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W Gilbert

Publications and source records attributed to W Gilbert.

At least 109 records · Page 6Linked to original sources
PubMed1981

Tissue-specific exposure of chromatin structure at the 5' terminus of the rat preproinsulin II gene.

The 5' terminus of the rat preproinsulin II gene exhibits a tissue-dependent DNase I sensitivity. Only in the chromatin froma pancreatic beta-cell tumor, but not in liver, spleen, kidney, or brain chromatin, is a region at and before the 5' end of the gene exposed to cleavage. The region of exposure extends 250-300 base pairs upstream from the 5' terminus of the preproinsulin mRNA. Such a region may allow control sequences special access to regulatory proteins.

Adenoma, Islet Cell↗
PubMed1981

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.

Antibody Specificity↗
PubMed1980

E. coli RNA polymerase interacts homologously with two different promoters.

We present and review experiments that identify points of close approach of the RNA polymerase to two promoters, lac UV5 and T7 A3. We identify the contacts to the phosphates along the DNA backbone, to the N7s of guanines in the major groove and the N3s of adenines in the minor groove, and to the methyl groups of thymines. These contacts to the two promoters are strikingly homologous in space, as shown on three-dimensional models, and identify major regions of interactions lying on one side of the DNA molecule (at -35 and -16), as well as further areas extending through the Pribnow box. Both promoters are unwound similarly by the polymerase, across a region of about twelve bases extending from the middle of the Pribnow box to just beyond the RNA start site. We discuss the areas of interaction in the context of promoter homologies and promoter mutations. The disposition of the contacts in space suggests a model for the pathway along which the RNA polymerase binds to promoters.

Adenine↗
PubMed1980

The evolution of genes: the chicken preproinsulin gene.

We have characterized a clone carrying a chicken preproinsulin gene, which is present in only one copy in the chicken genome. The gene contains two introns: a 3.5 kb intron interrupting the region encoding the connecting peptide and a 119 bp intron interrupting the DNA corresponding to the 5' non-coding region of the mRNA. This is similar to the structure of rat insulin gene II; therefore it represents the common ancestor. Since the rat insulin gene I lacks a 499 bp intron in the coding region, the rat genes have evolved by a recent gene duplication followed by loss of this intron in one copy. The divergences between insulin gene sequences, and also between globin genes, show that changes at introns and silent positions in coding regions appear very rapidly (7 X 10(-9) substitutions per nucleotide site per year), but that the accumulation of changes in these sites saturates, although not completely, after about 100 million years. From this we conclude that not all of these sites are neutral and that they do not behave as accurate evolutionary clocks over long periods of time. However, nucleotide substitutions leading to amino acid replacements are an excellent clock. Our analysis indicates that this clock is driven by selection.

Animals↗
PubMed1980

Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7.

Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Pribnow box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.

Base Sequence↗
PubMed1980

Eukaryotic signal sequence transports insulin antigen in Escherichia coli.

We made a series of plasmids with unique Pst restriction sites within or near the DNA that encodes the penicillinase signal sequence. Inserted DNA can be read in all three frames both within and immediately after the signal sequence. We cloned Pst-terminated DNA copies of the structural information for rat proinsulin and preproinsulin into these plasmids, forming a large number of hybrid penicillinase (bacterial) and insulin (eukaryotic) signal sequences. We then compared the levels of insulin antigen in the Escherichia coli periplasm with those inside the cells. We conclude that either the bacterial or the eukaryotic signal is sufficient to transport rat insulin antigen into the periplasmic space.

Amino Acid Sequence↗
PubMed1980

Bacteria mature preproinsulin to proinsulin.

By inserting the rat preproinsulin gene into the bacterial prepenicillinase gene, we formed a variety of hybrid bacterial-eukaryotic signal sequences attached to proinsulin. Among these were the four following constructions: rat proinsulin attached to the entire penicillinase signal sequence and rat preproinsulin fused to all of, to half of, or only to the first four amino acids of the bacterial signal sequence. In all four cases, more than 90% of the rat insulin antigen appeared in the periplasmic space. By immunoprecipitation and determination of the amino acid sequences of the radiolabeled products, we show that the bacteria correctly process both the bacterial and the eukaryotic signal sequences of these hybrid proteins. The cleavage of the eukaryotic signal by bacterial peptidase, in this case, generates proinsulin.

Amino Acid Sequence↗
PubMed1980

Chemical probes for higher-order structure in RNA.

Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution. Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing. The reactions work between 0 degrees C and 90 degrees C and at pH 4.5--8.5 in a variety of buffers. As an example we follow the progressive denaturation of yeast tRNAPhe terminally labeled with 32P as the tertiary and secondary structures sequentially melt out. A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved.

Alkylating Agents↗
PubMed1980

The structure of rat preproinsulin genes.

In rat there are two nonallelic insulins, I and II. We have cloned and sequenced double stranded cDNA copies of both preproinsulin mRNA I and II. Using the cloned sequence as probe, we established by the Southern blotting technique a restriction map of the two chromosomal genes. This map indicates that an intron exists within the insulin II gene. To examine this in more detail, we have isolated both genes from a library of rat DNA cloned in phage lambda. Restriction endonuclease analysis and direct DNA sequencing revealed that gene II contains two introns: a 490 base pair intron between the region encoding amino acids 38 and 39 of proinsulin, and a 119 base pair intron, which is 17 base pairs upstream from the initiation codon. Gene I is not interrupted within the protein coding region, but possesses an intron homologous to the 119 base pair intron of insulin II. We are studying the structure of insulin genes from other species to determine if the 490 base pair intron was lost or inserted in the duplicated gene. We have identified nuclear RNA molecules larger than preproinsulin mRNA which contain the transcribed intronic sequences. These molecules represent a new precursor in insulin biosynthesis.

Adenoma, Islet Cell↗
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↗