Proc. Nati Acad. Sci. USAVol. 79, pp. 7814-7818, December 1982Genetics

Histone H2A subtypes associate interchangeably in vivo withhistone H2B subtypes

(Saccharomyces cerevisiae/histone mutations)

DAVID KOLODRUBETZ, MARY C. RYKOWSKI, AND MICHAEL GRUNSTEIN

Molecular Biology Institute and Department of Biology, University of California, 405 Hilgard Avenue, Los Angeles, California 90024

Communicated by Emil L. Smith, September 15, 1982

ABSTRACT The yeast Saccharomyces cerevisiae contains twoprimary sequence subtypes of histone H2B (H2B1 and H2B2) andof H2A (H2A1 and H2A2). Mutants in each of the H2B subtypeshave been used to show previously that yeast cells lacking one orthe other, but not both, of the H2B proteins are viable. BecauseH2A protein interacts in the nucleosome with H2B, we wished todetermine whether specific H2A subtypes must interact with spe-cific H2B subtypes. We describe experiments in which frameshiftmutations were introduced into both ofthe H2A genes in vitro andthe mutant genes integrated into the yeast genome, replacing thewild-type H2A genes by a subsequent recombination. Using thesemutant (htal- and hta2i) strains we find that neither H2A genehas a unique essential function during any phase of the yeast lifecycle, although strains homozygous for htal- grow more slowly.However, one functional H2A gene is required for viability be-cause cells mutant in both H2A genes arrest at spore germinationprior to bud separation. By combining these H2A mutations withthe H2B mutations obtained previously, we show that all combi-nations ofH2A and H2B subtypes produce viable cells. From thesegenetic experiments and electrophoretic analysis of the histoneproteins of these mutants we conclude that the H2A subtypes canassociate interchangeably with the H2B subtypes.

All four core histones, H2A, H2B, H3, and H4, are present inthe yeast Saccharomyces cerevisiae (1). An analysis ofthe clonedgenes for H2A (called HTA) and H2B (called HTB) has revealedthat the haploid yeast genome contains only two copies of eachgene (2). Each HTA gene is adjacent to an HTB gene and thegene pairs are unlinked (2). The two HTB genes encode primarysequence variants (subtypes), each 130 amino acid residues inlength differing by 4 residues, all near the amino terminus (3).The two H2A proteins are 131 residues long and differ at only2 amino acid residues (4), both near the carboxyl end.

Our laboratory has examined the function of histone proteinsubtypes by using a combination of genetic and biochemicalapproaches. In a previous publication (5) we asked whether bothH2B subtypes were essential for a complete yeast life cycle. Aframeshift mutation was created in each of the HTB genes invitro and the wild-type genomic copy was replaced with thecorresponding mutated HTB gene. Studies with these mutantsshowed that yeast strains lacking either H2B1 or H2B2 (i.e.,htbl - or htb2- cells) undergo a complete life cycle. Therefore,neither H2B1 nor H2B2 has a unique essential function.

In this paper we address the question of H2A-H2B subtypeinteractions in vivo. Histones H2A and H2B interact with eachother to form part of the octameric structure of the nucleosomecore (6). Since there are two H2A and two H2B subtypes, per-haps there is an exclusive interaction between a specific H2Asubtype and a particular H2B subtype. Alternatively, each H2A

protein may associate randomly with either H2B subtype toform a functional H2A-H2B dimer. Either possibility is con-sistent with the experiments showing that cells mutant in HTB1or HTB2 are viable.

To determine if all combinations of H2A and H2B subtypesformed functional nucleosomes we first created frameshift mu-tations in each of the HTA genes. As with the HTB mutations,we found that either HTA1 or HTA2 alone can provide the H2Arequired for viability during any phase of the yeast life cycle.Using genetic and recombinant DNA techniques, we then con-structed all possible combinations of HTA (htal-, hta2-) andHTB (htbl-, htb2-) mutations in the same haploid cell. Geneticanalysis of these strains and analysis of their histone contentindicates that each H2A subtype can associate productively invivo with either H2B subtype.

MATERIALS AND METHODSNomenclature. To conform with the conventions of yeast

genetic nomenclature (7) we have adopted the following des-ignations for the loci involved in this work: the gene encodingH2A1 is called HTA1; H2A2 is HTA2; H2B1 is HTBJ; and H2B2is HTB2. The mutant alleles are written in lowercase to indicatethat they are recessive.

Strains and Culture Conditions. The relevant genotypes ofthe S. cerevisiae strains used are given in Table 1. Cells weregrown, mated, sporulated, and dissected as described (5, 8).Recombinant DNA Procedures. Southern blots and Maxam

and Gilbert DNA sequence analysis were done as detailed pre-viously (5, 9, 10). The in vitro mutagenesis of the HindIII sitesin the HTA genes has been described (5). Plasmid pDK2 wasconstructed in two steps. First, the hta2-1 -containing 1.37-kilo-base (kb) HindIII fragment was cloned into the unique HindIIIsite of the yeast vector YIp5 (11). The Hpa I/EcoRI fragmentof this plasmid then was replaced with the htb2-1-containingHpa I/EcoRI fragment from pMG101 (5) to generate pDK2(Fig. 1B). Plasmid pDK4 is the 2.5-kb HindIII fragment, con-taining htal -, cloned into the HindIII site of YIp5. The Sst I/BamHI fragment of pDK4 was replaced with the htbl -con-taining Sst I/BamHI fragment from plasmid pMR2 (5) to givepMR257 (Fig. 1B).

Yeast Transformation and Enrichment for Ura- Segre-gants. Plasmid pDK2, pDK4, or pMR257, which contains theyeast URA3 gene, was used to transform yeast strain NNY, andstable Ura+ transformants were isolated as described (5). StableUra+ isolates were enriched for Ura- segregants on orotic acid-ureidosuccinate plates as detailed previously (5).

Isolation and Analysis of Yeast Histones. Crude yeast chro-matin was isolated by using the procedure of Tonino and Rozijn(12) as modified by Mardian and Isenberg (13). Chromatin waswashed three times by Dounce B homogenization in 0.5% Non-

Abbreviation: kb, kilobase(s).

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Table 1. Strain listStrain Relevant genotypeNNY a trpl, ura3-52, his3-1*KY115 a ade2, ura3*GY401 a HTA1: :pDK4 (htal-1, URA3+), ura3-52GY402 a/a htal-1/HTA1, ura3/ura3GY403 a/a hta2-1/HTA2, ura3/ura3GY404 a/a hta2-1/HTA2, htb2-1/HTB2, ura3/ura3GY405 a/a htal-1/HTA1, htb2-1/HTB2, ura3/ura3GY406 a/a hta2-1/HTA2, htbl-1/HTBl, ura3/ura3GY407 a/a htal-1/HTA1, htbl-1/HTBl, ura3/ura3GY408 a/a htal-1/HTA1, hta2-1/HTA2, ura3/ura3

* Ref. 5.

idet P-40/10mM Tris HCI, pH 8.0/2mM EDTA/75mM NaCI/1 mM phenylmethylsulfonyl fluoride, shaking 20 min on ice andpelleting at 9,000 rpm for 10 min in a Beckman JA-20 rotor. Thepellet was rinsed twice by homogenization in the same bufferwithout Nonidet P-40 and was pelleted as before. The absor-bance of the last chromatin suspension at 260 nm was measuredin 1% NaDodSO4. The washed chromatin was resuspended toan A260 of40-80 per ml in micrococcal nuclease digestion buffer(10 mM Tris HCI, pH 7.4/0.5 mM phenylmethylsulfonyl flu-oride). One unit of micrococcal nuclease (Worthington) wasadded per 100 ,ul and the reaction was started by the additionof 1/100 vol of 0.1 M CaCl2. The digest was incubated for 30min at room temperature and then was brought to 4 mM inEDTA (pH 7.0) and centrifuged for 20 min in an Eppendorfmicrocentrifuge. Three microliters of this material was mixedwith 1 ,ul of loading buffer [0.05% pyronin Y/1% protaminesulfate (histone free, Sigma)/2.5% 2-mercaptoethanol/2.5% 2-mercaptoacetic acid/8 M urea] and run on an acid/urea gelaccording to Spiker (14), except that the stacking gel was poly-merized with 0.005% riboflavin and fluorescent light insteadof persulfate.

RESULTS AND DISCUSSIONMutant Construction. We constructed frameshift mutations

in the two HTA genes by in vitro mutagenesis of cloned copiesof the HTAI and HTA2. Each gene contains a recognition se-quence for the restriction endonuclease HindIII in the DNAsequence encoding amino acid residues 13-15. An insertion of4 base pairs was introduced into each HindIII site generatingframeshift mutations, which result in a termination codon atamino acid residue 19 in both genes (Fig. 1A). In addition, the4-base-pair insertion destroys the HindIII site. Thus, when themutant plasmid is digested with HindIII, the two fragmentspresent in the normal gene are absent and are replaced by alarger fragment whose size is the sum of the two smaller ones.This alteration in the restriction pattern allows us to determinethe genotype at each HTA locus. The mutant HTA gene re-striction fragments were subcloned into the yeast vector YIp5,which contains pBR322 sequences and the yeast URA3 gene(11). The inserts of the resulting plasmids, pDK4 and pDK2,are diagramed in Fig. 1B.

Portions of the plasmids pDK4 and pDK2, containing themutated HTAI and HTA2 genes, were subjected to sequenceanalysis; for each gene, the DNA contained the expected 4-base-pair insert (Fig. 1A). Thus, rather than coding for a protein of131 amino acid residues, both the HTA1 and HTA2 mutantgenes now cause premature termination, encoding polypep-tides only 18 residues long.

Next we wanted to replace the wild-type genomic HTA geneswith the mutant copies generated in vitro. The URA3 plasmidscontaining the mutant HTA genes were used to transform a

A

BpDK2

pDK4

pMR257

insertion

htal-or hta2-... GCT AAWX T|JTC TCA ATC TAGala lys ala ser phe ser ile TERM12

H2A wild type ... GCT AA|A TCT CAA TCT AGA TCT...ala lys ala ser gin ser arg ser12

10.41 0.97 1.1H H

YI - htb2 R YIp5

4 1.4 1.1H H

LIYIp5 hal S B YIp5

1.4 1.1H

Ylp5 htalI htblBI Yp5

FIG. 1. Sequence of mutantHTA genes and restriction maps of themutant plasmids. (A) The DNA and amino acid sequences of wild-typeand mutant HTA in the region mutagenized. Both HTA1 and HTA2have the same sequence in this region (4). (B)A partial restriction mapof the circular plasmids used in the strain constructions described inthe text. Shown are the positions of the HindI (H), Sst I (S), EcoRI(R), Hpa I (P), and BamHf (B) restriction sites. The locations of themutagenized Hindi sites in theHTA andHTB genes are denoted bythe vertical wavy lines. Distances between restriction sites are in kb.

ura3- haploid yeast strain and stable uracil prototrophs wereselected. These should result from integration of the plasmidinto the yeast chromosome at the appropriate HTA locus byhomologous recombination (15). Southern blot analysis of theUra+ transformants confirmed that integration had occurred atthe correct location (Fig. 2). Fig. 2A is a diagram ofthe expectedconfiguration ofEcoRI sites around HTA1 in the genome beforeand after integration ofplasmid pDK4. The 10.5-kb EcoRI frag-ment found in wild type should be replaced by two fragmentsin the integrant, one 14 kb and the other 4.4 kb. When DNAfrom a wild-type strain is cleaved with EcoRI and hybridizedto an HTA1 probe, one 10.5-kb band appears (Fig. 2B, lane 2).In the DNA from a stable Ura+ transformant, GY401, there aretwo EcoRI fragments, 14 kb and 4.4 kb, homologous to theHTA1 probe (Fig. 2B, lane 3). Therefore, the htal- plasmid hasintegrated at the yeast HTA1 locus in GY401.The next step involved excision of the wild-type HTA and

vector sequences, leaving the mutant hta- allele. Because theoriginal integration resulted in the duplication of the HTA geneand the incorporation of the URA3 gene at the HTA locus (Fig.2A), a spontaneous intrachromosomal recombination event canresult in the excision of the vector sequences and loss of theUra+ phenotype. Depending on where the crossover occurs,either the mutant or the wild-type HTA allele can be left in thegenome. Because the loss of a wild-type HTA gene in this stepmight be lethal, the stable Ura+ transformants were first matedwith strain KY115, which is wild type for HTAJ and HTA2.Thus, there is always a complementing wild-type HTA gene inthese diploid strains to ensure viability. We then grew thesediploids on orotic acid-ureidosuccinate plates that enrich forcells which have lost the Ura+ phenotype (5). The resultingUra- diploids were screened to determine which still containedthe mutant HTA gene. Because a HindIII restriction site iseliminated in the HTA mutants, the presence of the mutantallele can be scored by the appearance of a new HindIII frag-

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A is 10.5 kb- l

Integration

R htal R HTA I Ril 14 .b--- i4.4I

im - 14kb -4 -4.4kb

Bkb

C\jAd 0

z >- >-z C) ()

1i1.7- do

7.6- - w

56- -

2 67-

FIG. 2. Evidence for integration of pDK4 at the genomic HTA1locus. (A) Schematic of the expected arrangement ofHTA1 locus beforeand after integration of the mutant plasmid pDK4. EcoRI (R) restric-tion sites are shown. (B) Southern blot of EcoRI-digested DNA; fromthe strains indicated, hybridized to TRT-1 probe that contains HTA1as well as about 8 kb of flanking sequences (2). The first lane containsDNA size markers with sizes indicated. The 4.1-kb band seen in lane4 is from cross-hybridization of the probe to HTA2.

ment on Southern blots hybridized to the HTA-containingprobes.

Fig. 3 shows a Southern blot of HindIII-digested genomicDNA from one such Ura- segregant, GY402 (first lane), whichhas retained the 2.5-kb band expected for the mutant htal7.Because GY402 is heterozygous for htal -, the wild-typeHindIII bands also are present. In addition, this strain has re-gained the wild-type EcoRI restriction pattern, a single 10.5-kbband, confirming the loss ofthe plasmid DNA from the genome(Fig. 2B, GY402). Thus, in strain GY402, the htal - allele hassubstituted for a wild-type HTAJ gene. A similar analysis wasused to show that a wild-type copy ofHTA2 has been replacedwith the hta2- mutation in strain GY403 (data not presented).In this way we constructed two diploid strains, one heterozy-gous for htal- and one heterozygous for hta2-.

htal- and hta2 Mutants Are Viable. We used the htal- andhta2- mutants to address the following questions: Are haploidsmutant in htal- (or hta2) able to grow and mate? Are diploidshomozygous for htal- (or hta2) capable of growth and spor-ulation?To determine if an htal - haploid is viable, strain GY402 was

sporulated and the spores were dissected and allowed to ger-minate. Because the strain is heterozygous for htal-, thereshould be 2:2 segregation of htal - to HTAJ+. All four sporesin each ofthe seven tetrads that were examined germinated andgrew, indicating that the htal- haploid is viable. DNA isolatedfrom the spore clones was analyzed on Southern blots to de-termine the genotype ofeach spore. In Fig. 3 the last eight lanesto the right show HindIII endonuclease-digested DNA fromspores of two tetrads. As expected, in each tetrad there is 2:2

segregation of the mutant htal- (2.5 kb) and wild-type HTA1+(1.4 and 1.1 kb) HindIII restriction patterns. This proves thatan htalr haploid can grow mitotically. However, it was notedthat hta- haploids formed smaller colonies upon germinationthan did HTA1' haploid spores.

Strain GY403, heterozygous for hta2-, also was sporulatedand the germinated spores were examined for their HTA2 geno-type by Southern blot analysis (data not presented). Once again,all spores from the seven four-spored tetrads that were analyzedwere viable and there was 2:2 segregation of hta2- to HTA2'.This shows that an hta2- haploid also is viable.To determine if strains homozygous for htal - are capable of

sporulation, pairs of htal - haploids were mated to each otherto form homozygous diploids. These diploids were viable, al-though they grew more slowly than did wild type. The htal -diploids retained the ability to sporulate and thus complete thelife cycle. Diploids homozygous for hta2- also were constructedby mating pairs of various hta2- haploids to each other. Thesediploids grew normally and sporulated at normal frequencies.Thus, we conclude that neither histone H2A1 nor histone H2A2is uniquely required in the yeast life cycle.

htal- Strains Grow More Slowly than Do hta2- Strains. Asmentioned above, the only distinguishing characteristic ofeither HTA mutation is the slow growth of strains homozygousfor htal -. The htal - strains produce smaller colonies than dowild-type or hta2- strains when plated onto rich medium, evenafter continuous subculturing for more than 45 doublings. Fur-thermore, strains homozygous for htal - divide about 15-30%more slowly in liquid than do wild-type or hta2- strains (datanot presented). It is possible that the htal- slow-growth phe-notype is due not to the htal- mutation itself but to improperexcision ofthe plasmid. However, this is unlikely because htal -strains from several independent excisants were examined andall exhibited the slow-growth phenotype. Alternatively, thehtal - strains may be petite (respiratory deficient) strains. Pe-tites grow more slowly than do wild-type strains on glucose-con-taining medium but are unable to grow on nonfermentable car-

Tw-jO T it r froam>ro r

O o- ruhed GY402

*arUJr; r d C6 J

- mutant

gg1t* b * Newt-wt

FIG. 3. Analysis of segregation of spores arising from sporulationof HTAl/htal-. This Southern blot shows DNA from the indicatedstrains that was digested with HindM and hybridized to the HTA1-containingprobe TRT-1. Indicated are the 1.4- and 1.1-kbHindIllfrag-ments foundforthe wild-type (wt) configurationatHTAl andthe fused2.5-kb fragment found for the mutant configuration. The two otherbands are from hybridization of sequences that are outside HTAZ butcontained in the TRT-1 probe.

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bon sources, such as glycerol. The htal - strains grew on glyc-erol and therefore are not petite mutants.

Previous experiments with HTB mutants showed that htbl-spores also germinated to produce smaller colonies than didwild-type or htb2- strains on agar (5). Though the differencebetween htbl- and htb2- cells on agar plates was less pro-nounced after subculturing for more than 50 doublings, the col-ony sizes were still different. In liquid culture the differencesin growth rate were less evident and more strain dependent.Some htbl - strains grew as rapidly as did htb2- strains; otherscontinued to grow more slowly.

This phenotype for htal- and htbl- strains may reflect asubtle functional difference between H2A1 and H2A2 proteinsand between. H2B1 and H2B2 proteins.. It also is possible thatthis phenotypic difference between the two tandem gene pairsresults from differences in their mRNA transcription or turn-over (16). Our experiments cannot distinguish between thesepossibilities.

Cells Require at Least One HTA Gene. The viability of theHTA mutants indicates that either HTA gene alone is sufficientfor cell growth. However, the results presented so far also areconsistent with the possibility that no HTA is required for yeastcell growth. To test this, strain GY408-was constructed by mat-ing htalf haploids with hta2- cells. This strain, heterozygous

u')0

r-- -- a >I'lA

_~ U -htal

a -HTAl+0 _i HTB2+

(.00ac r

B

_ -htbF-

-hta2* _- HTB1+

'- -HTA2+

FIG. 4. Southern blot hybridization gels showing viability ofhtalhtb2- and hta2-htblf haploids. DNA from the appropriatestrains was restricted with HindIII restriction endonuclease and ana-lyzed by Southern blot hybridization. The DNA was transferred bidi-rectionally from the gel onto two nitrocellulose filters (9). One filterwas hybridized to a probe containing the 1.1-kb Hindm fragmentfrom the HTA1-HTB1 region (2) and the other to a probe containingthe 0.97-kb Hindm fragment from the HTA2-HTB2 region (2). Thefigure shows a photograph of the two resulting autoradiographs su-perimposed. (A) Southern analysis of haploid spore colonies (lanes a-d) from one tetrad resulting from the dissection of GY405. The loca-tions of the mutant 2.5-kb (htal) and 2.1-kb (htb2 ) HindIm bandsas well as the wild-type 1.1-kb (HTA1) and 0.97-kb (HTB2+) HindEbands are shown. (B) Analysis of four haploid spores (lanes a-d) fromone tetrad resulting from the dissection of GY406. The locations of themutant 2.4-kb (htbl) and 1.4-kb (hta2) HindiH bands as well as thewild-type 1.1-kb (HTB1) and 0.97-kb (HTA2+) bands are indicated.

for htal -and.hta2-, was sporulated and the genotypes of theviable spores were-determined by Southern blot analysis. One-fourth of the spores tested was expected to be htal-hta2- be-cause the two loci segregate independently. Of the 28 sporesanalyzed, 10 were htalEHTA2', 10 were HTAlhta2-, and 8were HTA1+HTA2+. None was htal -hta2- ..The pattern ofseg-regation of htal- and hta2l in the nine tetrads that were ex-amined allowed us to determine whichspores should have beenhtal-hta2-. In each case the double mutant spore, upon ger-mination, produced abud but arrested prior to separation ofthebud from the mother cell. This also is the phenotype observedfor an htbl-htb2- haploid (5). We. conclude that htal -hta2haploid cells are nonviable.

Testing the Interaction of Histone H2A and H2B Subtypes.Once the HTA mutants were constructed and characterized, wecould determine whether or not haploid strains containing amutation in one HTA gene and one HTB gene are viable. Forexample, it -is possible that the H2A1 protein might form func-tional nucleosomes only with H2B1. If this were the case, wewould expect that a haploid hta2 htbl- strain containing onlywild-type H2A1 and wild-type H2B2 proteins would be non-viable. Likewise, a cell may require some other combinationofH2A and H2B subtypes for viability. Therefore, we asked thequestion: Are there essential interactions between particularH2A and H2B histone protein subtypes?To combine the htal- and htb2- mutations we constructed

a diploid that was heterozygous for htal - and htb2- by matingtwo haploid strains, an htal HTB2+ haploid and an HTA1+htb2-strain. This diploid then was sporulated. Because the HTA1 andHTB2 loci are unlinked, we expect one-fourth of the haploidspores to be htal htb2-. Using Southern blot analysis we ex-amined a HindIII digest ofDNA from spore clones derived fromfour-spored tetrads. The presence of both the htal - band (2.5kb) and the htb2- band (2.07 kb) and the absence of the cor-responding wild-type bands (Fig. 4A, lane c) establishes theviability of the haploid strain htal htb2-. Thus, H2A2 does notpair only with H2B2 and H2B1 does not interact exclusivelywith H2A1.

In the same manner we constructed a diploid strain that washeterozygous for the mutations at the HTA2 and HTB1 loci. On

A o

a b c d 0

B q

a b c d 0

-mutant i

" -mutant

j;9 }wt - -w

FIG. 5. Viability- of htalfhtbl and hta2-htb2 haploids. DNAfrom the -appropriate strains was digested withHindm restriction en-donuclease and analyzed by Southern blot hybridization. (A) Southernanalysis of the haploid spores (lanes a-d) of a tetrad derived from thedissection of GY407. The hybridization probe used was the HTA1-HTB1-containing plasmid TRT-1 (2). The bands shown are themutant3.8-kb band (htalhtbl-) and the wild-type (wt) 1.1-kb band(HTAlHTBl+). The uppermost band found in all of the lanes is dueto hybridization of sequences that are outside HTAI and HTB1 butcontained in the TRT-1 probe. (B) Analysis of the haploid spores (lanesa-d) of a tetrad derived from the dissection of GY404. The hybridiza-tion probe used was theHTA2-HTB2-containing 0.97-kbHindll[ frag-ment (2). The bands indicated are the 2.5-kb mutant HindmI band(hta2-htb2-) and the 0.97-kb wild-type band (HTA2+HTB2+).

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a b c d e fg

1H2B2

NH2A

H3

*0O -.w. ; 4WE ....-

FIG. 6. Electrophoretic comparison of the histones from wild-type,and mutant cells. Coomassie blue-stained acid/urea gel of the histones

isolated from strains with the genotypes indicated below. The histone

bands are markedappropriately. The first lane (a) displays all four core

histones, including both H2B subtypes, from a wild-type strain. The

next two lanes show that both H2B subtypes are present in cells mu-

tant -in HTA1 (b) or HTA2 The last four lanes show that the ap-

propriate H2B subtypes are missing in cells mutant inHTB1, orHTB2.

Lanes: d, htal htb2-; e, hta2-htbli ; f, htal -htbl ; g, hWtahtb2-.

sporulation this strain yielded four-spored tetrads. Southern

blot analysis of the spores was done and the results are shown

in Fig. 4B. The segregants. hta2ThtbY-, hta2-HTBI',HTA2+htblJ7, HTA2'HTBI' are evident in lanes a-d; there-

fore, the, hta2-htbl mutant is clearly viable. From this result

we conclude that- there is no exclusive H2A,1-H2B2 or, H2A-

H2B2 interaction.

Because the, htal and htbl mutations are so closely linked,

in the genome (only 800 bases apart) (2), it would be extremelyrare to obtain an htal htbl haploid recombinant by using a

similar genetic approach. Therefore, this mutant was, con-

structed in vitro by using plasmid pMR257, which contains both

the htantmutation and thehtble- mutation (Fig. oB). This plas-mid was used to replace, the wild-type genomic copies. of bothHTAn and.HTB oby integration and excision. A Uray segregantthen was made-diploid, as described above, and sporulated;,thespore clones were analyzed by Southern analysis (Fig. 5A). The3.8-kb DNA fragment generated by HindILI digestion repre-

sents the mutant htal andhtbl - genes, both of which lack a

HindIII site. It is evident that there is the expected 2:2 seg-

regation ofthe mutant (lanes a and c) andwild-type (lanes b and

d) alleles. From this experiment it is clear that the htal2-htblhaploid cell is viable.

A similar approach was used to produce the hta2ihtb2 mu-

tant haploid strain by using pHasmidpDK2 (Fig. HB).Again,Southern analysis of haploid spores shows that anhta2chtb2khaploid is viable (Fig.8B, lanes a and d). The genetic experi-mments demonstrate that either histone H2A subtype can asso-

ciate productively with either H2B subtype because, in thesecrosses, the mutant cells are still able to divide mitotically.

These results were confirmed by an analysis of the histones

from the HTA mutants. For example, if there were an exclusive

interaction betweenH2Ai and Mh2B, then histone proteins

isolated from chromatin of an hta2- strain would contain onlyH2B1 protein rather than both H2B subtypes. Histone proteinswere isolated from an htal - strain (lane b) and an hta2- mutant(lane c) and were displayed by acid/urea gel electrophoresis(Fig. 6). As can be seen, both H2B subtypes are present in thetwo mutant strains. This confirms the genetic analysis. Fig. 6also shows that mutants in htbl - (lanes e and f) or htb2- (lanesd and g) are missing the appropriate H2B protein. BecauseH2A1 and H2A2 are not separable by electrophoresis, the ab-sence of only one H2A subtype cannot be corroborated bio-chemically. Fig. 6 shows different relative amounts ofH2B sub-types in the two HTA mutants. This may be due to somepreferential subtype-subtype interaction. Nevertheless, it isclear that a mutation in either HTAI or HTA2 does not resultin the absence of H2B1 or H2B2 from chromatin. Thus, al-though we cannot exclude subtle functional differences be-tween histone subtypes, the genetic and biochemical resultslead us to conclude that there is no exclusive interaction be-tween an H2A subtype and an H2B subtype.

We thank Shane Weber and other members of Dr. L. Isenberg's lab-oratory for suggesting the histone isolation procedure from chromatin.The research was supported by grants from the National Institutes ofHealth and the Cancer Research Coordinating Committee. D. K. wassupported in part by a National Research Service Award-National Can-cer Institute Postdoctoral Fellowship and by National Research ServiceAwardGM 07872. M.C. R. is a recipient ofa University Fellowship fromthe University of California, Los Angeles.

1. Brandt, W. F., Patterson, K. & von Holt, C. (1980) Eur. J.Biochemr. 110, 67-76.

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