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Biomedical subjects

W Gilbert

Publications and source records attributed to W Gilbert.

15 recordsLinked to original sources
PubMed1979

Hepatitis B virus genes and their expression in E. coli.

A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.

Amino Acids↗
PubMed1979

The structure and evolution of the two nonallelic rat preproinsulin genes.

In the rat, there are two nonallelic genes for preproinsulin. The insulin end products are very similar and are equally expressed. We have isolated clones carrying these genes and their flanking sequences, and characterized them by DNA sequencing and electron microscopic analysis. We have established the primary structure of the preproinsulin mRNAs and the signal peptides of these two proteins. One of the genes contains two introns: a 499 bp intron interrupting the region encoding the connecting peptide and a 119 bp intron interrupting the segment encoding the 5 noncoding region of the mRNA. The introns are transcribed and present in a preproinsulin mRNA precursor. The other gene possesses the smaller, but not the larger, of the two introns. Calculations based on the divergence of the two preproinsulin nucleotide and amino acid sequences indicate that these genes are the products of a recent duplication. Thus one of the genes gained or lost an intron since that time.

Amino Acid Sequence↗
PubMed1979

Pleiotropic mutations within two yeast mitochondrial cytochrome genes block mRNA processing.

The mRNAs from two yeast mitochondrial genes cob-box (cytochrome b) and oxi-3 (cytochrome oxidase 40,000 dalton subunit) are processed from large (7-10 kb) precursors. Certain mutations in each gene block the maturation of the RNAs from both genes at a variety of specific steps. The pleiotropic cytochrome b mutants seem to lack a functional trans-acting RNA required for the processing of both messengers. In contrast, the oxi-3 mutants may act by producing an activity that inhibits specific steps.

Cytochromes↗
PubMed1978

Molecular basis of base substitution hotspots in Escherichia coli.

In the lacI gene of Escherichia coli spontaneous base substituion hotspots occur at 5-methylcytosine residues. The hotspots disappear when the respective cytosines are not methylated. We suggest that the hotspots may result from the spontaneous deamination of 5-methylcytosine to thymine, which is not excised by the enzyme DNA-uracil glycosidase.

2-Aminopurine↗
PubMed1978

Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain.

We have determined the sequence of the DNA of a germ-line gene for the variable region of a mouse immunoglobulin light chain, the VlambdaII gene. The sequence confirms that the variable region gene lies on the DNA separated from the constant region. Hypervariable region codons appear in the germ-line sequence. A sequence for the hydrophobic leader, 19 amino acids that are cleaved from the amino terminus of the protein, appears near, but not continuous with, the light chain structural sequence: most of the leader sequence is separated from the rest of the gene by 93 bases of untranslated DNA.

Animals↗
PubMed1978

Immunological screening method to detect specific translation products.

We describe a very sensitive method to detect as antigens the presence of specific proteins within phage plaques or bacterial colonies. We coat plastic sheets with antibody molecules, expose the sheet to lysed bacteria so that a released antigen can bind, and then label the immobilized antigen with radioiodinated antibodies. Thus, the antigen is sandwiched between the antibodies attached to the plastic sheet and those carrying the radioactive label. Autoradiography then shows the positions of antigen-containing colonies or phage plaques. A few molecules of antigen released from each bacterial cell generatean adequate signal.

Antibody Specificity↗
PubMed1978

A bacterial clone synthesizing proinsulin.

We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli chi1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)-oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicillinase to the fourth amino acid, glutamine, of rat proinsulin.

Base Sequence↗
PubMed1977

Contacts between the lac repressor and the thymines in the lac operator.

We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.

Base Sequence↗
PubMed1977

A new method for sequencing DNA.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.

Adenine↗
PubMed1977

Rous sarcoma virus genome is terminally redundant: the 5' sequence.

When Rous sarcoma virus RNA is transcribed into DNA by the reverse transcriptase, a tRNA primer is elongated into DNA. The primer is near the 5' end of the virus genome; the first major DNA made is a "run-off" product extending 101 bases from the primer to the 5' end of the template. We have studied this DNA molecule to determine the sequence of the first 101 bases at the 5' end of the Rous sarcoma virus genome (Prague strain, subgroup C). Twenty-one bases at the extreme 5' end are also at the 3' end of the virus genome (see D. E. Schwartz, P. C. Zamecnik, and H. L. Weith, this issue, pp. 994-998), and thus this virus is terminally redundant. The existence of this sequence repetition immediately suggests mechanisms by which the growing DNA copy can jump from the 5' end to a 3' end of the template and become circular. The sequence also displays a possible ribosome binding site and enough secondary structure to permit a possible 5'-5' linkage of viral RNA molecules.

Avian Sarcoma Viruses↗
PubMed1977

Mapping adenines, guanines, and pyrimidines in RNA.

The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.

Adenine↗
PubMed1976

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

Coliphages↗