Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.     

Evidence for the location of high mobility group protein T in the internucleosomal linker regions of trout testis chromatin.

Antibodies against the trout testis non"histone chromosomal protein, high mobility group protein T (HMG-T), have been elicited in goats. The antiserum was shown to be specific for HMG-T and did not cross-react with histone 1 or with the other two trout testis HMG proteins, H6 and ubiquitin. Purified anti-HMG-T IgG was used to determine the location of HMG-T within chromatin subunits separated on sucrose gradients. Binding of fluorescent labeled anti-HMG-T to these subunits clearly supports the notion that this protein is associated not with the nucleosome core but rather with the internucleosomal linker regions, and previously suggested (Levy W., B., Wong, N.C.W., and Dixon, G. H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2810-2814).     

Heterogeneity of proteins resembling high-mobility-group protein HMG-T in trout testes nuclei.

Two proteins, HMG-T1 and HMG-T2, with electrophoretic mobilities and compositions similar to those of protein HMG-T, were isolated from trout testes nuclei. The isoelectric points of proteins HMG-T1, HMG-T2 and HMG-T differ. The first 20 residues of protein HMG-T2 have been sequenced and differ from protein HMG-T by only one residue.     

Comparison of the high-mobility-group chromosomal proteins in rainbow-trout (Salmo gairdnerii) liver and testis.

Chromatography and characterization of the proteins extracted by 5% (w/v) HClO4 from rainbow-trout (Salmo gairdnerii) liver and testis show that the two tissues present a characteristically different spectrum of high-mobility-group (HMG) proteins. A variant subfraction of HMG C is found in liver, but is not detectable in testis, where even the main fraction of HMG C is present in only very low quantity. A protein, F, which appears to be related to protein H6 has similarly been isolated only from liver and not from testis. Quantification of the HMG proteins in total 5%-HClO4 extracts of trout liver and testis nuclei shows that, in relation to DNA, levels of HMG T1 and T2, and D are more than 2-fold, and C, 20-fold higher in liver than in testis. However, these differences do not result merely from the sequential withdrawal of HMG proteins at the same time that histones are replaced by protamines in the developing spermatid, since in testis, at some stages of maturation, levels of H6 are almost 2-fold higher than in liver. The implications of these findings for the function of HMG proteins are discussed.     

Binding of HMG-T to trout testis chromatin

When ¹²⁵I-labeled HMG-T was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those $\text{in vivo}$, most of the radioactivity bound to the chromatin. Most labeled non-nuclear proteins which were tested did not bind. Four large cyanogen bromide fragments of HMG-T each bound, suggesting that HMG-T interacts with chromatin along most of its length. Trout testis chromatin contains two populations of HMG-T molecules which differ in their extractability with NaCl solutions; the ¹²⁵I-labeled protein equilibrated mainly with the more readily extracted population. HMG-T also bound to nuclease-treated chromatin, an observation with important implications for studies in which nucleases are employed to probe chromatin structure.     

Partial purification of transcriptionally active nucleosomes from trout testis cells.

Mononucleosomes (MN1) enriched in structural non-histone proteins and transcribed DNA sequences were obtained by limited digestion of trout testis nuclei with micrococcal nuclease followed by selective solubilization in 0.1 M NaCl. These monosomes consist of the four inner histones plus stoichiometric amounts of the non-histone protein H6, of the HMG group, complexed with 140 base pairs of DNA. Hybridization experiments indicate that MN1 DNA is enriched in sequences complementary to cytoplasmic polyadenylated RNA.     

Multiacetylated forms of H4 are found in a putative transcriptionally competent chromatin fraction from trout testis.

We have examined the distribution of acetylated histones derived from various trout testis chromatin fractions of different composition. Our results indicate that a chromatin fraction, preferentially solubilized by micrococcal nuclease, containing the bulk of the HMG proteins and similar to a fraction released from intact trout nuclei and previously shown to be enriched in transcribed DNA sequences also possesses high levels of multiacetylated species of H4. Histones 2A, 2B and 3 are also acetylated in this particular chromatin fraction. Monoacetylated species of the 4 inner nucleosomal histones appear to be characteristic of the nucleohistone portion of trout testis chromatin.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

池蝶蚌Sox2 基因在不同组织及不同月龄精巢中的表达

Sox 基因家族在胚胎发育过程和性别分化中起重要作用, 为研究池蝶蚌中Sox 基因的功能, 以人SRY基因HMG-box 保守区的序列设计简并引物, 以雌、雄池蝶蚌基因组DNA 和精巢cDNA 为模板进行扩增, 获得了2 个不完全相同的序列, 分别为DNA-HMG1、DNA-HMG2 和cDNA-HMG, 长度均为220 bp, 编码73个氨基酸。与人等物种Sox1、Sox2、Sox3 及Sox14 有很高的同源性, 雌雄个体之间没有序列差异性。采用RACE-PCR 扩增获得了池蝶蚌性腺Sox2 部分cDNA 片段, 长度为1774 bp, 该序列核苷酸与欧洲帽贝的SoxB和人类的Sox2 的同源性最高; 在部分开放阅读框249 个氨基酸残基中, 具有Sox 家族典型的HMG-box 结构域, 与人类、小鼠、原鸡和斑马鱼等Sox2 的HMG-box 同源性为98%。为了解该基因在各组织中的表达情况,采用实时荧光定量PCR 方法分析了外套膜、闭壳肌、鳃、肠、肝、肾、精巢和卵巢在内的8 种组织hs-Sox2的表达情况, 结果显示, hs-Sox2 基因在8 种组织中均有表达, 其中在肾脏中的表达量最高, 其次是肠与闭壳肌, 在雄性性腺中的表达量明显高于雌性性腺, 在肝脏中的表达量最低; 为了解hs-Sox2 在不同性腺发育时期的表达情况, 采用实时荧光定量PCR 方法分析了5 个不同月龄的精巢组织中hs-Sox2 的表达情况, 结果显示在39 月龄性腺的表达量最高, 其次是16 月龄性腺, 63 月龄蚌中的表达量最少。以上结果表明, hs-Sox2 基因可能参与了池蝶蚌精巢的发育及功能的维持。       

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.     

DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.     

Amino acid sequence homologies between the high-mobilitygroup proteins,HMG-T from trout testis and HMG-1 and-2 from calf thymus: is the poly-aspartic-glutamic acid polypeptide within the main chain?

The amino acid sequence of the N-terminal two-thirds of a trout high-mobility-group protein, HMG-T, has been determined as a continuous sequence of 174 residues out of a total for the whole molecule of 260 residues. When this sequence was compared with published sequences of long cyanogen bromide-derived peptides from the analogous calf-thymus proteins, HMG-1 and -2 (Walkeret al. , 1979), there was strong homology, with 60–70% identity of corresponding amino acid residues in the three proteins, the majority in lengthy identical runs. However, a discrepancy in the position of a highly acidic run of aspartic and glutamic residues suggests this region may not lie within the main polypeptide chain but may represent a separate chain or possibly a branched structure,     

70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences.

RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.     

Renaturation kinetics of cDNA complementary to cytoplamic polyadenylated RNA from rainbow trout testis. Accessibility of transcribed genes to pancreatic DNase.

We have determined the fraction of polyadenylated cytoplasmic RNA from trout testis complementary to unique and repetitive DNA. Some 21% of the cDNA probe representative of this RNA population renatures with rapid kinetics, characteristics of repetitive sequences. The major proportion of the cDNA renatures with unique sequence DNA. Experiments with fractionated cDNA probes allow us to conclude that, in trout testis, the most abundant polyadenylated mRNAs are not preferentially transcribed from repetitive DNA, as it has shown to be the case in two eukaryotic cell lines. Treatment of trout testis nuclei with DNase I, under conditions in which 10% of the total DNA is digested, preferentially depletes the DNA of sequences being transcribed into polyadenylated mRNA. These data confirm the results of H. Weintraub and M. Groundine [(1976) Science 193, 848-856] and those of A. Garel and R. Axel [(1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3966-3970] and suggest that the conformation of DNA in the active genes of chromatin is such that it is more susceptible to digestion by DNaseI.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.