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W Gilbert

Publications and source records attributed to W Gilbert.

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

Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells.

DNA can be encapsulated into lipid vesicles formed by sonication. The presence of a basic protein, lysozyme, enhances the incorporation 100-fold above the level expected by random trapping. This is demonstrated by the ability of the lipid vesicles to protect DNA from digestion with DNase. Such an enhancement of nuclei acid incorporation into vesicles by basic polypeptides and the sharply increased concentration of these macromolecules in the internal volume may have been advantageous in prebiotic evolution.

Biological Evolution↗
PubMed1986

Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate.

The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase is the carboxylate group of Glu-165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone phosphate or the 2-proton of (R)-glyceraldehyde 3-phosphate to yield the cis-enediol intermediate. Using the methods of site-directed mutagenesis, we have replaced Glu-165 by Asp. The three enzymes chicken isomerase from chicken muscle, wild-type chicken isomerase expressed in Escherichia coli, and mutant (Glu-165 to Asp) chicken isomerase expressed in E. coli have each been purified to homogeneity. The specific catalytic activities of the two wild-type isomerases are identical, while the specific activity of the mutant enzyme is reduced by a factor of about 1000. The observed kinetic differences do not derive from a change in mechanism in which the aspartate of the mutant enzyme acts as a general base through an intervening water molecule, because the D2O solvent isotope effects and the stoichiometries of inactivation with bromohydroxyacetone phosphate are identical for the wild-type and mutant enzymes. Using the range of isotopic experiments that were used to delineate the free-energy profile of the wild-type chicken enzyme, we here derive the complete energetics of the reaction catalyzed by the mutant protein. Comparison of the reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected. Each of the proton abstraction steps is about 1000-fold slower in the mutant enzyme. Evidently, the excision of a methylene group from the side chain of the essential glutamate has little effect on the free energies of the intermediate states but dramatically reduces the stabilities of the transition states for the chemical steps in the catalyzed reaction.

Aspartic Acid↗
PubMed1986

The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence.

We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase expressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six introns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals.

Animals↗
PubMed1985

B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo.

The mouse heavy chain immunoglobulin gene contains a tissue-specific enhancer. The enhancer and flanking sequences were studied in vivo by carrying out dimethyl sulfate protection experiments on living cells, in combination with genomic sequencing. Relative to reactions on naked DNA, there are changes (protections and enhancements) in the reactivity of guanine residues to dimethyl sulfate within the enhancer sequence in myeloma, B, and early B cells, whereas virtually no alterations appear in cells of non-B lineage. Most of the affected residues are in four clusters, in sequences homologous to the octamer 5'CAGGTGGC 3' (C, cytosine; A, adenine; G. guanine; T, thymine). The alterations in the pattern of G reactivity are consistent with the tissue-specific binding of molecules to the mouse immunoglobulin heavy chain enhancer.

Animals↗
PubMed1985

Rous sarcoma virus encodes a transcriptional activator.

Rous sarcoma virus expresses a transcriptional activator that affects the LTR as well as other promoters. We discern this activity as a stimulation of the transient expression of an LTR-promoted hybrid transcriptional unit and also of the rat preproinsulin II gene in transfected NIH 3T3 cells. We map the activity to an alternate reading frame in the p19-p10 region of the gag gene and identify a mRNA whose spliced structure would direct translation of this reading frame from the Pr76gag initiation codon. This mRNA probably differs from genomic RNA only by the 282 nucleotide splice. The predicted translation product is a 124 residue polypeptide; the first six amino acids arise from gag. The target for the action of this transcriptional modulator at the LTR lies between 111 and 620 nucleotides upstream of the cap site.

Animals↗
PubMed1985

Intron/exon structure of the chicken pyruvate kinase gene.

The chicken pyruvate kinase gene is interrupted by at least ten introns, including nine introns within the coding region. We compare the structure of this gene with the three-dimensional protein structure of the homologous cat muscle enzyme. The introns are not randomly placed--they divide the coding sequence into fairly uniformly sized pieces encoding discrete elements of secondary structure. The introns tend to fall at interruptions between stretches of alpha-helix or beta-sheet residues, and each of the six exons that contribute to the barrel-shaped central domain include one or two repeats of a simple unit, an alpha-helix plus a beta strand. This structure suggests that introns were not inserted into a previously uninterrupted coding sequence, but instead are products of the evolution of the first pyruvate kinase gene. We have found some sequence homology between a segment of pyruvate kinase and the structurally homologous mononucleotide binding fold of alcohol dehydrogenase. The superposition of these two regions aligns an intron from the maize alcohol dehydrogenase gene four nucleotides from an intron in the chicken pyruvate kinase gene.

Amino Acid Sequence↗
PubMed1985

Chicken triosephosphate isomerase complements an Escherichia coli deficiency.

We present the sequence of full-length chicken triosephosphate isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) mRNA based on the analysis of cDNA and genomic clones. To isolate cDNA clones encoding the enzyme, we screened a muscle cDNA library with radioactively labeled cDNA made from RNA that had been enriched by immunoselection of polysomes. We blocked the signal caused by contaminating species in the probe with cloned DNA corresponding to the contaminants. Screening a chicken genomic library with cDNA coding for triosephosphate isomerase led to the isolation of phage containing the entire gene, which we used to map the transcriptional start. When placed downstream from a hybrid trp-lac promoter, the cDNA encoding the chicken enzyme programs the synthesis of functional protein, as judged by enzymatic criteria and by complementation of an Escherichia coli mutant that is deficient in bacterial triosephosphate isomerase.

Animals↗
PubMed1985

Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.

Animals↗
PubMed1984

Antibodies of the secondary response can be expressed without switch recombination in normal mouse B cells.

A few percent of mouse splenocytes express isotypes characteristic of the secondary response together with IgM, and some cells express these isotypes alone. We isolated populations of small memory cells that express (i) IgM but not IgG1, (ii) IgM but not IgA, (iii) IgM and IgG1, (iv) IgM and IgA, and (v) IgG1 but not IgM. We have analyzed their DNA to show that there has been no switch recombination or deletion in the Ig constant region (C) genes. Using sandwich RNA hybridizations, we have found that cells expressing IgG1 contain nuclear RNAs that have both C mu and C gamma 1 sequences, and that cells expressing IgA contain nuclear RNAs that have both C mu and C alpha sequences. We propose that the expression of an isotype characteristic of the secondary response in memory cells is accomplished by alternative RNA processing of large (up to 180 kilobases) nuclear RNA transcripts that span the heavy chain gene locus.

Animals↗
PubMed1984

Genomic sequencing.

Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.

Animals↗
PubMed1984

Free and integrated recombinant murine leukemia virus DNAs appear in preleukemic thymuses of AKR/J mice.

We studied the appearance and structure of murine leukemia viral genomes in preleukemic AKR/J mice by Southern hybridization. Up to an average of one to two copies per thymocyte of unintegrated murine leukemia virus DNA appears in the thymuses of preleukemic mice beginning at 4 to 5 months of age and disappears in leukemic thymuses. The free viral genomes are absent in the spleens, livers, and brains of preleukemic mice. Using a series of ecotropic and nonecotropic murine leukemia virus hybridization probes, we showed that the unintegrated viral genomes are structurally analogous to those of recombinant mink cell focus-forming viruses that appear as proviruses in leukemic AKR thymocytes, suggesting that these free viral DNAs are the direct precursors to the leukemia-specific proviruses. The mosaic of ecotropic and nonecotropic sequences within these unintegrated viral DNAs varies from one preleukemic thymus to another but often appears structurally homogeneous within individual thymuses, indicating that often each thymus was being infected by a unique mink cell focus-forming virus. Analysis of high-molecular-weight DNA shows that recombinant proviruses reside in the chromosomal DNA of thymocytes within the preleukemic thymus, with the number rising to an average of several copies per thymocyte, but we do not detect any preferred integration sites. These results suggest that, in general, before the development of thymic leukemias in AKR mice there is a massive infection by a unique mink cell focus-forming virus which then integrates into many different sites of individual thymocytes, one of which grows out to become a tumor.

Animals↗
PubMed1983

Comparison of the methylation patterns of the two rat insulin genes.

We have investigated whether DNA methylation is involved in regulating the expression of the rat insulin genes. We studied cytosine methylation in and near the two insulin genes to examine the correlation of site-specific methylation with expression in different tissues and to compare the demethylation patterns of the two genes. For both genes, we found certain sites undermethylated only in insulin-producing tissues. However, the overall patterns of the two genes in expressing tissues are quite different. The insulin I gene is significantly less methylated than the insulin II gene in a tumor that makes equal amounts of the two insulin RNAs. In another tumor and cell line that make 5-fold higher levels of insulin I RNA than insulin II RNA, the changes in the methylation levels of the two genes do not correlate with the increased expression of insulin I. In nonexpressing tissues, the insulin I gene is completely methylated, while the insulin II gene is demethylated at a number of sites. Thus, the general level of methylation of the insulin genes does not correlate with their differential expression. Furthermore, the methylation at specific sites does not correlate either; some CG dinucleotides that appear to be important for one gene are not present in the other. Our results suggest that there is no specific control by methylation of the expression of the insulin genes in the rat.

Animals↗
PubMed1983

Isolation and mapping of cDNA hybridization probes specific for ecotropic and nonecotropic murine leukemia proviruses.

To study the structure of murine leukemia proviruses in AKR mice by Southern hybridization, we have isolated and mapped ecotropic AKV and nonecotropic MCF-derived cDNA restriction fragments. The ecotropic-specific probes originate from four regions of the AKV genome which include the corresponding recombinant region of two MCF viruses (VI-36 and 247). We also isolated two nonecotropic probes from the recombinant region of MCF V1-36. The probes were characterized by (i) mapping of restriction fragments at the 3' end of AKV and MCF V1-36 by a two-dimensional gel strategy, (ii) hybridization of restriction fragments to the related viral RNA genomes followed by electrophoresis, (iii) two-dimensional fingerprinting of single-stranded restriction fragments, and (iv) DNA sequence analysis of the ecotropic probes. The ecotropic AKV and nonecotropic MCF probes discriminate between two populations of endogenous murine leukemia viruses and show that the MCF viruses are not present in the germ line of AKR mice.

AKR murine leukemia virus↗