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G F Saunders

Publications and source records attributed to G F Saunders.

At least 109 records · Page 6Linked to original sources
PubMed1985

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.

Chromosome Banding↗
PubMed1984

Multiple origins of transcription for the human placental lactogen genes.

Transcriptional activity of the three human placental lactogen (hPL) genes was compared in vitro and their relation to the hPL RNA species obtained from term placenta was analyzed. We used in vitro transcription system containing the HeLa cell crude extract as the source for RNA polymerase II and initiation factors. Gene fragments of identical length of 0.96 kilobase pair made from hPL1, hPL3, and hPL4 each contained the 5' flanking sequence of 497 base pairs, the first exon, the first intron, and a portion of the second exon. More than 90% transcripts of the hPL1 template were 470 nucleotides long, indicating that transcription was initiated at the proposed cap site. hPL3 and hPL4 genes generated heterogeneous RNA products of about 430, 470, 520, and 680 nucleotides suggesting that multiple start points were recognized for RNA synthesis in vitro. alpha-Amanitin sensitivity of transcription indicated that the DNA-dependent RNA synthesis was carried out by RNA polymerase II. These results show that hPL1, hPL3, and hPL4 genes have functional promoters and multiple initiation sites for transcription. Primer extension analysis of the 5' termini of hPL RNA isolated from term placenta shows that 82-83% of the transcripts are initiated at a region 29 base pairs downstream from a "TATA" sequence. This origin is observed in vitro for the transcript 470 nucleotides long. An additional upstream initiation region (-53) accounts for 8% of transcripts in term placenta and corresponds to the origin for the in vitro transcript of 520 nucleotides. At least three other sites 15, 23, and 39 base pairs downstream from the major cap site are functional in vivo. The initiation site at +40 is utilized preferentially for transcription from hPL3 and hPL4 genes in vitro. We have mapped the different transcription origins on hPL genes.

Base Sequence↗
PubMed1984

Evolution of glucagon genes.

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(-9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.

Animals↗
PubMed1984

Localization of the human glucagon gene (GCG) to chromosome segment 2q36----37.

Hybridization of a 3H-labeled bovine glucagon cDNA plasmid to human metaphase chromosomes revealed significant labeling of the distal portion of the long arm of chromosome 2. A large portion (37%) of the cells analyzed exhibited labeling of the 2. A significant percentage (40%) of the labeled sites on the 2 were in segment 2q36----37. Therefore, the human glucagon gene (GCG), was assigned to this segment. Localization of the glucagon gene, whose chromosomal assignment was previously not known, demonstrates the general applicability of in situ hybridization as a powerful gene mapping technique.

Chromosome Mapping↗
PubMed1983

Two structurally different genes produce the same secreted human placental lactogen hormone.

Experiments were designed to determine if the members of the human placental lactogen (hPL) cluster, consisting of four nonallelic genes, are transcribed in term placenta. Taking advantage of differences in restriction endonuclease sites in the coding portions of the different hPL genes and thus of their putative cDNAs, we have identified two transcriptionally active nonallelic hPL genes in term placenta. The two expressed genes, which code for secreted hPL with identical amino acid sequences, are about equally represented in the hPL mRNA population.

Base Sequence↗
PubMed1983

Efficient extraction of RNA from mammalian tissue.

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.

Animals↗
PubMed1983

Mammalian pancreatic preproglucagon contains three glucagon-related peptides.

We have isolated cDNA clones encoding bovine pancreatic preproglucagon. Twenty-five putative preproglucagon clones were selected by screening 3,100 clones of a fetal bovine pancreas cDNA library with a synthetic oligodeoxynucleotide probe. The probe was a mixture of synthetic 17-base DNA oligomers constructed to correspond to the six carboxyl-terminal amino acids (residues 24-29) of mature glucagon. Restriction mapping of six of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1,200-base-pair DNA insert revealed that it contained an essentially full-length copy of glucagon mRNA. Analysis of the cDNA suggested a protein coding sequence of 540 nucleotides and 5'- and 3'-untranslated regions of 90 and 471 nucleotides, respectively. This cDNA sequence encoded a 20-amino acid signal sequence followed by one for glicentin, a 69-amino acid polypeptide containing an internal glucagon moiety that has been found in porcine intestines. Glicentin is followed by two additional glucagon-like peptides, each flanked by paired basic amino acids (Lys, Arg) characteristic of prohormone processing. These polypeptide sequences show striking homology with those for glucagon and other members of the glucagon family of peptides.

Amino Acid Sequence↗
PubMed1982

Transcriptional products of the human placental lactogen gene.

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.

Base Sequence↗
PubMed1982

Linkage arrangement of human placental lactogen and growth hormone genes.

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'- and 3'-specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.

Base Sequence↗
PubMed1982

Differential expression of selected genes in human leukemia leukocytes.

cDNA clones complementary to mRNA of cells from patients having chronic lymphocytic leukemia (CLL) were used to examine quantitative changes in the mRNA levels of specific genes in human leukemia leukocytes. Fourteen (of 400) CLL-positive clones that did not hybridize with placental mRNA were studied. Three of the 14 clones were highly represented in mRNA from CEM, a T-cell line. One clone was highly represented in mRNA from CLL and two were highly represented in mRNA from patients with chronic myelocytic leukemia. Ten clones were not significantly represented in normal leukocytes and spleen mRNA. We have identified several genes that are differentially expressed in human leukemia leukocytes.

Animals↗
PubMed1982

Chromosomal localization of the human placental lactogen-growth hormone gene cluster to 17q22-24.

Recombinant plasmid HCS-pBR322 containing a 550-base-pair (bp) insert of cDNA to human placental lactogen (hPL) mRNA was 3H-labeled by nick translation and hybridized in situ to human chromosome preparations in the presence of 10% dextran sulfate. A high percentage of cells (80%) were found to exhibit label on the distal end of the long arm of chromosome 17. Silver grains on this region constituted 25.5% of all labeled sites, allowing assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to 17q22-24. A gene copy number experiment showed that both genes are present in approximately three copies per haploid genome.

Chromosome Banding↗
PubMed1981

Insulin gene expression during development of the fetal bovine pancreas.

Poly(A+) RNA was isolated from the bovine pancreas at three stages of fetal development. Approximately 1% of the total RNA from first, second, and third trimester fetuses was polyadenylated, and the mean chain length of each RNA population was 1350 nucleotides. In cell-free protein synthesis experiments the concentration of insulin-immunoreactive translation products was 10.2%, 11.3%, and 9.7% for first, second, and third trimesters, respectively. Insulin mRNA sequences were estimated by transcription of insulin mRNA to [3H]cDNA and hybridization of cDNA with plasmid pI19 DNA containing rat proinsulin I sequences. Hybridization experiments gave insulin mRNA concentrations of 7.6%, 12.9%, and 3.9% for first, second, and third trimesters, respectively. These results show that insulin mRNA levels vary during development and become proportionally lower in third trimester, when the exocrine tissue is rapidly increasing in mass.

Animals↗
PubMed1981

Localization of single copy DNA sequences of G-banded human chromosomes by in situ hybridization.

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization in human metaphase chromosomes of the 3H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.

Azure Stains↗
PubMed1981

mRNA in human cells contains sequences complementary to the Alu family of repeated DNA.

Approximately one-half of the polysomal poly(A)+RNA from CCRF-CEM human lymphoblastoid cells associates at low R0t (10 M.sec) [where R0 is the initial concentration of RNA (M) and t is time (sec)] to form branched complexes detectable by electron microscopy. The complexes typically involve 2-16 molecules associated over double-stranded regions 120 +/- 30 base pairs long. Formation of such complexes suggests that poly(A)+RNA contains repeated-sequence elements that are highly represented in the mRNA population. Hybridization of polysomal poly(A)+RNA with a recombinant human DNA plasmid, p lambda H15C, which is shown to contain at least three regions complementary to two different members of the Alu family of DNA repeat sequences, showed a total of five regions where R loops are formed. The hybridized regions comprising these groups are 260 +/- 180, 240 +/- 170, 150 +/- 70, 180 +/- 60, and 180 +/- 80 base pairs long. The relative frequencies of R loops formed at these different sites indicate that sequences in this recombinant DNA are represented in the mRNA population at different frequencies. The hybridizing sequence of the RNA molecules is located near one terminus in 13% of the R loops and internally in 53% of the R loops. Surprisingly, 35% of the R loops apparently involve RNA molecules hybridized over their entire length of only 200 +/- 110 base pairs.

Cell Line↗
PubMed1981

Presence of a highly repetitive and widely dispersed DNA sequence in the human genome.

A genomic DNA library consisting of human DNA fragments about 18 kilobases long cloned in a bacteriophage lambda vector was found to contain a specific repeated DNA segment. The repeated sequence is present in greater than 95% of the genomic library, and selected clones contain at least two copies of the sequence. Our experiments indicate that this highly repetitive sequence (approximately 400,000 copies per haploid genome) is widely distributed in the human genome and is represented in the cytoplasmic polysomal mRNA. This sequence is homologous to the 300-base-pair Alu repeat family, the predominant repeat sequence in man.

Cloning, Molecular↗