• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Part I: Molecular and biochemical...
 Part II: Beginnings of an analysis...
 Appendix
 Bibliography
 Biographical sketch














Title: Studies in the transduction of a large F-prime, F14, by bacteriophage P1kc
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Permanent Link: http://ufdc.ufl.edu/UF00097487/00001
 Material Information
Title: Studies in the transduction of a large F-prime, F14, by bacteriophage P1kc
Physical Description: x, 113 leaves : ill. ; 28 cm.
Language: English
Creator: Hendrickson, Edwin R., 1949-
Copyright Date: 1977
 Subjects
Subject: Plasmids   ( lcsh )
Transduction   ( lcsh )
Microbial mutation breeding   ( lcsh )
Bacteriophages -- Experiments   ( lcsh )
Microbiology and Cell Science thesis Ph. D
Dissertations, Academic -- Microbiology and Cell Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 108-112.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Edwin R. Hendrickson.
 Record Information
Bibliographic ID: UF00097487
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000207531
oclc - 04068347
notis - AAX4329

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Abstract
        Page viii
        Page ix
        Page x
    Part I: Molecular and biochemical evidence for F14S in transductants made with bacteriophage P1 from F14 merogenote of Escherichia coli K12
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    Part II: Beginnings of an analysis of the mechanism of P1 transduction of a large F-prime, F14
        Page 66
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    Appendix
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    Bibliography
        Page 108
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    Biographical sketch
        Page 113
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Full Text















STUDIES IN THE TRANSDUCTION OF A LARGE
F-PRIME, F14, BY BACTERIOPHAGE Plke










By










Edwin R. Hendrickson


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





UNIVERSITY OF FLORIDA















ACKNOWLEDGEMENTS


I would like to give a special thanks to Dr. Dennis E. Duggan,

chairman of my graduate committee, for unlimited patience and

understanding, for his contribution in terms of discussions, sug-

gestions and encouragement, and most of all for his humor through-

out the period of this research.

Dr. Eiichi Ohtsubo of the State University of New York at

Stony Brook deserves acknowledgement for allowing me to study under

him and for sharing with me his vast wealth of knowledge in molecular

genetics. Hisdiscussions and contributions to this research are

greatly appreciated. I also wish to thank Dr. Hisako Ohtsubo for

her helpful discussions and her hospitality as a hostess during

my stay at Stony Brook.

A bid of thanks goes to Mr. Tom Yun and Dr. Daniel Vapnek of the

University of Georgia, who helped me in my initial attempts in

electron microscopy of DNA molecules. I also would like to thank

Drs. Gregory Erdos and Henry Aldrich for technical advice and

assistance with electron microscopy.

The other members of my graduate committee, Dr. Richard Boyce,

Dr. Neil Ingram, and Dr. Francis Davis, deserve thanks for thier

discussions and contributions in the course of this research.

I would like to acknowledge the financial support given to

me by the Department of Microbiology and Cell Science and the

University of Florida. Further, I wish to thank the American









Society for Microbiology for awarding me their President's Fellowship

Award which allowed me to study in Dr. Eiichi Ohtsubo's laboratory

in Stony Brook.

Mr. and Mrs. Richard J. Hendrickson, my parents, deserve

grateful recognition for their love, support, and guidance.

I would like to give a special thanks and acknowledgement to

Rene, my wife. whose love, support, patience, and humor helped me

through the duration of this research.
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS . . . . . . . . . . ii

LIST OF TABLES . . . . . . . ... . . v

LIST OF FIGURES . . . . . . . ... .. .vi

ABSTRACT . . . . . . . . .. . . . viii

PART I. MOLECULAR AND BIOCHEMICAL EVIDENCE FOR F14S IN
TRANSDUCTANTS MADE WITH BACTERIOPHAGE P1 FROM
F14 MEROGENOTE OF ESCHERICHIA COLI K12 . . 1

Introduction . . . . . . . . . 1

Materials and Methods . . . . . . 3

Results . . . . . . . . .. .11

Discussion . . . . . . . ... 63

PART II. BEGINNINGS OF AN ANALYSIS OF THE MECHANISM OF
P1 TRANSDUCTION OF A LARGE F-PRIME, F14 ... .66

Introduction . . . . . . . ... 66

Materials and Methods . . . . . . .68

Results . . . . . . . . .. ... . 72

Discussion . . . . . . . . 85

APPENDIX . . . . . . . . . . . 90

LITERATURE CITED . . . . . . . . .... 108

BIOGRAPHICAL SKETCH . . . . . . . .. .113














LIST OF TABLES


Page


PART I


PART


1. Escherichia coli K12 strains . . . . . . .

2. Size classes of circular DNA from X1254 and the
recAl- strains carrying the transduced F14s . .

3. EcoR1 fragments of DNAs from F14(P133), F14,
F16 and F310 . . . . . . . . . .


T II

1. Escherichia coli K12 strains . . . . . . .

2. Transduction vs. transformation as the mode of
transfer of the F14 . . . . . . . .

3. Effect of chelation on ability of P1-X1254 to
transduce F14 into AB1450 . . . . . . .


69


73


80















LIST OF FIGURES
Page
PART I

1. Structural map of F14 and its segregated subunits . 13

2. Electron micrograph of size class molecules in
the plasmid population of KF533 [F14 (P133)] . . 21

3. Heteroduplex structure of reference molecules (a)
Fins5 .8 and (b) F316 with F . . . . . 25

4. Electron micrograph of a self-renatured hetero-
duplex of FF14 P133)]/F14(P133) 1 . . . . . 27

5. Self-renatured structure: F[F14(P133)]/F14(P133)
(F class/F14 class) . . . . . . . .. 29

6. Out-of-register circular structure due to the
directly repeated sequences . . . . .... 32

7. Out-of-register structure involving the rrrA and
the rrnB gene sets found in a self-renatured
heteroduplex of F14(P133) . . . . . .... 35

8. Heteroduplex structure of F i /F14(P133) .... 38
ins58.8
9. Electron micrograph of F316/F14(P133) . . . ... 40

10. Heteroduplex structure of F316/F14(P133) . . ... 42

11. Electron micrograph of a heteroduplex structure
of F316/F14(P133) . . . . . . . ... 45

12. Heteroduplex structure of F316/F14(P133) . . ... 47

13. Electron micrograph of a heteroduplex structure
of F316/F14(P133) . . . . . . . ... 49

14. Heteroduplex structure of F316/F14(P133) . . .. 51

15. Agarose gel (0.5%) electrophoresis of the EcoRl
cleavage products of DNAs from F8(P6, F, F310,
F16, F14(P133) and F14 . . . . . ... 54









Page


16. EcoR1 generated fragments of F8(P6), F, F310,
F16,F14(P133) and F14 greater than 0.38 kb . . . 57

17. EcoR1 maps of bacterial DNA in the ilv region
and surrounding regions of F DNA on F14(P133),
F14, F16, F310 and F312 . . . . . ... 60


PART II

1. Nature of the F14 transduction . . . . ... 77

2. CsCL density centrifugation analysis of phage
particles . . . . . . . . . 84














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


STUDIES IN THE TRANSDUCTION OF A LARGE
F-PRIME, F14, BY BACTERIOPHAGE Pkce


By


Edwin R. Hendrickson

August, 1977


Chairman: Dennis E. Duggan, Ph.D.
Major Department: Microbiology and Cell Science


Previous genetic evidence has indicated the presence of "F14-like"

merogenotes in transductants made with P1 grown on AB1206, an F-prime

strain haploid for the region carried on the F14. Here we report

physical and biochemical evidences, which taken together demonstrate

that the transduced plasmids are indeed indistinguishable from F14.

Three size classes were found: 3.3 times 1311 kilobases (kb)].

2.3 times (217 kb), and 1.0 times (94.5 kb) the size of F. Hetero-

duplex analysis showed several structures present on the transduced

F14s that are also part of the F14 structure. They have all of the

F sequence (94.5 kb) which includes the ao directly repeated sequence

(IS3) of F. They also have another directly repeated sequence,

which occurs at the two junctions of F DNA with chromosomal DNA.

There is another directly repeated sequence that measures 5.2 kb









(rrnA and rrnB), which has a small 0.5 kb non-homology bubble (the

rsp spacers).

Heteroduplex analysis also revealed that the 1.0 times F size

class molecules in transduced F14 strains are F. This is consistent

with those found in the parental F14 strain's plasmid population.

The F has been proposed to form by intermolecular recombination events

between the sequences. Gel electrophoretic analysis of EcoR1

fragmented plasmid DNA showed that transduced F14s and parental F14

have identical gel patterns, sharing 44 bands (fragments greater

than 0.38 kb in size), indicating that their sequences are identical.

Comparisons of gel patterns with F and FiZvs were used to identify

the F. sequences, the iZv sequence and chromosomal sequences. Both

F14s also showed bands with identical molecular weight as the fragments

previously identified for the ppc-argECBH-bfe chromosomal region.

These data, taken together, support the genetic evidence that the

"F14-like" plasmids in P1 transductants are indistinguishable from F14.

The mode of genetic transfer of the F14 in these P1 Lysates is

transduction. This is a seemingly impossible transduction since the

F14 would appear to be too Large (205 x 106 daltons) to be transduced

by P1 (whose normal transducing particles carry 64 x 106 daltons);

however, the frequency of transduction of the F14 (7 x 10-8) is too

high to be transduced by multiple infection of transducing particles

carrying complementary fragments. A dose-response curve supports

the model of one transductional unit. Further studies revealed that

the unit is probably one transducing particle. Due to the amount of

DNA transduced, the transducing particle should be larger and/or

more dense. Such phage particles were not found by cesium chloride









density centrifugation, further, the capability of P1 to transduce

the F14 is lost during the centrifugation. A model is proposed for

the transduction of F14 by one particle.














PART I


MOLECULAR AND BIOCHEMICAL EVIDENCE FOR F14S IN TRANSDUCTANTS
MADE WITH BACTERIOPHAGE P1 FROM AN F14 MEROGENOTE OF
ESCHERICHIA COLI K 12


Introduction


P1 virulent particles carry a complement of linear deoxyribonucleic

acid (DNA) of 64 x 106 daltons or 97 kilobase pairs (kb) (Ikeda and

Tomizawa, 1965; Lee, Ohtsubo, Deonier and Davidson, 1974; Backmann,

Low and Taylor, 1976). Transducing particles carry a fragment of host

chromosomal DNA which usually has the same molecular size as the genome

of the virulent particles (Ikeda and Tomizawa, 1965; Lee et al., 1974;

Rae and Stodolsky, 1974; Rosner, 1975; Bachmann et at., 1976). But,

there have been several cases reported that suggest that P1 has trans-

duced F-primes that are larger than the DNA complement carried by

normal transducing particles, e.g. F8 (117 kb) and F14 (311 kb) (Ohtsubo,

1971; Pittard and Adelberg, Bacteriol. Proc., p. 138, 1963). The

case which we have been examining inl detail is the putative transduction

of F14. This very large F-merogenote is transduced at a frequency of
-B
7.0 x 108 per plaque forming unit (p.f.u.) (Hendrickson and Duggan,

1976). The transduced molecules are genetically indistinguishable

from the parental F14 in terms of (a) easily detectable genes trans-

ferred into recA recipients CilvEDAC, metE, rha, metB, argH), (b) the

order of transfer of genetic markers, and (c) the genetic distance

(time-of-entry) between proximal and distal markers (metB and ilvD)









(Hendrickson and Duggan, 1976). The genetic evidence obtained suggests

that a molecule more than three times the size (311 kb) of that normally

carried by P1 has been transduced. Whether by one particle or several,

the mechanism of such a seemingly unlikely transduction is of great

interest; but, since other explanations could be proposed to fit

the genetic data, firm physical and biochemical evidence for the

presence of F14 plasmids in transductants is needed before studies on

the mechanisms are justified.

The focus of the research reported here is to present the physical

and biochemical evidence for the presence of F1L plasmids in the

transductants. The identity of the physical structures of the trans-

duced F14s is determined by contour measurements of DNA molecules

on electron micrographs and by DNA heteroduplex analysis; the bio-

chemical identities of the molecules are determined by examining

gel electrophoresis patterns of restriction fragments.














Materials and Methods


Media.

All strains are routinely grown in Luria (L) broth (Luria and

Burrows, 1957) when a complex broth medium is required and with

the addition of 2.0% agar for complex plating medium. The Z broth

(L broth with 2.5 x 10-3 ;1 CaCL2) (Luria and Burrows, 1957) is

used to grow both the donor and recipients in transductions where

Pike is used as a vector. TheZ agar ( broth with 1% agar) is used

as a bottom agar for making and titering phage lysates. The SA-1 agar

(0.7% agar and 1.0% NaCd) is used as an overlay agar on Z plates

for plaque forming assays and for making lysates on plates

(Hendrickson and Duggan. 1976). Half-strength medium 56 (Adel-

berg and Burns, 1960) is a minimal medium routinely used for the

selection of recombinants and for the growth of F-merogenote strains

to prevent segregation of F-primes used in the study. All amino

acids, purines and pyrinidines are supplemented as required in

56/2 at final concentrations as described in Hendrickson and Duggan

(1976).


Bacterial Strains.

The bacterial strains used are described in Table 1. The haploid

F14 strain, X1254 (isolated from AB1206 in the laboratory of Roy

Curtiss III) (Pittard, Louitt and Adelberg, 1963; Ohtsubo, Deonier,

Lee and Davidson, 1974a; Hendrickson and Duggan, 1976) is used

as the donor strain in transduction experiments. It is also used as

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the source of F14 plasmid DNA in contour measurements of molecules,

heteroduplexing analysis and restriction enzyme mapping. Each

"F14-Like" merogenote strain in derived as previously described

(Hendrickson and Duggan, 1976). The "F14-Like" 2~oA strains are

used in time-of-entry (T.O.E.) matingsand as a source of plasmid

DNA.


Bacteriophage

The stock of Plke (Lennox, 1955) was obtained from Roy Curtiss

III (University of Alabama 'n Birmingham).


Production of Phage Lysates

All Pike Lysates are nade by the soft-layer method (Swanstrom

and Adams, 1951), using a simplified harvesting procedure (Marsh

and Duggan, 1972).


Time-of-Entry Matings

The procedure employed for time-of-entry matings is that of

Adelberg and Burns (1960) with modifications as described previously

(Hendrickson and Duggan, 1976) using nalidixic acid as a male counter-

selecting agent.


Transduction Procedure

Procedures are the same as described previously (Hendrickson

and Duggan, 1976).


Isolation of Plasmid DNAs

Plasmids were isolated using the method described by Sharp et al.

(1972). Some of the modifications made in the procedure for isolation









of F14 and transduced F14s were determined in this laboratory

and others were suggested in Ohtsubo et al. (1974a). Spheroplasts

were lysed at 370C not 00C. The shearing step was either omitted

or the DNA was gently sheared by a single slow passage (90 to 120

sec) through a 50 ml syringe without a needle. Both procedures

gave similar concentrations of covalently closed circular (CCC)

molecules. Since the lysate was very viscous, the NaOH was added

very slouly during the denaturation step as suggested by Ohtsubo

et aZ. (1974a) and Dean Rupp (personal communication), to allow

for sufficient mixing and time for the pH meter to respond to the

change in pH. Both a magnetic stirrer (1 rev/sec) and a teflon

policeman were used to ensure adequate mixing. The pH was

titrated to 12.2 and maintained for three to five minutes.

The sodium ion concentration was adjusted to 0.3 Ml for the most

effective absorption of single-stranded DNA on nitrocellulose

(Hercules, 1/4 sec). After the nitrocellulose step and removal

of nitrocellulose by centrifugation, the lysate was filtered through

glass wool. This was repeated again, after the DNA was pelleted

into the CsCI shelf. Both steps remove debris that would interfere

with the CsCl/ethidium bromide (EthBr) banding.

In the dye-buoyant density centrifugation, the density was

adjusted to 1.57 g/cc with CsCI and EthBr was added to a final

concentration of 500 pg/mL. The DNA was banded using a Beckman

Type 40 rotor for 36 hr at 35 krev/min. Since gentle shearing or

no shearing has been used, the upper band (linear and open circled

DNA) was broad and viscous and had to be removed with a capillary

micropipet because it interfered with the collection of the lower

band by dripping. The Type 40 rotor was also used in rebanding









of the pooled bands from the first run. The DNA is stored in the

dark at 4 C in the CsCL/EthBr solution.


Electron Microscope Methods

The basic protein film technique of Kteinschmidt (1963) was used

for mounting DNA samples for examination in the electron microscope.

Using the aqueous technique (Davis, Simon and Davidson, 1971).

the DNA was examined for contour size measurements and homogeniety.

Co EI (JC411) and F (which is naturally present in F14 samples

(Ohtsubo et at., 1974a)) were used as internal standards. Since all

of the plasmids were isolated in EthBr, it was unnecessary to remove

the dye.

The formation of heteroduplex molecules of transduced F14s and

F-prime plasmids of known sequences was carried out using the alka-

line-formamide technique (Davis et al., 1971; Sharp et al., 1972;

Ohtsubo et al., 1974a). Approximately 0.1 pg of each species of

DNA (ranging in concentration from 10 pg to 100 pg/ml depending on

plasmid used) were added to the heteroduplex mixture. The hetero-

duplex mixture consisted of 20 pl of 1 N NaOH plus the DNA samples

and double distilled H20 to 80 pL. The mixture was allowed to stand

four min before it was reneutralized with 20 pl of 1 M Tris-HOl

and 100 p1 of 0.2 M EDTA, pH 8.5. The DNA mixture was renatured in

the presence of 70% formanide under the conditions used by Sharp

et al. (1972).

Heteroduplexes were mounted by using the formamide technique

(Davis et al. 1971; Sharp et al., 1972). ColEI was used as the

dsDNA length standard (6.34 kb) and $X174 was used as the ssDNA

length standard (5.375 kb).









The heteroduplexed molecules were measured by a Numonics Graphics

length calculator. The contour lengths of covalently closed circular

molecules, enlarged on translucent paper were determined by a

Dietzgen Plan Measure. Electron micrographs were made using either

a Hitachi HU-11C electron microscope or a Phillips EM-201 electron

microscoDe.


Restriction Endonuclease Fragmentation and Gel Electrophoresis

For EcoR1 restriction nuclease cleavage one must first remove

the EthBr and concentrate the DNA (sample contained 10 to 20 pg/ml

DNA) (E. Ohtsubo, personal communication). The EthBr was removed

by five equal volume extractions with isopropanol equilabrated

with a saturated CsCI solution (the aqueous phase will contain

the DNA). The DNA sample (500 pL) was concentrated to 100 pg/ml

by ethanol precipitation, 3 parts 100 % ethanol to 1 part DNA

solution. The mixture was incubated in an ethanol-dry ice bath

for ten min. The ethanol-DNA solution was centrifuged for ten

min at 10 krev/min. The supernatant was poured off and the DNA

pellet was resuspended in 100% ethanol and reincubated in the

ethanol-dry ice bath for ten min. The ethanol-DNA was centrifuged

for ten min at 10 krev/min. All but approximately 0.1 ml of ethanol

was poured off. The remaining ethanol was removed by evaporation

on a lyophilizer. The remaining DNA pellet was resuspended in

TE buffer (0.01 M Tris, 0.001 M EDTA, pH 7.2).

The restriction endonuclease, EcoR1 (1.4 x 105 units/ml, Miles

Laboratories), cleavage of plasmid DNA was carried out using a

modification of Greeneet at. (1974). The incubation period was

two hr at 370C and the final DNA concentration was 80 lig/ml.









The method of Sharp et aZ. (1973) was used for agarose gel

electrophoresis of EcoR1 plasmid DNA fragments. Agarose (0.5% and

0.7%) gels were made with E buffer (0.4 M Tris, 0.02 M NaOAC,

0.003 M EDTA, 0.18 M NaCl, pH 8.0). Samples were then prepared

for loading onto the gel: 10 pl DNA sample, 10 pl E buffer,

4 pL dye solution (0.025% brompheno blue and 50% glycerol in E

buffer). Electrophoretic separation was accomplished by applying

100 V over 15 cm, allowing enough time for the tracking dye to

run 12 cm (four to six hr).

DNA bands were visualized by fluorescence over a long wave

ultraviolet light after staining the gels for one hr in E buffer

containing ethidium bromide (2 pg/ml) (Sharp, Snyder and Sambrook,

1973). Gels were photographed using a short wave ultraviolet

light and Polaroid 57 (ASA 3000) film or Polaroid 55 (p/n) film.

Four percent polyacrylamide gels in E buffer (4 gm acrylamide,

0.2 g N'N'-methyline bisacrylamide, 1.0 ml 10% NH4 persulfate,

50 pl TEMED in 100 ml of E buffer) were used to resolve the smaller

EcoR1 fragments (less than 2.5 kb). Electrophoretic separation

was carried out by applying 100 V over 15 cm, allowing enough time

for the tracking dye to travel eight cm (four to six hr). DNA

bands were visualized and photographed using the same procedure

as above.

Molecular lengths of EcoR1 DNA fragments were determined by

plotting Rf vs the tog of the molecular length (in kilobases).

The ten EcoR1 fragments of F8(P6) and the 19 EcoR1 fragments

generated from F were used as standards in estimating the molecular

length of other DNA fragments in the same gel (E. Ohtsubo, personal

communication; Childs et aZ., 1977).















Results


Genetic Properties and Contour Measurements of F14

The F14 is an F-prime that is harbored in an Escherichia coli

K12 strain, AB1206. This strain is haploid for all or most of the

genes carried on the F14 chromosomal sequence (Pittard et al., 1963;

Pittard and Ramakrishnan, 1964; Glansdorf, 1976; Ohtsubo et al.,

1974a; Deonier, Ohtsubo, Lee and Davidson, 1974). AB1206 transfers

ilvEDAC+, metE+, rha metB+ and argH+ to both recA+ and recA

(Ohtsubo et al., 1974a; Hendrickson and Duggan, 1976). The F14

carries these genetic markers and are mapped as shown in Figure 1

(Pittard et al., 1963; Glansdorf, 1967; Ohtsubo et al., 1974a).

The bacterial sequence on F14 consists of 210.8 10 kb and is

indicated in the structure map (Figure 1) as sawtoothed lines.

The 94.5 kb F sequence indicated in the map by smooth lines is

complete on F14. However, there is a 5.7 kb sequence, y6 (2.8 to

8.5 F), which occurs only once on F, this is directly repeated on

the F14. This sequence occurs at each of the junctions of F DNA

with chromosomal DNA which are labelled as 8.5 F/OB and 210.8

B/2.8 F, respectively. The molecular length of F14 is therefore

311 kb (Ohtsubo et al., 1974a).

The F14 has another sequence, aB, that is repeated twice in its

F sequence [this is also true for F (Davidson, Deonier, Hu and

Ohtsubo, 1975)]. They map at 93.2 to 94.5 F and 13.7 to 15.0 F.

The F14 also has two ribosomal RNA gene sets, rrnA and rrnB. The




























l,-- ) t -' 3 I
toN-r0 tooH-'o to
0 C- D0 C ---C D,


-U*- C C
tfl N- 0 C-.- 0 CO

o 0 *- u LL '- H

Ct toi C > 0 L

V) Or- OLC D
ao o i *r- **-to- i C- o
. 3 aU )
t -) L 0 C -0 0 -C-
o 0 CL uOoL 0M

DO C t U"- to to
CD C *- El o ul -
E L 0 -m t a I- E
to. L4 -'2t t u

~mOL ma c 0
-i- 03 OO t to
-Cr mto 0 a)
C4-' Q-'tOto-- C t
3 o iL m nl
A Z h co ( ro to
0-AU -r C (D T- Ll
toO-3 t NC 01


4 oo-o o m NC
tW0 Q) tO o
0,tr- a), 4- 'X 6 at to
4 .- *- A 0 c-
A C' 003-'rt- O4-
-- C 0 *E 0: 4
t Vo0 0O t-
Uo 0 t 'v0 AO

04 (D r m ct o

SO -C 01 C
tO-' -toO E LAtof > C-


vO -C OA QC 0
C 0 -C w0EC -

0-0 C0
S O--i C- '-0 to t4-
O 3 E t 0 U t
'-i X0 0 -0 CO



3 C U)E Ul
. r-' o C 0 t
to-CO -- L 0tC
E- ) ( V -c C

-0 > 0 L) L ( -
D 0 C A -Co *
3 L 0 C O3D + a
u ^O ( 3 ll C

3 CL CL L wO
0- Q. O '- 3 C o
n4 Cto t -C o 0 tmC
v) E 0 vO. 0 *- -'






0

LL




















































0
sk

















































-0
3
C)




c,

C)


C,
U)















0
C






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ar
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u_





















































Q 'VVL~5N
."' /









DNA sequences of r16A and r163, and r23A and r238 are identical.

The spacer sequences, rspA and rspB, are different (Ohtsubo et al.,

1974a; Deonier Pt al, 1974; Ohtsubo, Soil, Deonier, Lee and

Davidson, 1974c; Davidson et al., 1975).

Plasmid DNA extracts of F14 strains contain at least three

classes of molecules: 3.3, 2.3 and 1.0 times the length of F.

These have the respective molecular lengths of 311 kb, 216.5 kb

and 94.5 kb. By heteroduplex mapping, these molecules are identi-

fied as the F14, the F14 chromosomal sequence plus one copy of y5

(F14AF subunit), and the F sex factor, respectively (Ohtsubo et al.,

1974a).

It has been long observed that AB1206 will lose its donor

ability for F14 markers after Long storage, but retain its F

characters. Since there are three plasmids, F14, F14AF and F,

present in F14 plasmid extracts, it has been postulated that F14

segregated into the two smaller molecules, F and the F14AF subunit

(Figure 1). The interpretation is that the instability of F14

is due to reciprocal recombination between the two yv sequences

on F14. This 5.7 kb sequence has been proposed to be a hot spot

for F recombination (Ohtsubo et al., 1974a; Davidson et al., 1975).

The repeated sequences (1.3 kb) could also produce segregated

molecules of similar class sizes, but recombination probably occurs

at a frequency too low to be observed (Davidson et al., 1975).

This sequence has been shown to be active in reciprocal recombination

in some hosts of F152 (Ohtsubo et al., 1974a).

The genetic properties of X1254 (genetic markers, F14 markers

transferred and kinetics of transfer of F14 markers) are the same









as those described for AB1206 by other investigators (Pittard

et al., 1963; Pittard and Ramakrishnan, 1964; Glansdorf, 1967;

Ohtsubo et al., 1974a). The plasmid DNA isolated from x1254 in

this study contains three classes of molecules (Table 2); they

are the same size classes as reported by Ohtsubo et at. (1974a),

3.3, 2.3 and 1.0 times F. Their relative frequency of occurrence

in the DNA mixture is less than those reported by Ohtsubo et al.

(1974a) because we did not use the x-ray isolation method to obtain

open circles (Sharp et al., 1972; Ohtsubo et al., 1974a). The

method of isolation of supercoiled circular molecules favored the

isolation of the smaller F class molecules due to the greater

probability of nicking in the larger molecules. Based on transfer

kinetics, genes transferred and the contour measurements of the

plasmid population we conclude that the donor F14 from x1254 is

the same as the F14 previously described by Pittard et al. (1963)

and Ohtsubo et al. (1974a). Data from studies reported below do

not contradict this conclusion.


Genetic Properties of Transduced F14s and Their Contour Measurements

Three recA strains, KF436, KF532 and KF533, carrying transduced

F14 plasmids are used in this study. The transduced F14s in these

strains are designated F14(P36), F14(P132) and F14(P133), respec-

tively. F14(P36) in KF436 has been described previously (Hendrickson

and Duggan, 1976). The other transduced F14s, F14(P132) and

F14(P133), transfer F14 genetic markers (ilvD, metE, rha, metB and

argH) to both recA+ and recA- strains (AB1472, KF104 and KF2201).

F14 (P133)'s time-of-entry of F14's proximal marker (metB), and

distal marker (iZvD) are similar to those described previously

















0


0 0 00
LJ-,r 0Uin

C, C


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C d

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N
+1+1

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o


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No














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o 02
























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D. 0. 0 0. 0- 0.


u o 00( 0 n01 U 4 000
c- ,n-oo oo co c c c
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>-




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C
o
-C

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c0














-C
-C




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00
C





















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oc
c
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ma


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(Hendrickson and Duggan, 1976). It has a delay in transfer of the

proximal marker, metB, when compared to the transfer of this marker

from AB1206. The time-of-entry of netB is approximately 17 min,

which is similar to the time-of-entry of this marker on the F14

harbored in a recA strain (Hendrickson and Duggan, 1976). The

most distal marker tested, ilvD, comes in five min after metB;

this is characteristic of the parental F14, whether it is harbored

in AB1206 (X1254), in recA+ diploid or in recA- diploid strains

(Pittard and Adelberg, 1963; Hendrickson and Duggan, 1976).

The plasmid DNA extracted from each of the recA strains

carrying transduced F14s fall into three size classes (Table 2)

(Figure 2). The smallest class of molecules has been shown to be

of the same size as F, 94.5 kb. For these measurements ColEI was

used as a reference molecule. The other two size classes are either

of the F14 class (311 kb) or of the F14AF subunit class (216.5 kb).

For these measurements the F class molecules were used as reference

molecules.

The genetic data (Hendrickson and Duggan, 1976) and the contour

length classes of the "F14-like" transductants are similar to those

determined for the parental F14 and are indistinguishable from F14.

In the isolations of each of the different transduced F14

plasmid DNA, the frequency of occurrence of each size class differed

in the final preparation. This was also true for independent

isolations of the same plasmid DNA. The differences in the frequency

of occurrence is probably due to slight variations in the isolation

procedure and not to the plasmidDNA distribution in vivo. The

plasmid DNA extracted from F14(P133), as shown in Table 2, gave a



































Figure 2. Electron micrograph of size class molecules in the
plasmid population of KF533 [F14(P133)]. Note the
presence of the 3.3 times (lower left-hand corner),
2.3 times (upper right-hand corner) and 1.0 times
F (lower right-hand corner) molecules present in the
sample.















..........- .. ... . . ..



. . .. . .



--
I i
..->1 -.


'' -"" '"L--L" "'." -". .- : ,

,.. +. .I .. .. .. ..

... .L . -


. .. ., . .. :-
,','-- '.. , ,"" ,+.~~~~- I ". ., -.+.'+ +:.-, .
F .. ...

- .i .

.: _,' UL-
7 + ,1 :" : '+4:. + + + +

~ ~~ ~~~i. ++. + .,-/.

i i r'" ''rI '.!+.
;A ,, .+. j









higher frequency of the F14 class to F class molecules than any

other isolation. It also had the highest concentration of DNA

(20 pg/ml). F14(P133) was used as the representative of transduced

F14s in all further experiments.

We have shown that both the parental F14 strain (X1254) and

the strains carrying "F14-Like" plasmids from transductants con-

tain the same sizes of plasmid DNA. These molecules, even though

they are of the same contour lengths, could be of slightly or

greatly different base sequences. It seems necessary to show that

the molecules of DNA that had been transduced are the same as

those in the parental F14 strain by criteria other than that pre-

viously discussed. One method for demonstrating sequence identity

isheteroduplex analysis.


Heteroduplex Analysis

To determine if the relevant structure of the molecules from

the different size classes of transduced F14 derivatives were the

same as the structures of the different molecules from size classes

of parental F14, several heteroduplex experiments were carried out.

Using F. (Palchaudhuri, Maas and Ohtsubo, 1976), and one of the
tns 58.8
FiZv's, F316, generated from F14 by P1 transduction (Pittard and

Adelberg, 1963; Ramakrishnan and Adelberg, 1965; Marsh and Duggan,

1972; Lee, Ohtsubo, Deonier and Davidson, 1974), as reference

molecules in heteroduplex analysis, we proposed to determine the

structure of the F class molecules and the structures of the

junctions of F DNA and chromosomal DNA on the F14 class molecules.

A self-renatured sample, that is, the F14 class molecules hetero-

duplexed with the F class or the F14AF subunit class from the same









plasmid isolation would demonstrate that both of the size classes

were subunits of the transduced F14 (Ohtsubo et al,, 1974A; Deonier

et al., 1974).


(i) The Reference Molecules

F. and F316 are well characterized F-primes. They appear
7-ns58.8
in Figure 3 as heteroduplex molecules with F to indicate their

structure. Fns58.8 has the complete F sequence (94.5 kb) plus an

insertion at 58.8 kb (1.3 kb) giving it a molecular length of 95.8

kb (Palchaudhuri et al., 1976). F316 is a deletion mutant of F14

and carries a small insertion (0.8 kb) at 78.6F. It has a deletion

in the F14 sequence from 6.9B to 11.4F. This can be depicted in a

heteroduplex with F (Figure 3) as a 6.9 kb insertion of chromosomal

DNA (OB to 6.9B) and a 2.9 kb deletion of F DNA (8.5 to 11.4F)

(Lee et al., 1974). F316 has a molecular length of 99.8 kb.


(ii) Self-renatured Heteroduplex Structures

For this experiment a sample of plasmid DNA from KF533CF14(P133)j

was denatured and then self-renatured. We had expected, in addition

to homoduplexes of the three size classes, heteroduplex structures

between the three size classes. However, because of multiple nicked

strands in the F14 class and the F14AF subunit class, we were unable

to find complete homoduplexed molecules or heteroduplexed structures

in these size classes. Others who have studied the FIt did not

find heteroduplexed structures of these molecules at a high frequency

(E. Ohtsubo, personal communication). We did, however, find some

complete circular structures with the F class molecules (Figures 4

and 5). The structure consisted of a covalently closed double stranded






























a
0
0 0



-O 0

t-I to



C a)




\ Cs


-D-



0' -t



0 L) I
o -i0

c-



to Ca 3
0 0
(U
OC.


C) 0 1-1
L- O- t

-- r 0
CI C



0 0
C *
to -0 0 U-
o Q **--0
3 0.- 0 CO
4- L C N-



POX




CLo r. n
tr L-


-o



0) 3








U)





25


LL



>1 -0




C0' u
co y/vu- \



r ^

































Figure 4. Electron micrograph of a self-renatured hetero-
duplex of FLF14(P133)] /F14(P133). Interpretation
is depicted in Figure 5. See text for explanation.

















JJ -.,.4

,r' .r 698: n- \U/I

y. U'-. ". 1N:
3-. ..,- N.


-. u /i)

K.
S .. Lc. *
..'t1J *





























-o
lU (D
0 -1 COO
u 0
S-0 c c -= o
.- 0 4-' 0
w( > u -SE
o .C -C 0
(Um O )
O U r 0

-4 cJ'C '- I .. x-
r U 3--C 1CO-
0- T- CL a
0 -C C I X a>

Lu 0 -O 2 C

(D 'oC4-a-n


00)0 -0>0
C0 (0) ci a) +
Lo4- -4L u -' O




-'- -o a E Cn a
LLn I bn+0






I mO .0-
u L (U 0 0 a


M *0 C 0 C
r >i 3 - O O







r- (000 i (CO)
mi C 0 CO u
L4 0 (U E C -( C
--. joC OC




i a 3 .0 Ci o






LO 0 iC 0 -




4JC ;0a0 a CJ
00 *- ir- c o 0 c -

(6 034-CO(U03
4C c0 Ci0
CO ar c- 0 0 ar- ci
to - Q- a


n cn 0-C L --7 (O
U C. *3 U 0( U ( :


SL(U0 C C-C r ) -
S41 L. J U.O '-*- r
UC +O ciO-'


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n3
0 C o
+J t l+ r














































cn



ul









DNA (dsDNA) circle plus a point junction from which two single

stranded DNAs (ssDNA) emanated. The closed dsONA circle has a

molecular Length of 94.5 1.0 kb. The single strands were viable

in Length and were never seen linked to one another. We propose

that this is the F class molecule deplexed to the F segment of a

broken F14 class molecule with the remaining single stranded portion

missing, as depicted in Figure 5. This structure demonstrates

that the F class was part of the structure of the F14 class mole-

cules.

The other heteroduplexes formed were out-of-register structures.

This phenomenon occurs when DNA molecules have directly repeated

sequences (Ohtsubo et al., 1974a). The F14 has three directly

repeated sequences as shown in Figure 1). Ohtsubo et al. (1974a)

found three heteroduplex structures that demonstrated duplexing

between the directly repeated sequences of either aB or y6, or both.

Among the self-renatured heteroduplex of F14(P133) we found one of

these structures as interpreted in Figure 6. We deduced this struc-

ture to be a circular structure that has two fixed-length, double-

forked duplexes involving the y6 (2.8 to 8.5F) and aS (93.2 to 94.5F

and 13.7 to 15.OF) sequences. There are two single stranded segments

completing the circle measuring 2.8 kb (0 to 2.8F) and 5.2 kb

(8.5 to 13.7F). respectively. The other single strands emanating

from the junction of the duplexes are chromosomal DNA sequences

from the 2.8 to 8.5F duplex and F DNA sequences from the VB duplex.

The 5.2 kb (8.5 to 13.7F) segment was broken in the molecule shown

here, but the rest of the structure is exactly as described by

Ohtsubo et al. (1974a). This structure suggests that the aB sequence






























O -0




0) C CD
f0

CLC
a 4-A 0

cm -
QC (a



0 t
d0 0


SQ) -



*r- 0
at







L 0 0 4'


0 0 0

an o

D Oe 2
C -C C
CO









(- 0 En


3 (J
4- m w
O C)












00




en - i.
u (11 Q. aO
m C) 0









I (0
Uo
t )













D~a
0)LV
















V)
I,,
-a -
C

C



u..
o .


I -





u 0 \


OC \
'- W/

c, //
." U //
\ ) LL
L-. C


u-

0LI









and the y6 (2.8 to 8.5F) are directly repeated in the F14(P133)

sequence as it is in the F14.

The other sequences repeated in F14 are the rRNA gene sets.

The r16 and r23 DNA are identical in sequence, but the spacers

(rspA and rspB) differ in sequence. If these genes were to duplex,

one would see a double-forked duplex. 5.3 kb in length, with a

small non-homology loop of 0.3 kb (the spacers) dividing the total

duplex into two sub-regions of 1.9 kb and 3.1 kb (r16 and r23,

respectively). We found such an out-of-register structure among the

self-renatured structures of F14(P133) (Figure 7). This confirms

that the sequence of F14(P133) has both of the rRNA gene sets

(rrnA and rrnB) on its structure as found on F14.

The self-renatured structures confirm three points about the

transduced F14s [F14(P133)]: (a) the F class molecules are part

of the structure of F14 class molecules, (b) F14(P133) has directly

repeated sequences similar to aB and y6 (2.8 to 8.5F) sequences

found on F14 and (c) there is a directly repeated sequence with

the characteristics of a heteroduplex between rrnA and rrnB as

found on F14.

We also heteroduplexed F14(P133) with F14. This would be

the same as the self-renatured heteroduplex, if the structure

of F14 and F14(P133) were the same. We saw no new structures in

this heteroduplex that were not found in the self-renaturation.






























o
C









-u
c-
0

4-






CD































ar- 1
m
C






41
Ec




-D
C


icn









C 4
o



> a

-u
0




0 C
LCJ

1- -o




3











CC


0 *-



























CM



CM


'0


Ll



CN


)r


r23A r r23B


rspA rspB


r I 16B


C
L.
C


0
r)
4








C)
U-

LZ


UJ


o




-o
(N







CO
(N


r 16A













C)






co


r









(iii) Fins58.8 Heteroduplex Structures

Two different structures can be predicted when Fi 8.8 is
ins 58.8
heteroduplexed with the plasmid molecules of F14(P133). The first

structure is identical to a Fins58.8/F heteroduplex (Figure 3),

that is, a complete duplexed molecule with a 1.3 kb insertion at

58.8F. We found several molecules that had this structure. These

results confirm that the F size class molecule in the transduced

F14 plasmid population is F. The second structure is a completely

duplexed circle with a 1.3 kb insertion (58.8F) with two single

strands emanating from a point junction between 38.5 and 44.2 kb

due to branch migration (see Figure 5) between the two y6 (2.8 to

8.5F) sequences. We only found structures with the junction at

2.8F (Figure 8). There were very few complete circles in this

preparation. These results confirm that F is a part of the F14(P133)

sequence and that the 2.8 to 8.5F sequence may be on the right hand

junction of the F DNA/chromosomal DNA junction.


(iv) F316 Heteroduplex Structures

The structure of the heteroduplex F316/F14(P133) is shown in

Figures 9 and 10. This was a complete duplex circle with two single

strands emerging from a point junction: 30.3 0.5 kb from a 0.8 kb

insertion loop. This structure is the same structure one would

find in a F316/F14 heteroduplex. This structure confirms that

F14(P133) has the 6.9 kb chromosomal sequence that includes ilvE and

part of ilvD, the 2.8 to 8.5F sequence on the left junction and

a sequence that is similar to the F sequence of F14.

























-o --

X C (L mO
C 0 -C E
-' 0 4- 00

3 -0 0. 00

.C U.. + .C 0
>. L) C L-
- C C 3 .C


CD 0) 0 00 C
. r* C O E
a rn 4-' t L + 4-
EC u 0L
OO > C 3 C-
U CiO 24-' 0
-' *-s 0U -
0 U 4-'-C 3
m ur u t:
C 4-OL-0
) C CC 4I

*r- 4- *- i (u





m -tC .C X 0
0 0 O -(
y) (D 0
r- C 0) E

0
:. -r- 0 ) U C

I- 0 Q ) C E -
f- C a )U C L
'o 2m o 0
0) a) 0 c )




< -u S. -- a

i C O) 1u C4 C


2 (n ( -- )
0 w m m c m
0C *r C C

C D 3 0C c


aU) 00 n
L U 3 U 4-



0 L. 0 (L 0 0 2
0 0Ci 4-' 0 C U)

02.-- C 00
L OJ C-
L 0iL200.
0-1 0 2O 2 L2 3
2 3l > M )0

K LL( V)L

-- + C* O 00 4- C
.- C COiC U




IL 0 003 Ca n U
0i- I-' ) 0 C2





00

3






38













LL


LC.







co
CC
C.L











00





U-


LL


ob0-

LL
co 4
\6 c
\ \ m^ /




































Figure 9. Electron micrograph of F316/F14(P133). Interpre-
tation is depicted in Figure 10. See text for
explanation.




40





















.. ;. ; ...

. .'** ,,'t ,-
... J.;,.,: .
WON





low. Am

F>.:~: fjy. -

5 i rn y '-.


Y3


..v p'.-*toy
, ~ ~ ~ ~ ".' ..:-.~:
I,:~ P~. i.~ .:




























Vo 0

'4- +4
X a)
(0 a) -C
C C+4



C

*:- 3 X
o a u c
( 0
CC 0
c c a


L OU CO 3

41 o0O




o o
09
4 -C








*0 i4
















-0 0 C
0 -0
) *. C. U. .
-O
c0







Sc O





0LL0 D C
01 ru



C C



O1 V'.
^ r 01 L-c


u 0 U 1 '



Q C3



















I-

LL.






CO
0-
1C)


'11










Another structure found in this heteroduplex is shown in

Figures 11 and 12. This was a linear structure with the 78.6F

insertion loop. At a distance of 24.4 kb from the insertion loop

there was a substitution Loop with two arms having lengths of 6.9

kg and 2.9 kb, respectively. The 6.9 kb arm would correspond to

the chromosomal sequence of F316, carrying the ilvE gene. The

2.9 kb arm would be the F DNA (8.5 to 11.4F). This structure

would be expected in a F316/F heteroduplex, as shown in Figure 3.

This suggests once again, that the F sequence on the F14(P133) is

similar to F and that the F class molecules may be subunits of

F14(P133) as F is a subunit of the F14 (Ohtsubo et al., 1974a).

The structure in Figures 13 and 14 was also found in the

F316/F14(P133) heteroduplex. This was a linear duplex molecule

with a substitution loop with two arms having lengths of 6.9 kb

and 2.9 kb, respectively, followed by a duplex region having a

length of 5.7 kb and two single strands emanating from its other

end. This 5.7 kb duplexed region is interpreted as an out-of-

register structure between F316 and F14(P133), involving the Y262

(2.8 to 8.5F) sequence on the right-hand junction of F14(P133)

and y151 (2.8 to 8.5F) of F316. The 6.9 kb substitution arm is

the chromosomal DNA segment of F14(P133). The single strands

emerging from the other end are the chromosomal sequence of

F14(P133) and the F sequence from F316; this single stranded

point junction indicates the 210.8B/2.8F junction of F14(P133)

in a complete F14/F316 heteroduplex. This structure and the previous

structure demonstrate that the F14(P133) has the directly repeated




































Figure 11. Electron micrograph of a heteroduplex structure
of F316/F14(P133). See Figure 12 and text for
explanation.





45














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Figure 13. Electron micrograph of a heteroduplex structure
of F316/F14(P133). This is an out-of-register
structure. See Figure 14 and text for explanation.

































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sequence of 2.8 to 8.5F on both of its F DNA and chromosomal DNA

junctions, identical to the F14 (Ohtsubo et al., 1974a).

To this point we have established three general facts about the

transduced F14 structure. Firstly, there are three size classes

found among plasmid DNA isolated from recA strains carrying these

plasmids; each size is identical to those of the F14, 3.3, 2.3 and

1.0 times the size of F (Ohtsubo et al., 1974a). Secondly, the F

class molecules are part of the transduced F14's structure. And

thirdly, the F DNA/chromosomal DNA junctions have directly repeated

y6 (2.8 to 8.5F) sequences, identical to the structure of F14. We

have not been able to show the relationship of the remaining structure

of the F14 class molecules to the F14 because we did not have any

other reference DNA molecules carrying sequences of the F14 chromo-

somal segment.


Gel Electrophoresis of EcoR1 Fragments

It has been demonstrated that certain restriction endonucleases

(such as EcoR1) will make double stranded breaks at specific sequences

(Hedgepeth, Goodman and Boyer, 1972). They will cleave homologous

DNA into specific and reproducible fragments. Gel electrophoresis

has been used on an analytical scale to separate these fragments

into bands based on molecular weight (length) (Sharpe, Snyder and

Sambrook, 1973).

EcoR1 cleavage of F14 and related plasmids F, F310 and F16 could

be used to identify the EcoR1 fragments of a transduced F14 by gel

electrophoresis. Figure 15 shows typical EcoRI fragments in an

agarose gel. The restriction fragment pattern of F14(P133) is

































Figure 15. Agarose gel (0.5%) electrophoresis of the EcoR1
cleavage products of DNAs from (Left to right)
(a) F8(P6), (b) F, (c) F310, (d) F16, (e) F14(P133)
and (f) F14. Explanation and interpretation of
bands are in Figure 16.



































r j
C .

EEuo










identical to that of F14. Figure 16 and Table 3 summarize the data

obtained in 0.7% and 0.5% agarose gels, and 4.0% polysacrylamide gels.

We have identified 44 bands shared by both F14(P133) and F14. They

range in size from 0.38 kb to 26 kb and account for 297.5 kb of 311 kb

of F14. We can identify all 18 fragments of F greater than 0.38 kb

(E. Ohtsubo and H. Ohtsubo, personal communication; Skurry, Nagaish

and Clark, 1976; Childs, H. Ohtsubo, E. Ohtsubo, Sonnenberg and

Freundlich, 1977), demonstrating, once again, that the F size class

molecules are F.

Childs et al. (1977) have mapped EcoR1 fragments of F310 and F312

which are F14 deletion mutants, carrying chromosomal sequences in

the ilv region. Both carry a large fragment, x24 (11.9 kb), which has

a restriction site at 4.7F and 8.1B (F14 coordinates). This fragment

contains the 8.5F/OB junction and all the chromosomal DNA through the

ilvA gene. Our results show that F16, F14 and F14(P133) have the

same x24 fragment, further evidence of the structure of this junction

in transduced F14s.

The restriction map of the ilv region also shows four other frag-

ments: x27, x28, x29 and x30 (1.25 kb, 1.05 kb, 0.87 kb and 0.63 kb,

respectively) (Figure 17) (Childs et al., 1977). These fragments

account for the sequence from the ilv region to 12.2B, F16, F14 and

F14(P133) carry all of these fragments.

F16 has two additional fragments as seen in Figure 16, x32 amd x33

(13.8 kb and 6.9 kb, respectively). The x33 fragment is also shared

with F14 and F14(P133). This fragment must, therefore, be the next

segment in the clockwise direction from the ilv region. The chromo-

somal region through 19.1B can now be identified on F14 and F14(P133).
























Figure 16. EcoR1 generated fragments of F8(P6), F, F310,
F16, F14(P133) and F14 greater than 0.38 kb.
These patterns were derived from electrophoresis
in 0.5% and 0.7% agarose gels, and in 4% poly-
acrylamide gels. F fragments are denoted by
broken Lines and in decreasing order or molecular
weight (MW) by fl-f18 (E. Ohtsubo and H. Ohtsubo,
personal communication; Childs et al., 1977).
Chromosomal fragments are denoted by solid lines
and numbers preceded by x. Fragments x24 and
x27-x30 denote chromosomal DNA in Filv's in de-
creasing order of MW (Childs et al., 1977).
Fragments x32 and x33 denote two new Filv chromo-
somal fragments found on F16 in decreasing MW.
F14 and F14(P133) share fragments x24, x33, and
x27-x30 with Filv's. The remaining chromosomal
fragments are denoted by x34-x53 in decreasing
order of MW. F8(P6) fragments were the internal
standards used in all gels. They are denoted by
solid lines and are numbered 1-10 in decreasing
order of MW.










F8(P6)


1 -


F16 F14(P133) F14


fl
f2
2 f-

3 f4
3 5
f5
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f7



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6 f9
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/x35\
x36
x37
x38
Sx3 39;^

---x4 --9
- - - - -


x24


x33 -


-- xx45


fll
7 f12 ----
f12





8 f13 --
9 fl -
fl5 -----


fl6
7 -----


x26
x27 --
x28-

x29 __-

x30


----- JL
x52


--- x53


10 f 18----


F F310



















x x x I XX I I I I I x I I x x x I





x x x x x x x x x x x x x x x x x
r~i
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x x x xx x x x x x x x x x x x x x x x x x x x x x x x




























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The x32 fragment of F16 accounts for the remainder of the chromosomal

region through the 28.48/35.8F junction to 40.3F, which is the EcoR1

restriction site between fragments f7 and f5 on the EcoR1 map of F

(E. Ohtsubo and H. Ohtsubo, personal communication). For obvious

reasons, F14 and F14(P133) do not carry this fragment. The chromo-

somal region fragments x37, x46, x49 and x51, shared by F14 and

F14(P133), can also be tentatively identified by molecular Lengths.

The ppc-argECBH-bfe region, which is found on the F14, have four

EcoR1 fragments (Devine, Moran, Jederlinic, Mazaitis and Vogel, 1977).

The F14 EcoR1 fragments that correspond to these chromosomal fragments

can be arranged as they appear clockwise on the chromosomal map as

follows: x49, x37, x46 and x51 (2.1 kb, 17.3 kb, 2.7 kb and 1.5 kb,

respectively). The x37 fragment carries the ppc gene and argECBH

region. The x51 fragment carries bfe (Devine et aZ.,1977). The

EcoR1 site, left of the bfe gene, is probably in or near the rnB

gene set.

Since EcoR1 sites occur in the y6 (2.8 to 8.5F) sequence, one

should not see any new fragments generated from the F14AF subunit

size class. This subunit shares identical EcoR1 cleavage sites near

the 8.5F/OB and 210.8B/2.8F y6-chromosomal junctions with F14, the

4.7F site and the 4.OF site, respectively (Skurry et al., 1976;

Childs et al., 1977). The 4.7F site attributes to the formation of the

x24 fragment of F14 which should be found on the F14AF subunit.

The 4.OF site generates the f16 fragment of F and the fragment counter-

clockwise into the chromosomal DNA the rmB region; both fragments

should be found on the F14AB subunit. There are no other EcoR1 sites

in y6 because the next F fragment counterclockwise from f16 is f12,




62




2.3 kb in length (EcoR1 site is at 1.7F). The f16 fragment is also

the only EcoR1 fragment that is repeated twice in the F14 structure.

F14 and F14(P133) had identical restriction fragment gel patterns in

all of the gels run. Any fragments smaller than 0.38 kb could not be

determined. This included f19 of F. These results indicate that

sequences of F14 and F14s transduced by P1 are indistinguishable.

There appears to be no detectable DNA sequences deleted from F14(P133).















Discussion


The transduction of an F-merogenote with properties similar to F14,

using P1 lysates made on AB1206(x1254), the F14 haploid strain, was

suggested by Pittard and Adelberg (Bacteriol. Proc., p. 138, 1963) and

later supporting evidence was reported by Hendrickson and Duggan

(1976). The transduction recipient strains could transfer ItvD,

MetB+ and ArgH+ to recA strains. In addition, they could transfer

other F14 markers (iZvEAC, metE, and rha). It is conceivable that the

transduction could have occurred with extensive deletions in the

F-merogenote in order to be packaged by P1, but, the genetic evidence

does not support this. Transfer kinetic analysis showed that all of

the transduced F-merogenotes examined were indistinguishable from the

parental F14 in the order of transfer and the genetic distance between

the proximal (argH, metB) and distal (ilvD) markers. These results

suggest that the entire F14 or at least a major protion of the F14

may have been transduced.

The physical and molecular data presented in this report support

the earlier genetic evidence that "F14-like" merogenotes are indistin-

guishable from the parental F14 by three criteria. 1) Each of the

three "F14-like" merogenotes examined had three size classes of plasmids,

3.3, 2.3 and 1.0 times F, as did the parental F14. 2) Heteroduplex

analysis revealed that (a) the 1.0 times F size molecules in "F14-Like"

DNA extracts are F, (b) the F sequence is part of the F14 size class

molecule, (c) the "F14-like" merogenotes have three directly repeated










sequences: the y6 (2.8 to 8.5F) sequence, the aB set of F, and the rRNA

DNA sequences (r16 and r23) with heterologous spacers of the rRNA gene

sets (rnA and rmB), (d) the structures of the F DNA-chromosomal DNA

junctions on the transduced "F14-like" merogenotes have the yA (2.8 to

8.5F) sequence present on the F DNA side of each junction, and (e) the

left hand junction (8.5F/OB) has the first 6.9 kb sequence that contains

ilvE, iZvG and part of the ilvD sequence found on F316. All of these

properties and structures were found to be unique characteristics of

the F14 (Ohtsubo et al., 1974a). 3) The gel patterns of EcoR1

fragments, generated from an "F14-Like" merogenote, F14(P133), demon-

strated that the DNA sequence of F14(P133) is indistinguishable from

that of parental F14; the two plasmids yielded identical gel fragment

patterns. In all, we could account for 297.5 kb of the 311 kb sequence

of F14. We could identify F EcoR1 fragments, fl through f18, demonstrating

F to be a subunit of both plasmids. We also found that F14 and F14(P133)

shared identical fragments with two of the Filv+ deletion mutants of

F14, F16 and F310. These included the ilv region fragments, x27, x28,

x29 and x30, and the x24 fragment. The latter encompassed the 8.5F/OB

junction. They also shared the x33 fragment with F16, a 6.9 kb fragment

clockwise from the ilv region. Other fragments of the F14 chromosomal

sequence were identified by thier size when compared to EcoR1 fragments

of the ppc-argECBH-bfe region, which were determined by Devine et al.

(1977). These fragments were x37, x46, x49 and x51.

The physical evidence presented here and the genetic evidence

reported earlier (Hendrickson and Duggan, 1976) demonstrate that the

transduced "F14-like" merogenotes are indistinguishable from the F14.

These data do not say how the F14 is packaged and transduced by P1 when





65



Lysates were grown on AB1206(X1254). They only show that F14s were

found in recipients (previously F ) after transductions using the

P1'AB1206(x1254) lysates. Some preliminary evidence for the mechanism

of transduction is reported in Part II.















PART II

BEGINNING OF AN ANALYSIS OF THE MECHANISM OF
P1 TRANSDUCTION OF A LARGE F-PRIME, F14


Introduction


Pittard, using a P1 lysate prepared on AB1206, an Sseherichia

coli K12 strain that harbors the F14 and is haploid for the chromo-

somal region carried on it, reported that he transduced an F-prime

that would conjugally transfer proximal and distal markers of the

F14 (Pittard and Adelberg, Bacteriol. Proc., p. 138, 1963). Trans-

ductants from such matings were characterized and the F-primes

generated were shown to be genetically indistinguishable from the

F14 (Hendrickson and Duggan, 1976). In the accompanying paper

(Part I) we have shown evidence that these "F14-like" plasmids are

physically and molecularly indistinguishable from the F14.

The intriguing question to be asked is how did P1 transduce

this pLasmid that is at least three times larger (205 x 106 daltons)

than the DNA found in the transducing particles (64 x 106 daltons).

Ohtsubo (1971) also reported the transduction of F3 by P1, which is

larger (77.4 x 106 daltons) than the P1 genome. Neither of the

transductions would conform to the headfull" hypothesis for

packaging DNA proposed by Streisinger, Emrich and Stahl (1967) if

the P1 particle used in these transductions were the same size as

the normal size phage capsid (1.472 g cm-3 or 86 nm head). As









previously reported, the genetic transfer of F14 with P1 lysates

were carried out in the presence of natidixic acid and deoxyribonu-

clease (DNase) to rule out conjugation and transformation, respec-

tively. Neither of these had any effect on the efficiency of genetic

transfer (Hendrickson and Duggan, 1976).

One further suggestion has been that P1 phage particles may

play a role in the transformation of these plasmid DNA, acting as

phage "helpers," as shown by Kaiser and Hogness (1960) using phage

lambda. The calcium concentration used in the transduction procedure

is near the lower limits of that used in transformation (Cohen,

Chang and Hsu, 1972). In this paper we present further evidence

that the mode of genetic transfer is transduction. Other objectives

are to determine the number of particles involved in the transduction

and the influence of calcium on the transduction, and to find and

characterize the phage particles involved.















Materials and Methods


rMedia

All strains and phage lysates were grown on the media described

previously (Hendrickson and Duggan, 1976 and Part I). The selective

and minimal media used in the selection of recombinants were the

same as described previously.


Bacterial Strains

The bacterial strains used are shown in Table 1.


Bacteriophage

The bacteriophage used was the generalized transducing phage

P1ke (Lennox. 1955) obtained from Roy Curtiss III (University of

Alabama in Birmingham).


Production of Phage Lysates

The methods used for the production of P1 lysates were described

previously (Marsh and Duggan, 1972; Hendrickson and Duggan, 1976).


Transduction Procedures

The recipient strains were grown to a concentration of 2 x 10
-3
cells/ml in Z broth containing 2.8 x 10 M CaCI2. Transductions

were performed at a multiplicity of exposure (m.o.e.) of 1.0. incu-

bated for 20 min at 370C, chilled, centrifuged, resusoended in 56/2

buffer and plated on selective media. When we looked for the co-

transduction of F14 markers iZvD+ metB+ and argH+, selection was
















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made for all three markers at once. The presence of F14 among

transductants was tested by cross-streak complenentation with ilvD+,
+ +
metB argH cotransductants against an F that was ilvD metB ,

argH and recA .


Transformation

Two procedures were used. The first was the same as the trans-

duction procedure described above. The other was the procedure of

Cohen et al. (1972). The cells were grown in minimal medium to a

concentration of 1 x 10 cells/ml. The CaCL, concentration used

was 0.03 11. The DNA concentrations were 20 pg/ml.


DNA Isolation for Transformation

The procedure used was modified from Cohen and Miller (1969).

Cultures were grown to a final concentration of 2 x 109 cells/mL

in L broth or 56/2 minimal medium. Cells were washed twice in

Tris-HCL buffer (0.05 H, pH 8.0). Cells were resuspended in 25%

sucrose, 0.05 il Tris-HCl, 0.075 M EDTA, pl 8.0. Lysozyme was added

to a final concentration of 1.0 mg/ml, and the mixture was incubated

for ten min at 370C. Spheroplasts were treated with 1.3 ml of 5%

Brij-56 and RNase was added (50 pg/ml). This mixture was incubated

at 600C for 60 min. Sodium lauryl sulfate (0.2%) was added and

the mixture was incubated for five min at 250C to achieve a complete

lysis. The lysates were mixed with equal volume of phenol (equili-

brated with 25% sucrose buffer), containing 0.08% hydroxyquinoline.

This was repeated three times. The final DNA solutions, 150 pg/ml,

were dialyzed six times against 0.02 M Tris-HCl, 0.001 M EDTA, 0.02

M NaCI, oH 8.0.









Density Centrifugation of Phage Particles

Approximately 1 x 1010 p.f.u. were suspended in 4.5 ml of CsCL

solution buffered with 0.01 M Tris (pH 7.5) at a final density of
-3
1.473 g cm-3 This mixture was centrifuged in a Beckman SW39L

rotor for 36 hr at 23 krev/min at 150C. Ten drop fractions from

the gradients were collected in 0.3 ml of Z broth. Activity of

p.f.u. was determined by appropriate dilutions of the CsCI Z

broth fractions, using KF2201 as an indicator strain. Distribution

of transducing capability was determined by adding 0.2 mt of the

test strain (2 x 109 cells/ml) to one-tenth ml of each CsCL

fraction. This mixture was incubated at 370C for 30 min. One-

tenth ml of the mixture was added to three ml of SA-1, plated on

selective medium, incubated for 48 hr at 370C and tested for

appropriate markers and/or F14-mediated transfer.


Procedure Showing the Effect of Ca++ on the Transduction of F14

The transduction procedure was the same as previously described
++
except for chelation and restoration of Ca to lysates. The

lysates were treated with EDTA (2.8 x 10-3 M) to chelate the Ca .

These treatments were left for three hr, for 48 hr and for three

weeks prior to their use to make sure EDTA had no detrimental effect
++
on the phage particle. The Ca concentration was restored to

2.8 x 10-3 M when the lysates were mixed with the cells for trans-

duction.















Results


Mode of Genetic Transfer

We had previously ruled out conjugation and provided evidence

against transformation as the modes of genetic transfer of F14 during

transduction (Hendrickson and Duggan, 1976). Since then, it has been

suggested that (a) P1 may, in some way, protect the DNA in the

Lysate from the action of DNase. and (b) P1 may act as a "helper"

phage in the transformation of F14. These suggestions were deemed

worth investigating since the calcium concentration in these trans-

ductions is 3 mM, one-tenth the optimum calcium concentration used

in the transformation of E. coZi (Cohen et al., 1972).

Accordingly, we first carried out the transformation experiments

under the transductional conditions and then under the optimal

conditions for E. coli transformation (Cohen et aZ., 1972). The DNA

used was isolated from the haploid F14 donor strain, x1254. The

P1 donor used was AB1472 (ilvD, metB and argH ).

The results of the first experiment (Table 2) showed that a P1

Lysate prepared on x1254 could transfer ilvD metB argH but not

his+ since X1254 is his-. P1 lysates prepared on AB1472 could trans-

duce AB1450 for his+ but not for ilvD+, metB or argH because both

donor and recipient are mutant in these genes. When DNA from X1254

(ilvD+, metB+, argH ) was added to the latter lysate the results

did not change, indicating that P1 particles did not "help" in trans-

formation of ilvD metB argH The his marker could only come



















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from transduction since x1254 has the same mutation in the histidine

operon (his-i) as the recipient, A81450. Transformation also did

not occur when P1 was left out of the mixture. The P1"x1254 lysate

used as a control, had an F14 transductional frequency similar to

that reported earlier (Hendrickson and Duggan, 1976). It can be

concluded that transformation, under the experimental conditions

used, does not contribute to the genetic transfer of F14.

We tested the transformability of AB1450 using the calcium

treatment procedure of Cohen et al. (1972). AB1450 was transformed

with DNA from F-prime strains (F42, F14 and F115) for plasmid

Linked markers at a frequency of 10-7. Thus, AB1450 can be trans-

formed, but not under the condition of P1 transduction used in

these studies.


Determination of the Number of Phage Units Needed for Transduction

F14 has a molecular weight of 205 x 106 daltons (311 kb) (Ohtsubo,

Deonier, Lee and Davidson, 1974a). This is 3.2 times larger than

the transducing fragment usually packaged by P1, 64 x 106 daltons

(Ikeda and Tomizawa, 1965 and 1968). To be packaged within P1 virions,

the F14 would need to be fragmented before or during encapsidation.

The F14 might then be genetically reconstructed in recipients receiv-

ing contiguous fragments from several virions. This would

require at Least three, but probably four, transducing particles

carrying complementary fragments. Ikeda and Tomizawa (1965) estimated

that there are 0.05 to 0.5% transducing particles in a lysate. With

a m.o.e. of one and 109 p.f.u. per transduction, there were, at most,

5 x 106 transducing particles present in the transducing mixture.









Since the transducing efficiency for any one marker on the chromosome

is approximately 104, there are approximately 105 transducing particles

carrying any one segment of the chromosome. This should be true for

F14 segments, but we found F14 markers to be transduced at one-half

the frequency of chromosomal markers (Hendrickson and Duggan, 1976).

Using the Poisson distribution equation, it was determined that the pro-

bability of one cell being infected with four random particles carrying

contiguous fragments is 1 x 1015. The probability of being infected by

three random particles carrying the F14 fragments is 4.5 x 10-2. The

probability of being transduced by three or four specific particles

carrying all of F14 would be less. However, the transducing efficiency

-S
of the F14 in our lysates was 7 x 10-8

The requirement of multiple infection by P1 particles carrying

fragments for the reconstruction of the F14 was experimentally deter-

mined by using a dose-response curve similar to the one describe by

Rae and Stodolsky (1974). The results are presented as a plot of the

log of the transductants versus the log of the virion concentrations

in Figure 1. Transduction of F14 among the ilvD, metB, argH cotrans-

ductants was determined by cross-streak matings (Hendrickson and

Duggan, 1976). The slopes of the dose-response curve used to

determine the mechanism for the transduction of F14 is approximately

one, indicating one virion (or one unit, which will be discussed

in the next section) transduced the F14. This curve was repeated

several times; the slope of the curve varied from 0.85 to 1.4.

The slope of the dose-response curve for the cotransduction of the

F14 markers, iZvD, metB and argH, is also one, indicating they are

packaged together in one particle. As expected, dose-response curves























Figure 1. Nature of the F14 transduction. KF117 was grown to a
concentration of 2 x 109 cells/ml. Selected markers
were prPD, ilvD and iZvD, metB, arpH. The F14 trans-
ductants were determined by using a replica plate
method with the iZvD, m'etB, H transductants onto F
lawns. The curve is based on the Poisson distribution
equation for predicting the quantitative aspects of
infection:
-m k
e m
(1) P(k) = k!

n = multiplicity of infection
k = number of particles infecting same cell
P(k) = proportion of cells infected by k
transducing particles (transductants)

Taking the natural log (In) of equation (1), it
follows:

(2) In P(k) = -m + k In m In k!

-m and k! are constants = c

(3) In P(k) = k In m + c

k = slope of curve = number particles

Dotted Lines are drawn at slope (S) of 1, 2 and 3. Virion
concentration is based on o.f.u./ml. Recipient concentration
was kept constant, 2 x 109 cells/ml.
































































7

log virion


8 9

concentration


S :3
I,
iI
I I
I
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i i ,


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o
a

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-0

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1












of individual markers, iZvD (from F14) and purD (from the chromosome)

have slopes indicating that their transduction involves one particle.

It may be noted that purD was transduced at a lower frequency than

ilvD. This is contrary to the usual observation of a higher trans-

ducing efficiency of chromosomal markers compared to F14 markers.

This may be explained by the close proximity of purD to the AB1206(X1254)

chromosomal deletion, A(83 to 88 min), giving the transduced purD

fragment regions of non-homology with the non-deleted chromosomal

sequence of the recipient.


The Effect of Calcium on the Transduction of F14

One interpretation of the slope of one in the dose-response curve

for the transduction of F14 is that it indicates the necessity of one

transductional unit; this one unit may be a single virion or an aggre-

gate of virions. Karamata (1970) noted that the calcium concentration

in phage P1 lysates caused the phage particles to aggregate. Harriman

(1972) later, using prophages A, 21 and 186 as genetic markers to study

the production of P1 transducing particles in a single burst, demonstrated

that transducing particles carrying different sections of the chromosome

can be formed within the same bacterium. He further showed that the

chance that two markers which are too far apart to be transduced will be

packaged within the same cell increases with the proximity of the markers.

One could conceive that in order to transduce the large F14,

aggregates may be formed in the presence of Ca (Karamata, 1970) that

consist of three or four transducing particles, produced from a single burst,

carrying contiguous fragments of the F14. The particles from one of these










aggregates can then simultaneously infect the same bacterium. Once in

the cell the contiguous fragments can fuse together (end to end)

similar to the chromosomal fusion model of Stodolsky (1973) and form

the complete F14.

If we could disaggregate the phage particles by removing Ca and

other bivalent ions, there should be a loss of, or a decrease in the

transducing efficiency of the Lysates for the F14. Lysates of P1'X1254

were treated with DETA (2.8 x 103 M) to remove Ca from the lysate

in an attempt to break up aggregates of phage particles. The Lysate

used was treated with EDTA for three weeks, for 48 hr or for three hr

prior to being tested for transductional capability. Calcium ions were
-3
added back to the usual concentration (2.8 x 10 M CaCl2) for trans-

duction. The lysate did not show any loss in its ability to transduce
++
any markers or F14 when Ca was removed and readded, regardless of

the period of time in EDTA (Table 3). All selected markers were

transduced at their usual efficiency.
++
It may be asked if the chelation of Ca actually dispersed

aggregated particles. Karamata (1970) noted that when he decreased the

concentration of Ca by dialysis, the number of aggregates de-

creased to a level where they were barely detectable by ultracen-

trifugation in CsCI or seen in the electron microscope. Since

he also noted that Loss of plaqueing efficiency is correlated to the

formation of aggregates, the p.f.u. should increase if phage particles

disaggregate with the addition of EDTA to the Lysate. The plaqueing

efficiency of a P1 Lysate increased as much at 200% (1 x 101 to

3 x 110 p.f.u./ml) and transducing efficiency of chromosomal markers

increased from 10 to 60% after the lysate was treated with EDTA.























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These results suggest that more particles were available for infection

and transduction after Ca was removed by chelation. No detectable

increase in the transductional frequency of the F14 markers was noted.

These results do not support the model which suggests that the

transduction of F14 occurs by means of an aggregate of transducing

particles. This leaves the possibility that the one transductional

unit indicated in the dose-response curve (Figure 1) could be one

virion, possibly Larger and more dense than the normal phage particles.


The Search for a Large and/or More Dense Phage Particle

If one virion may package the entire F14, as suggested above,

it should be large enough in volume to accommodate three times the

DNA in normal P1 particles. Considering the density and molecular

weights of head and tail proteins (Walker and Anderson, 1970) and

applying the architectural principles for virus particles (Caspar

and Klug, 1962), the density of such a phage particle should be

greater than the density of the infectious particles.

Morphological variants have been found by Anderson and Walker

(1960), Ikeda and Tomizawa (1965) and Karamata (1970); they

observed two kinds of P1 particles that differed in size (density),

1.472 g cm and 1.433 g cm Walker and Anderson (1970) reported
-3
four morphological variants, those reported earlier, 1.472 g cm

(a capsid diameter of 65 nm), and two newer ones with capsid dia-

meters of 47 nm and 74 nm. Since various sized P1 particles have

been seen (the larger the capsid diameter, the more dense the

phage particle), it seemed worth while to look for the existence

of a larger, more dense phage.










A P1 Lysate made on X1254 was subjected to buoyant density centri-

fugation in CsCL. After centrifugation, fractions were tested for

plaque forming units (p.f.u.) and for the transducing capability of
+ + +
chromosomal markers (leu ) and F14 markers (iZvD only, and ilvD+,
+ + + + +
metB ariH together). The ilvD o metB argH cotransductants

were tested for the F14 characteristics by replica plating onto F

lawns. The results are shown in Figure 2. We did not find a more

dense phage particle in the gradient. The one transductant, in

four runs of this experiment, that would transfer F14 markers was

found at 1.473 g cm- (the density of p.f.u.). The transducing
+ + +
particles carrying iZvD metB and argH appear to be a little heavier
+ + -3
than Zeu or ilvD (0.003 g cm ).

Others have reported a loss (up to 60%) in the number of infectious

and transducing particles after centrifugation in CsCL (Ting, 1962;

Ikeda and Tomizawa, 1965). About 40% of the p.f.u. added to our

gradients were recovered; further, only about 17% of the particles

transducing Zeu+, 2.2% of the particles transducing iZvD+ and 2.0% of
+ + +
the particles transducing ilvD metB argH were recovered. Ting (1962)

reported that phage P1 is not sensitive to osmotic shock when diluted

from CsCI, but loses it viability when stored in CsCl. It may be

that larger, more dense phage particles (if they exist) are more sensitive

to CsC---possibly even osmotically shocked. Therefore, these particles,

presumably low in number (based on the transducing efficiency

of F14) may never be found after being centrifuged in CsCL.





























Figure 2. CsCI density centrifugation analysis of phage particles.
Ten drop fractions were collected and infectious particles
were determined using KF2201 as an indicator strain. Leu
transductants were determined with C600. iZvD metB and
argH transduction was measured with AB1450.





84






_-3
density gcm3
1.500 1.460 1.440
I I I I 1 I
ilv D,iLetB,argH A-A
ilvD -
Lu
9--6 p.f.u. 0--o -
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fraction


number
















Discussion


We have shown that the "F14-Like" transductants (Pittard and

Adelberg, Bacteriol. Proc., p. 138, 1963; Hendrickson and Duggan,

1976) carry a plasmid indistinguishable from the F14 (Part I).

In this study we have provided evidence supporting our contention

that the mode of genetic transfer is transduction and that the

transduction of F14 appears to be by one particle. Unfortunately,

we have not yet found this particle.

Ohtsubo (1971) studied P1 transduction of another F-prime, F8,

Larger (77.4 x 106 daltons) than the P1 genome. Among ga+ trans-

ductants, he found plasmids which were indistinguishable from F8.

He was not successful in finding a larger or more dense particle

carrying the F8. Most of the Fgal's were transduced in the 1.472 g
-o
cm peak. However, he found that 100% of the gal+ transductants from

the denser region of the gradient proceeding this peak, had received Fgal .

Rae and Stodolsky (1974) and Rosner (1975) have reported studies

on the transduction of P1 prophages carrying chromosomal genes,

Pldlac and P1-pro, respectively. The prophage in each case was

too large to be packaged in the single 86 nm P1 capsid. They found

that the prophage was transduced by two or more particles, and comple-

mentary fragments or circular derivatives were reassembled within

the transductant. Neither of them found evidence for Larger or more

dense particles.










The DNA in transducing particles carrying just chromosomal genes

or F-prime genes differs from transducing particles carrying prophage

genomes linked to chromosomal genes in their mode of replication

before being packaged. The P1 prophage, upon induction, will replicate

its own DNA extensively into concateneric structures, followed by

headfull" packaging of smaller linear molecules (Rosner, 1975). F-

primes exist as one to two copies per cell (Jacob and Monod, 1961:

Revel, 1965) and are not induced to replicate while P1 is packaging

DNA (Ikeda and Tomizawa, 1965). Therefore, there will be a higher

frequency of copies or partial copies of the prophage genome packaged

than copies or partial copies of the F-prine genome packaged.

How does a P1 package a genome as large as F8 or F14 and transduce

it at frequencies higher than multiple particle transductions? One

explanation is that there exists a phage particle, which is similar

to those described for phage T4 (Cummings, Chapman, Delong and Couse,

1973; Bijlenga, Aebi and Kellenberger, 1976), that has a head large

enough to package the F14. The headfull" hypothesis of Streisinger

et al. (1967) was thought to hold true for P1 since P1 DNA is ter-

minally redundant and circularly permuted (Ozeki and Ikeda, 1968;

Scott, 1968). This theory was supported by the findings that morpho-

logical variants packaged DNA molecules which are proportional to the

size of their capsids (Ikeda and Tomizawa, 1965; Walker and Anderson,

1970; Karamata, 1970). Since the smaller and regular size P1

have "headfuls," it seems reasonable that there is some relationship

between the cleavage of DNA and the closing of the head. However,

Walker and Anderson (1970) found evidence that is at variance with

the present theories of packaging DNA proposed for lambda and T4;










they found that some phage particles from each class of morphological

variants of P1 were only partially filled with DNA.

Two models are generally invoked to explain the packaging of DNA

into a phane head (a) the DNA is condensed into a compact body

that is then surrounded by a coat of protein, (b) the empty protein

shell is first assembled and subsequently filled with DNA. Lambda

and T4 DNA have been shoun to follow the protein shell model.

Both have nucleases that cleave the DNA when a headfull" is achieved

(Hohn, 1975; Uhlenhopp, Zimm and Cummings, 1974). DNA packaging

in both cases is specific because one does not find T4 or lambda

packaging host DNA (except when bacterial DNA is covalently linked

to lambda DNA specialized transduction). The empty shell theory,

therefore, appears to be a specific mechanism for packaging DNA in

some kinds of phages.

The P1 encaosidation mechanism is not as specific as T4 in

packaging DNA since about 0.5% of the P1 particles can carry bacterial

DNA (Ikeda and Tomizawa, 1965; Ozeki and Ikeda, 1968). How such

packaging can occur is a matter of conjecture at this time. As a

proposed model, consider P1 infecting a cell, then replicating its

DNA into concatemeric structures. The concatemers of DNA are then

condensed into a compact form to be packaged. Capsid protein

subunits are constructed around the condensing DNA, conforming to the

icosahedral construction principles (Caspar and Klug, 1962). The

size of the capsid formed (86 nm, 65 nm, 47 nm or some other size

capsid that conforms to Pi's architecture) will be determined by

the amount of DNA condensed while the capsid is being formed. The

rate of the DNA condensing reaction will determine the size of the










capsid formed. This must be an inherent rate since 90% of the capsids

are of the 86 nm size. An endonuclease (associated with the newly

formed capsid) may cleave the DNA just before this capsid is completed.

This could explain full heads in the morphological variants. Partial

headfuls (Anderson and Walker, 1970) may also be explained by this

model. They may be formed from condensed DNA of various sizes left

over from the above packaging process. The DNA may be packaged in a

capsid that is indeed, large enough to package the DNA. However: the

capsid, conforming to the icosahedral triangulation architecture,

will carry less than 100% of its standard "headful" when it is

completed.

Packaging of large plasmids by P1 may be explained by this

mechanism. A plasmid that is in between stages of replication pro-

bably exists in a supercoiled state. This plasmid may already be

compact enough for the capsid to condense around it, again conforming

to icosahedral architecture. The other possibility is that a plasmid,

being supercoiled rather than linear, may be more efficiently con-

densed into a more compact unit, resulting in the packaging of more

DNA into any given size icosahedral capsid. In either mechanism,

plasmids in a supercoiled, compact state may not be susceptible to

cleavage by an endonuclease coupled to the packaging process; thus

the head continues to grow until the plasmid is completely packaged.

Either case may explain the transduction of F14 or F8. P1 can carry

circles of DNA covalently closed DNA circles have been found in

DNA from P1 particles (Yun and Vapnek, personal communication).

Perhaps large phages were not found for the prophages, PldZac or P1-pro,

because their DNA, upon induction, form linear concatemeric structures

that would produce and fill normal (86 nm) heads as linear genomes.









F-prime deletion mutants of F14 and F8 formed from P1 transduction

(Pittard and Adelberg, 1963; Ramakrishnan and Adelberg, 1965;

Ohtsubo, 1970; Marsh and Duggan, 1972; Lee, Ohtsubo, Deonier and

Davidson, 1974), may be produced from molecules that are replicating.

Under these conditions the supercoiled DNA may become relaxed, allowing

it to come under the influence of the condensing process and to be

cut by a proposed capsid-linked endonuclease into headfull" size

DNA for packaging. This mechanism might also hold true for the encap-

sidation of chromosomal DNA; P1 may begin packaging at relaxed regions

of the chromosome and continue encapsidating adjacent regions, explaining

the observation that transducing particles for adjacent sections of

the host chromosome mature within the same bacterium (Harriman, 1972).

We have shown that the F14 is present in the transductants (Part I)

and that the mechanism of genetic transfer is transduction. We

have begun an analysis of the mechanism of transduction (one particle

seems to be involved in the transduction) and have proposed a working

model that encompasses our findings. The continuing study will be on

the mechanism of this seemingly impossible transduction, particularly

the nature of the transducing particles involved.
















APPENDIX


Isolation of Plasmid DNA


The procedure used in the isolation of plasmid DNA is similar

to the one described by Sharp et al. (1972). Bacterial cultures

are grown to late Logarithmic phase [approximately 109 cells/ml:

(A590 = 0.85)] in two Liters of L broth (56/2 medium is used for the

growth of all "F14-like" strains). The cells are centrifuged at

10,000 x g for ten min and washed twice in Tris/EDTA/saline buffer,

pH 8.5 (TEN) (0.05 M Tris, 0.005 M EDTA, 0.05 M NaCI) (Sharp, Hsu,

Ohtsubo and Davidson, 1972). The pellet is resuspended in a sphero-

plast forming mixture of TEN, containing 0.1 g/ml of sucrose,

1.0 mg/ml lysozyme and 0.1 mg/ml RNase. This mixture is incubated

at 370C for ten min. The suspension is chilled in an ice bath, and

the spheroplasts are lysed by adding 50 ml of 2% sodium lauryl

sarcosinate solution in TEN. Spheroplasts of F14 and "F14-like"

strains are lysed at 37 C and not 0 C. After several minutes a

clear lysate is formed; the resulting viscous lysate is mixed and

chromosomal DNA sheared by slowly forcing 50 ml batches of the

Lysate (30 to 45 sec) through the orifice (1.5 mm diam.) of a

50 ml disposable syringe. This is repeated three to four times.

For F14 and "F14-Like" plasmids this step is skipped or the DNA is

gently sheared by a single slow passage (90 to 120 sec) through the

50 ml syringe. Both procedures gave similar concentrations of CCC

molecules. The sheared lysate is brought to room temperature and

90




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