Herpes simplex virus type 1 variant a sequence generated by recombination and breakage of the a sequence in defined regions, including the one involved in recombination.

0022-538X/93/095685-07$02.00/0

Copyright X)1993,American

Society

for

Microbiology

Herpes

Simplex Virus

Type

1

Variant

a

Sequence

Generated

by

Recombination and Breakage of the

a

Sequence

in

Defined

Regions, Including the One

Involved in Recombination

KENICHI UMENE

Department of

Virology,

Faculty of Medicine, Kyushu University 60, Fukuoka 812 Japan Received 7 April 1993/Accepted 15 June 1993

Aherpessimplex virus type 1clone, GN29, having

exclusively

thevarianta sequencewasisolated. Thisa

sequence was composed of unique (U) and

directly

repeated (DR) elements DR1, Ub, (DR2)14, Ucd, Ubd,

(DR2),,

DR4n2, and Uc and was assumed to be generated by recombination between sites in Ub and Uc.

UnusualDNAfragments containingparts of theasequence, presentin the DNApreparationsofGN29,were

molecularly

cloned.Almostalltermini oftheclonedunusualDNAfragmentsweresituatedindefinedregions assumedtoberecombinogenic: (i)asiteintheinvertedrepeatof theL component, (ii) DR1, (iii)DR2,(iv)the DR4stretch,and(v) the novel recombination stretchinthe variantasequenceof GN29.The terminiofunusual DNAfragments,

possibly

produced by strand breaks,canserve asfreeDNA endstoinitiaterecombinationof

thea sequence. These results support the model ofdouble-strand-break repairfor recombination of the a

sequence. Sequence-specific enhancement of the recombination ofthea sequence

probably

depends on the

presenceofrecombinogenic elements apt tobreak, such asDR2 repeatsand the DR4stretch.

The linear 155-kb genomeofherpes simplex virustype 1 (HSV-1) consists of two covalently linked components, L andS (16).The shortsequence a is repeated directlyatboth ends of the genomeand at the L-S junction (5, 12, 13, 16). The region spanning the L-S junction isinherently recombi-nogenic, and thea sequence could beahotspotfor

recom-bination (1-3, 17, 22, 25, 28-30). The a sequence contains unique (U) and directlyrepeated (DR)sequenceelements of DR1, Ub, (DR2)n, DR4 stretch, and Uc and is flanked by DR1 (5, 12, 25). Thelengths of thea sequences of various

strainsrange from 220to 550 bp anddepend mainlyon the number ofreiterationsof DR2 (5, 12, 14, 21, 25, 27, 28).

Isolation of an HSV-1 clone, GN29, with the variant a

sequence. The difference in lengths of the a sequence of

different HSV-1 strains became evident with Southern hy-bridization analyses of HSV-1 DNAs digested with SmaI, usingthe0.175-kb SmaIfragment ofTW14containing most of theasequence astheprobe (25). When theasequenceof HSV-1 strain K52 was analyzed, four SmaI fragments of 0.175, 0.245, 0.26,and 0.27kbwere detected(27) (lane 1 of Fig. la). To isolate HSV-1 clones having an a sequence generatingoneSmaIfragment, 30single-plaque cloneswere

isolated fromK52 and the DNAswere analyzed (23). One

clone had ana sequence generating one SmaI fragment of

0.26 kb andwasnamedGN16 (lane2 ofFig. la). Two clones

hada sequences generatingone SmaI fragment of 0.27 kb,

andone wasnamedGN28(lane 3ofFig. la). Thirteen clones

hadasequencesgeneratingtwoSmaI fragments of 0.175 and 0.245 kb, and one was named GN29 (lane 4 of Fig. la). Fourteen other clones had a sequences generating three

SmaI fragments. To separate clones of the 0.175-kb SmaI fragment from those of the 0.245-kb SmaI fragment, 85 single-plaquecloneswereisolated fromGN29.All85 clones hadtwoSmaIfragmentsof0.175 and 0.245kb. The failureto isolate an HSV-1 clone having one of the two SmaI frag-ments of0.175 and 0.245 kb suggested that the two SmaI fragments had derived from one a sequence having an

additional SmaI site. One DraI site is present on the a

sequence, andapairof Dralfragments correspondingtothe

a sequence are detected by Southern hybridization (5, 12, 14, 25, 28). One is the fragment of unit length of the a

sequence,and the other isshorterby16.5bpbecause of the

cleavage of DR1 (13). DNAs of K52 derivatives digested with DraI were analyzed by Southern hybridization (Fig. lb).DraIfragments ofGN29were0.46 and0.48kb inlength, longerthan those ofGN16(0.29and 0.31kb)and GN28(0.30 and 0.32 kb), while SmaI fragments of GN29 (0.175 and 0.245 kb) were shorter than those of GN16 (0.26 kb) and

GN28(0.27 kb) (Fig. la andb). The resultsof DraIanalyses support the assumption that two SmaI fragments of 0.175 and 0.245kb of GN29 hadderived fromone asequence.The restrictionfragments derived from the endof the L

compo-nent of the HSV-1 genome appear as a set of ladder-like fragments, and the interval between two neighboring frag-ments of the ladder corresponds to the length of one a

sequence (5, 12, 13, 16). When DNAs of K52 derivatives digested with KpnIwere analyzed by Southern hybridiza-tion,theinterval of the ladder ofGN29waslongerthan those of GN16 andGN28.Therefore,thelengthof theasequence

ofGN29was assumedtobelongerthan those ofGN16 and GN28.

Thea sequences of K52 derivatives and characterization.

DraI fragments corresponding to thea sequence ofGN16, GN28, andGN29wererecovered fromacrylamide gelsand

weremolecularly clonedintotheSmaIsiteofplasmidvector pUC18 (24). After subcloning of the cloned fragments into both phage vectors M13mplO and M13mpll, nucleotide

sequences of the a sequences were determined by the dideoxynucleotide chain termination procedure using Bca Best DNA polymerase (Takara ShuzoCo., Kyoto, Japan), whichwas obtained from the thermophileBacillus cardot-enax and functions best at 65 to 75°C. Thea sequence of GN28was338bpinlengthwith 14copiesofDR2 andwas ordinaryinstructure(Fig. 2a).TheasequenceofGN16was

327bpinlengthwith 13copiesof DR2 andwasthesame as

that of GN28, except for the copy number ofDR2. The a

sequenceof GN29was 476bp inlength and had twoDR2

arrays(Fig. 2b). Theasequenceof GN29wasassumed to be

5685

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(a)

M 1 2 3 4

- - m

- __ m

(b)

1 2 3 4

(c)

2 3 4 M

0.603

0,310

0.281 0.271

0.234

-

0.194

' 0.118

FIG. 1. Southern hybridization profiles of a sequences of K52 derivatives of HSV-1. HSV-1 DNAs digested with SmaI (a) and DraI (b and c) were electrophoresed in a 5% acrylamide gel, transferredto anylon membrane, and hybridized with a32P-labeled 0.175-kbSmaIfragmentof pUK340(25).Lanes: 1, K52; 2, GN16; 3, GN28;4,GN29.Theautoradiograms afterprolongedexposures are shown inpanelc.Lane M is a marker mixture ofHaeIIIdigests of 4X174phage DNA(21).Sizes of fragments are shown in kilobases.

DRI Ulb

I

DRI Ub

II E J

DR2 DR4n2 Uc DR1

Ub Lk

DR2 DR4n2aUtd DR2 DR4n2 Uc DRI

R1c Smal

Dral I

FIG. 3. Model for generation of the a sequenceof GN29 with

twoDR2arrays.TheasequenceofGN29(III)wasassumedtobe generated by recombination between the site on Uc of the a sequence having14copiesof DR2(I)and the site on Ub of the a sequencehaving 5 copies ofDR2(II). The novel recombination site onthea sequence ofGN29(Rec) is indicatedby a closed circle. Cleavage sites of SmaI andDraI onthea sequenceofGN29 are shown(III).

generated by recombination between a site on Uc (corre-sponding to nucleotide [nt] 311 of GN28) and the site on Ub (corresponding to nt 76 of GN28) (Fig. 3). A cytosine residue, the derivation of which was unclear, was present at nt 312

(Fig.

2b). This residue was diagnostic of the novel recombination site on the a sequence of GN29. A new SmaI sitewasgenerated at nt310to315

(Fig. 2b).

Thenucleotide sequences

of

DR4 stretches of the K52

derivatives

resem-bled those of DR4n (25), and the DR4 stretch of K52 derivatives was named DR4n2. When one-step

growth

curvesof GN28 and GN29wereconstructed,no differences were evident (25).

UnusualDNAfragments containingasequences presentin the DNA preparationsof GN29.Additional faint bandswere detected, with prolonged exposures,ontheSouthern blotof GN29 DNAsdigested with DraI (lane4ofFig. lc). Toclone the unusual DNAfragments of GN29, DNAs of GN29were treated with Klenow fragment, digested with DraI, and electrophoresed ina5%acrylamide gel and the region of the gel correspondingtoDNAfragments of 0.2to0.4 kbwas cut out. DNAs wereextracted from thegel and clonedinto the

(a) GN28

1 30 60 90

CCGCGGGGGGCCCGGGCTGCCCGCCGCCGCGCTTTAAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGGCCCGTTTTTGG

DR1 SmaI Ub DraI SmaI

98 253 270 300 312

GGTCTGGG (CGCTCCTCCCC),4CGCCTTTTTCGGCCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCC

DR2 DR4n2 Uc

330 338

TTTTTTGCGCGCGCGCACGC

(b) GN29

-31 -1

(Inverted repeat of L component) AGTGCTTGCCTGTCTAACTCGCTAGTCTCGG IR2

1 30 60 90

CCGCGGGGGGCCCGGGCTGCCCGCCGCCGCGCTTTAAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGGCCCGTTTTTGG

DR1 SmaI Ub DraI SmaI

98 253

GGTCTGGG (CGCTCCTCCCC),4CG

DR2 DR

330 335 CGTTTTTGGGGTCTGGG 450

270 300 312

GCCTTTTTCGGCCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCC

R4n2 391

Uc 420

Rec SmaI

(CGCTCCTCCCC) CGCCTTTTTCGGCCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCCGGACCG

496

Uc

CCGCCCGCCTTTTTTGCGCGCGCGCACGCCCGCGGGGGGCCCGGGCTT

DR1 SmaI

FIG. 2. Nucleotide sequences of a sequences ofGN28(a)and GN29 (b). Nucleotide numbers start at the left end of the left DR1 and terminateattherightend ofUc(GN28inpanela)andattheright endof therightDR1(GN29inpanel b).Theleftendof eachcomponent of theasequenceis indicated. The DR4stretch ofK52 derivatives was named DR4n2. DR1 and DR2 are underlined. Nucleotide sequences ofmajor invertedrepeatsof theL componentadjoiningtotheasequence arealso shown inpanelb and numbered -1to -31.IR2defined in strain F is underlined (5, 12). The cleavage sites ofSmaI and DraI are shown. The cytosine residue at nt 312, diagnostic ofnovel recombination of theasequenceofGN29, is underlined(Rec).

DR2 DR4n2

476

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[image:2.612.57.298.74.203.2] [image:2.612.317.556.74.194.2] [image:2.612.129.491.440.664.2]

(a) pUK359-1 (363 bp)

(253)

CTCCTCCCC (CGCTCCTCCCC ),CGCCTTTTTCG

DR2 DR4n2

(330)(335) (253)

CGTTTTTGGGGTCTGGG (CGCTCCTCCCC)14CG

DR2 DF

(312) GCCCGG

Rec

(270) (300) (312)

GGCCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCC

Uc

(270) (300)

Rec

GCCTTTTTCGGCCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACC

R4n2

(b) pUK359-2 (384 bp)

(253) (270)

CCC (CGCTCCTCCCC)12CGCCTTTTTCGGCCCCGCCCCC

DR2 DR4n2

(330)(335) (253 or 391)

TTGGGGTCTGGG (CGCTCCTCCCC)14CGCCTTTTTCGG

DR4n2

Uc

(300) (312)

CCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCCCGTTT

Uc Rec

300 312

CCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCCCGTTTTTGG

Uc

(269 or 407)

TTTCGGCCCCG

(d) pUK361-5 (212 bp)

(391)

CCCC (CGCTCCTCCCC)I2CGCCTT

DR2 DR4n2

(466)

TGCGC

(e) pUK360-4 (250 bp)

(279) (300)

Rec

(420)

(312) (330) (335

CCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCCCGTTTTTGGGGTCTGGG

Uc (270)

Rec

(300) (312)

330 335 (253 or 391)

GGTCTGGG (CGCTCCTCCCC),4CGCCTT

DR2 DR4n2

(450)

5) (253)

; (CGCTCCTCCCC)5CGCCTTTTTCGGCCC

DR2 DR4n2

(330)(335)

CGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCCGGGGCCCGTTTTTGGGGTCTGGG

Uc Rec

(CGCTCCTCCCC ) DR2

(f) pUK360-2 (290 bp)

(60) (71) (79) (90) (98)

AAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGGGCCGTTTTTGGGGTCTGGG

(291 or 429)

CGCCTTTTTCGGCCCCGCCCCCCACGCCCGCCGCGCGCG

DR4n2 Uc

(CGCTCCTCCCC),7

DR2

FIG. 4. Nucleotidesequences ofcloned, unusual DNAfragments containinga sequencesnotdirectly derived from thea sequenceof

GN29. Thenamesofhybrid plasmids carryingthe unusual DNAfragments containingasequences areshown,andlengthsof the insert DNA

areinparentheses. The nucleotide numbers shown in parentheses indicate those of corresponding regionsontheasequenceof GN29 (Fig. 2b).DR2is underlined. Thecytosine residueat nt312, diagnostic of novel recombination of theasequenceofGN29, is underlined (Rec).The

rightendof insertDNAofpUK360-2 (f)wasthe Dralsite. Thecopynumberof the DR2arrayofpUK360-2 (f)was17and differed from those

oftwoDR2arraysof GN29.

SmaI site of pUC18. Nucleotide sequences of the insert DNAs of16hybrid plasmidsweredetermined. Structuresof theunusual DNAfragments assumedtobederiveddirectly fromtheasequenceofGN29aresummarized in Table1,and

structures not drawn from the a sequence of GN29 are

shown inFig. 4. Of the 16 unusual DNAfragments studied,

two (pUK362-3 and pUK360-2) had termini generated by DraIdigestion,but terminiof theother 14 unusualfragments werenotthosegenerated bythe DraIdigestion.Theunusual DNAfragments without the DraIterminuswerenot

gener-ated byDraI digestion offull-length HSV-1 genomes and probablynotderivedfrom DNAspackagedwithin the virus particle. Uptothispoint, the HSV-1 DNAswere prepared as follows (crude preparation). The infected cultures (cells

and medium) were centrifuged at 54,000 x g for 2 h. The pelletsweresuspended, sonicated, andcentrifugedat5,000

rpm 2,300 xgfor 5 min.The supernatantwaslayered over aglycerolstepgradientandcentrifugedat81,000x gfor 1 h (7, 26). The pellet containing nucleocapsids was digested with proteinase K and then was phenol extracted. Two modificationsweremadetoincrease theproportionofDNAs derived from viralnucleocapsids (purified preparation).One modification was the centrifugation of infected cultures at 2,300 x g for 5 min after the cytopathic effect became apparent, and thesupernatantswerecentrifugedat54,000x g for 2 h. The other modification was the digestion of

nucleocapsid preparations with DNase I afterglycerol step gradient centrifugation.Unusual bandsdetected in the crude

(c) pUK361-2 (235 bp)

(272)

PTTTCGGCCCGCCCCCCACGCCCGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCCTTTTT

uc

(3 (DraI)

16)

(253 or 391)

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Mt

o H~~~~~~~~~~~~~

0 Z4)~~~~0U,00

4-4 ~ ~ ~

0 0 0 ~~~.o0 '-~~~M

0 0 H '-'o ~~~~~~o(NOE-0 ~~~ 0

0 o o ~~~~Eo oE

0 c~~~ 0 ~~ ~ ~ ~ 00

0.~~~~~0 0E- E0

-~~ H 0 ~~E- 0 4)4

.~~~~~~ 0~- 0 . U

0~~~ ~ ~ ~~~~~~~~~c

O 0 ~ 0~

* H 0 E0 0

0 0 r

* 0 ~~~~~~~~~~~~.0

0

0 AC

0I* .- *N 0 H0 O) r

0 0 m.0

0 H~~~~~~~~~~~~

o~~~~~~~~~~~~~ H~~~~~~~c

4) ri 0 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~csr

04 ~ ~

C) 0 0

0~~~~~~~~'~

,0 0~m4 0 C 04

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TABLE 2. Distribution of termini of cloned unusual DNAfragmentsonthe a sequence and the IRof the L component

Region of HSV-1 genome or Nucleotide position Plasmid(s) with:

domain of thea sequence of terminia Left termini Righttermini

Inverted repeatof L component -31(1 nt before IR2) pUK362-1,pUK362-2, pUK362-4,pUK362-5, pUK363-4*

asequence

DR1 490(correspondingto14) pUK360-3

494(correspondingto18) pUK363-1*

Ub 21(adjoining DR1) pUK363-1*, pUK363-3*

36(cleavagesite ofDraI) pUK362-3, pUK360-2

333(2ntbeforeDR2) pUK362-3

DR2 1st' pUK360-1

3rd pUK359-1

5th pUK360-3

7th pUK361-1

8th pUK361-5

9th pUK359-2

11th pUK361-3, pUK361-4 pUK360-4

DR4n2 264 or 402 pUK359-2

269 or 407 pUK361-2

272(correspondingto410) pUK361-2

414(correspondingto276) pUK363-3*

416(correspondingto278) pUK360-1

279(correspondingto417) pUK360-4

Uc 291or429 pUK360-2

441 pUK361-1

313(recombination stretch) pUK362-1, pUK362-2,

pUK362-4, pUK362-5, pUK363-4*

314(recombination stretch) pUK359-1

450(relatedtothe novelrecombination) pUK361-3, pUK361-4

466 pUK361-5

a Nucleotide positionsarethose of theasequence and theinvertedrepeat of the L componentofGN29(Fig. 2b).

bPositions within one DR2 elementof11bpareindicated.

preparations

were

hardly

visible inthe

purified preparations

by

Southern hybridization.

Formolecularcloning of longer unusual DNA

fragments,

the

purified preparation

of GN29 DNAwastreated with Klenow

fragment

and electrophore-sed in a

5%

acrylamide gel

and the

region

of the

gel

corresponding

toDNA

fragments

of 0.35 to0.6 kbwas cut out. Three

hybrid plasmids containing

a sequences were obtained from the

purified preparation,

and their

designa-tionsweremarked with asterisks

(pUK363-1*, pUK363-3*,

and

pUK363-4*).

Thenucleotide sequences of insert DNAs of these

hybrid plasmids

were

determined

(Table 1).

pUK363-1*

containedan

almost-full-length

a sequence, ex-cept for 2

bp

ofDR1. The insert DNA of

pUK363-4*

was the same asthose of four other

plasmids derived

from the crude

preparation (pUK362-1,

pUK362-2, pUK362-4, and

pUK362-5) (Table 1).

Distribution of the termini of molecularly cloned unusual DNA

fragments

in each domain of thea sequence is sum-marized in Table 2.

(i)

Five termini were not within the a sequence but rather were at nt -31 of the

major

inverted repeatof the L component,nearinverted repeat 2

(IR2) (12).

(ii)

Termini of DNA fragments cloned in pUK360-3,

pUK363-1*,

and

pUK363-3*

wereinor

adjoining

DR1. The

right

terminus of

pUK363-1*

corresponds

to the authentic

cleavage

terminus

by

cleavage-packaging machinery.

Left termini of

pUK363-1*

andpUK363-3*were1bp shorter than the authentic

cleavage

product.

Spontaneous deletions

re-moving

one copyof the DR1 element from thea sequence werefound in variant viruses and suggest involvement of the

cleavage

of DR1 in

generation

of the deletions

(11,

20).

These observations support the

hypothesis

that the

site-specific

DNA breaks induced

by

the

cleavage-packaging

systemstimulate the initiation of recombination

(4, 8, 15, 18,

28). (iii)

Nine terminiwerein

DR2,

and the

right

terminus of

pUK362-3

in Ubwas 2

bp

before DR2. The termini in the DR2 arraywere notrestrictedto a

specific

site in the DR2 sequence;

rather, they

seemedtobedistributed

randomly

in the DR2 element. These terminiwereassumedtobe gener-atedmostly by nuclear

endonucleolytic activity

specifically

cleaving

sequencesof non-B DNA

conformation,

suchasthe DR2 array

(31-33). Thus,

strand breaks necessary for recom-bination of the a sequence could be introduced

by

the

DR2-specific

nuclease.

(iv)

Six terminiwere in DR4n2 and withinastretch of 16

bp

(nt

264to279ornt402to

417).

Four typesof DR4

stretch,

i.e., DR4, DR3.5, DR4t,

and

DR4n,

have beenidentified

(5, 12, 14, 25,

28).

DR4and DR3.5were reiterated

(12, 14).

The

heterogeneity

andreiteration of the DR4 stretch could be due to recombination of the DR4

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stretch, and theDR4n2 DR4 stretch of K52 derivatives was assumed to be derived from DR4n by recombination.These findingssupport recombinogenicity of the DR4 stretch, and the DR4 element of strain F enhanced the recombinational activity of thea sequence (3). (v) Five termini were at nt 313 and oneterminus was at nt 314 in the novel recombination stretch of the a sequence of GN29. The right termini of pUK361-3 andpUK361-4 were at nt 450inthe region ofUc and corresponded to the stretch involved in generation of the novel recombination site. The region around the novel recombination site was assumed to be recombinogenic, as shown bythe recombination event generating the variant a sequence of GN29 and strand breaks in the novel recom-bination stretch detected as termini of unusual DNA fragments. Three termini of pUK360-2, pUK361-1, and pUK361-5 were in Uc and were not directly related to the novel recombination. Thus, almost all termini of the cloned unusual DNA fragments were situated in the definedregions, i.e., the site in the major inverted repeat of the L component,

DR1,

DR2, the DR4 stretch, and the novel recombination stretch,which may have recombination potential (Table 2). The recombination between a sequences occurs by stan-dard homology-dependent generalized recombination, and activity that may mediate recombination between a se-quences ina site-specific manner was detected (2, 9, 17). The double-strand-break repair model for recombination has been proposed (10, 19). Amplification of the a sequence of HSV-1 was explained by the model proposed for the cleav-age-packaging process, based on the double-strand-break repair mechanism (6). Double-strand break is an efficient initiator of homologous recombination, and it has been suggested that free DNA ends find an intact homologous sequence and use it as a template for the repair of its sequence, via DNA synthesis and ligation. The generation of free DNA ends of DR1 by the cleavage-packaging system and cleavage of the DR2 array by a virus-induced nuclear endonuclease have been reported (13, 32). Results in the present study support these findings. The generation of breakage in the DR4 stretch and in the novel recombination stretch and nucleotide sequences of termini ofcleaved DR2 elements are described for the first time in thepresent study. These findings support the double-strand-break repair model for recombination of the a sequence. The presence of recombinogenic elements such as DR2 and DR4 stretch in the a sequence would explain thesequence-specific increase in the frequency of recombination of the a sequence by the standard homology-dependent generalized

recombination

(2, 9, 17).

Gratitude is extended to M. Ohara for assistance withthe prepa-ration of this report.

Part of this study was supported by grants from theMinistryof Education, Science, and Culture of Japan.

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