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Biomedical subjects

S H Hardin

Publications and source records attributed to S H Hardin.

9 recordsLinked to original sources
PubMed1997

Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen.

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.

Animals↗
PubMed1996

Octamer-primed cycle sequencing: design of an optimized primer library.

This paper describes a novel method of primer walking using octamer oligonucleotides to prime DNA sequencing reactions. Octamer sequencing is compatible with isotopic and fluorescent sequencing chemistry, reaction conditions are optimized such that the samples can be processed in parallel, and the procedure has the potential to be automated. This strategy is faster than the traditional primer walking sequencing strategy, as the existence of a primer library allows immediate access to a primer for the next sequencing reaction, eliminating delays associated with designing and synthesizing gene-specific primers. The octamer library is comprised of optimized sequencing primers, such that octamer sequencing yields results equivalent to or better than traditional primer walking. This technology is more economical because gene-specific sequencing primers, the major cost in the reaction, are replaced by an optimized subset of frequently occurring octamers that are able to prime multiple reactions.

Anabaena↗
PubMed1995

Complete sequence of the binary vector Bin 19.

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11,777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.

Base Sequence↗
PubMed1995

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.

Amino Acid Sequence↗
PubMed1993

Sequence of the Candida albicans erg7 gene.

The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.

Amino Acid Sequence↗
PubMed1988

Spec2 genes of Strongylocentrotus purpuratus. Structure and differential expression in embryonic aboral ectoderm cells.

Members of the Spec gene family are expressed during embryonic development of the sea urchin, Strongylocentrotus purpuratus. The family encodes proteins related to the calmodulin/troponin C/myosin light chain group of calcium binding proteins and one gene, Spec1, has been studied extensively in our laboratory. In this paper, we analyze other members of the family, collectively termed Spec2 genes. We make use of several hybridization probes derived from Spec1 and Spec2 cDNA clones, which recognize different members of the family. Genomic DNA gel blot and slot blot analyses show that there are approximately eight Spec genes in the S. purpuratus genome. The structures of three Spec2 genes, Spec2a, Spec2c and Spec2d, are described. A 60 kb (kb = 10(3) bases or base-pairs) region of the genome contains the linked Spec1-Spec2c genes and two separate 20 kb regions contain the Spec2a and Spec2d genes. Six members of a repetitive sequence family are dispersed at various locations among the genes. The transcriptional initiation sites of the three Spec2 genes are mapped, and 400 to 500 base-pairs of 5'-flanking DNA sequenced. All three Spec2 genes initiate transcription approximately 120 base-pairs upstream from the 3' end of the first exon. In contrast, the 5' end of the Spec1 transcript begins about 107 base-pairs farther upstream, so it contains 5' untranslated sequences that correspond to non-transcribed 5'-flanking sequences of the Spec2 genes. There is little similarity among the sequences upstream from the CAP site of the Spec2 genes except the TATA consensus sequence and a repeating trinucleotide, AAC. Measurements of Spec mRNA levels during embryogenesis show that Spec1 mRNA begins to accumulate at the early blastula stage and is the most abundant; Spec2a/Spec2c mRNAs begin accumulating several hours later at the late blastula-early gastrula stage and reach about 40 to 60% the levels of Spec1; and Spec2d mRNAs accumulate mostly during the gastrula and pluteus stages with levels reaching only 2% those of Spec1. In situ hybridization with probes that recognize either all Spec2 mRNAs or only Spec2d mRNAs show that, like Spec1, these mRNAs are restricted to aboral ectoderm cells and their precursors. The Spec gene family represents a group of related genes whose mRNAs all accumulate in the same cell type but at different times and to different levels during embryogenesis.

Animals↗
PubMed1987

Use of consensus oligonucleotides for detecting and isolating nucleic acids encoding calcium binding domains of the troponin C superfamily.

Proteins belonging to the troponin C superfamily (troponin C, calmodulin, myosin light chains, and parvalbumin) are involved in a wide variety of cellular activities mediated by calcium ions. Most of these proteins bind ionic calcium, and all have calcium binding domains that are conserved to some extent at the nucleic acid level. We made use of the conservation in the third calcium binding domain to synthesize two consensus sequence oligonucleotide probes, one 43 bases and the other 25 bases long. By using cDNA and genomic clones encoding calmodulin, troponin C, parvalbumin, and the sea urchin Spec proteins, we show that these probes hybridize with nucleic acid sequences representing calcium binding domains. In an RNA gel blot analysis of embryonic RNA from the sea urchin Strongylocentrotus purpuratus, we show that transcripts which have previously been shown to encode troponin C like proteins hybridize with the consensus sequence probes. Screening sea urchin cDNA and genomic libraries with the 43-base consensus oligonucleotide shows that the probe can be used to isolate cloned nucleic acids. Two such genomic clones from a Lytechinus pictus library were isolated and characterized. One clone encodes part of an L. pictus calmodulin gene, and the other encodes a member of the superfamily that has not been characterized previously. The consensus oligonucleotides should be valuable probes in the diagnosis and isolation of nucleic acids encoding proteins of the troponin C superfamily.

Amino Acid Sequence↗
PubMed1985

Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.

We have identified and characterized the structure of the Spec1 gene in the sea urchin Strongylocentrotus purpuratus. In earlier studies we demonstrated that a small family of messenger RNAs, termed Spec mRNAs for S. purpuratus ectodermal mRNAs, begins to accumulate 20 hours after fertilization in ectoderm cells of the sea urchin embryo. The Spec mRNAs code for a group of low molecular weight proteins belonging to the troponin C superfamily. Spec1 transcripts, the predominant mRNAs of the family, are heterogeneous in their 3' untranslated sequences but code for a single protein, recently shown to be a calcium-binding protein. Spec complementary DNA clones were used to isolate genomic clones from two lambda libraries. These genomic clones comprise a 41 kb (kb = 10(3) bases or base-pairs) region of the S. purpuratus genome and contain a Spec1 gene closely linked to another Spec gene, Spec2c. The Spec1 gene is 10.3 kb in length and contains six exons. The genomic clones containing the Spec1 gene can be placed into two groups based on restriction fragment length differences and differences in hybridization strengths using probes derived from Spec1 3' untranslated regions. Evidence that these groups probably correspond to two alleles of the Spec1 gene was obtained by probing genomic DNA blots of sperm DNA from different individuals with 3' untranslated sequences of Spec1 complementary DNA clones. These blots show that two of the Spec1 mRNAs we have characterized, and probably a third, are alleles of the Spec1 gene. Thus, there appears to be a single polymorphic Spec1 gene in the sea urchin genome. We used S1 protection and primer extension procedures to map the 5' end of the Spec1 gene. Results from these experiments indicate that the initiation of transcription of the Spec1 mRNA begins at an A residue 220 bases from the 3' end of the first exon. Adding support to this claim, cannonical T-A-T-A and C-A-A-T sequences, indicative of many eukaryotic promoters, are found 23 bases and 60 bases upstream from this site, respectively. Analysis of sequences within a few kb of the Spec1 gene show that there are five members of a repetitive sequence family near the gene, three upstream and two downstream. The 5' leader sequence of another Spec mRNA, Spec2a, also contains a member of this repeat family.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗