Biomedical subjects

J M Hardwick

Publications and source records attributed to J M Hardwick.

62 records · Page 4Linked to original sources

PubMed1988

A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen.

Several Epstein-Barr virus (EBV) early promoters respond to a new EBV transactivator encoded by BRLF1, designated R. Transactivation was measured in chloramphenicol acetyltransferase assays on Raji, BHK, and Vero cells that were cotransfected with the transactivator and target promoters linked to the cat gene. The divergent promoter of BamHI-H was particularly responsive to R transactivation. This large promoter region consists of a leftward TATA box for the NotI repeat gene (BHLF1) and a probable rightward TATA box for the EA-R gene (BHRF1) separated by 940 base pairs of unusual sequence complexity. Sequences within this divergent promoter region appear to confer inducibility by EBV transactivators R and Z (BZLF1). The Z transactivator stimulated expression in both the leftward and rightward directions, and R stimulated expression primarily in the rightward direction, but the MS transactivator (BMLF1) had no activity in either direction. The adenovirus E3 promoter also responded to the R transactivator, but several other herpesvirus and human promoters were nonresponsive. When the divergent promoter was linked to the EA-R gene as it is in the EBV genome, the R and Z transactivators also induced the expression of EA-R in cotransfected cells. This cytoplasmic early antigen is encoded by BHRF1 and may be anchored in intracellular membranes by a carboxy-terminal transmembrane region.

Adenoviridae↗

PubMed1986

Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.

Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface.

Amino Acid Sequence↗

PubMed1984

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.

Affinity Labels↗

PubMed1983

Alterations in the transport and processing of Rous sarcoma virus envelope glycoproteins mutated in the signal and anchor regions.

The env gene of Rous sarcoma virus codes for two glycoproteins which are located on the surface of infectious virions. Subcloning of these coding sequences in the place of the late region of SV40 DNA has allowed the expression of a normally glycosylated, functionally active glycoprotein complex on the surface of monkey cells. Through the use of site-directed mutagenesis, the role of specific amino acids in the signal peptide, signal peptidase cleavage site, and membrane anchor region have been investigated. Amino-terminal mutations have shown that deletion of the signal peptidase cleavage site along with one or two amino acids of the hydrophobic signal peptide results in the synthesis of an unglycosylated, uncleaved, and presumably cytoplasmically located precursor. Nevertheless, changing the signal peptidase cleavage site from ala/asp to ala/asn does not block the translocation of the glycoprotein across the membrane or the action of the peptidase. At the other end of the molecule, carboxy-terminal mutations have shown that the deletion of the hydrophobic membrane anchor region is not sufficient for the secretion of the truncated glycoprotein. Interpretations of these results based on recent models for protein transport and secretion are discussed.

Amino Acid Sequence↗

PubMed1981

Rous sarcoma virus mutant LA3382 is defective in virion glycoprotein assembly.

LA3382 is a temperature-sensitive replication-defective mutant of Rous sarcoma virus that contains four active mutations. In this study we performed experiments to determine the biochemical defect that blocks the synthesis of infections virus late in the replication cycle. At the nonpermissive temperature (41 degrees C) cells infected with LA3382 synthesized virus particles which were noninfectious and exhibited significant reductions in the amounts of gp85 and gp37 present in the virions. An analysis of the intracellular viral polypeptides indicated that the precursor of the viral glycoproteins (Pr95) were synthesized normally but underwent cleavage at a reduced rate at the restrictive temperature. Pr95 did not accumulate in infected cells ans was inserted into mutant virions at 41 degrees C; however, Pr95 was cleaved in such a way that gp85 was released from the viruses and could be detected in the supernatant medium by immunoprecipitation. The virus-free glycoprotein was indistinguishable from wild-type gp85 and may have been released due to an anomalous cleavage. Pulse-chase experiments also indicated that the Pr180 polyprotein precursor of the reverse transcriptase was cleaved to the active form of the enzyme more slowly at 41 degrees C in LA3382-infected cells. This accounted for the twofold lower level of polymerase activity found in mutant virions at 41 degrees C, defect which probably did not account for the observed 20- to 50-fold reduction in infectivity. Furthermore, the replication defect was not complemented by an env deletion mutant Rous sarcoma virus [RSV(-)[, which should complement a pol defect. Therefore, we conclude that the major lesion that impairs replication in LA3382 is within the env gene.

Avian Sarcoma Viruses↗

PubMed1979

Role of bilateral adrenalectomy (and oophorectomy) in the management of patients with metastatic breast cancer.

One hundred consecutive patients who underwent bilateral adrenalectomy for metastatic breast cancer with at least a 2 year "tumor-free" interval and with symptomatic bony, local-regional, pleural, or discrete pulmonary or mediastinal metastases had objective remission rates of 65 per cent at 6 months, 48 per cent at 12 months, 28 per cent at 18 months, and 16 per cent at 24 months.

Adrenalectomy↗

PubMed1979

Disulfide bonds in Sendai virus glycoproteins.

The precursor of fusion protein (Fo) in Sendai virus growth in Vero cells can be cleaved by trypsin to forms F1 and F2, which can be resolved on SDS-polyacrylamide gels. However, if disulfide bonds are preserved during electrophoresis, F1 and F2 remain linked together even after trypsin treatment (F). Sendai virus growth in embryonated chicken eggs does not contain the precursor Fo. However, an F protein was found for Sendai virus grown in eggs when disulfide bonds were preserved during electrophoresis. The hemagglutinin-neuraminidase (HN) glycoproteins also appear to be disulfide-linked to form large complexes which are observed on SDS-polyacrylamide gels of nonreduced samples.

Chemical Phenomena↗

PubMed1978

Glycoproteins of measles virus under reducing and nonreducing conditions.

Measles virus has two glycoproteins. The larger glycoprotein (HA) is composed of 76,000-dalton subunits that are bound by disulfide bonds. The smaller glycoprotein (F) appears to contain a glucosamine-rich portion that is linked to an unglycosylated protein by disulfide bonds.

Electrophoresis, Polyacrylamide Gel↗