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Biomedical subjects

M S Urdea

Publications and source records attributed to M S Urdea.

At least 73 records · Page 4Linked to original sources
PubMed1987

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

Chromosome Mapping↗
PubMed1987

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.

Amino Acid Sequence↗
PubMed1986

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.

Amino Acid Sequence↗
PubMed1986

Isolation of the yeast calmodulin gene: calmodulin is an essential protein.

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.

Amino Acid Sequence↗
PubMed1986

A chemical 5'-phosphorylation of oligodeoxyribonucleotides.

To circumvent the use of T4 polynucleotide kinase and ATP for the 5'-phosphorylation of synthetic oligodeoxyribonucleotides after deprotection and purification, we have developed a new chemical phosphorylation reagent that can be used on automated DNA synthesis instruments. The phosphite-derived compound, bis(beta-cyanoethoxy)-N,N-diisopropylaminophosphine, is coupled to a solid-supported fragment and oxidized under the same conditions as nucleoside phosphoramidites. The resultant 5'-phosphate oligomer is then fully deprotected in standard ammonium hydroxide solution at elevated temperature. Reverse-phase, high-pressure liquid chromatography, silica thin-layer chromatography, and 31P-nuclear magnetic resonance spectra of chemically phosphorylated thymidine were compared with a variety of phosphorus derivatives of thymidine. All evidence suggested that the nucleoside was converted to the 5'-phosphate form quantitatively with the reagent. Moreover, an excellent enzymatic ligation reaction was obtained with a chemically phosphorylated oligonucleotide.

DNA Ligases↗
PubMed1986

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.

Adrenal Hyperplasia, Congenital↗
PubMed1985

Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization.

Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo-B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2.

Amino Acid Sequence↗
PubMed1985

Homology requirements for recombination in Escherichia coli.

The DNA sequence homology required for recombination in Escherichia coli has been determined by measuring the recombination frequency between insulin DNA in a miniplasmid pi VX and a homologous sequence in a bacteriophage lambda vector. A minimum of approximately equal to 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homologous DNA is increased from 20 base pairs to 74 base pairs and an apparently linear increase with longer DNA segments. Mismatches within a homologous segment can dramatically decrease the frequency of recombination. Thus, the process of recombination is sensitive to the length of precisely base-paired segments between recombining homologues.

Animals↗
PubMed1985

Enzymatic purification of chemically synthesized oligodeoxyribonucleotides prior to removal from a solid-support.

A novel combined solid-phase DNA synthesis and solid-phase DNA purification strategy is described. Removal of all N-, cap-, and phosphate protecting groups while maintaining a 5'-blocking group on the final product followed by a 5'- specific exonuclease digestion of failure sequences with the synthetic DNA still attached to the solid phase yields an essentially pure oligomer.

DNA↗
PubMed1985

Base modification and cloning efficiency of oligodeoxyribonucleotides synthesized by the phosphoramidite method: methyl versus cyanoethyl phosphorus protection.

We have found large oligodeoxyribonucleotides (50-120 bases) synthesized with N,N-dialkylmethylphosphoramidites to have considerably lower cloning efficiencies than biological DNA. As evidenced by HPLC analysis, these large fragments contain as much as 30% thymidine conversion to 3-methylthymidine. If cyanoethyl- instead of methyl-phosphorous protection is employed, as anticipated, no thymidine methylation is noted. More importantly, large fragments produced by N,N-diisopropylcyanoethyl-phosphoramidite coupling show cloning efficiencies indistinguishable from biological DNA.

Amides↗
PubMed1984

The complete nucleotide sequence for rabbit muscle aldolase A messenger RNA.

The complete nucleotide sequence of rabbit muscle aldolase mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 1375 nucleotides, exclusive of the poly(A) tails. The 5'-untranslated region contains 62 bases including a potential ribosome-binding site. The 3'-untranslated region is 221 bases long. The remaining 1092 nucleotides are in an open reading frame coding for 364 amino acids. There is a single methionine residue preceding the NH2-terminal proline. The deduced amino acid sequence corrects a number of discrepancies between published structures as well as assignments previously missed. The sequences of the cloned cDNAs suggests there may be microheterogeneity in the messenger RNA population.

Amino Acid Sequence↗
PubMed1984

Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.

Amino Acid Sequence↗
PubMed1984

Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing.

Synthetic oligodeoxyribonucleotides were used as primers for sequencing DNA by the dideoxy chain termination method. We report that the use of unpurified preparations of such oligonucleotides decreases the time involved in preparing the primers and yields a sequencing reaction of comparable quality to that obtained with pure preparations. It is possible to use unpurified primers if the yield per coupling step during manual or machine synthesis of oligonucleotides is greater than 98%. Also, a method for the rapid purification and desalting of synthetic DNAs is presented for cases in which the coupling is less efficient. Unpurified primers have been used to determine the sequence of a 1987-bp fragment with a single M13 recombinant clone. This DNA was first sequenced using the universal M13 primer. Subsequently, a primer homologous to the 3' end of the newly obtained sequence was synthesized and used to extend the sequence. New primers were synthesized until the sequence of the whole fragment was determined. Because only one clone is necessary, this method can be used to obtain the nucleotide sequence of large DNA fragments in a relatively short time and in an orderly fashion, simplifying analysis of the data.

Base Sequence↗
PubMed1984

Construction and evaluation of an instrument for the automated synthesis of oligodeoxyribonucleotides.

The details for constructing an easily used and maintained laboratory instrument capable of routine oligodeoxyribonucleotide synthesis are presented. The synthesizer consists of relatively inexpensive, commercially available components and is controlled by an Apple IIe computer. Since reagents are distributed from pressurized reservoirs through a liquid manifold by opening solenoid-activated valves, no pump is required. More than 600 oligomers containing up to 122 bases have been produced with a condensation cycle time of approximately 15 min with apparent coupling yields of 98.5%. A unique bidirectional flow reactor and controlled reagent distribution system provide for rapid mass transfer during solvent and reactant equilibrations and for long-term stability of reagent solutions. Aspects of the system should also find use in other solid-phase synthetic and analytical strategies.

Automation↗
PubMed1983

Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.

Amino Acid Sequence↗