Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the 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 one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

Histone gene organization of fission yeast: a common upstream sequence.

Histone genes of the fission yeast Schizosaccharomyces pombe were cloned from Charon 4A and cosmid gene libraries by hybridization, and their nucleotide sequences were determined. The genome of S. pombe has a single, isolated H2A, a pair of H2A-H2B and three pairs of H3-H4 (one H2B, two H2A and three each of H3 and H4). This non-assorted histone gene organization is distinct from that of the budding yeast which has two pairs of H2A-H2B and H3-H4. The predicted amino acid sequences of S. pombe histone H2As, H3s and H4s were identical except for three residue changes in H2As. Compared with those os S. cerevisiae and human, variable residues were clustered near the NH2- and COOH-terminal regions of H2A and H2B. Sequence homologies to the two organisms were roughly the same in H2A (79-83%), H3 (92-93%) and H4 (91%), but differed in H2B (82% to S. cerevisiae and 68% to human). The coding sequences in pairs of S. pombe histone genes were divergently directed. A 17-bp long highly homologous sequence (AACCCT box) that had internal 6-bp direct repeats was present in the intergene spacer sequences or in the 5' upstream region of all the cloned histone genes. A possible regulatory role of the common upstream sequence for histone gene expression is discussed.     

Sequence comparisons of non-allelic late histone genes and their early stage counterparts. Evidence for gene conversion within the sea urchin late stage gene family

We have determined the nucleotide sequence of sea urchin (Lytechinus pictus) late stage H3 and H4 histone genes contained on the clone pLpH3H4 -21 and of the early stage H3 gene contained on the plasmid pLpA . Comparison of these differentially regulated histone genes with each other and with other L. pictus late and early stage histone H3 and H4 genes previously sequenced confirms that members of each histone gene family (early and late) are more homologous to each other than they are to members of other histone gene families. The spacer regions between two late H3-H4 gene pairs on the clones pLpH3H4 -19 and pLpH3H4 -21 have diverged to the point where they are no longer homologous. However, comparative analysis of the 5' flanking DNA has identified a sequence 5'C-T-C-A-T-G-T-A-T-T3' upstream of both late H4 genes and another, 5'A-G-A-T-T-C-A3', upstream of both H3 genes. Except for a short conserved sequence near the initiation codon, the transcribed 5' leaders of the late mRNAs differ in length and sequence in the two non-allelic late histone gene pairs. This divergence contrasts with the 95 to 96% conservation found between late histone gene coding sequences. The results suggest that there is intergenic exchange in the germline among members of the late histone gene family and that the unit of exchange is the individual gene rather than the heterotypic dimer which includes the common spacer DNA.     

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.     

Histone genes of the sea urchin (S. purpuratus) cloned in E. coli: Order,polarity, and strandedness of the five histone-coding and spacer regions

Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I, Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions.Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNAs have little sequence homology to other genes. Exonuclease III digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5′-3′ polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of the S. purpuratus histone gene repeat unit:

     

A rat histone H4 gene closely associated with the testis-specific H1t gene

A rat histone H4 gene closely associated with the testis-specific H1t gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the H1t gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5'-consensus sequence elements is unique for an H4 gene. S1-nuclease protection analyses reveal that both the H4 and H1t mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Ag1.2.3). During a 1-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis.     

Molecular cloning and nucleotide sequence of a variant wheat histone H4 gene

To determine whether there is structural variation among histone H4 genes in wheat, one (TH091) of the H4 genes that had been cloned from a wheat genomic DNA library was sequenced and compared with another H4 gene (TH011) which we had described previously [Tabata et al., Nucl. Acids Res. 11 (1983) 5865-5865]. Nucleotide sequence analysis revealed that there are 17 nucleotide replacements in the protein-coding region of two H4 genes, causing only one amino acid substitution: a glycine at position 4 (from the N terminus) in TH011 was replaced by an aspartic acid in TH091. S1 mapping, using total nuclear RNA from germinated seeds, indicated that the H4 gene was transcribed in vivo.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

Variations in the organization of human genomic DNA segments containing H1 histone genes

We have isolated from a lambda Ch4A library four human genomic DNA segments containing H1 histone genes. Analysis of the representation and organization of histone coding sequences indicates that three of these cloned DNA segments contain both core and H1 histone genes. One of the cloned human H1 histone genes has no core histone genes in close proximity.     

Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.     

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electron microscopy

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pSp2 and pSp17 have been mapped by hybridizing total histone messenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2–B3, where B4, B1, and B2–3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.     

Differential expression of two clusters of mouse histone genes

The mouse histone mRNAs coded for by three different cloned DNA fragments have been characterized. Two of these cloned DNA fragments, MM221 and MM291, located on chromosome 13, code for H3, H2b and H2a histone mRNAs, which are expressed at low levels in cultured mouse cells and fetal mice. The other DNA fragment, MM614, located on chromosome 3, codes for an H3 and an H2a mRNA, which are expressed at high levels in these cells. The mRNAs for each histone protein share common coding region sequences, while the untranslated regions of all the genes have diverged significantly, as judged by S1 nuclease mapping. Amino acid substitutions in some H3, H2a and H2b proteins are detected as internal cleavages in the S1 nuclease maps. All of these genes code for replication variant histone mRNAs, which are regulated in parallel with DNA synthesis.     

An unusual evolutionary behaviour of a sea urchin histone gene cluster

DNA sequences of cloned histone coding sequences and spacers of sea urchin species that diverged long ago in evolution were compared. The highly repeated H4 and H3 genes active during early embryogenesis had evolved (in their silent sites) at a rate (0.5-0.6% base changes/Myr) similar to single-copy protein-coding genes and nearly as fast as spacer DNA (0.7% base changes/Myr) and unique DNA. Thus, evolution in the major histone genes conforms to a universal evolutionary clock based on the rate of base sequence change. By contrast, the H4 and H3 coding sequences and a non-transcribed spacer of the DNA clone h19 of Psammechinus miliaris show an exceptionally low rate of sequence evolution only 1/100 to 1/200 that predicted from the clock hypothesis. According to the classical model of gene inheritance, the h19 DNA sequences in the Psammechinus genome require unusual conservation mechanisms by selection at the level of the gene and spacer sequences. An alternative explanation could be recent horizontal gene transfer of a histone gene cluster from the very distantly related Strongylocentrotus dröbachiensis to the P. miliaris genome.