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D V Goeddel

Publications and source records attributed to D V Goeddel.

177 records · Page 10Linked to original sources
PubMed1977

Studies on gene control regions. 2. Enzymatic joining of chemically synthesized lactose operator deoxyribonucleic acid segments.

The T4 polynucleotide ligase catalyzed joining of six chemically synthesized deoxypolynucleotides corresponding to lactose operator DNA has been investigated. Joining was studied using various combinations of segments. Joining reactions involving multiple sites and the formation of duplex operator DNA were complete in a few hours. Joining reactions involving a single site and the formation of only one strand of operator DNA required several days and repeated annealing in order to go to completion. These studies have permitted the synthesis on a preparative scale (several nanomoles) of operator duplexes and operator single strands.

Coliphages↗
PubMed1977

Cloning of chemically synthesized lactose operators.

Recombinant DNA molecules, constructed from the ColE1-Mk5 hybrid plasmid PMB9 and a chemically synthesized wild-type lactose operator segment, have been used to transform Escherichia coli. Up to 10% of the transformants (selected for the tetracycline-resistance property of PMB9) are partially constitutive for the lactose operon enzyme beta-galactosidase. In vitro studies demonstrate that these partially constitutive transformants contain plasmid DNA molecules which carry one or more lactose operators, and which will bind purified lactose repressor. Preliminary results with some modified operator sequences are also presented.

Coliphages↗
PubMed1977

Binding of synthetic lactose operator DNAs to lactose represessors.

The nitrocellulose filter assay was used to study the interactions of wild-type (SQ) and tight-binding (QX86) lac repressors with synthetic lac operators 21 and 26 base pairs long. The repressor binding properties of both operators were very similar, indicating that both contain the same specific repressor recognition sites. The repressor-operator association rate constants (k(a)) were more sensitive than dissociation rate constants (k(d)) to changes in ionic strength. The responses of both k(a) and k(d) to ionic strength were relatively small compared to the effects previously observed with lambdah80dlac as operator DNA. These results suggest that under natural conditions there are electrostatic interactions between lac repressor and DNA regions outside of the 26 base pair operator sequence. Association rate constants for SQ repressor with either operator are higher than have been predicted for diffusion-limited reactions. We postulate that long-range electrostatic attractions between repressor and operator accelerate the association reaction. The presence of nonoperator DNA decreased association rate constants, the effect being more noticeable at an ionic strength of 0.05 M than at 0.20 M. Nonoperator DNA reduced k(a) values for associations involving QX86 repressor to a greater extent than for those with SQ repressor. The two types of repressors also had different rate constants for interactions with synthetic operators. The values for k(a) and k(d) were both higher with SQ repressor than with QX86 repressor. However, the rate constants were more sensitive to ionic strength when the repressor used was QX86.

Chemical Phenomena↗
PubMed1977

Studies of gene control regions. III. Binding of synthetic and modified synthetic lac operator DNAs to lactose repressor.

Chemically synthesized lactose operator DNA was tested for binding with lactose repressor protein. These operator DNAs were found to (1) bind specifically to lactose SQ repressor as measured by release of binding with the inducing ligand isopropyl-beta-D-thiogalactoside, (2) have dissociation half-lives of 37 seconds (21 base-paired duplex) and 46 seconds (26 base-paired duplex) and (3) have dissociation half-lives with x86 repressor of 9 minutes (21 base-paired duplex) and 18 minutes (26 base paired duplex). Modified operators containing 5-bromodeoxyuridine and deoxyuridine at specific sites were also prepared. These analogs bound both repressors about as tightly as the wild type sequence.

Base Sequence↗
PubMedDate not supplied

Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene.

Analysis of the organization and nucleotide sequence of two human loci related to the transforming gene of Kirsten murine sarcoma virus establishes one as a functional gene and the other as a processed pseudogene. The two final coding exons of the functional gene seem to have arisen by duplication. Differentially spliced mRNAs incorporating one or other of the duplicated exons probably served as the intermediates by which the viral transforming gene and the pseudogene were generated. This suggests that the functional gene may specify either of two related polypeptides depending on the pattern of RNA splicing.

Amino Acid Sequence↗
PubMedDate not supplied

Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations.

Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.

Alleles↗
PubMedDate not supplied

Comparative biochemical properties of normal and activated human ras p21 protein.

Human Ha-ras1 cDNAs encoding normal and activated p21 polypeptides have been efficiently expressed in Escherichia coli and the biochemical activities associated with each polypeptide compared. In addition to the guanine nucleotide binding activity, normal p21 displays a GTPase activity which is selectively impaired by a mutation which activates its oncogenic potential.

Cloning, Molecular↗
PubMedDate not supplied

Biological properties of human c-Ha-ras1 genes mutated at codon 12.

Vertebrate genomes contain proto-oncogenes whose enhanced expression or alteration by mutation seems to be involved in the development of naturally occurring tumours. These activated genes, usually assayed by their ability to induce the malignant transformation of NIH 3T3 cells, are frequently related to the ras oncogene of Harvey (Ha-ras) or Kirsten (Ki-ras) murine sarcoma viruses, or a third member of this family (N-ras). Activation involves point mutation which often affect codon 12 (refs 16-26) of the encoded 21,000-molecular weight polypeptide (p21). To provide insight into structural requirements involved in p21 activation, we have now constructed 20 mutant c-Ha-ras1 genes by in vitro mutagenesis, each encoding a different amino acid at codon 12. Analysis of rat fibroblasts transfected with these altered genes demonstrates that all amino acids except glycine (which is encoded by normal cellular ras genes) and proline at position 12 activate p21, suggesting a requirement for an alpha-helical structure in this region of the polypeptide. The morphological phenotype of cells transformed by the activated genes can, however, depend on the particular amino acid at this position.

Amino Acid Sequence↗
PubMedDate not supplied

Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin.

Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, a lymphokine that has similar biological properties. Recombinant tumour necrosis factor can be obtained by expression of its complementary DNA in Escherichia coli and induces the haemorrhagic necrosis of transplanted methylcholanthrene-induced sarcomas in syngeneic mice.

Amino Acid Sequence↗
PubMedDate not supplied

Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells.

The partial amino-acid sequence of purified human transforming growth factor-beta (TGF-beta) was used to identify a series of cDNA clones encoding the protein. The cDNA sequence indicates that the 112-amino acid monomeric form of the natural TGF-beta homodimer is derived proteolytically from a much longer precursor polypeptide which may be secreted. TGF-beta messenger RNA is synthesized in various normal and transformed cells.

Amino Acid Sequence↗
PubMedDate not supplied

Tumour necrosis factors alpha and beta inhibit virus replication and synergize with interferons.

Tumour necrosis factor (TNF) and lymphotoxin were initially described as tumoricidal proteins that are produced by activated macrophages and lymphocytes, respectively. Since TNF and lymphotoxin are structurally related, bind to the same cell surface receptor and have indistinguishable biological activities, they have been designated as TNF-alpha and TNF-beta, respectively. The multiple activities of these molecules indicate their importance in immunoregulative responses. Here we report that both TNF-alpha and TNF-beta have antiviral activity and synergize with interferons (IFNs) in the induction of resistance to both RNA and DNA virus infection in diverse cell types. These effects of TNFs are not due to the induction of IFN synthesis. Virus-infected cells are selectively killed by TNFs and this activity is accelerated by IFN-gamma. The production of TNFs is induced by viruses, further suggesting the importance of TNFs in the physiological antiviral response.

Animals↗
PubMedDate not supplied

Sequence, genomic organization, and chromosome localization of the mouse TRADD gene.

Signals triggered by tumor necrosis factor (TNF) are mediated by its two receptors: the 55 kDa TNF receptor one (TNF-R1) and the 75 kDa TNF receptor two (TNF-R2). Activation of TNF-R1 induces cell death, NF-kappa B activation, inflammatory response and anti-viral activity, while TNF-R2 mainly stimulates cell proliferation and NF-kappa B activation. The TNF-R2-associated factor TRAF2 has been shown to mediate activation of NF-kappa B by TNF-R2 and CD40. The human TNF-R1-associated death domain protein (TRADD) induces cell death and NF-kappa B activation when overexpressed. Here we describe the cloning and gene structure of the mouse homolog of TRADD. Mouse TRADD shares 75% overall amino acid sequence identity with human TRADD, suggesting high conservation of function. Mouse TRADD specifically interacts with wild type TNF-R1 but not with a truncated mutant TNF-R1 lacking its C-terminal 20 amino acids. Like human TRADD, mouse TRADD also induces activation of NF-kappa B and cell death. The expression of TRADD in mouse embryo appears developmentally regulated. The mTRADD gene contains four exons, with the fourth exon encoding all of the death domain. The mouse TRADD gene was localized to the distal region of chromosome 8.

Amino Acid Sequence↗