The structure of the bacterophage alpha DNA molecule : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

11£ BACJ'ERIQJHAGB ALPHA 1NA _M)l.BaJLE

A

thesis presented

in partial fulfibwnt of the requiTements

for the degTee of Ibetor of AU.losqmy

in Biochad.stJy

at

Musey University

WIUIAM DAVID _SIJM'CN 1969

ABSTRACI'

Selective predpitaticm with polyethylene glycol is an efficient

•thod for c:mcentrating and }Urifying bacteriophage a and other }ilages.

The�·

^{ea WA mlecule has} a molecular weight of 33 millim.

When prepared by phenol extraction of _crude phage suspensions, it c:mtains many single-strand breaks. When prepared

by

pheml extract­

im of purified phage, it contains appl'Old.mately one t'811domly-located single-strand brealc per molecule. The n.nber ^of single-strand breaks

can be further reduced by changing the condi tims _{of the} phenol extracticm.

The c:mplementary s i�le strands _of u INA an be separated by

MAl< chrma.top-aphy folloried by

self-

^anne

aling

and hydraxylapatite chl"'l\atography, but this proc:e<llre results in xtenSive breakage of the strands. An alternative procedure _{has been} develq>ed using CsCl gr�ent

amtrlfug�

^{in the presence of} polyguanylic acid (polyG) to give an efficient separatim of the intact strands

in 100

^�g.

quanti ties ^•

Both the L strand and the H stnmd of a IJt\ fom COAiplexes with polyG, although to different extents. The PolyG biniiJW sites in the L strand appear to be canfintd to a small segment having a similar buoymt density to the H stnnd.

Seq\Mnces of cmseaative pyrimidine rmcleotidet of _all lqths up to 13 have been detected in diphcylaaine·fondc add digests of

a INA_. There 11 a slight general t:endetcy _towuds clustering of the pyrimidine J'I.ICleotides, sequences _of lengths 1·4 being _present at

p.

(ii)

belcw rmdom frequencies, md longer sequences being present at above randaa frequencies. These some general features are famd in diphe.lsdne digests of the separated H and L strands. The distri­

bution of pyrimidine mcleotide sequence lengths in 11 INA does _not appear to follrM a rhythmic code of the _type fO\.Dld in mA phages.

Preliminary analyses have been made of the lmgest pyrimidine wcleotide sequences in e1 INA, and _of the distrlbutim ^of various sequences between the two strands.

The dialysis of pyrimidine deoxyoligcmucleotides was iltV'esti­

gated, and fOllld to be strmgly influeneed by cytosine _ccmtent. This may reflect an urrusual cmfcmruaticm of cytosine-rich oligcnucleotides

at low ionic strength. Gel filtration was fcnmd to provide a satisfact­

ory method for the preliminary fracticnation of diphenylanine digests

en the basis _at chain length.

It is a pleasure to ac:knowledge the debt to my supel"Visor • Dr.

G.B. Petersen, who pTOVided advice, inforution and encouragement at all stages of this lr«)l"k .

I also greatly indebted to PrGfessor ^R.D. Batt for his aapport,

and to Dr. G.W. Butler, Director, Plant Olemlatry Division D.S.I .R., for providing laboratory space and facilities.

I have _been supported by awards frail the liliversi ty Grants Colmdttee _and the lippenberger Memorial Tl'ult Fund.

p.(tv}

Title Page Abstract

Aclcncwledgtments Table of Contents Abbnwiatims List of Tables Ust of Pigures IN'ImllJCTI �

TABLB OP OM"ENTS

'lbe St111cture of Phage mA Molecules Sequena Detetminatim in _mA

1he Aims of the present work

ME'l'HOO)

Phage and Bacterial Strains Bulk Growth of Phage Alpha Phage ftue:ys

Growth of Radioactive ^Phages l'tleml Bxtractim of Phag• 00

Blectrcm

MiCTOScopy of Phages

^{md Phage I.NA}

Boun

dary

Sedimentatim _{of mA}

Zane SedimentatiGn through SUcrose

Gradients Alkali Denaturation

^{of INA}

Hmdling

of Single•stl'Mlded mA Shearing _{of IWA}

AmoaliqJ of m\

MAJ( Olrala

tography

HydJ"CCIlYlapati

te

am.atography Density Gradient Ceatrlfuaina Dipheayl..tne Digests

carrier Dlpsts

paae (1) (ii) (iv) (v) (viii)

(ix) (xi)

1.

3.

s.

13.

15.

16.

18.

19.

20.

n.

22.

24.

u.

ICil·Bxdumae Chrcauatogrephy _of OligCI'WCleotides Urea Solutions

Dualting _of Oligc:mucleotides

Trietbyl81111101lium Bicarbonate _Buffet'S Dephosphorylated Digests

Paper OlrcmatoJnphy of O

l

igonucleotides Phosphate Estimations

Radioactivity BstimatiCilS Counting _of

llp ;2p

^Ratios

Ul trariolet Absorption Mtasunments Nucleotide Sequence Fnquend.es

RESULTS

p.(vi)

page

26.

28.

29.

30.

:n.

32.

33.

35.

36.

37.

1he

^{GrClfth and} Purificatioo. of Bacteriophage Alpha 38.

'lbe Preparatic:11 _of Strand-Intact Alpha INA 40 . Separation

of the

Ccmplanentaty Strands of Alpha TNA 48.

'fhe

J.blecular

^{Anatawy of Alpha IJ.fA 53.}

Pyrimidine Nucleotide

Sequences

ⁱⁿ Al}ita DNA _57.

Pyrimidim rtlcleotide Sequences in the Separated

�_lementary Strands _of Alpha 00 61.

DISOJSSI<Jf

The f.blerul.ar Stl'UCtlmt of Alpha rHA

Sequences of ConSecutive

Pyrild

^dine tllcleotldes in Alpha 1NA

BIBLIOGRAPift'

APPBNDIX I.

The Use of

Polyethyl.,. Glycol

^{for :the} ConCI!IIltratim and PurlflcatiCil of Bac:terl.opbages fl and T7

64.

70.

77.

API'BNDIX II _•

Dialysis of Pyr1mJ.d.iM Oligodeoxymcleotides

APPENDIX Ill _•

Gel Piltratim of Pyrimidine Deoxyoligonucleotides

ABBREVIATIOO

.. alpha

p.f .u. pltqUe ^... forming units

INA, RNA deaxyri.bmucleic: ac:id, rlbcmscleic: ad.d L strand pJrine-rlch _strand of alpha _rNA

tl strand pyrimidine-rldl strand ^of alpha INA

�... deoxyrlbcmuclease

A,G,C,T adenine, guanine, cytosine, thymine polyG polyriboJUSl)'liC: acid

polyiG, polyUG capolymer ^of guanylic acid _vitb imsinic acid,

PEG polyethylene glycol uridylic acid

MU smc

EI11'A tris ssc SP PB

OOAE­

TCA PPO

POPOP Py p

d A,

n,

^c

,,. p so

I, M

P.S.P.

s.s.

aethylated albunin adlorbed on kieselguhr sodium diethyldi thiocarbaute

diaminoethanetetra-acetic add tris (bydl'altymethyl) aminaaethane

0.15 M N.Cl, O. 015 _{M Sodi1.11l} cit.rate (pH 7)

saline phosphate _buffer pH 6.8, eantaining

o.os

^{M phosphate}

phosphate buffer _pH ⁷ _.o

N ,N-diethylamim-ethyl- trlchloroacetic add 2,5-diphenyloxazole

1,4-bis-(5-phenylaxazolyl-2)-benzene pyrimidine mcleotide

esterified phosphate (S' to the left, 3' to the right)

deoxy-

absorbmc.., l"efractive 11'ldex, cxtlnctiort intrinsic viscosity, denli _ty

sedimeutatim coefficient at infinl _te dilutiaa imic stnmath, molarlty

percatage sequence frequccy stan.dal'd eJTOl"

LIST OP TABLBS

�� �·

1. Preclpi tatim of Alage Alpha by Low Cmcentmtions

of Polyethylene Glycol 3!-39

2. Recovery of Ultraviolet Absorptian after Precipitation

of Phage AlJila by PEG 39·40

3. Re<:onty of _p. f .u. after Precipi tatim of Phage Alpha

by PEG 39·40

4.

Base-canpositlats of the

Separated

^{H and L Strzds}

of Alpha

l'K\ 48-49

5. Pen:entage Sequence Frequencies of Inorganic Phosphate and Isopliths of Culin Length 1-6 in Diphenylamine

Digests of Alpha I:NA 57-58

6. Percentage Distributim of the I _{semen of} Olain Length

Grcups 1-6 in Dlphenylandne Digests of Alpha mA Si-58 7. Percentage Sequence Frequend.es of the Pyrimidine

Nucleotide Sequences of Length 1-6 in Diphenylamine Digests of Alpha tNA. ^as Cclnpared with a Random

Distribution 57-58

8. Percentage Sequence Frequencl.es of the Longest

Pyrimidine Nucleotide Sequences in Alpha _mA ^59-60 9. The Distributim of IsopU ths _{of Chain}

l.Atngth

1-7 in the Separated Strands of Alpha _fNA 61-62 10. The Distribution of Isomers of _Olain Length 1-5 in

Dlpbenyl.aldne Digests of the Separated Strmds of

Alpha IHA ^. 62-63

11. Ratio of the Distribution of Sane Sequences _of Pyrimidine Nucleotides Between the two Strands

of Alpha INA 62-63

12. Ratio of the Distribution of Scne Sequences of 'PyriJddine Nuc:leotides BetNeen the two Strands

of Alpha �. u _Measured by a Double Label

Method

^63-64

13. «:ytosine Cmtent _of the Isopllths of

Lenath

^1·6

in Diphenylamine Digests of

Alph

^{a mA}

and

^{B • .,u. INA 74·75}

LlBRARY M/.SSEY UNIVERS!iY

table

Appendix

^{I. 1. Preclpi taticn}

of Phage fi by

^Low

p .

(X)

page

Ccncentratims of Polyetbylene Glycol Appe_rdix I • p _.1 Appe_tdix I. 2. Recovwny of Phage f1 After

PTed.pi taticn by Polyethylene Glycol Appendix II. 1. Reductim of Specific: Activity m

Re-dialysis Appemlx _11. p.4

LIST OP Pl(IJRES

figure page

1. PBG

Gradient Elution of Phage Alpha ^{fl'Oft a Celite}

Cohal 39·40

2. The Ultraviolet Absorptim Speet:nlrl of Purified

Phage

Alpha

^39-40

3 . Elect1'CI'l Mieroscopy of

Phage Alpha Prepared

by

^PBG precipi tatim 39-40

4. Maasu'ftlllent

of the

J.blecular Weight

of

^Native

Alpha mA by Zone Sedimentatim 41-42

s. Zme

Seclimentatim. and lkuldary Sedimentatim _of

Nicked Alpm mA 43-44

6. Routine Assays for 00 Intactness

by Boundary

Sedimentaticn 4-t-45

1. The DJ.s tribution _of Strand Breakage Between ^the

H and L Strands _of Phage Alpha IJ.lA «-45 8. _Zme Sedimentatim of Phage Lambda ₀₀ After

Incubation with Intact md Osmotically-lysed

Alpha Phage 45·46

9. Ba.mdary Sedimentation _of Denatured Alpha�

Showing 75\ Strand- Intactness 46-47

10. Stnlnd-Intactness _a11d

the

Tempenltun of Phem1

Extraction 46·47

11. Gradient Blutim MAX

Clrcmatography

^{of Denatured}

Alpha INA _49·50

u. CsCl Gradient Cattrifuging _of Practicm fratl _MAK

Cohal Chrcmatography

^49·50

13. MAl

Olraaatognphy

^{of Denatured} Alpha mA by

Interrupted Gradient Elutim 49-50

14. Hydroxylapatite OtJ'\W&tography of _a

Self-annealed

L

Stnnd

^{Practim of Al}ivl mA 49-50}

15. Zcae SeclimentatiCD _{of H} sd L Strmds

of

^{Alpha tNA}

Prepared by Cohlm 0\ranatography 49·50

p.(xii)

figure page

16. CsaSO.. Dtnsity Gradient Centrifuaing of Mera.aric

Ccllplexes of Denatured Alpha INA Sl-52

17.

Effect

^of

PolyG

^{on the} Buoyant Dlftsi ty of

Denatured Alpha rHA Sl-52

18. Separatim and Assay _of Po

l

yG Cctnplexes of the

H and L Strmds of Alpha tNA by Preparative CsO

Gradient CentrifugJ.na 52·53

19. Zone Sedimentation of _H and L Strands of Alpha

tm Prepared by

CsCl

Ghdient Centrifuging 52-53 20.

Analytical

^{CsCl Gradiat} C'Antrlfuging of Donatured

Alpha mA .. PolyG Ccmplexes S3·54

21. CsCl Gradient Cattrifuging of the "Third Band"

of Denatured Alpha _mA in the Presence _of PolyG 53· 54 22. _CsC. Gradient Centrifuging of the Isolated L Strands

of Alpha mA in the Presence of

PolyG

^{54· SS}

23. CsCl Gradient Centrifuging _of Purified

l2p_labelled

H md L Strand Prwparatians of AlJ:i\a lNA SS-56 24. C.Cl Gradi.-tt C.entrifuging of Sheared and Unsheared

Denaturvd Alpha INA iD the Pl'esenc:a of PolyG

25. CsCl Gradient Centrifug� of Sheared and Unsheared Isolated H met L Strmc:J. of Alpha INA in the

Presence of PolyG

26. Separation of Isopli1hs by Im-exdumge Cllronatognphy 57·58 27. Separation of lsaners of Ouain Length 1-2

by

^Im•

exchange Omaatography

28. Separation of Isomers of Olain Length 3·4 by Ian·

exchmp <lmllatography

29. Separatim of Iscaen of Ctain Length S-6 by Im·

exchange Olmutography

lO. Separatian of the Longest Isopliths froa a

DJ.phenylalne Digest

of Phage Alpha

^{INA SI-59}

31.

�ticn

_{of Iscarrs of Olain} Lenath Bight from pbcmyladne Digest of Phage Alpha tNA

figufe pap 32. Separatim _of llaaen of Olain

IAmgth

^{Nim 58-59}

33. Separatim _of Isomen of Chain I..enath Ten

34 . Separatim of Isc:aera of Oaain

Length

^Bleven 35. Separaticm of Isomers of Oudn

Length

^Twelve

36 . SeparatiCI'l _of IsCDers of Oudn _Length

Thirteen

37. Rechro�atography of the

Tbymine-ric:h

lsomen of

Length

^{Eleven fran a} Diphenylaine Digest of

Phage Alpha IHA 59-60

38. SeparatiCI'l of �horylated Oligonucleotides

Prall a Diphenylamine _Digest of Phage Alpha _mA

by 2-Dimenlienal Paper Olroutography 60-61

39. Alternative Bxpl.mati(mS _{far the} Origin of

Sinlle-stnnd Breaks in Purified Alpua INA

65-66

40. 1be Distributim of

Pyrimidine

^{and Purine}

Isopliths in _Sale Mic:robial Polymcleotides 71-72

Appendix ^I. 1. _PBG

Gradient

^{Elutim of Phage T1 from}

a CeUte _Q)llJM1 Appendix I. p.l

Appendix

^{I. 2.}

Appendix

^{I. 3.}

Appendix I.

4.

lllec:t'I'Cil Ml.croscopy of Phage T1 Prepared by _PHG Pred.pi tatiat Blectral Mleroscopy of Phage _T7

Prepared by Diffeftlltial Centrifuging Blec:trm Mleroscopy of _Phap f1

Prepared by

^{Selective Preeipl tation}

with PEG

Appendix II. 1. Oli�d.es in 1he Dift\Jaate

Appendix

^{II. p.l-2}

Appendix II. 2.

Separaticn

^{of Isop11 ths}

in

^{a Mixed} DlJilnrladue Digest of Baeterl.ophap

1NA.

Appen;Jix

^{I I. p. 2· 3}

p. (xiv)

figun page

Appendix II. 3. Olain

IAngth

ald the Loss

of

Oligodeox.ynucleotides lming Dialysis Appendix II. p.Z^-3

Appemu n.

^{4 . CytosiJW Ccmtent} and the Loss of

Oligodeoxyrucleotides n&ring

^{Dialysis Appendix 11. p . l-4}

Appendix II • S. Base Compositim _{and the} Effect of Clain Length cm _the Dialysis _of Oligodeaxynucleotides

Appendix II • 6. Dialysis _of Purified Tetranucleotides

Against Distilled Water Appendix II. p.S-6 Appendix II . 7. Dialysis of Purified Pentanucleotides

Against 0.1 M Triethylaomonium

bicubmate Appendix II. _p. 7-8

I

Appendix Ill. 1. Gel Filtration of _Mixed

Pentanucleotides Appendix III. p . l-Z Appendix xn. a. A Base �itian Effect an

Gel PiltratiCil at pH 2.6

Appendix Ill. 3 . Preliminuy Practicmatim of

I

Diphenyllllline Digests by Gel ^I

Filtratian Appendix Ill. p.Z-3

I

Appendix Ill. 4. Gel Piltratim of a Simple _Mi.x1:ure of OligCI'IUCleotides m

Sephadex

^GSO

Appendix III • S. Effect of the TriethylartllDlium (Tea _•) Catian m

the

Gel Pil traticm _of

Ollgcmueleotides Appendix _Ill. p.l·