Search PubMed⌕ Search

Biomedical subjects

G F Saunders

Publications and source records attributed to G F Saunders.

At least 127 records · Page 7Linked to original sources
PubMed1981

Localization of the human insulin gene to the distal end of the short arm of chromosome 11.

The human insulin gene was mapped by hybridization in situ of 3H-labeled recombinant plasmid pHIG900 to chromosome preparations in the presence of 10% dextran sulfate. pHIG900 contains a 900-base pair insert of human genomic DNA that includes the coding region for the C peptide and intervening sequence 2. A significant percentage of hybridized cells (34%) exhibited silver grains on the distal end of the short arm (band p15) of chromosome 11. Furthermore, silver grains on this region represented 26% of all chromosomal label. These results demonstrate chromosomal localization of the human insulin gene to 11p15.

Chromosome Mapping↗
PubMed1980

Use of RNA polymerase as an enzymatic probe of nucleosomal structure.

Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.

Cell Nucleus↗
PubMed1980

Dinucleotide priming of RNA synthesis.

Initiation of RNA synthesis with preformed dinucleotides as primers and either a bacterial or a eukaryotic DNA-dependent RNA polymerase was used to probe sequences on DNA or chromatin that are available for transcription. Using saturating levels of Escherichia coli RNA polymerase or wheat germ RNA polymerase II to transcribe free DNA, a preference was noted for the dinucleotides GpA, GpG, ApG, and UpG for initiation of RNA synthesis. When saturating levels of E. coli RNA polymerase were used to transcribe chromatin, a little preference was noted for UpA and UpC; wheat germ RNA polymerase II, on the other hand, utilized the dinucleotides GpA, ApG, ApA, and UpG more efficiently. Under conditions of limiting amounts of enzyme to transcribe chromatin, wheat germ RNA polymerase preferred to initiate RNA synthesis with the dinucleotide primers GpA, ApA, ApC, and UpG; E. coli RNA polymerase preferred GpA and GpG. These results emphasize both the role of the configuration of the template and the role of the enzyme in sequence selection for initiation of transcription.

Chromatin↗
PubMed1979

Isolation and characterization of preproinsulin mRNA from fetal bovine pancreatic islets.

Total polyadenylated RNA prepared from isolated islets of fetal bovine pancreas was physically characterized. Incubation of this poly A+ RNA with a cell-free protein-synthesizing system derived from wheat germ results in the synthesis of insulin-immunoreactive polypeptide identical in size to that described earlier, 11 200 daltons (Lomedico et al. (1977) J. Biol. Chem. 252, 7971--7978). This material comprised approximately 22% of the total 3H-labeled translation products. Compared to poly A+ RNA from the total pancreas, we conclude that islet mRNA is enriched in proinsulin mRNA.

Animals↗
PubMed1978

Transcription of nucleosomes from human chromatin.

Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes.

Binding Sites↗
PubMed1977

Immunological and chemical characterization of bovine preproinsulin.

Fetal bovine pancreatic poly(A)-containing RNA directs the synthesis of an insulin immunoreactive polypeptide that is larger than proinsulin, preproinsulin, in the wheat germ cell-free translation system. We have characterized this peptide in detail both immunologically and chemically and have shown that it is 2500 daltons larger than bovine proinsulin (8700 daltons), possesses both insulin and bovine C-peptide-specific antigenic determinants, and contains all the tryptic peptides found in bovine proinsulin. Preproinsulin synthesized in the wheat germ cell-free system was precipitated with approximately 4-fold greater efficiency by bovine proinsulin antiserum than by insulin antiserum. Additional evidence was obtained which indicated that the preprotein folds and undergoes correct sulfhydryl oxidation less efficiently than proinsulin, perhaps due to the presence of the hydrophobic NH2-terminal extension. Automated sequential Edman degradation of bovine preproinsulin revealed the presence of an additional NH2-terminal sequence of 23 residues, preceding the B chain segment of proinsulin. The positions of 6 of the 7 leucine residues found in the bovine preproinsulin extension were identical to those reported previously for the rat preproinsulins. This close sequence similarity between the extensions of the bovine and rat preproinsulins supports the hypothesis that these molecules fulfill similar biosynthetic functions in vivo.

Amino Acid Sequence↗
PubMed1977

Cell-free modulation of proinsulin synthesis.

In vivo, glucose preferentially stimulates proinsulin biosynthesis; at least part of this process is independent of new RNA synthesis and is accompanied by increases in the overall rate of polypeptide chain initiation. The cell-free translation of proinsulin messenger RNA is very sensitive to changes in the protein-synthesizing system. Proinsulin synthesis is preferentially inhibited by the addition of increasing quantities of polyadenylate-containing RNA from the fetal bovine pancreas or by the addition of the drug, aurintricarboxylic acid, which blocks polypeptide chain initiation. These results suggest that proinsulin messenger RNA completes less efficiently for rate controlling initiation factors. We propose that glucose stimulates proinsulin biosynthesis by allowing the less competitive proinsulin messenger RNA to be translated more efficiently.

Aurintricarboxylic Acid↗
PubMed1977

Salt-induced structural changes in nucleosomes.

Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococcal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes was examined in the presence and absence of 10 mM MgCl2. Nucleosomes and oligonucleosomes are insoluble over a broad range of salt concentration. Nucleosomes are insoluble in larger than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 allowing analyses of changes in nucleosomal DNA by C.D. spectroscopy. Nucleosomes are insoluble in less than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 as demonstrated by turbidity measurements. We conclude that the insolubility of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.

Ammonium Sulfate↗
PubMed1977

Effect of bleomycin on the synthesis and function of RNA.

Bleomycin inhibits cellular RNA synthesis and the inhibition is nonspecific. The ratio of polyadenylate- [poly(A)] containing RNA to non-poly(A)-containing RNA in the drug-treated human lymphocytic cells, line Wil2, was the same as that in untreated cells. Poly(A) RNA isolated from untreated cells was used as a template for reverse transcriptase to synthesize complementary DNA, which was then used as a probe to assay the sequence diversity of poly(A)RNA's from treated and untreated cells. It was found that essentially all of the poly(A) RNA's in the untreated cells were also present in the treated cells. The effect of bleomycin on the biological activity of messenger RNA (mRNA) was tested with globin mRNA in a wheat germ embryo translation system. Although bleomycin inhibited protein synthesis at high concentrations, the inhibition was not due to a modification of mRNA. This was evidenced by the fact that no decrease in the ability of mRNA to function in the test system was found when globin mRNA was pretreated with high concentrations of bleomycin followed by removal of the drug.

Bleomycin↗
PubMed1976

Inverted repetitive sequences in the human genome.

A specific class of DNA sequences, the inverted repetitive sequences, forms hairpin-like structures in denatured DNA by the folding back of a single linear chain. The reassociation process of these sequences is unimolecular and the rate is extremely fast. Inverted repetitive sequences comprise 6% of the total human genome. They appear to be heterogeneous in length with an overall average length of 190 nucleotides. The inverted sequences are represented in almost all families of repetition frequencies, highly repetitive as well as very few copies per genome. They are not localized at unique sites on metaphase chromosomes. It is estimated that there are approximately 2 X 10(6) inverted repeats per haploid human genome. The biological function of this class of sequences is unknown.

Base Sequence↗
PubMed1976

Abundancy and diversity of mRNA sequences in human leukocytes.

The messenger RNA populations in two haematopoietic tissues were compared with respect to number of different sequences present and their relative abundancies. This was accomplished by hybridising complementary DNA to the cytoplasmic polyadenylated RNA from which it was transcribed and to heterologous polyadenylated RNA. The hybridisation kinetics were essentially the same in the homologous hybridisation. However, in heterologous hybridisation some differences were observed in the high abundancy messengers.

Base Sequence↗
PubMed1976

Template properties of human DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II has been purified from lymphocytes of patients with chronic lymphocytic leukemia (CLL). Form II polymerase and Escherichia coli RNA polymerase have been used to study the transcription of human DNA and chromatin. The hybridization kinetics of the transcripts of DNA and chromatin by the human polymerase are quite different; chromatin transcripts hybridized to DNA at a much slower rate than DNA transcripts, whereas the transcripts of DNA and chromatin by bacterial polymerase have similar hybridization kinetics.

Centrifugation, Density Gradient↗
PubMed1976

Template restriction in human chromatin.

DNA-dependent RNA polymerase from Escherchia coli was used to transcribe chromatin from human leukocytes and purified human DNA. RNA was labeled at the 5' terminus with either [gamma-32P]ATP or [gamma-32P]GTP and internally with [3H]UTP. Determination of the average chain length of the RNA molecules by the ratio of moles of 3H-labeled nucleotide incorporated to moles 32P-labeled nucleotide incorporated showed that the size of the transcript of purified DNA was about 2 1/2 times greater than those from chromatin. The percentage of chains initiated with ATP and GTP was observed to vary with the template, the ATP to GTP ratio being greater on chromatin. The kinetics of 3H and 32P hybridization of transcripts of purified DNA showed hybridization primarily to nonrepetitive sequences. Transcripts from the chromatin templates when hybridized to DNA showed a larger proportion of RNase resistance of the 32P-termini at low Cot's.

Chromatin↗
PubMed1976

Presence of messenger specifying sequences in the DNA of chromatin subunits.

Messenger-specifying sequences in subunits of human lymphocyte chromatin were detected by hybridization of DNA complementary to cytoplasmic polyadenylylated RNA with DNA isolated from the subunits. Comparison of the kinetics and extents of hybridization of complementary DNA with chromatin subunit DNA and with nuclear DNA showed that most of the repetitive sequences and single copy sequences in mRNA are present in chromatin subunits. This result indicates that inclusion of a DNA sequence into the subunit does not prevent its transcription in vivo.

Chromatin↗