Mapping the euchromatic long arm of the human Y chromosome

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
    List of Figures
        Page vii
    Abbreviations
        Page viii
    Abstract
        Page ix
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    Chapter 1. Introduction
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    Chapter 2. Deletion mapping of the euchromatic long arm of the human Y chromosome
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    Chapter 3. Evolutionary comparisons of sequences mapping to the human Y chromosome
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    Chapter 4. Long-range analysis of sequences mapping to the distal euchromatic long arm of the human Y chromosome
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    Chapter 5. Conclusions and future directions
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    References
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    Biographical sketch
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Full Text












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.