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J Sambrook

Publications and source records attributed to J Sambrook.

At least 55 records · Page 3Linked to original sources
PubMed1985

SV40 T antigen and the exocytotic pathway.

A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (approximately 5 X 10(6) molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. By contrast, HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains one potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.

Amino Acid Sequence↗
PubMed1985

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport.

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.

Animals↗
PubMed1985

An efficient method for introducing macromolecules into living cells.

The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.

Animals↗
PubMed1984

Cytotoxic T lymphocyte recognition of the influenza hemagglutinin gene product expressed by DNA-mediated gene transfer.

We have used the technique of DNA-mediated gene transfer to examine cytotoxic T lymphocyte (CTL) recognition of the product of the cloned A/JAPAN/305/57 hemagglutinin (HA) gene in murine (L929) cells. Using both heterogeneous and homogeneous (clonal) populations of type A influenza-specific CTL, we have demonstrated that the HA molecule can serve as a target antigen for both the subtype-specific and the cross-reactive subpopulations of influenza-specific CTL. Our results also raise the possibility that other virus-specified polypeptides may serve as target molecules for cross-reactive CTL.

Animals↗
PubMed1983

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

Animals↗
PubMed1982

Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein.

The DNA sequences encoding the amino-terminal signal peptide or the carboxy-terminal hydrophobic anchor have been deleted from a cloned gene coding for the haemagglutinin (HA) of influenza virus. The wild-type gene has previously been shown to be expressed with high efficiency from simian virus 40 (SV40)-HA recombinant vectors into a fully glycosylated protein that is displayed on the infected cell's surface in an antigenically and biologically active form. The anchor-minus HA also is glycosylated but is secreted efficiently into the medium. By contrast, the signal-minus HA is produced only at low levels, is not glycosylated and is located intracellularly.

Cell Compartmentation↗
PubMed1981

Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene.

By replacing either the eight early or the late genes of SV40 with a cloned copy of the influenza virus haemagglutinin gene we have constructed recombinant viruses which, in infected cells, express large quantities of haemagglutinin. This glycoprotein, over 10(8) molecules of which are produced per cell, is identical in molecular weight to authentic influenza virus haemagglutinin, accumulates at the cell surface and displays haemabsorbing activity.

Cloning, Molecular↗
PubMed1980

Integration of viral DNA sequences in cells transformed by adenovirus 2 or SV40.

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonculease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

Adenoviruses, Human↗
PubMed1980

Origin-defective mutants of SV40.

We have described a new technique for the isolation and propagation of nonconditionally lethal mutants. We have used this method to generate mutants of SV40 that contain a defective origin of DNA replication. Using these mutants, we have established the following: (1) SV40 DNA replication and early transcription are functionally separate. (2) A functional viral origin of DNA replication is not necessary for the maintenance of transformation. (3) The lack of the origin of SV40 DNA replication does not affect the efficiency of transformation when nonpermissive cells are transfected by DNA using the calcium technique.

Animals↗
PubMed1979

Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA.

Fiber mRNA of adenovirus 2 has been used as a template for RNA-dependent DNA polymerase. The resulting cDNA/RNA hybrids have been inserted at the Pst I site of the plasmid vector pBR322 after A:T tailing. One recombinant plasmid, pJAW 43, has been characterized in detail and shown to contain sequences from the main body of fiber mRNA, the three leaders common to most late adenoviral mRNAs and a fourth leader found in some species of fiber mRNA. The complete DNA sequence of the leader region has been determined and does not contain the initiation codon AUG, although this codon does occur immediately downstream from the junction between the fourth leader and the main body of the fiber mRNA. The first leader (map coordinate 16.6) is 41 nucleotides long, the second (from 19.6) is 71 nucleotides, the third (from 26.6) is 88 nucleotides and the fourth (from 78.5) is 181 nucleotides. The location of junctions between viral leaders and intervening sequences has been determined by reference, where possible, to sequences of the adenovirus 2 genome. Although the presence of short repeated sequences at the boundaries of intervening sequences and leaders makes it impossible to locate the splice point unambiguously, all of the leader-intervening sequence junctions can be arranged to stress a common feature--the presence of the dinucleotides GT and AG at the 5' and 3' ends, respectively, of the intervening sequences. This prototype sequence, which has also been recognized at or near the splice points in other eucaryotic systems, is possibly part of a larger unit which serves as a recognition site for specific excision-ligation events that ultimately lead to the production of mature mRNAs.

Adenoviruses, Human↗
PubMed1979

Transformation of rat cells by the hybrid virus Ad2(2+) HEY.

A set of four isogenic rat cell lines transformed by Ad22+ HEY have been studied. While all of the cell lines synthesize SV40 T antigen, only one expresses adenovirus 2 T antigen: none expresses SV40 V antigen or adenovirus 2 fibre antigen. Three cell lines contain 1 to 2 virus equivalents of SV40 and adenoviral sequences per diploid quantity of rate cell DNA and the fourth line contains five copies of SV40 and 20 copies of the adenovirus genome. At least three of the cell lines contain DNA sequences from the helper adenovirus 2 in addition to sequences from the Ad2+ HEY genome. The patterns of integrated virus sequences are complex suggesting multiple insertions of both adenovirus and SV40 DNA sequences. SV40 can be rescued from three cell lines by fusion with permissive cells.

Adenoviruses, Human↗
PubMed1978

The isolation of defective variants of simian virus 40 whose genomes contain sequences derived from adenovirus 2 DNA.

A new set of hybrid viruses has been isolated whose closed circular genomes 5 to 6 kB in size, contain DNA sequences derived in part from adenoviruses 2 and in part from SV40. The structure of these genomes is complex, but in the simplest case, analyses by restriction endonuclease digestion and hybridization indicate that the adenovirus 2 DNA is present as a continuous block, of maximum size 2.8 kB. Different hybrids contain sequences derived from different segments of the adenovirus 2 genome.

Adenoviridae↗