Biomedical subjects
Publications and source records attributed to W Gilbert.
We report the sequence of the single chicken triosephosphate isomerase gene and its flanking regions. The 3-kilobase-long gene is composed of seven similarly sized exons and six introns. By using crystallographic and sequence data, we argue that this ancient gene was originally assembled from the genetic antecedents of exons.
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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.
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.
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.
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.
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.
We present the 9312 nucleotide sequence of the Prague C (Pr-C) strain of Rous sarcoma virus (RSV). A comparison of known protein sequences with the nucleotide sequence allows assignment of the coding regions for the gag, pol, env and src genes. The gag gene is terminated by an amber stop codon and is contained within a different reading frame than is the pol gene. The pol and env genes overlap. The sequences surrounding the src gene in the Pr-C and Schmidt-Ruppin (SR-A) strains of RSV have been compared, and they reveal that an element, E, of approximately 153 nucleotides is present on the 3' side of the src gene in Pr-C, and on the 5' side in SR-A. We hypothesize that E was part of a duplicated region of over 250 nucleotides flanking the src gene in an ancestral RSV, and that differential deletion of one copy of E led to its positional difference in Pr-C and SR-A.
We have determined the cDNA sequence corresponding to chicken muscle pyruvate kinase mRNA; the predicted coding region spans 529 amino acids and establishes the complete amino acid sequence for the vertebrate enzyme. We demonstrate that the level of mRNA for this enzyme is under developmental control and suggest a structural model for the protein kinase-mediated regulation of the mammalian liver isozyme. We report a method for the direct analysis of, and the preparation of cDNA probes from, mRNA which has been fractionated on methylmercury/agarose gels.
We have examined the patterns of heavy-chain immunoglobulin gene rearrangement and provirus integration in seven murine leukemia virus-induced thymic leukemias from AKR/J mice. In five of the seven tumors examined, there were between two and five rearrangements of the heavy-chain immunoglobulin J (JH) segment. Except for one case, the rearranged JH segments are present in less than one copy per cell, indicating that these tumors contain subpopulations of thymocytes with differing JH rearrangements. Nevertheless, each tumor is probably of monoclonal origin because the cells in a given tumor contain a common set of randomly integrated murine leukemia proviruses. Our results indicate that the JH segments rearranged within a few cell divisions after tumor cell proliferation began and may, therefore, identify a specific stage in T-cell differentiation when tumorigenesis occurs.
We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.
We report the nucleotide sequence of the 3' half of the ecotropic murine leukemia virus AKV genome. To obtain a preliminary sequence, we developed a sequencing strategy whereby a nested set of restriction fragments is chemically modified prior to gel purification and strand scission. The sequence defines the genetic map of the 3' half of AKV and locates recombinant regions previously identified in structural analyses of MCF viruses.
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We study the biosynthesis of preproinsulin and proinsulin in Escherichia coli by pulse-chase experiments. When E. coli is transformed with plasmids bearing a gene consisting of a fusion of preproinsulin DNA to part or to all of the DNA encoding a bacterial signal sequence, a variety of hybrid preproinsulin molecules can be made. Molecules with largely complete hybrid signal sequences are secreted into the periplasm. Molecules with defective signal sequences, lacking the hydrophobic core, are not secreted and remain in the cytoplasm. Such molecules are degraded with a 2-min half-life, whereas the molecules transported to the periplasm are at least 10 times more stable. A full-length preproinsulin precursor appears transiently before the signal sequence is cleaved off by the bacterial signal peptidase, and we propose a model to account for this.
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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.