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A D Riggs

Publications and source records attributed to A D Riggs.

At least 91 records · Page 5Linked to original sources
PubMed1984

Sequence of the promoter region of the gene for human X-linked 3-phosphoglycerate kinase.

We have determined the sequence of an 812-bp BamHI-EcoRI restriction fragment containing the 5' region of the human gene for PGK (3-phosphoglycerate kinase or ATP:3-phospho-D-glycerate 1-phosphotransferase; EC 2.7.2.3). The fragment contains 450 bp 5' to three start points for transcription (located by primer extension and S1 nuclease mapping), a leader sequence 85-94 bp long, the first exon of gene (65 bp), and part of the first intron. The promoter region is extremely G + C-rich, lacks a TATA box, and has an 8-bp direct repeat. A comparison of the promoter region for PGK with other promoters on the X-chromosome reveals homology with the promoter for HPRT, but not with the operator for factor IX.

Base Sequence↗
PubMed1984

Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli.

Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.

Amino Acid Sequence↗
PubMed1984

Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.

We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.

Antigens, Viral↗
PubMed1983

Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.

We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation. Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides. A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen. One clone among 2,500 gave a strong positive signal. Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting of a mixture of four sequences. The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes. DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.

Amino Acid Sequence↗
PubMed1982

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Amino Acid Sequence↗
PubMed1981

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

Amino Acid Sequence↗
PubMed1980

Equilibrium binding of inducer to lac repressor.operator DNA complex.

The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases the affinity of the protein for inducer molecules. The maximum change observed is a 20-fold increase in the concentration of inducer necessary for half-saturation of the protein. The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating amounts of operator DNA fragments were compared to curves predicted by several different models. Using the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to discriminate between the various models. The models which accurately predicted the inducer binding curves and conformed to the measured values for the other parameters were Monod-Wyman-Changeux and Koshland type models with the following features: 1) positive cooperativity in the binding of inducer to the repressor . operator DNA complex; 2) two sites for the binding of the operator DNA fragment to repressor protein; 3) effect of binding of each operator DNA fragment on all four subunits of the repressor. These models are consistent with all of the available data, and they indicate that the binding of the first two inducer molecules accounts for greater than or equal to 60% of the difference in affinities between free and induced repressor for operator DNA; in vivo this difference should be sufficient for induction to occur.

DNA, Bacterial↗
PubMed1980

DNA methylation and gene function.

In most higher organisms, DNA is modified after synthesis by the enzymatic conversion of many cytosine residues to 5-methylcytosine. For several years, control of gene activity by DNA methylation has been recognized as a logically attractive possibility, but experimental support has proved elusive. However, there is now reason to believe, from recent studies, that DNA methylation is a key element in the hierarchy of control mechanisms that govern vertebrate gene function and differentiation.

Animals↗
PubMed1980

Chemical DNA synthesis and recombinant DNA studies.

Chemically synthesized DNA has been used in many recombinant DNA studies. These uses have included the total synthesis and cloning of functional genes, the cloning and expression of natural genes, and editing of changing genes by directed mutation.

Base Sequence↗
PubMed1980

Large-scale purification of two forms of active lac operator from plasmids.

A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.

Chromatography, Gel↗
PubMed1979

Methylation of DNA in mouse early embryos, teratocarcinoma cells and adult tissues of mouse and rabbit.

The distribution and amount of 5-methylcytosine (5-MeCyt) in DNA was measured for early embryos of mouse strain CF1 (2 to 4 cell stage to blastocyst) and mouse teratocarcinoma cells. In each case, the pattern of methylation was examined by use of the restriction enzymes Hha I and HPA II HPA II, which cut DNA at the sites 5'GCGC and 5'CCGG respectively, when the cytosines at these sites are not methylated. Mouse embryo DNA was found to have the same level of methylation as adult mouse tissues, and no changes in methylation were seen during differentiation of the teratocarcinoma cells. The ratio of 5-MeCyt/Cyt in DNA was measured by high performance liquid chromatography for the differentiating teratocarcinoma cells and for several adult mouse and rabbit tissues. The variation between tissues or between teratocarcinoma cells at different stages of differentiation was less than 10 percent. These results are discussed in view of proposals that 5-MeCyt plays a role in differentiation.

Animals↗