Nucleotide sequence of the chi recombinational hot spot chi +D in bacteriophage lambda.

JOURNAL OFVIROLOGY,Jan.1981,p.336-342 Vol. 37,No. 1 0022-538X/81/010336-07$02.00/0

Nucleotide

Sequence of the Chi Recombinational

Hot

Spot

X+D

in

Bacteriophage

Lambda

GERALD R. SMITH,`* MICHAELCOMB,' DENNIS W. SCHULTZ,' DONNA L. DANIELS,2 AND FREDERICKR. BLATTNER2

Institute of MolecularBiology, University of Oregon, Eugene, Oregon97403,'and Laboratory of Genetics, McArdle Laboratory, University of Wisconsin, Madison, Wisconsin537062

Chisites inbacteriophageAstimulate recombination promotedby the RecBC

pathway of Escherichia coli. Mutations which create these sites occur at four widelyseparated loci inA. We report here the nucleotidesequence surrounding the site ofoneoftheseloci,

XD,

locatednearthe S gene. The mutationscreating the activeChi site, designated

X+D,

aretransversions

fromG

to

T.

Thismutation, like the

X+B

and

X+C

mutations previously analyzed, leads to a nucleotide

sequence common toallthree activeChi sites.

Generalized recombination in

bacteriophage

A isstimulated

by

special sites,

called

Chi,

when recombinationproceedsvia theRecBCpathway

of the host ofA, Escherichia coli

(see

reference

24for a review).Onehypothesis ofthe

mecha-nism ofthisstimulationsupposes thatChisites are

special

nucleotide sequences

recognized by

oneof theenzymes intheRecBC

pathway.

To substantiate this

hypothesis,

wehaveexamined thenucleotidesequences

surrounding

Chi sites todetermine whether Chiisa

unique

nucleotide

sequence(22, 23). Extensivesequencehomology

wasfound in thesequences of twowidely

sepa-ratedChi sitesinA(22).Fromthoseanalyseswe

could not concludehowmuchofthehomology

wasessential for Chi activity. Acomparison of

other sequences containing Chi would aid in

revealing

the extent ofthe common sequence

necessary forChi. Inthis paper we report the

nucleotidesequence of a third ChisiteinA and

compare that sequence with the two Chi

se-quencespreviouslydetermined.

Wild-type A is devoid of Chi,but mutations

which create Chi occur at the four

well-sepa-rated loci shown in Fig. 1 (10, 25). Sequence

analysis of the

XB

and xC loci was possible

because of theavailability of deletionmutations

with endpointsnear the Chi sites. These

dele-tions allowed thegenetic and physical mapping

essential for locating an interval smallenough

for convenient nucleotide sequence analysis.

Themutations creating Chi at the

XB

and

XC

lociwere found to be single-base-pair changes (22, 23).

Asimilaranalysisofthe

XA

and

XD

loci was

notimmediately possible since these loci are in

regions of genes essential for A growth. The

availabilityof A DNAfragmentswithendpoints

near

XD

andinserted into self-replicating

plas-midsallowedthegeneticandphysical mapping

and thenucleotidesequenceanalysisofxD

re-portedhere.

(Weuseherethenomenclature of Maloneet al.[12].

X+

designatesthegenotype of theactive formof Chi sitesin A.X°designates theinactive form.

XA, xB,

xC, and

XD

designate the lociin

Awhere the

X'

mutationsmay arise. Chi refers

tothephenotype ofthesites.)

MATERIALS

AND METHODS

Bacterial strains. E. colistrainswith Aprophage

deletions extending from galthrough attL and into theQtoSregionwereisolated and characterizedby Shapiro andAdhya (19). More detailed mappingwas

described by Herskowitz and Signer (11). Strains MS5061 and MS509wereobtained from thecollection of Frank Stahl (University of Oregon), and strains MS1118 and MS1153wereobtainedfrom the collec-tion ofJeffrey Roberts (Cornell University). Strain

594 (28) is lacgal Strr and doesnotsuppressamber (sus) mutations.

E. coli strains withplasmidsinto whichfragments

ofA DNA were inserted were obtained from other

investigatorsasfollows. From David Wilson (Cornell University) weobtained strainD264, which contains plasmidpDW1,aderivative ofplasmid pBR322 (26)

carrying EcoRI fragment F ofA (27) with a BamHI

linker attachedtotheright cohesive end.Inaddition tobearingthisplasmid,strain D264 isHfrC thi-1trpR rbs-115 leu. From Jeffrey Roberts and Michael Schechtman (CornellUniversity) we obtainedstrain

Q,which containsplasmidpOPII-APQ,aderivative of

plasmid pMB9 (3) carrying a fragment of the lac operon withUV5 mutation (21) and EcoRIfragment Eof A(27)bearingthe nin-5 mutation(5).Inaddition tobearingthisplasmid, strainQ carriesadeletionof

the lac-proregionofthechromosomeand anepisome

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SEQUENCE OF

X'D

RECOMBINATIONAL HOT SPOT 337

Xloci

?genome

Right end of

XA XB XC XD

A t t , Jt t ~~~~~~cIt~ t R,

0.0 0.2 OA :0.6 5.8 1*i

I"MS"deletions

Eco8[ / / Hindm EcoRI H

A P. S

R0

0.8 0.9 1.0\'

ALDNA Tragments L !I\

inserted into plosmids / \

pRK112

PDW1

XD

/HaM A/UIl HhaI

15 AATTCATTAGTAATAGTTACGCTGCGGCCTTTTACACATGACCTTCGTGAAA TGGCAGGAGGTCGCGCTAACAACCTCCT 3

A3'CT TAAGTAATCATTATCAATGCGACGCCGGAAAATGTGTACTGGAAGCACTTTCc CCACCGTCCTCCAGCGCGATTGTTGGAGGA 5

EcoRI m

;inX+Dmutants

FIG. 1. Genetic andphysical map of phage A, showing the location and nucleotide sequence of theXD

locus. Oneach line,physicaldistances aredrawnto scale. The top line shows thefour lociat which X+

mutations arise, genes A, J, cI, and R, and theprophage attachment site att atthe indicatedfractional

distances from the left end of the A chromosome (7). Theextentsof prophage deletions in strains MS509,

MS1118,MS1153, and MS5061, usedtomapXD(Table1),aredrawnrelativetothepositions indicatedonthe

top line. The second line shows therightendofA, includingtheXD locus, genes cIIthrough R, and the positions of certain endonuclease cleavage sites. The extents ofA DNA fragments generated by these endonucleasesareshown relativetothe second line. Thesefragmentsarepresent in the indicatedplasmids,

whichwereusedtomapXD(Table2). The bottom line shows the nucleotide sequence surrounding the

X'D

mutations, which alter the basepairoutlined(Fig.2).Hyphensareomittedforclarity. Brackets above and below the line indicaterecognitionsequencesforthe indicated endonucleases.AluI* isacleavage site present only in DNAfromX Dmutants(Fig. 3).1andrrefertotheconventional stranddesignationsinA.

with thelacIQmutation (15). FromWilliamReznikoff

(University of Wisconsin) we obtained plasmid

pRK112A, aderivative ofplasmid ColElbearing the kanamycin and neomycinresistance determinants of Tn5 (1) and HindIII-HindII fragment A2 of A (18) bearing the susS7 mutation(9). Strain C600(thr-1 leu-6thi-1 lacYl supE44 tonA21) wastransformed with

thisplasmidDNAtogeneratestrain FB1034.

Phage strains. Aint4 red3 gam210X+D123susS7 and XtsJ15 red3gam210 c126X+D124wereobtained from JeanCrasemann(University ofOregon). Ab1453

X+D123 susR5 was obtained from Frank and Mary

Stahl(UniversityofOregon).Otherphageswerefrom

ourcollectionor werefromcrossesbetween themand those listed above. susP and susR mutationsare de-scribedbyCampbell (4).

Phage crosses. Cellscontainingplasmidsor

par-tially deleted prophages were grown at 37°C in a

mediumcontaining1%(wt/vol)tryptone (Difco Lab-oratories, Detroit, Mich.) and0.5%(wt/vol) NaClin

water,supplementedwith 0.1%(wt/vol)maltose.Ata celldensity ofabout 3x 108/nl,fivephagespercell

wereadded.After 20 minat37°Cwithoutshaking,to allow phage adsorption, theinfectedcellswerediluted 1:1,000 inthe above medium.Afteraeration at37°C for 2h,CHC13wasaddedtokillremainingcells.Sus+

recombinants were selected on strain 594 on plates

containing1%(wt/vol) Trypticase (BBL Microbiology

Systems,Cockeysville,Md.), 0.5% NaCl, and1%agar

(Difco). Sus+ recombinants were scored for Chi as

describedinfootnoteaof Tables1and2.

Enzymes. Sources of some of the enzymes have been describedpreviously (22, 23). In addition, endo-nuclease MspI was purchased from New England BioLabs,Beverly,Mass.

Preparation of DNA and DNA sequence

anal-ysis.Wild-type

(k0D)

DNAwaspreparedfromAcI60

susS7 (22). Mutant

(W+D)

DNA wasprepared from

AcI857X+D123susS7 and fromAb2red3 gam210 cI26

X+D124susS7.Samples ofapproximately 100 agwere

digestedfor18 h at37°Cwith20Uof endonuclease

EcoRIinareaction volume of1.0 ml. Afterphenol

extraction and ethanol precipitation, the DNA was

labeled withpolynucleotide kinase by the exchange

procedure(2), usingapproximately5Uof kinasein a

reactionvolume of100pl.Reactionwasfor90minat 37°C. Afterprecipitation ofthe DNA withethanolin

thepresence of ammoniumacetate(14),the DNAwas digested for 12 hat 37°Cwith 8U of endonuclease

MspIinareaction volumeof 50pl. Aftertheaddition ofsucroseand markerdyes,the DNAwassubjected

to electrophoresis in a 7% polyacrylamide gel

(Bio-RadLaboratories,Richmond,Calif.)in Ebuffer (20) untilxylenecyanolFF hadmigratedabout7 cm.The third mostrapidlymigratingband,at apositionabout

two-thirdsof thewayfrom theorigintoxylenecyanol VOL. 37, 1981

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338 SMITH ET AL.

FF, was detected byautoradiography, electroeluted,

andsubjectedtonucleotidesequenceanalysis (13, 14).

RESULTS

Geneticandphysicallocation ofXDlocus.

Theprecise location of the

XD

locus was

essen-tial toidentifyaregionof Asufficientlysmall for

convenient DNA sequenceanalysis. Stahletal.

(25) mapped the A+D123 allele to a position in

orbetweengenesQ and S.Wemapped thisand

another

X+D

allele

(X+D124),

both of

sponta-neous origin (J. Crasemann, personal

commu-nication), between the endpoints of two dele-tionsby usingavariation oftheprocedure de-scribed by Stahl et al. (25).

X+D

phages

with

susS or susR mutations were grown in cells

containing partially deleted

prophages

from

which

S'

or R+ could be rescued (Fig. 1). The

simultaneousrescueof the

X°

allele froma

given

prophage indicated that the deletion in that

prophage had not removed the

XD

locus.

Table 1 shows that the

XD

locus is located

betweenthe endpointsofdeletions MS1153 (or MS1118)andMS509.X°wasrescuedfrom

dele-tions MS5061, MS1118, and MS1153, but not

fromMS509, whether

S'

or R+ was the selected

rescuedallele. Similar results wereobtained in

crosseswith

X+D123

and

X+D124,

whichsuggest

thatthese two

X+

mutations arelocatedatthe

same site or neareach other. (The nucleotide

sequenceanalysis reported below demonstrated

that these two mutations occurred at the same basepair.) These results did not,however, allow

us toconclude whether

XD

islocatedinQ, in S,

orbetweenthe two genes.

Tolocate the

XD

locuson thephysical map of

A,

weutilizedplasmids

containing fragments

of

TABLE 1. Rescue ofXDfrom partially deleted prophagesa

Pro- Fraction of

Chi'

among follow-phage

_paeDeletion

Deletion ends_ends ingselectedrecombinants:

dele-

betweenb:

S+

tion bewen

strain

X+D123

X+D124

WM=

MS5061 Q21andQts7 74/141 317/469 189/347

MS1118 Qts7and S3 176/310 280/419 72/119 MS1153 Qts7 and S3 67/117 409/592 116/182 MS509 S3and S2 0/370 0/448 0/111

aX+DsusSorX+DsusRphagesweregrown intheindicated bacterialstrains asdescribedinthetext. Sus+recombinants wereselectedon strain 594 andscoredbyaninspectionof the plaquesize for the Chi phenotype (10). The data indicate the number ofChio(small-plaque phenotype) recombinantsout of thetotalnumber ofrecombinantsinspected. Representative plaques from eachcrosswererestreakedonfreshplates to verifythe Chiphenotype.Complete genotypes of the infecting phagewereint4red3gam210X+D123susS7,b1453X+D123 susR5,and b2red3gam210 cI857X+D124susS7.

'Fromreference11.

A DNA from the Q-S region (Fig. 1). The

end-points of these fragments were generated by

endonucleases whose cleavage sites have been

located on thephysical map (6, 16, 18, 27). The

rescue of the X° allele from these fragments

wouldlocate

XD

relative to thesecleavage sites.

This rescue was accomplished similarly to the

rescue from theprophages, via linkage to a

se-lectable marker (susP+, susS+,orsusR+).

Table 2 showsthat

XD

is located very near

EcoRI site 5 and to therightof it.AmongSusR+

phages generated by recombination with

plas-mid pRK112, 12% have rescued

X0.

This result

shows that

XD

is located on the3,600-base-pair

fragment A2 (18) generated by endonucleases

HindIII and HindII. This fragment contains

EcoRI site 5 (18, 27). We crossed

X+D

Sus-phages with the twoEcoRI-generated fragments

extending to the left (pOPII-APQ) and to the

right (pDW1) of this site. No X° recombinants

were found among 5,000 P+ recombinants

gen-erated by recombination with plasmid

pOPII-APQ. On the otherhand, asmall number ofX°

recombinants were found withplasmid pDW1.

Theseresults show that

XD

islocated on EcoRI

fragmentF(27), whichextendstotherightfrom

EcoRI site 5. Taken together, the results in

Table 2 show that

XD

isbetweenthe EcoRI site

and the HindII site located about 2,800 base

pairs to its right. From the lowfrequency of X°

rescue from plasmid pDW1 and the high

fre-quency of

X0

rescuefrom plasmid pRK112, we

concludedthat

XD

islocated very near theright

of EcoRI site5.

Nucleotide sequence of the

XD

locus. To determine the nucleotide sequence of the

XD

locus, weprepared DNA labeledatEcoRIsite 5andextending rightwardabout 350 basepairs

to an MspI site. This fragment contains the

regiondeducedabovetocontainthe

XD

locus.

Figure 2 shows the results of nucleotide

se-quenceanalysis from

wild-type

(X°),

X+D123,

and

X+D124

DNAs. The sequences obtained are identicalfor all threeDNAs,except forthe 55th nucleotide to the right of the EcoRI cleavage

I

site(5'G-A-A-T-T-C 3'; measured on the I

strand). In wild-type DNA (AcI60 susS7), this

nucleotide isG, whereas in

X+D123

and

X+D124

DNAs, thisnucleotide is T. Sequence analysis

of another wild-type

(X0)

strain (AcI72) showed

a Gatnucleotide 55 (data not shown). In

addi-tion, ourwild-type sequence in this region agreed

with thatdetermined by F. Sanger and A.

Coul-son for their strain of AcI857 susS7 (personal

communication). We concluded that the X+D

mutation is the

change

G -- T 55 nucleotides

from the EcoRI site on the Istrand.

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SEQUENCE OF x+D RECOMBINATIONAL HOT SPOT 339

A G C+T C A G C+T C

6-a

FIG. 2. Nucleotidesequencedetermination. (Left)

X'D124

sequenceextendingabout40nucleotides

right-wardfromtheEcoRI site shown in the bottom lineofFig.1.Identicalsequenceswereobtainedwithwild-type (X°)and

X'D123

DNAs(datanotshown).(Middle)Wild-type (X°) sequence extending rightward fromnucleotide 38toabout nucleotide85, relativetothe EcoRIcleavagesite.(Right)

X+D124

sequencecorrespondingtothe

wild-typesequence in the middlepanel.Anidenticalsequencewasobtained with

X+D123

DNA(datanot shown). Arrows indicate thepointoftheX+Dmutation. Thesequencedeterminationwasbythemethodof

Maxam andGilbert(13, 14).Cleavageatspecificbases isindicatedatthetopofeachlane. Fortheleft panel,

cleavedsamplesweresubjectedtoelectrophoresis througha20%polyacrylamide geluntilxylene

cyanol

FF

(designatedX) hadmigrated11.5cm.Forthe middle andrightpanels,electrophoresis wasthrougha12% polyacrylamidegeluntilxylenecyanolFF hadmigrated34cm.Migrationwasfromtoptobottom. Further detailsonthepreparationofthelabeled DNA andonthesequencedeterminationaregiveninthetextand

references cited there.

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340 SMITH ET AL.

TABLE 2. RescueofXDfrom plasmids carrying fragmentsof ADNAa

Re-Bacte- Extent of A bin- Fraction rial Plasmid DNAfragment' ants ofChi°

strain

se-lected FB1034 pRK112 HindIII-HindII R+ 51/432 Q pOPII-XPQ EcoRI-EcoRI P+ 0/5,000

D264 pDW1 EcoRI-cosc S+ 3/500

D264 pDW1 EcoRI-cos R+ 4/4,000

aX+D123phagewithsusP, susS,orsusR mutationswere

[image:5.496.64.250.74.190.2]

grown in theindicatedbacteria, andSus+ recombinants were selected and scored for the Chiphenotypeas described in Table 1, footnote a. Complete genotypes of the infecting phage wereint4 red3 gam210X+D123susS7,b1453X+D123susR5, and red3 gam210 cI26susP80X+D123.Incontrolcrosseswith

Xe

phage (red3 gam210 cI26 susS7 or int4 red3 gam210

susP80), noChi+ (large-plaque)Sus+ recombinants were found (data not shown),indicating that the X DNA fragments in the plasmids did not contain aX+Dmutation.

'SeeFig. 1.

cTherightcohesive end of X.

CreationofanAluI

cleavage

site

by

the

X+D

mutation. The nucleotide sequences

re-ported above predict that the

X+D

mutation

creates arecognitionsequenceforendonuclease

AluI, d(5' A-G-C-T 3') (17). We verified this prediction bydigestion oftheend-labeledDNA fragments used for sequence

analysis.

Figure 3

shows that digestion of the

X+D123

fragmenrt

with AluI produces a labeled

fragment

of the sizeexpected fromthe

position

oftheAluI rec-ognitionsequencerelative tothe other endonu-cleaserecognitionsites inFig. 1. Thewild-type

(X0)

fragment

is resistantto

digestion

with AluI

under the sameconditions. We concluded that

the

X+D

mutations, like the

X+C

mutations and

some

X+B

mutations, create an AluI recognition

site (22, 23).

DISCUSSION

The resultspresented heredemonstrate that

X+D

mutationsare

single-base-pair changes,

like

the

X+B

and

X+C

mutations analyzed previously.

The active

X+D

hot spot is created by the

trans-version G -- T (Fig. 1).

X+B

can be created

eitherby the tranversion C-+ G or bydeletion

of this C (22), whereas

X+C

is created by the

transversion A T

(23).

The

X'

mutations

change sequences in A which are almost the Chi

sequence into the correct, active Chisequence.

Acomparison of the nucleotidesequences at

the

X+B,

X+C,

and

X+D

loci sheds somelight on

the sequence which defines Chi. As shown in

Fig. 4, the octamer 5' G-C-T-G-G-T-G-G 3' is

present at all three loci.When the twopossible

activeChi sequences at the

XB

locus are taken

intoaccount, this octamer is the longest

contin-uous nucleotide sequence present at all three loci.At eachlocus, the

X+

mutation changesa

baseto create this octamer(Fig. 4). If Chi isa

unique nucleotide sequence, this octamer may well be Chi. Two other

possibilities

must be considered, however. Perhaps Chi is a unique

sequence, but onlya portion ofthe octamer is

Chi;orperhaps Chiis avariablesequencewhich includes the octamer.

The extent and precise nucleotide sequence

requirement of Chi can be determined via an

examination of mutations which create or

de-stroy anactive Chisequence. Inthe

alignment

ofsequencesshown inFig.4, wecould conclude

thattwo nucleotides are essential to Chi.

X+B

mutationslead to aGatthe leftmostpositionin

the octamer underlined in Fig. 4. A C at this

position abolishes Chi activity (22).

X+C

and

X+D

mutations leadtoaTatthethird

position

in the octamer. An Aor a G at this

position

is

FIG. 3. Creation ofanAluIcleavage site by

X'D

mutations. DNAfr-agmentslabeledatthe EcoRI site shown in the bottom line ofFig. 1 and extending

rightwardto thefirst MspI cleavagesite(about350

base pairs) were digested with endonuclease Alul.

Thedigestwassubjectedto electrophoresisthrough

an8%polyacrylamide gelinEbuffer (20). Migration

wasfr-om top to bottom. (a)

X'D123

DNA; (b)

wild-type (X") DNA. Arrows indicate thepositionsofthe

origin (0), migration of xylene cyanolFF (X9, and

migration of bromophenolblue(B). Thepositionsof

labeled

fr-agments

produced by digestionwith

endo-nuclease HhaI (arrow 1) and with endonuclease HaeIII(arrow 2)weredetermined ina separate

ex-periment underidentical conditions by noting their

migrationrelative to thedyes. The relative sizes of

thesefr-agmentsareshowninFig.1.Asmallamount

oflabeledDNAfr-om

X'D123

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X+D

X B121 5'ACAATGAGTGGCAGATATAGGCTGGTGGTTCAGGCGGCGCATTTTTAT 3' X B132 5'AACAATGAGTGGCAGATATAGCTGGTGGTTCAGGCGGCGCATTTTTAT 3' XC157 5'CGTTGATAAGTCGCAGATCAGCTGGTGGAAGAGGGACTGGATTCCAAA 3' XVD123 5'TACACATGACCTTCGTGAAAGCTGGTGGCAGGAGGTCGCGCTAACAAC 3'

FIG. 4. Comparison of nucleotide sequences at the X+B, x+C, and X+D loci. Portions of the I-strand

sequencessurrounding the indicatedX+mutations are shown. Two different sequences, exemplified by the

X+B121

and

X+B132

mutations, are found at theX+Blocus(22). The

X+C

sequence isfromreference 23, and the

X+Dsequence isfrom Fig.1and 2.Thesequences are aligned such that theunderlined octamer is coincident for the four sequences (seetext).Hyphens are omitted for clarity.

ineffective (23; Fig. 2). These observations are

consistent withtheview thatChiis, orcontains,

the uniqueoctamer indicated in Fig. 4. Verifi-cation of thishypothesis could be achieved by analysis ofmoremutationswhich createChior by the isolation andanalysisofmutations which destroy Chi. If Chiislimitedtothe octamer,all ofthese base changes would occur within the octamer.

It is interesting that the octameris oriented in thesame directionatthe three

X+

loci pres-ently analyzed. That is, the octamer shown is

present on the I strand of A atall three active

Chisites. Fauldsetal. (8) have shown thatthe

inversion of ChiwithinA inactivates Chi. Yagil et al. (E. Yagil, N. Dower, D.

Chattoraj,

M.

Stahl,

C. Pierson, andF. W.

Stahl, Genetics,

in

press)haveshown thatthe active orientation of

the Chi site that they used, resulting from a

spontaneousmutationin thetransposonTn5,is

the samethroughout the right 60%ofA.Ifthe

octamerisatleastpart of

Chi,

wewould

antici-patethat it would be present in the same ori-entation ateach

position

in

A,

as wehave ob-served.

ACKNOWVLEDGMENTS

We areparticularlyindebtedtoJeffrey Roberts,Michael

Schechtman,DavidWilson,and William Reznikoff forsending

ustheirplasmidscontainingfragmentsof ADNAand infor-mation about them beforepublication.Wealso thank Jean

Crasemann, JeffreyRoberts,MaryStahl,and Frank Stahl for phage and bacterial strains from their collectionsandFred

Sanger and Alan Coulson for unpublished sequences. We

gratefullyacknowledgeourmanycolleaguesintheInstitute ofMolecularBiologyfor manyhelpfuldiscussions and unpub-lished data.

This research was supported by Public Health Service grantsAI13079andGM26798from the National Institutes of HealthtoG.R.S. and Public Health Service grant GM21812 from theNational Institutes of HealthtoF.R.B.

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