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D V Goeddel

Publications and source records attributed to D V Goeddel.

At least 127 records · Page 7Linked to original sources
PubMed1987

Structure and nucleotide sequence of a Drosophila melanogaster protein kinase C gene.

Genomic and cDNA clones encoding a Drosophila melanogaster protein kinase C (PKC) homologue were identified using a bovine PKC cDNA probe. The cDNA clones contain a single open reading frame that encodes a 639 amino acid, 75-kd protein having extensive homology with bovine, human and rat PKC and homology with the kinase domains of other serine, threonine and tyrosine kinases. The Drosophila PKC gene is localized to region 53E of chromosome 2. The gene spans approximately 20 kb and contains at least 14 exons. Messenger RNA for PKC could not be detected in 0-3 h Drosophila embryos. Adult flies contain three PKC transcripts of 4.3, 4.0 and 2.4 kb.

Amino Acid Sequence↗
PubMed1987

Heterologous expression and characterization of the human R-ras gene product.

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.

Alleles↗
PubMed1986

Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta.

A human interleukin 1 beta (IL 1 beta) cDNA probe was utilized to identify a homologous murine cDNA clone. The murine cDNA encodes a 269-residue protein which is 67% homologous to human IL 1 beta. The murine sequence was engineered for expression in mammalian cells and directs the synthesis of biologically active IL 1. This protein, termed murine IL 1 beta, is only 22% homologous with the previously described murine IL 1 sequence. Both IL 1 alpha and IL 1 beta are encoded by single genes, but IL 1 beta mRNA is about fivefold more abundant in a stimulated macrophage cell line.

Amino Acid Sequence↗
PubMed1986

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes.

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.

Animals↗
PubMed1986

Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation.

Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar.

Animals↗
PubMed1986

The murine transforming growth factor-beta precursor.

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.

Amino Acid Sequence↗
PubMed1985

Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization.

Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent).

Amino Acid Sequence↗
PubMed1985

Human tumor necrosis factor. Production, purification, and characterization.

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.

Amino Acid Sequence↗
PubMed1985

Structure and chromosomal localization of the human lymphotoxin gene.

We have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a 32P-labeled 116 bp synthetic DNA fragment whose sequence was based on the NH2-terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human-murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified.

Amino Acid Sequence↗
PubMed1985

Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.

A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids. The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein. There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site. The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the partition function of Escherichia coli plasmid ColE1.

The DNA nucleotide sequence of a 382-bp Hpa II fragment containing cer (ColE1 resolution) function responsible for ColE1 plasmid stability in dividing Escherichia coli was determined. The partition (par) region of pSC101 and the cer region have similar biological functions, as they both maintain plasmid stability through plasmid monomerization. Both regions contain 40- to 70-bp hairpin-loop structures that resemble bidirectional transcription terminators and share sequence homology with each other. Deletion mapping of the cer fragment shows that sequences extending beyond both sides of the terminator-like structure are also involved in the plasmid partition process.

Bacteriocin Plasmids↗
PubMed1985

Two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides.

The classical human interferon-alpha (HuIFN-alpha) gene family is estimated to consist of 15 or more nonallelic members which encode proteins sharing greater than 77% amino acid sequence homology. Low-stringency hybridization with a HuIFN-alpha cDNA probe permitted the isolation of two distinct classes of bovine IFN-alpha genes. The first subfamily (class I) is more closely related to the known HuIFN-alpha genes than to the second subfamily (class II) of bovine IFN-alpha genes. Extensive analysis of the human genome has revealed a HuIFN-alpha gene subfamily corresponding to the class II bovine IFN-alpha genes. The class I human and bovine IFN-alpha genes encode mature IFN polypeptides of 165 to 166 amino acids, whereas the class II IFN-alpha genes encode 172 amino acid proteins. Expression in Escherichia coli of members of both gene subfamilies results in polypeptides having potent antiviral activity. In contrast to previous studies which found no evidence of class II IFN-alpha protein or mRNA expression, we demonstrate that the class I and class II IFN-alpha genes are coordinately induced in response to viral infection.

Animals↗
PubMed1985

Interferon-gamma enhances induction of lymphotoxin in recombinant interleukin 2-stimulated peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant human interleukin 2 (rIL 2) to secrete lymphotoxin (LT) and interferon-gamma (IFN-gamma). Induction of both LT and IFN-gamma by rIL 2 is regulated at the transcriptional level. Treatment of PBMC with rIL 2 in combination with recombinant human IFN-gamma (rIFN-gamma) resulted in an earlier appearance of LT mRNA and an augmented production of LT than did rIL 2 treatment alone. IFN-gamma alone had no effect on production of either LT or its mRNA. PBMC cultured in the presence of rIL 2 plus anti-rIFN-gamma resulted in decreased LT production. Only a 15-min incubation of PBMC with rIL 2 is needed to stimulate LT production, whereas 3 hr is necessary for IFN-gamma production. These results suggest that rIL 2, in addition to being a T cell growth factor, may exhibit other activities through induction of LT and IFN-gamma.

Animals↗
PubMed1985

Effect of interleukin 2, interferon-gamma, and mitogens on the production of tumor necrosis factors alpha and beta.

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant interleukin 2 and mitogens to secrete two distinct cytotoxic polypeptides, tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta), previously called lymphotoxin. Treatment of PBMC with recombinant human interleukin 2 (rIL 2) or mitogens in combination with recombinant human interferon-gamma (rIFN-gamma) resulted in augmented production of both TNF-alpha and TNF-beta. rIFN-gamma alone had no effect on production of either cytotoxic polypeptide. TNF-alpha was produced within 2 to 3 hr after induction and was the major cytotoxin produced by PBMC during the first 48 hr of culture, after which time TNF-beta became the predominant species. TNF-beta was first secreted into the media after 8 hr of induction. Enhanced levels of both TNF-alpha and TNF-beta were seen when the PBMC were separated into adherent and nonadherent cells. Both TNF-alpha and TNF-beta were induced in different tumor cell lines of hematopoietic origin. The results demonstrate that the production of TNF-alpha and TNF-beta can be enhanced by two lymphokines, IL 2 and IFN-gamma.

Animals↗
PubMed1984

The amino acid sequence of murine p53 determined from a c-DNA clone.

A c-DNA clone containing the complete sequence information for the murine p53 protein, from embryonal carcinoma cells, has been isolated. The nucleotide sequence of this clone reveals an open reading frame encoding a protein of 390 amino acids with a molecular weight of 43,364 Da. The NH2-terminal domain of this protein is acidic whereas the carboxyl terminus is rich in basic amino acid residues. These terminal domains are separated by a proline-rich, hydrophobic run of amino acids. Proline comprises approximately 10% of the total amino acid residues. Two tryptic peptides, derived from p53 protein radiolabeled with either methionine or proline, were purified and the position of these labeled residues in the peptide was determined. The positions of three methionine and five proline residues in these two peptides matched the amino acid sequence of the predicted open reading frame determined from the c-DNA clone.

Amino Acid Sequence↗
PubMed1984

Human transforming growth factor-alpha: precursor structure and expression in E. coli.

Transforming growth factor-alpha (TGF-alpha) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-alpha from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-alpha mRNA. A cloned TGF-alpha cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-alpha produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.

Adenocarcinoma↗