Single-step isolation and sequencing of vasopressin and oxytocin precursors.

The pre- and post-Golgi processing of preprovasopressin and prepro-oxytocin was evaluated by microsequencing for incorporated radiolabel. 35S-Cysteine and 3H-fucose were microinjected into rat supraoptic nuclei (SON), and proteins and peptides related to the biosynthesis of vasopressin (VP) and oxytocin (OT) were isolated at various times from the supraoptic nuclei and neural lobe by employing a one-step procedure of high performance liquid chromatography (HPLC). These proteins and peptides were recognized through their binding to specific antibodies against VP, OT, and rat neurophysins (RNPs), and by their binding to ConA-Sepharose. Two immunoreactive glycoproteins related to VP biosynthesis were recovered from the SON and both contained fucose and had a 35S-cysteine placement consistent with the location of the hormone sequence at the N-terminus. SDS-electrophoresis revealed the major protein form to be 21,000 daltons and the minor protein form to be 19,000 daltons. One nonglycosylated protein of 16,000 daltons related to oxytocin biosynthesis was recovered from the SON, and this protein also had a 35S-cysteine placement consistent with an N-terminal OT sequence. These data provide the first sequential evidence that prior to, or shortly after, packaging in the Golgi the preprohormones of VP and OT have lost their entire leader-peptide structures.     

Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison

A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA.     

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.     

The chicken HMG-17 gene is dispensable for cell growth in vitro.

HMG-17 is a highly conserved and ubiquitous nonhistone chromosomal protein that binds to nucleosome core particles. HMG-17 and HMG-14 form a family of chromosomal proteins that have been reported to bind preferentially to regions of active chromatin structure. To study the functional role of the single-copy chicken HMG-17 gene, null mutants were generated by targeted gene disruption in a chicken lymphoid cell line, DT40. Heterozygous and homozygous null mutant cell lines were generated by two independent selection strategies. Heterozygous null mutant lines produced about half the normal level of HMG-17 protein, and homozygous null lines produced no detectable HMG-17. No significant changes in cell phenotype were observed in cells harboring either singly or doubly disrupted HMG-17 genes, and no compensatory changes in HMG-14 or histone protein levels were observed. It is concluded that HMG-17 protein is not required for normal growth of avian cell lines in vitro, nor does the absence of HMG-17 protein lead to any major changes in cellular phenotype, at least in lymphoid cells.     

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.     

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.     

Neither HMG-14a nor HMG-17 gene function is required for growth of chicken DT40 cells or maintenance of DNaseI-hypersensitive sites.

HMG-14 and HMG-17 form a family of ubiquitous non-histone chromosomal proteins and have been reported to bind preferentially to regions of active chromatin structure. Our previous studies demonstrated that the chicken HMG-17 gene is dispensable for normal growth of the DT40 chicken lymphoid cell line. Here it is shown that the major chicken HMG-14 gene,HMG-14a, is also dispensable and, moreover, that DT40-derived cells lacking both HMG-17 and HMG-14a proteins show no obvious change in phenotype with respect to the parental DT40 cells. Furthermore, no compensatory changes in HMG-14b or histone protein levels were observed in cells lacking both HMG-14a and HMG-17, nor were any alterations detected in such hallmarks of chromatin structure as DNaseI-hypersensitive sites or micrococcal nuclease digestion patterns. It is concluded that the HMG-14a and HMG-17 proteins are not required for normal growth of avian cell linesin vitro, nor for the maintenance of DNaseI-hypersensitive sites in chromatin.     

Single-step isolation and resolution of pancreatic carboxypeptidases A and B.

Carboxypeptidases A and B have been isolated individually from aqueous extracts of mammalian pancreatic acetone powders by affinity chromatography on [N-(epsilon-aminocaproyl)-p-aminobenzyl]succinyl-Sepharose 4B (CABS-Sepharose). The affinity ligand was synthesized from DL-benzylsuccinic acid, purified, and characterized by UV absorption and NMR spectroscopy. Both enzymes from the various species were homogeneous by NaDodSO4-polyacrylamide gel electrophoresis and displayed high specific activities. No cross contamination of one enzyme species with the other was found. The ease of synthesis of the ligand from its commercially available precursor, its stability, and the mild elution conditions render CABS-Sepharose an excellent affinity support for the single-column isolation of both carboxypeptidases A and B. The procedures extend the utility of this resin previously demonstrated for carboxypeptidase A from human pancreatic juice [Peterson, L. M., Sokolovsky, M., & Vallee, B. L. (1976) Biochemistry, 15, 2501]. The use of CABS-Sepharose as a general affinity matrix for the isolation of metallocarboxypeptidases is suggested.     

Chromosomal protein HMG-14 is overexpressed in Down syndrome.

The physical phenotype of Down syndrome, one of the most prevalent genetic disorders, results from an extra copy of regions q22.1 to q22.3 of chromosome 21 in cells of affected individuals. The gene coding for chromosomal protein HMG-14 is among the limited number of genes, coding for known functions, which has been mapped to this region of chromosome 21. Here we report a gene dosage effect on the expression of HMG-14 in both cultured cells and brain tissue samples obtained from Down syndrome patients. The putative role of HMG-14 in the structure of active chromatin raises the possibility that elevated levels of this protein may be a contributing factor in the etiology of Down syndrome.     

Effect of high mobility group nonhistone proteins HMG-20 (ubiquitin) and HMG-17 on histone deacetylase activity assayed in vitro.

We have used a method previously described by Reeves and Candido (1) to partially release histone deacetylase from cell nuclei together with putative regulators of the enzyme. Histone deacetylase released from testis cell nuclei and its putative regulators were separated by gel filtration in Sepharose 6B. A peak of low molecular weight contains a heat-stable factor that stimulate histone deacetylase in vitro. Many of the properties of the activator coincide with those of the protein HMG-20 (ubiquitin). Ubiquitin isolated from testis cell nuclei stimulated histone deacetylase in vitro. It has been suggested that HMG-17 partially inhibits histone deacetylase in Fried cell nuclei (2). In our system, HMG-17 shows no inhibitory effect on histone deacetylase activity     

Immunochemical detection of chromosomal protein HMG-17 in chromatin subunits

Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1.     

Immunofractionation of DNA sequences associated with HMG-17 in chromatin

Antibodies specific for chromosomal protein HMG-17 were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to purify chromatin segments enriched in HMG-17. The DNA was purified from both the nucleosomal fraction which was bound to the column and from the fraction which was not bound, and examined with DNA probes representing repetitive DNA, non-transcribed genes, transcribed genes and inducible genes. The results suggest that HMG-17 is preferentially associated with DNA sequences coding for genes, regardless of whether they are transcribed, and therefore support the notion that HMG-17 confers specific structural characteristics on selected regions in the genome.     

Chromosomal protein HMG-17. Complete human cDNA sequence and evidence for a multigene family

Antibodies elicited against chromosomal protein HMG-17, purified from calf, were used to screen a human lambda gt11 cDNA expression library and isolate the full length cDNA coding for this protein. Sequence analysis reveals that the nucleotide distribution along this cDNA is highly asymmetric. The amino acid sequence, deduced from the reading frame, reveals that the human HMG-17 is, respectively, 96 and 92% homologous with the calf and chicken protein. The amino acid substitution are conservative suggesting evolutionary constraints on the conformation of the protein. The human genome contains 35-50 HMG-17 gene copies which, as revealed by Southern analysis, are distributed at several loci. Northern analysis of total RNA isolated from 3 human cell lines, indicates that each cell contains a single-size mRNA coding for this protein. Nucleotide sequences which cross-hybridize, under stringent conditions, with the human HMG-17 cDNA are present in the genome of rodents and absent from the genomes of sea urchin, Drosophila, and yeast. The availability of a probe for the HMG-17 gene may help elucidate the cellular role of this protein which may confer specific conformations to transcribable regions in the genome.     

HMG-2 protein in developing rat brain cells

• 1.1. The distribution of HMG-2 protein was followed in unfractionated rat brain cells at different stages of development. Its amount gradually decreased and reached the lowest level in the terminally differentiated and non-proliferating cells.
• 2.2. In isolated oligodendrocyte nuclei the changes in the content of HMG-2 followed the same pattern of distribution which corresponded to their stage of development and proliferative activity, while in the terminally differentiated and non-proliferating cortical neurons a substantial amount of HMG-2 protein was present up to the twenty-eighth postnatal day.
• 3.3. In the presence of anti-HMG-2 antibodies the DNA synthetic activity of oligodendrocyte nuclei in vitro was significantly decreased. The treatment with antibodies affected mainly the DNA replicative activity of the nuclei, while their DNA repair activity remained unchanged.