Evolution and sequence analysis of a human Y-chromosomal DNA fragment.

A Y-chromosomal DNA fragment has been isolated from a human Y-Charon 21A recombinant library. Evolutionary analysis of 1F5 indicates that the size and sequence of this fragment have been conserved in higher primates. Deletion mapping and in situ hybridization analysis have localized 1F5 to the middle euchromatic portion of the long arm of the human Y chromosome at Yq11.2. Sequence analysis revealed the presence of an atypical Alu element and two regions rich in polypyrimidine-polypurine residues.

Immunocytochemical localization of an H2A variant in the spermatogenic cells of the mouse.

Immunocytochemical localization of protein "A," an H2A variant, has been carried out in the adult, neonatal, and embryonic spermatogenic cells of the mouse using the peroxidase-antiperoxidase technique. The results indicate an apparent enrichment of this protein in the meiotic cells of the adult testis. In addition, T-prospermatogonia present in the neonatal mouse and 16-day-old embryos were found to be immunoreactive. By contrast, Sertoli cells and other somatic elements of the neonatal and embryonic gonads were only weakly immunoreactive. These data suggest potential usefulness of protein "A" as a nuclear marker of the embryonic spermatogenic cells.

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis.

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.

Immunocytochemical localization of a histone H2A variant in the mammalian nucleolar chromatin.

The distribution of protein "A", a minor variant of H2A present in the mouse testis, was studied in the liver and brain nuclei using peroxidase-antiperoxidase technique. The data presented here suggest that nucleolar-associated chromatin is highly enriched in protein "A". Microspectrophotometric measurements corroborate the immunocytochemical data. The regional differentiation in the eukaryotic chromatin, therefore, may involve qualitative changes in the histone composition.

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse.

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH4)2SO4 precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.

Preparative electrophoresis of histones in solubilizable polyacrylamide gels.

Histones from the mouse testis have been fractionated on 17% acrylamide gels containing 0.19% Bis-acrylylcystamine (BAC), 25 M urea and 0.4% Lubrol-WX at pH 2.7. Polyacrylamide gels which contain BAC, a cross-linking agent with disulfide bonds, can be solubilized in the presence of 2-mercaptoethanol, at pH 8.3. Polymerization is carried out at 40 degrees C and in the presence of 6.5--7.5% tetramethylethylenediamine (TEMED) at pH 8.3. Gels containing high acrylamide concentration (greater than 15%) are not soluble if polymerized at lower pH or TEMED concentrations. Following electrophoresis, the gels are stained with Coomassie brilliant blue R for the visualization of protein bands. The stained protein bands are excised and adjusted to pH 8.3 prior to their solubilization. The histones are recovered by ion-exchange chromatography and are free of soluble gel components and dye. Two variants of histone H2B (H2B . 1 and H2B . 2) have been isolated at approx. 95% purity using this single purification step.

Bacterial peptides with C-terminal similarities to bovine neurotensin.

Using radioimmunoassay, peptides resembling the C-terminal region of bovine neurotensin (NT) have been demonstrated in acid/acetone extracts of Rhodopseudomonas palustris, Escherichia coli, and Caulaobacter crescentus. The NT-like bacterial components were shown to behave as peptides of small molecular weight (less than 2000) which were stable to acid and heat but labile to proteolytic digestion. In the radioimmunoassay toward NT they displayed dose-response curves parallel to standard and gave results indicating a competitive type of interaction with NT binding sites on antibody. The bacterial extracts did not register in a control radioimmunoassay toward rat luteinizing hormone. Some of the NT-like immunoreactivity could also be bound to an retrieved from anti-NT-antibody-Sepharose preparations. Since the C-terminal region of NT constitutes its biologically active core, these results suggest that presence of biologically important congeners of NT in bacteria.

Immunochemical characterization of neurotensin-like peptides in chicken.

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4-6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (greater than 200 fold than in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained form brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These finding also suggest the exstence of an NT-family of peptides serving multiple biological roles.