Useful proteins from recombinant bacteria.
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Animals↗
Biomedical subjects
W Gilbert
Publications and source records attributed to W Gilbert.
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↗
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↗
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↗
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↗
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↗
The synthesis of insulin in bacteria: a model for the production of medically useful proteins in prokaryotic cells.
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Amino Acid Sequence↗
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↗
DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator.
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Animals↗
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↗
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↗
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↗
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↗
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↗
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↗
Promotor region for yeast 5S ribosomal RNA.
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Base Sequence↗
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↗