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D V Goeddel

Publications and source records attributed to D V Goeddel.

At least 145 records · Page 8Linked to original sources
PubMed1983

Expression of the human interferon-gamma cDNA in yeast.

Several plasmids which direct the expression of human interferon-gamma (IFN-gamma) cDNA in the yeast Saccharomyces cerevisiae under the transcriptional control of the 3-phosphoglycerate kinase (PGK) promoter have been constructed. Up to 25 x 10(6) units per liter culture of biologically active IFN-gamma are synthesized in yeast transformed with these expression plasmids. The amount of IFN-gamma mRNA in yeast transformed with the IFN-gamma expression plasmids is much lower than the amount of PGK mRNA present when the PGK gene and promoter are inserted on a similar plasmid. The major and three minor sites of transcription initiation have been localized. The major starting point of the IFN-gamma mRNA is at 4 basepairs upstream of the PGK mRNA start. All transcription starts map at an ApG as does PGK mRNA. The yeast IFN-gamma mRNA terminates in a specific segment of the 3' untranslated region of the IFN-gamma cDNA. The polyadenylation site at about 182-184 nucleotides 3' of the IFN-gamma stop codon is preceded by a sequence which is very similar to a sequence proposed to be involved in transcription termination and polyadenylation (49).

Cloning, Molecular↗
PubMed1983

Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue.

DNA sequence analysis of the activated oncogene from the T24 human bladder carcinoma line and two alleles of its normal cellular progenitor (c-Ha-ras-1) indicates that the genes encompass at least four exons, and that only a single point mutation residing within the first exon distinguishes the coding region of both alleles of the normal gene from their activated counterpart. Both versions of the gene encode a protein which is predicted to differ from the corresponding viral gene product at three amino acid residues, one of which was previously shown to represent the major site of phosphorylation of the viral polypeptide.

Base Sequence↗
PubMed1983

Human immune interferon gene is located on chromosome 12.

A cDNA clone for human immune interferon (IFN-gamma) gene sequences, plasmid p69, was used to chromosomally map the IFN-gamma gene by detecting human IFN-gamma gene sequences in DNA isolated from human-rodent somatic cell hybrids. We were able to map the IFN-gamma gene by correlating the human chromosomes present in these hybrids with the human specific 8.8 and 2.0 kilobase pair fragments produced by EcoRI digestion of genomic DNA. Southern blot analysis of 37 hybrid cell lines indicated that the gene for IFN-gamma was on human chromosome 12. A hybrid containing a portion of chromosome 12 localized the IFN-gamma gene to the p1205 leads to qter region.

Animals↗
PubMed1983

Secretion of human interferons by yeast.

Plasmids were constructed to direct synthesis of the human interferons IFN-alpha 1, IFN-alpha 2, and IFN-gamma in the yeast Saccharomyces cerevisiae. Expression of IFN genes containing coding sequences for secretion signals resulted in the secretion of IFN activity. A large proportion of the IFN-alpha 1 and IFN-alpha 2 isolated from the yeast cell growth media had the same amino termini as the natural mature interferons, suggesting a removal of the signal sequences identical to that of human cells. These results show that a lower eukaryote, such as yeast, can utilize and process a human signal sequence.

Cloning, Molecular↗
PubMed1983

Rabies virus glycoprotein analogs: biosynthesis in Escherichia coli.

The surface of rabies virus is composed of an approximately 60,000 dalton glycoprotein, in which most of the antigenic and immunogenic determinants of the virus reside. We have constructed plasmids for the direct expression in Escherichia coli of the mature full length rabies glycoprotein gene and also for the expression of a glycoprotein gene which has been truncated to exclude the coding region for a hydrophobic, possibly transmembrane, domain of the protein. Escherichia coli harboring the plasmids synthesize analog proteins which conform by several biochemical and antigenic criteria to rabies glycoprotein.

Amino Acid Sequence↗
PubMed1983

Human immune interferon (IFN-gamma) gene sequence and structure.

Human IFN-gamma is encoded by a single gene which, in contrast to the IFN-alpha and IFN-beta genes, contains three introns. This gene has been isolated from a recombinant human-gamma gene library, and the entire gene and 5' and 3' flanking regions have been sequenced. Southern hybridizations failed to show any polymorphism of the gene or its flanking regions. The first intron contains a repetitive element which is unrelated to the Alu family repeat.

Amino Acid Sequence↗
PubMed1983

Cloning and expression of murine immune interferon cDNA.

The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136 amino acids long, 10 amino acids shorter than human IFN-gamma. The nucleotide homology between the murine and human IFN-gamma genes is 60-65%, whereas the encoded proteins are only 40% homologous. Murine IFN-gamma cDNA was expressed in Escherichia coli under trp promoter control.

Amino Acid Sequence↗
PubMed1983

Efficient bacterial expression of bovine and porcine growth hormones.

cDNAs prepared using poly(A)mRNA from pituitaries and containing the coding sequences for bovine and porcine growth hormones (bGH and pGH) were cloned in bacteria. The primary structures of the peptide hormones derived from the nucleotide sequences of the respective cDNAs show approximately 90% homology. The cloned cDNAs were modified using synthetic DNA to construct expression vectors for efficient bacterial production of the mature animal growth hormones.

Animals↗
PubMed1982

Structure of the human immune interferon gene.

Sequence determination of cloned cDNAs and genes of the three classes of interferon (IFN-alpha, -beta and -gamma) has revealed more than a dozen members of the human IFN-alpha gene family and a single gene for IFN-beta. These genes are found on chromosome 9 and contain no introns. We recently reported that the 146-amino acid sequence of mature IFN-gamma deduced from the nucleotide sequence of a cloned cDNA was quite unrelated to those of the other IFNs, and that the gene for IFN-gamma contains at least one intron. We now describe the isolation, characterization and DNA sequence of the human IFN-gamma gene. It contains three introns, a repetitive DNA element, and is not highly polymorphic. All our evidence to date and the present data suggest that this is the only gene for IFN-gamma and that the resolution of IFN-gamma into two components is probably the result of post-translational processing of the protein.

Amino Acid Sequence↗
PubMed1982

Human interferon gamma is encoded by a single class of mRNA.

Polyadenylated RNA from human peripheral blood lymphocytes and spleen cells, treated with different inducers for IFN-gamma production, was fractionated on denaturing sucrose gradients. Two IFN-gamma mRNA peaks at 12S and 16S were consistently observed. Nucleotide sequence analysis of cDNA clones showed that the 12S IFN-gamma mRNA from the different sources is identical to the gel fractionated 18S IFN-gamma mRNA which gave rise to the IFN-gamma cDNA clone p69 (1). Nucleotide sequence analysis of several IFN-gamma cDNA clones showed the presence of a CGA (Arg) codon at position 140 of mature IFN-gamma in contrast with the CAA (Gln) codon, which is found in p69 (1). Specifically primed cDNA extension on total induced polyadenylated RNA revealed the presence of a single mRNA species having a 5' untranslated region of 125-130 nucleotides. The nucleotide sequence of this region has been obtained. These data suggest that a single human IFN-gamma gene, which has very little polymorphism, gives rise to a single size class of mRNA.

Base Sequence↗
PubMed1982

Increased synthesis in E. coli of fibroblast and leukocyte interferons through alterations in ribosome binding sites.

Eleven chimeric plasmids have been constructed which direct the synthesis of mature human fibroblast (IFN-beta 1) or leukocyte interferon (IFN-alpha A) proteins under the control of the E. coli trp promoter. The plasmids differ with respect to the nucleotide spacing between the Shine-Dalgarno sequence of the trp leader and the ATG translation start signal of the interferon genes. By utilizing a unique Xba I endonuclease site located within the spacer region of the expression plasmids, the spacings were altered from 2-10 nucleotides or 7-15 nucleotides for the fibroblast and leukocyte interferon expression plasmids, respectively. The optimal spacing for expression, as determined by interferon assay, is 9 nucleotides for both types of transcripts, despite differences in nucleotide sequence within the spacer region and downstream from the AUG initiator. Yields of IFN-alpha A varied about six-fold, while among the different IFN-beta 1 expression plasmids a range of more than 100-fold in interferon production was observed. The difference in the range of variation between the IFN-alpha A and IFN-beta 1 plasmids is attributed partly to changes in messenger RNA secondary structure within the ribosome binding sites which affect message half-life.

Binding Sites↗
PubMed1982

Carboxyterminal region of hybrid leukocyte interferons affects antiviral specificity.

Four hybrid human leukocyte interferon (IFN-alpha) genes have been constructed and expressed in Escherichia coli using molecular cloning methods. Plasmids containing genes encoding human interferons, IFN-alpha A, IFN-alpha D, IFN-alpha I and several hybrids of the different IFN-alpha genes (formed by in vitro recombination at common restriction endonuclease sites located within the DNA sequence encoding mature polypeptides) joined identically to an E. coli trp promoter gave rise to bacterial-produced interferons with distinctly different antiviral activities. The expression plasmid which directs the synthesis of IFN-alpha I was constructed using a gene isolated from a human genomic library in a manner similar to the previous expression of IFN-alpha A and IFN-alpha D cDNA clones. The use of a cell-free transcription-translation system has allowed the calculation of the specific activities of IFN-alpha made from isolated DNA fragments containing these hybrid IFN-alpha genes. These bacterially-derived interferons vary considerably in their ability to inhibit vesicular stomatitis virus (VSV) in different mammalian cells. The results show that the cloned hybrid interferons have unique antiviral activities when compared with the parent interferons, and they demonstrate that more active IFN-alpha s can be made using recombinant DNA techniques.

Amino Acid Sequence↗
PubMed1981

Antiviral activities of hybrids of two major human leukocyte interferons.

Four hybrid human leukocyte interferon (LeIF or IFN-alpha) genes have been constructed by in vitro recombination of LeIF-A (IFN-alpha 2) and LeIF-D (IFN-alpha 1) genes at common restriction endonuclease sites located within their coding regions. These hybrid genes have been expressed in E. coli under trp promoter control. The interferons produced [LeIF-AD (BglII), -AD (PvuII), -DA (BglII), -DA (PvuII)] have antiviral properties distinct from the parental molecules LeIF-A and -D, varying considerably in their abilities to inhibit plaque formation by different viruses in a range of mammalian cells. All six of the cloned LeIFs exhibit the heat stability, pH 2 stability and antigenic specificity of natural leukocyte interferons.

Animals↗
PubMed1981

Expression of a human gene for interferon in yeast.

A DNA sequence coding for mature human leukocyte interferon D (LeIF-D) was linked with DNA fragments of the 5'-flanking sequences of the Saccharomyces cerevisiae (yeast) alcohol dehydrogenase I gene in a plasmid capable of autonomous replication and selection in both yeast and Escherichia coli. Yeast cells transformed by these plasmids synthesize up to 1 x 10(6) molecules of biologically active LeIF-D per cell.

Alcohol Oxidoreductases↗