Cell, Vol. 18, 1261-l

Isolation

271, December

1979,

Copyright

0 1979

by MIT

of Yeast Histone Genes H2A and H2B

Lynna Hereford,* Karen Fahrner, John Woolford, Jr.? and Michael Rosbash Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University Waltham, Massachusetts 02254 David B. KabackS Division of Chemistry California Institute of Technology Pasadena, California 91125

Summary Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within th8 haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from on8 another in the yeast genome by a minimum diStanC8 of 35-60 kb. Sequence homology between the two copies iS restricted to the g8n8S for H2A and H2B; the spacer DNA between th8 genes is nonhomologous. In both copies, th8 genes for H2A and H2B ar8 divergently transcribed. In addition, both plasmids cod8 for other nonhistone proteins. Sequences coding for hiStOneS H3 and H4 have not been detected in the immediate vicinity of th8 genes for H2A and H2B. Introduction Characterization of histone genes from a variety of metazoans has led to the generalization that these genes constitute a tandemly repeated gene family. For example, in Drosophila they are approximately 100 fold (Lifton et al., 1977) and in sea urchins 200300 fold (Kedes and Birnstiel, 1971) repeated. If only these two organisms are considered, it might be assumed that repetition frequency is correlated with genome size. This simple conclusion does not appear to be the case, since organisms with much larger genomes than sea urchins, such as chickens or Xenopus, have a much lower histone gene repetition frequency-10 (Crawford et al., 1979) and 20-50 (Jacob, Malachuski and Birnstiel, 1976), respectively. It is probable, although unproven, that the number of histone genes is related to a variety of biological parameters, such as the need for differing amounts of histones or histone mRNAs during early cleavage l To whom correspondence should be addressed. t Present address: Carnegie-Mellon University, Mellon Institute. 4400 Fifth Avenue. Pittsburgh, Pennsylvania 15213. $ Present address: Department of Microbiology, CMDNJ-New Jersey Medical School, 100 Bergen Street, Newark, New Jersey 07103.

divisions, the maximal rate or rates of cell division and/or the preferential synthesis of subsets of the gene family. The nature of the repeating unit has been best characterized in sea urchins (Cohn, Lowry and Kedes, 1976; Schaffner et al., 1976; Wu et al, 1976) and Drosophila (Lifton et al., 19771, where the genes have been cloned. Each repeating unit is comprised of one copy of each of the five histone genes. A similar organization has been reported for other organisms (Freigan, Marchionni and Kedes, 1976; Crawford et al, 1979); it is therefore probable that this basic repeating unit is a common feature of metazoan histone gene organization. Significant differences between Drosophila and sea urchin histone gene organization do, however, exist. These differences include the order of the genes within the repeat, the direction of transcription of the genes and the lengths of the spacer DNAs separating them. The significance of these differences remains to be determined. Although there is a substantial amount of information on metazoan histone genes, nothing is yet known about the organization of histone genes in lower eucaryotes. The organization of histone genes in these organisms is of interest not only for evolutionary considerations, but also for an understanding of the significance of the repetition frequency of the histone genes. For example, if histone gene repetition frequency is a direct function of the rate of cell divisionor, more precisely, of the length of the S period-it might be asked what would be the minimum number of histone genes necessary to support DNA replication. A simple way of looking at this problem is to compare mammalian cells and yeast. L cells have an S phase approximately 20 times longer than yeast [8 hr (Perry and Kelley, 1973) versus 25 min (Hartwell, 197411 and a genome approximately 200 fold larger. L cells must therefore synthesize 10 times as many histones per cell per unit time as yeast. The repetition frequency of histone genes in human cells is approximately 30-40 (Wilson and Melli, 1977). Therefore, assuming that histone gene transcription and translation are roughly equivalent in the two organisms, histone genes need not be very repeated in yeast. Indeed, as we show in this communication, this is close to the actual situation. Isolation of clones for yeast histone genes H2A and H2B reveals that there are only two copies of these genes within the haploid yeast genome. Moreover, these copies are not tandem repeats; rather, they are separated from one another by a minimum of 35-60 kb. In addition, we also show first, that the homology between the two copies is restricted to the genes for H2A and H2B; second, that these genes are divergently transcribed; and third, that each copy codes for additional nonhistone proteins but not histones H3 and H4.

Cell 1262

Results Identification of Histone Plasmids We have previously shown that yeast histone mRNA is almost exclusively polyadenylated (L. Hereford and K. Fahrner, unpublished observations). In addition, we found that the small poly(A) RNA fraction used to identify ribosomal protein-containing plasmids (see accompanying paper, Woolford, Hereford and ROSbash, 1979) was also enriched for histone mRNA. Figure 1 shows the in vitro translation products of this mRNA fraction, separated on the 20 gel system originally described by Spiker, Key and Wakum (1976) and used by Mardian and lsenberg (1976) for the purification of yeast histones. In Figure 1 A are shown purified yeast histones which were supplied by D. Lohr, and in Figure 1 B, the in vitro translation products of the enriched histone mRNA fraction. As can be seen, all four histones are clearly visible by in vitro translation. From the 100 recombinant clones described in the accompanying paper which hybridized strongly to enriched poly(A) mRNA, three plasmids (TRTl, TRT2, TRT3) containing the histone genes H2A and H2B were found. No clones were found which contained the genes for histones H3 and H4. These results will be dealt with in more detail below (see Discussion). Plasmids containing the histone genes H2A and H2B were identified by in vitro translation of mRNAs purified by R loop hybridization to plasmid DNA (Woolford and Rosbash, 1979). When these translation products were analyzed on the 2D gel system described in

Figure 1, all three plasmids were shown to code for the histones H2A and H2B (Figure 2). No in vitro translation products corresponding to either histones H3 and/or H4 were seen, although one of the plasmids, TRT3, codes for an additional basic protein (indicated by arrow). Because this gel system resolves well only basic proteins, the in vitro translation products were also analyzed on 15% Laemmli gels (Laemmli, 1971). These data, presented in Figure 3, are consistent with the two-dimensional gel analysisthat is, all three plasmids are complementary to mRNAs for histones H2A and H2B. TRTl codes for a third protein (protein 1) which has an apparent molecular weight of 30,000 daltons. TRT3 also codes for an additional protein (protein 2) which is not well resolved in this particular experiment, but is more apparent in Figure 6. The molecular weight of this protein is 6000 daltons. We have not determined whether the minor basic protein coded for by TRT3 (Figure 2) is identical to protein 2 or whether they are coded for by two separate genes, both of which are present on TRT3. Restricted Homology of TRTl , TRTP and TRT3 Since the three plasmids which we had isolated were constructed from randomly sheared DNA (Petes et al., 19761, we initially presumed that they contained overlapping DNA sequences. This presumption was only partially correct. Restriction mapping of the plasmids with a variety of enzymes indicated that while TRTP and TRT3 do contain overlapping sequences, TRTl does not contain restriction sites which overlap with

Acid Urea fT.c E 3

Figure

1. In Vitro Translation

(A) HiStOne (1976).

markers;

of Histone

mRNA

(B) in vitro translation

products

of 7-l

OS poly(A)’

RNA run on the 20 gel system

described

by Spiker,

Key and Wakum

Yeast Histone 1263

Acid

Figure

Genes

Urea

2. Identification

The R looped in vitro products of pMBg.

of Histone-Containing translation

products

Plasmids of TFtTl , 2 and 3 run on the same gel system

those in either TRT2 or TRT3 (Figure 4). For convenience, we have given all Hind III fragments the alphabetical designations indicated in Figure 4, and we will refer to them here and in subsequent experiments by these designations. Both TRT2 and TRT3 contain the F’ fragment. TRT3 has an A:T join (A:T sequences used to link eucaryotic DNA to plasmid DNA) in the E’ fragment and TRT2 has an A:T join in the C’ fragment, as determined both by DNA blotting (Southern, 1975) and by electron microscopy (data not shown). The restriction map of TRTl , however, is distinct from that of either TRT2 or TRT3. Since both TRTl and TRT2,3 contain the genes for H2A and H2B, they should exhibit sequence homology despite the lack of identical restriction sites. Indeed, by DNA blotting, homologous regions exist and are within the E, F and G Hind Ill fragments of TRTl and the E’, F’ and G’ fragments of TRT2,3 (data not shown). To further define the regions of homology, heteroduplexes were formed and analyzed by electron

as shown

in Figure

1. The control

gel is the translation

microscopy. Typical heteroduplexes between TRTI and TRT3 are shown in Figures 5b and 5c and are summarized in 5a. Only two regions of homology are visible, and these regions lie within the homologous Hind Ill fragments (that is, fragments E, F and G for TRTI and E’, F’ and G’ for TRT2,3). The two regions are approximately 470 and 380 bp in length and, as we shall demonstrate, correspond to the genes for H2A and H2B. respectively. Heteroduplexes between TRTl and TRTP give comparable results (data not shown); only two regions of homology are observed. The only difference in this case is that the homologous region corresponding to the H2B gene is only 200 bp long due to the fact that TRT2 has an A:T join in the middle of this sequence. Order of Genes To order the genes within the plasmids we analyzed the in vitro translation products of mRNA complementary to each of the Hind Ill fragments subcloned from

Cell 1264

TRT-I

A

BCD

E

F

G

H

I1 1 E’ I, I II E’ F’ G’ H’

I’

TRT-2 I A’

8’

C’

D’

TRT-3

I

_/H2B ‘H2A

Prot 2-

Figure

3. Additional

Proteins

Coded

for by Histone

Plasmids

15% Laemmli gels of the R looped in vitro translation products of TRTl , 2 and 3. Also shown are the endogenous synthesis (END01 and in vitro translation of total yeast RNA (TOTAL).

TRT1,2,3. These data are presented in Figure 6. The TRTl E fragment codes for H2A, F codes for H2A and H2B and G codes for H2B. In addition, the D and E fragments both code for protein 1. The gene order must therefore be protein 1, H2A, H2B. For TRT2 and TRT3, E’ codes for H2A, F’ codes for H2A and H2B and G’ codes for H2A and protein 2. The order of the genes in TRTP and TRT3 must therefore be H2A, H2B, protein 2. Although these experiments unambiguously order the genes, they also reveal ambiguities. The first is that the D’ fragment of TRT2,3 appears to code for H2A. Since this fragment does not crosshybridize with TRTl (data not shown), the two copies of the H2A gene may contain regions of nonhomology. The second ambiguity arises from the presence of what appears to be an additional protein coded for by both the E and E’ fragments. We have found no R loops corresponding to this protein (see below). We do not know whether this putative R loop was not detected or whether this protein is the result of an in vitro translation artifact. To determine accurately the position and structure of the H2A and H2B genes, R loops formed by hybridizing total poly(A)+ RNA to each of the three plasmids were analyzed by electron microscopy (Kaback, Angerer and Davidson, 1979). Sal l-linearized TRTl DNA contained two R loops present at high concentrations.

Figure

4. Restriction

Maps

J’

IkB

BAM RI SST PST HIND

--rot

1

I

of TRTI , TRTP and TRT3

The positions and sizes of these two R loops, within the limits of error, appeared to be symmetrically arranged around a central axis, which prevented proper orientation with relation to the restriction site (data not shown). Plasmid TRTl was therefore cut with Barn HI, which generates three fragments (Figure 41, and R loops formed with this mixture of fragments (Figure 7). All the R loops were present on the 6.34 kb fragment. The two R loops corresponding to sequences complementary to H2A and H2B mRNAs were present on 94% and 73% of the molecules observed, respectively. The R loops were separated by 800 + 100 bp of DNA. To determine the direction of transcription of the genes, we took advantage of the fact that yeast histone mRNA is polyadenylated (L. Hereford and K. Fahrner, unpublished observations). The free 3’ poly(A) end of the RNA in the R loops could therefore be identified by hybridization to a polyBr(dU) tail polymerized on a microcolicin El plasmid DNA (Bender et al., 1978). The direction of transcription as determined by this method was divergent (Figures 7b and 7~). An additional R loop was observed in 8% of the molecules. Presumably, this R loop is formed with a less abundant RNA. The Y-3 orientation of this R loop was also determined (Figure 7d). The data summarized in Figure 7a show the positions of the R loops compared to the Hind Ill map. The two major R loops are present within the H2A and H2B coding regions (Figures 6A and 7a) and therefore identify the positions of the two genes. The position of the third minor (that is, frequency) R loop lies within the region (Hind’ Ill fragments D and E) which codes for protein 1 (Figure 6A). The positions of the H2A and H2B R loops correspond exactly (k 100 bp) to that predicted by the heteroduplex analysis presented above. Barn HI-linearized TRT3 also contains two major (high frequency) R loops. The two R loops, corresponding to sequences complementary to H2A and H2B mRNAs, were present on 100% and 51% of the observed molecules, respectively (Figure 8). AB with TRTl, the coding assignments were determined by comparing the relative positions of the genes to the

Yeast Histone 1265

Genes

a.

HET

\

Figure

5. Heteroduplexes

between

TRTB and TRTl

Sequences

(a) Schematic diagram showing results of heteroduplex analysis, Vertical arrows denote the Barn HI sites. A:T join positions and pMBQ vector (hatch marks) are also labeled. Lengths are results of measurements made on 16 heteroduplex molecules. (b) Purified 6.34 kb Barn HI fragment of TRTl annealed to Barn HI-linearized TRT3 and spread from 50% formamide where AT linkers are annealed. (c) As in (b), except spreading was from 70% formamide. where the A:T joins are single-stranded. The 6.34 kb Barn HI fragment of TRTI was used because TRTB is cloned in inverse orientation relative to TRTl

mapped and subcloned Hind III fragments, and they are summarized in Figure 8a. As in TRTl, the sequences complementary to H2A and H26 mRNAs are both approximately 500 nucleotides long, separated by 670 -+ 70 nucleotides and divergently transcribed (Figures 8a-8d). In addition to sequences complementary to H2A and H2B mRNA, TRT3 contains three other R loops present at much lower frequencies. These R loops denote the positions of other, presumably much less abundant mRNAs (for example, Figures 8e and 8f). One of the transcription products, present in 11% of the molecules, maps very close to the 3’ end of the H2B gene and appears to be transcribed from the opposite strand (Figure 8f). The DNA sequence between the two genes appears to be very short, leading to two distinct R loops joined by an unmeasurably short duplex in about 50% of the molecules (data not shown). The other 50% contains a single loop structure with DNA:RNA hybrids on opposing sides of the loop (Figure 8f). The two coding sequences are probably separated by fewer than 100 bp, and may indeed be much closer. This R loop almost certainly corresponds to the third in vitro translation product, protein

2, coded for by TRT3 (compare Figures 8a and 6b). The other additional R loops, 3 and 4, were present at much lower frequencies, approximately 2% and 4%, respectively, of the observed molecules. Presumably because of this low concentration, we have not been able to detect them by in vitro translation. R loops were also formed with TRT2 (data not shown). The R loop map of TRT2 is consistent with that of TRT3 and is summarized in Figure 8a. In agreement with the heteroduplex analysis, the H2B R loop of TRT2 is small, due to the presence of the A:T join in the middle of the H2B gene. No additional R loops were found on TRT2. Given the number of molecules examined (37) R loops at a frequency of lo%, comparable to those corresponding to proteins 1 and 2, would have been detected. Arrangement of Histone Genes in the Yeast Genome Since TRTl and TRT2 share limited homology, the question arose as to whether their sequences were contiguous within the yeast genome-that is, whether they were organized in a tandem repeating unit. To answer this question, we analyzed a series of Southern blots (Southern, 1975) in which haploid genomic

Cell 1266

A.

TRTI

8. TRT2.3 A’ B’ C’

-Protein

0’

E’ F’ G’

H’

I’ J’

I

,H28 ‘H2A Fin Figure

6. Order

15% Laemmli

of Genes

within Plasmids

gels of Hind III subcloned

DNAs R looped

Q

and translated

H2A

H,

A I 0

,Bj,D+ I 2

E

+

I 4

7. R Loop Map of TRTl

(ENW-endogenous

H2B m uF i 1 6

kb

Figure

in vitro. (A) TRTI ; (B) TRT2,3.

synthesis).

TRT I G

+

t B

’ B

H 1 IO

1 12S

f

B

DNA

R loops, formed by hybridizing total poly(A)+ RNA to Barn HI-cut TRTl , were formed and analyzed as described in Experimental Procedures. RNA is denoted by the dashed lines. R loops seen at high frequency, presumably due to relatively abundant RNA species, are denoted by heavy dashed lines. (a) Schematic summary of results. Hind Ill sites are indicated by arrows above the line and are given the same alphabetical designations as R in Figure 4. Barn HI (B) and Sal I (S) sites are indicated by arrows below the line. Direction of transcription is 5 ’ + 3’. (b) Molecules containing loops from H2A and H2B RNA. The free poly(A) tail from the H2A RNA is hybridized to a polyBr(dWtailed microcolicin El plasmid. (c) Same as (b) but the H2B RNA is hybridized to the 3’ end label. (d) Microcolicin El plasmid with two tails hybridized to minor R loop 1 on one molecule and an H2A R loop on a second molecule allowing determination of the direction of transcription of R loop 1.

DNA, digested with various restriction enzymes, was hybridized with each of the three histone clones, to determine whether each of the plasmids hybridizes to a unique set of restriction fragments or whether they share any restriction fragments in common. The latter

result would be evidence in favor of contiguous or tandem array of the genes. TRTP and TRT3 contain a region of genomic DNA which has three Eco RI sites; they should therefore hybridize to four genomic RI fragments, one of which

Yeast Histone 1267

Genes

a H2A H2B TRT3 n 1*11

TRT2 + A’ + I

0

I

B’

2

+

I

4

C’ +D$ E’ + F’ + G’+ H’ + I I

6

8

I

IO

I’

I

12

i I

J

14

kb

Figure

8. R Loop Maps of TRTP and TRT3

DNA

R loops were formed and analyzed as described in Experimental Procedures. (a) Schematic summary of results, as in Figure 7a. (b) Molecule containing R loops from H2A and H2B RNA. The free p(A) tail from the H2A RNA is hybridized to a polyBr(dU)-tailed microcolicin El plasmid. (c) Same as (a) but the H26 RNA is hybridized to the 3’ end label. (d) 3’ poly(A) ends of both H2A and H2B RNA molecules are hybridized to a single Col Et label, (e, f) Molecules containing R loops seen at lower frequencies, in addition to the H2A and HZB R loops. The insert in (f) interprets the fine structure of the R loops with sequences complementary to the H2B gene and gene 2. As described in the text, R loops were also formed and analyzed between p(A) RNA and Barn HI-cleaved TRTP. and the data are included in the summary in (a).

is in common (Figure 4). The genomic DNA contained in TRTl has no Eco RI sites and should, therefore, hybridize to a single genomic RI fragment. If TRT2,3

were closely linked to TRTi in the genome, they would share one genomic RI fragment-that is, the single RI fragment homologous to TRTl would be identical to

Cell 1268

one of the terminal RI fragments (a fragment interrupted by the A:T joins) of TRT2,3. The hybridization patterns of TRT1,2 and 3 to Eco RI-digested genomic DNA are shown in Figure 9. TRTP and TRT3 hybridize to four Eco RI restriction fragments and contain, as predicted, one Eco RI fragment in common. TRTl hybridizes to a single fragment unique from the four fragments which define TRT2 and TRT3. Within the limits defined by the size of Eco RI fragments, therefore, the two sets of histone genes are not linked. It should also be noted that the plasmids cross-hybridize (dotted lines) as predicted. Both TRT2 and TRT3 cross-hybridize to the single TRTI Eco RI fragment, while TRTl cross-hybridizes to the Eco RI fragment common to both TRT2 and TRT3. Based on the data presented above, it is known that this fragment contains the histone genes. We have used the above approach with four restriction enzymes, singly or in combination, to generate the genomic restriction maps summarized in Figure 10. The major conclusion of this analysis is that TRTl and TRT3 are not contiguous in the genome. Our estimate of the minimum distances separating them is between 35 and 60 kb. Repetition of Histone Genes Although TRTl and TRT2,3 are not contiguous, the possibility exists that either or both of them could be tandemly repeated. Alternatively, TRTl and TRT2,3 are the only two copies of the histone genes H2A and H2B in the yeast genome. Two lines of evidence argue that the latter alternative is the correct one. The first comes from the data summarized in Figure 10 and is based on the minimum size of such a putative tandem repeat unit. If we consider TRTl, we know from the work presented above that there is a single copy of the H2A and H2B genes within the 6.34 kb fragment. When TRTl is hybridized to Barn HI-cut genomic DNA, there is hybridization to three fragments-the expected 6.34 kb fragment, and two additional frag-

13KB

ments of approximately 40 and 17 kb (Figure 10). Since neither of these two fragments cross-hybridizes with TRT2 or TRT3, they do not contain additional copies of the H2A or H2B genes and therefore must represent flanking sequences. The question then is whether these are tandem copies of the 6.34 kb Barn HI fragment between these flanking sequences. This is not the case, since the 6.34 kb fragment is flanked by Eco RI and Pst I sites which cut in both the 17 kb and 40 kb Barn HI fragments. Since the 6.34 fragment is not itself tandemly repeated, therefore, the minimum repeat size must be the sum of the three Barn HI fragments, or approximately 64 kb. Since this size is approximately an order of magnitude greater than the histone repeat size of either Drosophila (Lifton et al., 1977) or sea urchin (Cohn et al., 1976; Schaffner et al., 1976) it is improbable that TRTl is tandemly repeated. Similar reasoning can be applied to TRT2,3. If TRT2,3 is repeated, then the minimum repeat size is 47 kb or the maximal genomic lengths we have determined. Again, this large size argues against the possibility that TRT2,3 is tandemly duplicated. The second line of evidence in support of the conclusion that TRTl and TRT2,3 are present as singlecopy genomic sequences comes from an estimation of the copy number of these genes. A typical experiment is shown in Figure 11, in which nick-translated TRTl has been hybridized to Sst l-cut genomic DNA and varying genomic equivalents of Sst l-cut TRTl . The two Sst I fragments (indicated by arrows) which flank the genes for H2A and H2B give a signal with genomic DNA which is most comparable to one copy of plasmid DNA per haploid genome. Similar experiments have been performed with TRT2 with the same result (data not shown). Discussion The histone genes which we have isolated from display a variety of features which are novel compared to the histone genes of sea urchins et al., 1976; Schaffner et al., 1976; Wu et al.,

-IIKB

TRT-I

H2AHZB

-----

3.5 KB

-2.75 -2.1

TRT3 Figure 9. Genomic RI TRTl , TRTP and TRT3

KB KB

TRT2 Restriction

lOLB

TRT I Fragments

Complementary

to

Figure

10. Genomic

The vertical

Restriction

lines delineate

I

Maps of TRTl

and TRT2,3

the ends of the cloned

sequences.

yeast when (Cohn 1976)

Yeast Histone 1269

Genes

E

TRT3, TRT2 I TRTII

0 c

I

z

0

3 6 IO

Figure

11,

Quantitation of experiment of plasmid.

of TRTl see text.

Copy

Number

1, 3.6,

10 are numbers

of genome

or Drosophila (Lifton et al., 1977). Many of these features derive from the structural organization of the genes, which is summarized in Figure 12. As in Drosophila, H2A and H2B are adjacent to one another and are divergently transcribed. Unlike both Drosophila and sea urchins, however, the spacer DNA is not conserved. Also of note is the presence of genes other than t-l3 and H4 within close proximity to H2A and H2B. This is likely to be a consequence of the lack of tandem repetition of the histone genes and the high proportion of the yeast genome [40% (Hereford and Rosbash, 1977; Kaback et al., 1979)] which is transcribed. Since, on average, every 2-3 kb of yeast DNA should contain an mRNA complementary region, these other genes may simply code for random proteins. Alternatively, they may be proteins with functions related to or coordinately controlled with histones. Although we have not obtained clones which code for H3 and H4, we can set some limit on the minimum distance which must separate them for H2A and H2B. In the case of TRT2,3 there is approximately 5 kb on either side of H2A and H2B which does not contain the genes for H3 and H4 (Figure 12). In the case of TRTl (Figure 12) there is approximately 5 kb of DNA which does not contain H3 and H4 on one side, and approximately 0.70 kb on the other side. We have recently obtained additional cloned sequences which have allowed us to examine 5 kb beyond this 0.70 kb region, and the genes for H3 and H4 are still absent

4

6

8

IO

12

kb Figure 5’ +

For details equivalents

2

12.

Sequence

3’. direction

Organization

of Histone

Genes

H2A and H2B

of transcription.

(L. Hereford and K. Fahrner, unpublished observations). We therefore conclude that, in the case of both TRTl and TRT2,3, the genes for H3 and H4 are not closely linked to the genes for H2A and H2B. The most striking finding of this analysis is that there are only two copies of the genes for H2A and H2B within the haploid yeast genome and that these copies are widely separated from one another. Three lines of evidence support the conclusion that there are only two copies. First, reconstruction experiments argue that both TRTI and TRT2,3 are single-copy DNA sequences (Figure 11). Second, we have never observed hybridization to genomic restriction fragments other than those predicted by the restriction maps of the plasmids and the cross-hybridization of the plasmids with one another. There is therefore no evidence for additional dispersed copies of these genes within the genome. The third line of evidence derives from the minimum length of the repeating unit. If either TRTl or TRT2,3 were tandemly repeated, the minimum repeat sizes would be 64 kb and 47 kb, respectively. Since these sizes are an order of magnitude greater than that of the histone gene repeats of either sea urchin or Drosophila, they argue, albeit corrobaratively, that neither TRTl or TRT2,3 is tandemly duplicated. These three lines of evidence, taken together, strongly suggest that there are only two copies of histone genes H2A and H2B in the yeast genome. The conclusion that the two copies are widely separated from one another derives from the nonoverlapping genomic maps of the two plasmids. These distances (35-60 kb) are necessarily a lower limit. Using a genetic approach, we have confirmed this initial conclusion and extended our estimates of the minimum distances which separate the two gene copies. Two haploid yeast strains have been found with a different genomic restriction site for both TRTI and TRT2,3. Construction of a diploid from these two strains and analysis of the restriction patterns in meiotic segregants reveals that TRTl and TRT2,3 segregate independently from one another (L. Hereford and K. Fahrner, unpublished data). These data support the idea that the two copies of the histone genes are very far apart [a minimum of 100-200 kb (Byers and Goetsch, 1975)] and may indeed be on separate chromosomes. Since there are only two copies of the genes for H2A and H2B in the yeast genome, it is of interest to determine whether both copies are transcriptionally

Cell 1270

active. It is known that in the sea urchin different histone mRNAs are synthesized (Newrock et al., 1977). This differential synthesis has been correlated with different stages of development, although, strictly speaking, the dependency of the two phenomena has not been proven. Yeast, in contrast to sea urchins (or anything else, for that matter), has a very limited repertoire of developmental options. It does, however, exhibit two distinct phases in its life cycle, mitosis and meiosis (sporulation). One tantalizing possibility is that there is differential expression of the two copies of H2A and H2B during these two phases. Alternatively, the two copies of H2A and H2B could simply be the result of gene duplication. Gene duplication in yeast is not unprecedented. Yeast has two cytochrome C genes, ISO-I -cytochrome C and ISO-2-cytochrome C (Sherman et al., 1978). The genes are on different chromosomes and most (95%) of the cytochrome C gene is produced by the ISO- -cytochrome C gene. Interestingly, one of the ribosomal protein genes is also duplicated in the yeast genome (Woolford et al., 1979). If gene duplication is responsible for the two H2A and H2B gene clusters, then it will be important to determine whether both copies are expressed and, if so, whether the expression is disproportionate. The importance of determining the level of expression of each histone gene set derives from the fact that either disproportionate or differential synthesis would make the yeast histone genes H2A and H2B effectively single-copy genes. As such, they would provide a system in which histone genes can be analyzed genetically, a situation which is almost certainly impossible in metazoans. Either conditional mutants within the genes themselves or regulatory mutants would provide invaluable tools with which to study a variety of phenomena associated with histones, such as their coordinate synthesis vis a vis one another and DNA replication, as well as the histone assembly process. Experimental

Procedures

Yeast Strains The yeast strains were 2622 (a, adei, his.5 lysl 1, leu2, ural gall) and A364A ((1. adel, ade2. Ural, his7. try1 , lys2). Cells were grown in modified YM-1 as previously described (Hartwell. 1967). Yeast RNA rind DNA Isolation Total poly(A)+RNA and polysomal poly(A)+ RNA were isolated from strain A364A as previously described (Hereford and Rosbash. 1977). DNA was isolated as follows. Cells were incubated in 0.2 M Tris. 5% P-mercaptoethanol for 30 min at room temperature. The cells were spun down and resuspended in sorbitol, 0.04 M KPOl (pti 6.8). Zymolase 5000 (Kirin Brewery Co.) was added to a final concentration of 2.5 mg/ml wet weight cells, and the cells were incubated at 30°C for 30 min. The spheroplasts were spun down and then lysed by incubation at 60°C for 30 min in lysing buffer [0.2 M NaCI. 0.1 M EDTA. 5% SDS 50 mM Tris (pH 8.5) 10 Kg/ml pronase]. Nucleic acid was extracted twice with T.E. [IO mM Tris. 1 mM EDTA (pH 6.3)] -saturated phenol/chloroform (1 :l). DNA was precipitated by the addition of 2 vol of 95% ethanol, removed with forceps, immersed

twice in 70% ethanol, dried, then redissolved in T.E. The DNA was then incubated for 30 min at 37°C with 100 y/ml ribonuclease. followed by incubation for the same time and at the same temperature with 10 gg/ml pronase. The extraction and precipitation steps described above were repeated. The DNA was resuspended and stored in T.E. at a concentration of 1 mg/ml. Plasmid Identification The plasmid bank was that constructed by Petes et al. (1978). CotonY screening has been described by Woolford et al. (1979). The R looping procedure (Woolford and Flosbash. 19791, as wett as the tn vitro translation and gel systems used, have also been described (Woolford et al., 1979). Characterization of Plasmid DNA8 The conditions used for restriction enzymes were those described in the New England Biolabs catalogue. Southern blots were done according to published procedures (Southern, 1975) in 8 X SSC at 58°C. DNA was labeled by the “nick translation” procedure of Maniatis. Jeffrey and Kleid (1975). R Loop Formation for Electron Microrcopy Plasmid DNA preparations were cross-linked once per 2-5 kb by adding trioxsalen (I Ag/ml for 50 &ml DNA) and irradiating with long wavelength ultraviolet light (2.5 min at 45 ergs mm-’ set-‘1 as previously described (Kaback et al., 1979). The crosslinked DNA (10 pa/ml) was hybridized to total poly(A)+ or polysomal poly(A)+ RNA (100 pa/ml) in 70% formamide, 0.1 M PIPES (pH 7.2), 0.01 M EDTA. 0.4 M NaCl at 53°C for 24 hr. as described. At the RNA concentration used, abundant or moderately abundant species of RNA should form R loops at a high frequency. The R loops were stabilized by treatment with 1 M glyoxal for 2 hr at 12°C (Kaback et al., 1979) and the mixture was dialyzed and passed over a Sepharose 28 column at 4’C. This procedure separates the unhybridized RNA from the R loop-containing DNA, providing fields in the electron microscope with little contaminating free RNA, and permits the labeling of the free 3’ poly(A) tails in the R loops with a polyBr(dU) electron microscope label. R loop containing DNA was prepared and spread for examination in the electron microscope as described by Davis, Simon and Davidson (1971). Heteroduplex Formation Heteroduplex formation was carried out as previously described (Davis et al.. 1979) at 42OC in 50% formamide. which prevents selfannealing of the A:T linkers in the plasmid DNA. This is necessary to prevent formation of underwound structures due to winding constraints imposed by the inverted repeat A:T structures (Broker, Soll and Chow, 1977). Spreading was done from 50% formamide. or where necessary from 70% formamide, which eliminated any duplexes in the A:T linker regions, often simplifying the heteroduplex structure. 6X 174 single-stranded viral or double-stranded RFII DNAs (5386 bp) (Sanger et al., 1977) were used as size standards in all microscopy experiments. The 6.34 kb Barn HI TRTl restriction fragment was purified from 0.7% agarose gels as described by Tabak and Flavell(l978). Acknowledgments We would like to thank Dr. Norman Davidson for his advice and encouragement and Lynn Golden for helpful discussions. This work was supported by three National Institute of General Medical Sciences Public Health Service grants. L.H. was also supported by a New England Medical Foundation Fellowship. D.K. and J.W. were supported by NIH Postdoctoral Fellowships. M.R. is a recipient of an NIH Research Career Development Award. This is contribution No. 6091 from the Department of Chemistry, California Institute of Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

September

4, 1979

Yeast 1271

Histone

Genes

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