal was much weaker than that detected by
1F5 and 49f (see Figure 4-1, d-f). Second, the weak 200 kb
fragment detected by BAY2-lla was absent from double digests
involving Mlu I, while the strong 200 kb fragment seen with
1F5 and 49f remained visible in Mlu I double digests with
both BssH II and Ksp I (Figure 4-2, d-f). Third, the same
set of double digest blots was sequentially hybridized with
BAY2-lla and 1F5. The absence on these blots of the 200 kb
band in the BAY2-lla hybridization, followed by the
detection of a strong 200 kb fragment by 1F5 indicated that
the different intensities of the fragments detected on the

95
single digest blot were not the result of differing
conditions of hybridization, but that the fragments
themselves were different.
Restriction maps covering hundreds of kilobase pairs
were constructed for each enzyme in a manner analogous to
conventional restriction mapping. To establish a composite
long-range restriction map of interval 7, double digests
were performed with combinations of BssH II, Ksp I, and
Mlu I (see Table 4-1). Two blots were required to include
all enzyme combinations and controls, and two sets of these
blots were produced and sequentially hybridized with probes
from interval 7. Multiple fragments were detected by each
probe in most double digestions, however, most of these
fragments were the same sizes as fragments produced by one
or the other of the enzymes when used singly (see Table 4-1)
and were therefore assumed to be single digestion products.
Because of the complexity of the banding patterns produced
at each of the loci, the double digest data were only used
to corroborate the relative positions of the maps produced
from the single digests. Southern blot data from double
digests of the Oxen cell line are presented in Figure 4-2.
The rates of migration on the gels from which the multiple
blots were produced varied, even under standardized
conditions. Banding patterns from different autoradiograms
could be superimposed upon one another (for example, 1F5 and

96
BAY3-8) and molecular weights estimated from different
autoradiograms were comparable.
Physical Linkage of 4B-2. GMGY1. BAY3-8. BAY2-lla. 1F5. and
49f.
All probes apparently shared a common 820 kb Mlu I
fragment, simplifying the positioning of the BssH II and
Mlu I maps relative to one another. A proposed long-range
restriction map covering interval 7 and some of interval 6
on the Y chromosome long arm is presented in Figure 4-3.
This preliminary map is based upon the assumption that all
six probes detecting an 820 kb Mlu I restriction fragment
were recognizing a single fragment. Probes 4B-2, BAY2-7a,
BAY25a, and BAYl-8b, from interval 6, were hybridized to a
different set of PFGE Southern blots than that shown in
Figure 4-1 in the effort to establish physical linkage of
intervals 6 and 7 (not shown). Hybridizations of those
initial blots indicated that various Ksp I and BssH II
fragments were shared between these loci and the loci in
interval 7. 4B-2 appeared to overlap the most with loci in
interval 7 using the blots analyzed in Figures 4-1 and 4-2
and was therefore included in the mapping analyses to
indicate the directions of the centromere and telomere. In
the map presented here, the order of loci is: cenDYS15
(4B-2), DYS12 (GMGY1), DYS194 (BAY3-8), DYS193 (BAY2-lla),
DYS128 (1F5), DYS1 (49f)tel.

Figure 4-3. Proposed long-range map of interval 7 on the long arm of the Y chromosome.
The composite map at the top indicates the relative positions of the probes (not to
scale). Fragments detected by each probe in Mlu I and BssH II digests are arranged below
the composite map in their proposed order and relative degree of overlap. The Ksp I
fragments indicated in brackets below do not indicate their arrangements relative to one
another. Ksp I sites are not included on this composite proposed map due to an inability
to establish the relationship among individual fragments. B: BssH II, K: Ksp I, M: Mlu I.

4B-2 GMGY1
J L
BAY3-8,2-lla

J I IJ
1F5 49f

l l l ll
telomere>
r
420
I
J 300 1
J 550
I
360
120
J 420 L
I
J 510
J 550
-* 760
T~
i 300
J 780
J 820
J 630
J 200
1 200
u 20
60
I 900
i 780
CD
00
370
320
250
190
480
150
-100 kb
BssH II
HKsP 1
1I Mlu I

99
The distance between the endmost loci of the map
presented in Figure 4-3 is approximately 820 kb, with the
entire map covering some 1600 kb of distal Yqll. Because of
the complexity of the Southern blot data compiled in Table
4-1, the single digest products are included below the
composite map in Figure 4-3. The fragments produced by each
enzyme are drawn beneath the probes which detect them.
Several trial maps were generated for each of the
single enzyme digestion products. The ordering of the loci
relative to one another was dictated by the Mlu I and
BssH II fragments. The Ksp I fragments were not useful for
ordering the loci because of the absence of shared fragments
detected at different loci (see Table 4-1). Several
orientations for some loci were suggested by the BssH II
data, but only one was consistent with the linkage required
by the Mlu I data. Once the probe order was established,
the individual maps were aligned. Initially the BssH II
fragments appeared to be explainable by at least two maps
which differed in overall length, but scale drawings of each
could not be aligned in such a manner that all probes would
coexist on an 820 kb Mlu I fragment. An early version of
the BssH II map placed 4B-2 and GMGY1 approximately 180 kb
closer to BAY3-8 and BAY2-lla. This placement was
inconsistent with the presence of the 630 kb Mlu I fragment
detected by BAY3-8 and BAY2-lla that was not detected by any
of the other probes. The presence of this fragment placed

100
severe restrictions on the placement of loci on the Mlu I
map which required that all loci share the previously
mentioned 820 kb fragment. This fragment was considered to
be common to all the probes because of its relatively
invariant intensity on autoradiograms of all blots analyzed
during the study. Another consideration was that the
calculated size of the area in question (6 euchromatic
intervals covering 6-10 Mb would each be approximately 1-1.6
Mb in length) would be unlikely to support two such
fragments. The proposed map also would not support another
820 kb Mlu I fragment in an overlapping orientation. As
can be seen from the products of single enzyme digestion
below the composite map, very little shifting of the single
maps relative to one another is allowed. Probably less than
a 20 kb shift in either direction would be tolerated, well
within the 10% resolution of a restriction map covering
over 1 Mb. The strength of this map lies in the consistency
of probe order and site placement imposed upon it by the
combined single digest data.
Because of the constraints imposed upon the production
of a composite map from the three single digest maps by the
Mlu I data, the double digests were needed only to
corroborate the placements of the maps relative to one
another. The double digests served to strengthen the map
again by confirming the probe order already deduced. Often,
a fragment of a particular size detected by one or more

101
probes could be explained on the map by a variety of sites.
These variations could involve the choice of left and right
restriction sites (1,2 or 2,1) and/or the placement of the
fragment along the map. The extremely intense 550 kb band
detected in BssH II/Mlu I double digests by BAY3-8 and
BAY2-lla on different blots may represent a number of
overlapping 550 kb fragments that vary with respect to their
right and left restriction sites and their relative map
positions. Observations such as these served to further
strengthen the relative placements of the three individual
maps that went to form the composite map.
The map does not address the question of why the
numerous other fragments presumably possible were not
detected. The double digest data suggest that many more
fragments than those listed in Table 4-1 were indeed
produced. In Figure 4-2, faint smears in many of the lanes
appeared to be bands. They were not included in the
analyses because their lack of intensity and resolution was
considered to be inadequate for positive identification as a
restriction fragment. Even the 780 kb BssH II fragment
detected by BAY3-8, BAY2-lla, 1F5, and 49f was barely
adequate for positive identification in the blot shown in
Figure 4-1. This band was included in the analysis because
it had produced a stronger signal in blots prepared from
Oxen cells harvested at a different time, and was therefore

102
considered to be due to variable methylation in different
clonal populations of cells.
Discussion
Pulsed field gel electrophoresis allows for the
correlation of genetic and physical distances. In the case
of the euchromatic long arm of the Y chromosome, genetic
distances cannot be ascertained because of the absence of
recombination. PFGE was used to establish the order and
physical positions of five loci mapping to the most distal
euchromatic region, interval 7, defined by the deletion
mapping panel in Chapter 2. A sixth locus, mapping to
interval 6, was included in the proposed restriction map to
establish the orientation of the map with regard to the Y
chromosome centromere and telomere. The purpose of this
study was to provide physical landmarks required to further
characterize this genetically interesting region of the Y
chromosome.
The AZF locus lies in the region distal to 4B-2
(Johnson et al., 1989) which anchors the proximal border of
the map presented here, while 49f localizes to the distal
end of the map. 49f and other more proximal loci have been
shown to be deleted in a number of individuals with
azoospermia (Vergnaud et al., 1986; Johnson et al., 1989;
Nakahori et al., 1991). In additional hybridization
analyses of individuals with deletions of distal Yqll, 49f
has been reported as the most distal locus in a series of

103
loci studied (Kotecki et al. 1991; Nakahori et al., 1991).
To date, euchromatic loci have not been detected distal to
49f, which may place this locus at the distal end of the
region associated with the AZF gene. If 49f is truly distal
to AZF, then 4B-2 and 49f would bound that locus, indicating
that the gene may lie on the 820 kb Mlu I fragment common to
49f and 4B-2.
Clusters of infrequently cleaving restriction sites
associated with HTF islands are not evident in the map in
Figure 4-3. Restriction sites for the endonucleases used in
this study appear to be distributed randomly throughout the
region. None of the infrequently cleaving restriction
enzymes detected distributions of fragments similar to those
produced by the others; thus indicating that HTF islands
were not in the vicinity. This finding is unlike the
prediction by Brown and Bird (1986) that most sites for
infrequently cleaving endonucleases should be concentrated
into HTF islands and not be distributed randomly. Long-
range restriction maps constructed from the short arm of the
Y and from other chromosomes bear out their prediction of
the island association of these sites (Pritchard et al.,
1987; Sargent et al., 1989; Li et al., 1990; Henke et al.,
1991; Kirkilionis et al., 1991). As expected for sequences
associated with islands, both 1F5 and 49f detected common
non-Y-linked fragments in Ksp I and BssH II digests (see
Figure 4-1, e and f) and thereby served as internal controls

104
for islands. The test hybridizations of pulsed-field blots
with 19b also demonstrated the existence of the MIC2-
associated islands. The absence of HTF islands should not
be construed to mean that genes are likewise absent from
distal Yqll, but only that the region lacks island-
associated genes.
In addition to the unexpectedly random distribution of
double CG enzyme restriction sites, sites for Not I were
undetected. The Not I fragment recognized by probe 19b from
the pseudoautosomal region was detected using these blots,
indicating that the Not I digests and DNA transfers were
successful. The apparent absence of Not I sites might be
due either to methylation of sites in the region or to a
lack of sites.
Methylation was expected to be encountered in Yqll, so
the restriction endonuclease Ksp I was included in the
analyses because it was reported by the manufacturer,
Boehringer Mannheim Biochemicals, to be a methylation-
insensitive isoschizomer of Sac II. Southern blot analyses
of male, Oxen, and female DNA restricted with either of
these enzymes and separated in parallel on the same gel
demonstrated identical banding patterns (not shown). Mlu I,
with a recognition sequence of ACGCGT, was chosen for the
analyses in case the CG-only enzymes Not I, Ksp I, and
BssH II should detect restriction fragments of the same
sizes. Mlu I was expected to cleave somewhat more

105
frequently than the other enzymes because of its recognition
sequence, and did produce more numerous restriction
fragments than either Ksp I or BssH II in most cases.
The proposed long-range restriction map presented
represents the most parsimonious arrangement of restriction
sites accounting for all of the fragments produced by the
single enzyme digestions, and at the same time confining all
six loci to an area of no more than 820 kb as indicated by
the Mlu I data. The map represents the first description of
mammalian genomic DNA that is devoid of HTF islands in an
area covering more than 1500 kb. Although these results
were not expected, the nonrecombinant nature of Yqll
represents a unique situation in the genome as well.
Because all genes are not linked to HTF islands, this map
indicates that genes in distal Yq may not be associated with
clusters of unmethylated CpG dinucleotides. The absence of
this segment of the Y chromosome is associated with abnormal
spermatogenesis, and implies the presence of at least one
gene. Evidence gathered from several laboratories,
including that from this study, indicates that the
spermatogenesis locus, AZF, may reside within the region for
which a map is now presented. This map provides a starting
point from which a detailed characterization of this
important area may begin.

CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
The studies in this dissertation present a model for
the long arm of the human Y chromosome. In Chapter 2, a
mapping panel is described that was assembled from the Human
Genetic Mutant Cell Repository (NIGMS). Using this panel, a
series of novel and previously described Y chromosomal
sequences were mapped. The cell lines in the panel divided
the Y chromosome into eight intervals, six of which are in
the euchromatic long arm. Where comparisons can be made
between this deletion map and those from other laboratories,
they are consistent with one another. These results
indicate that the panel can be used for more precise
deletion mapping of the long arm of the Y.
The results of the comparative evolutionary analyses
described in Chapter 3 were unexpected in that eleven
sequences mapping to various intervals of the human Y
chromosome were found to be retained on the Y chromosomes of
the hominid primates: chimpanzees, gorillas, and orangutans.
Although unexpected relative to sequences previously
studied, this sequence conservation appears to parallel the
cytogenetic similarities seen among higher primate Y
chromosomes. The sequences conserved on the Y chromosomes
106

107
of the hominid primates differed from most sequences for
which comparative mapping data were gathered, in that most
of the sequences used in this study were exclusively Y-
linked in humans. Whereas all of these sequences are
unlikely to be protein-encoding genes, their exclusive Y-
linkage in humans and conservation on the Y chromosomes of
related species suggests some functional significance.
The long-range map presented in Chapter 4 demonstrates
that sequences within different intervals may be ordered and
that the different intervals defined by the mapping panel
may be linked to one another by long-range mapping
techniques. The random distribution of infrequently
cleaving restriction endonuclease sites seen in distal Yq
was unexpected, as was the apparent lack of sites in the
region for the enzyme, Not I. The probes from the most
distal euchromatic interval were chosen for study because of
their proximity to the region associated with the AZF
gene(s). If preliminary indications about the location of
AZF gathered from several laboratories are correct, this
locus may be contained on the Mlu I fragment bounded by
probes 4B-2 and 49f.
In future studies, the mapping panel presented in
Chapter 2 may serve two purposes. First, on the basis of
its demonstrated suitability for mapping sequences to
different intervals on Yq, the cell lines may be
incorporated into future mapping analyses. Second, probes

108
previously localized to Yqll can now be sublocalized with
these cell lines to facilitate the production of a common
deletion map of the long arm. A cooperative mapping effort
would greatly facilitate the production of a deletion map of
the human Y chromosome that is composed from common probes
and common cell lines.
To further address the origins of the human Y
chromosome, a greater number of Y-linked probes need to be
mapped comparatively and categorized as to their X- or
autosomal-linkage in humans and their subsequent association
or non-association with primate Y chromosomes. The two
postulates addressing the origin of the chromosome need not
be mutually exclusive; certain portions of the human Y
chromosome may indeed be composed of recently transposed
sequences, while others have a more ancient association with
the Y. It will be interesting to investigate whether the
different classes of sequences have intermixed or have
remained together in large blocks on the human Y, or
conversely on other great ape Y chromosomes. The only
evidence touching on the long-range distribution of
sequences relates to the probe 2:13 (Cooke et al., 1984), an
X-Y homologous sequence that is only found on the X
chromosomes of chimpanzees and gorillas. The X and Y
homology was shown to extend for at least 50 kb in humans
(Bickmore and Cooke, 1987). The continuity of this block
with other sequences such as DXYS1 and DYS22 that show a

109
similar pattern and date of arrival (Page et al., 1984;
Koenig et al., 1985) has not been addressed. Additional
important experiments indicated by the strong conservation
of GMGYl-related sequences in mammalian species involves
investigating whether or not the autosomal allele of GMGY1
represents a functional gene.
A long-range map that includes the more proximal
intervals may demonstrate the presence of HTF islands or
continue to demonstrate the pattern of randomly distributed
sites for infrequently cleaving enzymes seen in the most
distal interval. It may be important to demonstrate a point
where these randomly distributed sites give way to an
arrangement of sites into islands as seen on the short arm
of the Y and on other chromosomes. Although such a long-
range genomic restriction map is subject to methylation,
maps from other chromosomes have proved useful for
comparisons to maps generated from yeast artificial
chromosomes (YACs). A YAC-derived map of the human Y
chromosome would be unaffected by the methylation
encountered on the Y chromosome at the genomic level. YACs
may indicate whether the randomly distributed infrequently
cleaving restriction sites mapped in interval 7 are present
on very few chromosomes, or that the sites are present on
all the Y chromosomes, but are not detectable at the genomic
level because of methylation.

110
This project represents a starting point from which to
generate a composite molecular map of the human Y chromosome
long arm. A panel of cell lines useful for producing a
common Yq deletion map has been introduced in Chapter 2.
The feasibility of linking intervals in which the order and
physical positions of individual probes was established, has
been demonstrated in Chapter 4. Continuing on in that
manner, the remaining intervals may be connected and the
resultant molecular map correlated with the cytogenetic map.
The conservation of sequences on the Y chromosomes of
closely related species seen in Chapter 3, coupled with the
unique arrangement of infrequently cleaving restriction
enzyme sites observed in Chapter 4, suggests the intriguing
possibility that these observations are interconnected. A
restriction map of the entire long arm of the Y chromosome
may be of use in elucidating the extent of that
relationship, as well as in ultimately detecting other genes
that are thought to reside there.

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Yunis, J., Prakash, 0. 1982. The origin of man: a
chromosomal pictorial legacy. Science 215:1525-1529.
Zeuthen, E., Nielsen, J. 1973. Pericentric Y inversion in
the general population. Humangenetik 19:265-270.

BIOGRAPHICAL SKETCH
Beverly Steele Allen was born to Frank Beverly Steele
and Marguerite Irvin Steele in Birmingham, Alabama. She
graduated from Miami Palmetto Senior High School in 1974,
and received a Bachelor of Arts degree cum laude in
microbiology from the University of South Florida in 1977.
In the same year, she married Herbert Allen, Jr. and began
her career as a medical technologist. In January, 1986, she
entered the Ph.D. program in the Department of Biochemistry
and Molecular Biology at the University of Florida under the
direction of Dr. Harry Ostrer. After receiving her doctoral
degree in May, 1992, she will begin a postdoctoral
fellowship in the Department of Medicine, Division of
Oncology, at the University of Florida.
121

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor oi^Philosophy.
Thomas P. Yarn
Assistant Pro
Biochemistry'a
Biology
I certify that I have read this study and tfta£_An my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Harry bstrer
Associate Professor of
Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Philip J. Atai£is C
Professor^of'Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Harry £J. Nick
Associate Professor of
Biochemistry and Molecular
Biology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Susan A. Moyer
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Edward E. Wakeland
Professor of Pathology and
Laboratory Medicine
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy^
May, 1992
Dean, College of Medicine
' 'r,~ s* *
Dean, Graduate School



49
chromosomes was needed to serve as a starting point for the
more detailed mapping of a specific region of the Y
chromosome that will follow.
In this chapter, I have introduced a reference panel of
Y chromosome translocations and deletions that others may
use for the generation of standardized maps. The data
presented here were used to produce a deletion map of the
euchromatic long arm of the human Y chromosome that is
divided into six intervals. This deletion map is consistent
with maps and in situ hybridization data that were generated
in other laboratories, although exact correlations are
difficult to make due to the different probes and cell lines
used in those studies. In the course of regionally mapping
29 newly isolated Y chromosomal sequences, an enrichment for
long arm sequences and an apparent paucity of GC-rich
islands was noted in the clones taken from a flow-sorted Y
chromosome library. The distribution of Y-linked sequences
obtained from the library was consistent with the
distributions noted in other laboratories.
The cell lines in the panel were chosen after meeting
certain criteria. Only unbalanced Y chromosomal
translocations were chosen so that the absence of a Y-linked
band would be indicative of an abnormal Y chromosome. Only
cell lines that were stable in culture were used to ensure
their suitability for molecular analyses. For example,
GM 3774 was rejected for use in the panel after cytogenetic


REFERENCES Ill
BIOGRAPHICAL SKETCH 121
V


51
chromosome. Oosthuizen, however, has observed that 80% of
probes isolated from this library that identified
exclusively Y-linked sequences mapped to Yqll.23, indicating
a "nonrandom chromosomal distribution of randomly isolated
probes" (Oosthuizen et al.. Human Gene Mapping 9, 1987).
An alternate explanation would suggest that the Y
chromosomal fragment in GM 2730 was formed by a complex
deletion/inversion event such that only distal short arm
sequences are retained, as evidenced by the retention of the
SRY gene. Complex cytogenetic events involving the Y have
been described, and most were determined only after
molecular analyses because of the inability to resolve these
events at the microscopic level (Zeuthen and Nielsen, 1973;
Affara et al., 1986; Page et al., 1990). This explanation
would allow for the regional localization of some of the
twelve most proximally mapping probes to the proximal Y
short arm.
The second discrepancy between a karyotype reported by
NIGMS and the molecular data from this study involves cell
line GM 0118. At the cytogenetic level, GM 0118 appears to
retain a large portion of the Y chromosome. Judging by the
size of the additional material on 15p, some portion of Yq
euchromatin should have been retained. However, none of the
probes used in the analyses detected any euchromatic
sequences. A closer look at the translocation chromosome
(Figure 2-1) reveals a banding pattern strongly similar to


LIST OF TABLES
Table 2-1. Y chromosome linkage of BAY clones .... 32
Table 2-2. Genome Data Base D-number assignments ... 48
Table 3-1. Interspecies comparisons of Southern blot
data 65
Table 4-1. High molecular weight restriction fragments
detected by distal Ygll probes 88


BIOGRAPHICAL SKETCH
Beverly Steele Allen was born to Frank Beverly Steele
and Marguerite Irvin Steele in Birmingham, Alabama. She
graduated from Miami Palmetto Senior High School in 1974,
and received a Bachelor of Arts degree cum laude in
microbiology from the University of South Florida in 1977.
In the same year, she married Herbert Allen, Jr. and began
her career as a medical technologist. In January, 1986, she
entered the Ph.D. program in the Department of Biochemistry
and Molecular Biology at the University of Florida under the
direction of Dr. Harry Ostrer. After receiving her doctoral
degree in May, 1992, she will begin a postdoctoral
fellowship in the Department of Medicine, Division of
Oncology, at the University of Florida.
121


89
Table 4-1continued
Bss
Ksp
Mlu
B/K B/M
K/M
PROBE
1F5 900
820
900 (820)
(960)
(780}
780
(780)
(820)
(550}
630
(780)
200
(590)
710
(550)
630
470
200
440
200
49f 900
820
(900) 820
820
(780}
(550}
630
(550)
200
(590)
200
60
(550)
60
(20)
470
(20)
200
60
(20)
Band sizes are in
kb.
Enzymes
are Bss: BssH II
, Ksp
Mlu: Mlu I. B/K:
BssH
II and
Ksp I, B/M: BssH
II anc
Ksp I,
Mlu I,
K/M: Ksp I and Mlu I. Weakly hybridizing fragments are
indicated in parentheses. Restriction enzyme fragments
recognized by multiple probes are underlined for the single
digests.