H1t Gene Research Articles

Structure and expression of the human gene encoding testicular H1 histone (H1t)

The gene coding for the human H 1t histone, a testis-specific H1 subtype, was isolated from a genomic library using a human somatic H1 gene as a hybridization probe. The corresponding mRNA is not polyadenylated and encodes a 206-amino-acid protein. Sequence analysis and S1 nuclease mapping of the human H1t gene reveals that the 5' flanking region contains several consensus promoter elements, as described for somatic, i.e., S-phase-dependent H1 subtype genes. The 3' region includes the stem-and-loop structure necessary for mRNA processing of most histone mRNAs. Northern blot analysis with RNAs from different human tissues and cell lines revealed that only testicular RNA hybridized with this gene probe.

Structural and functional analysis of the rat testis‐specific histone H1t gene

A 6.86 kb rat genomic DNA fragment containing the testis-specific histone H1t gene and the histone H4t gene has been sequenced. S1-nuclease protection analyses of total cellular RNA from rat liver and testis showed that histone H1t mRNA was present only in testis. Examination of various highly enriched populations of rat testis cell types revealed that H1t mRNA was found exclusively in a fraction enriched in pachytene spermatocytes. When protein, DNA interactions within the proximal promoter region of the histone H1t gene were examined by electrophoretic mobility shift assays, only minor differences were found in mobility shift patterns of the H1t promoter in assays comparing binding of nuclear proteins from pachytene spermatocytes and early spermatids. However, major differences in binding were observed upon comparing nuclear proteins from rat pachytene spermatocytes to liver. Comparison of binding patterns of rat testis, rat hepatoma H4 cells, HeLa cells, and COS-1 cells also revealed dramatic differences. Transcriptional activity of the histone H1t promoter was examined by measuring H1t promoted chloramphenicol acetyltransferase (CAT) mRNA levels in transient expression assays in transfected rat hepatoma H4 cells, HeLa cells, and COS-1 cells. These assays revealed that the histone H1t promoted CAT gene functioned poorly in HeLa cells and COS-1 cells compared to expression with the parent SV40 promoted vector pSV2CAT. The H1t promoted CAT gene apparently did not work at all in transfected rat hepatoma H4 cells, which is consistent with testis germinal cell specific expression of the histone H1t gene.

Comparison of the structural organization and expression of germinal and somatic rat histone H4 genes

A rat somatic histone H4 gene was isolated by screening a rat genomic library using a cloned cell-cycle-regulated human histone H4 gene as a probe. The somatic histone H4 gene was subcloned and the nucleotide sequence was determined. The structural organization and expression of the somatic histone H4 gene and the rat germinal histone H4t gene were compared. Although the predicted amino-acid sequences of the two histones were identical, 49 out of 102 codons differed. The leader sequence of the germinal histone H4t mRNA was 17 bases compared to 40 bases for the somatic histone H4 mRNA, and the 3′ terminal sequence of the germinal histone H4t mRNA was 52 bases compared to 75 bases for the somatic histone H4 mRNA. The germinal histone H4 gene also lacked a consensus purine-rich motif which was present in the 5′ noncoding region of the somatic histone H4 gene. Northern blot analyses and S1-nuclease protection analyses revealed that the germinal histone H4t and H1t genes were expressed during spermatogenesis in rat pachytene spermatocytes, and the somatic histone H4 gene was expressed only in nongerminal rat cells and tissues. The histone H4t gene was also expressed in some other rat cell types. The differences in expression of the histone H4t and H1t genes may reflect differences in transcription, differences in turnover rates of the mRNAs, or a combination of these factors.

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.

Isolation of the gene for the testis-specific H1 histone variant H1t.

H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.