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Biomedical subjects

H Garoff

Publications and source records attributed to H Garoff.

At least 91 records · Page 5Linked to original sources
PubMed1982

Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells.

The three structural proteins of Semliki Forest virus--i.e., the capsid, p62, and E1 proteins--were expressed in baby hamster kidney cells from cloned DNA transcribed from the virus-specific 4.1-kilobase mRNA. The cDNA was engineered into an expression vector developed by others [Mulligan, R. C. & Berg, P. (1980) Science 209, 1422--1427] downstream from the simian virus 40 early promoter and was introduced into cell nuclei by microneedle injection. Immunofluorescence analysis of injected cells showed that the capsid protein was located in the cell cytoplasm, whereas the membrane proteins were associated with cellular membranes. The p62 protein was shown to be transported from the rough endoplasmic reticulum to the plasma membrane, whereas the E1 protein remained in the rough endoplasmic reticulum.

Animals↗
PubMed1982

Nucleotide sequence at the junction between the nonstructural and the structural genes of the semliki forest virus genome.

The nucleotide sequence at the junction between the nonstructural and the structural genes of the Semliki Forest virus 42S RNA genome has been determined from cloned cDNA. With the aid of S1-mapping, we have located the 5' end of the viral 26S RNA on this sequence. The 26S RNA is homologous to the 3' end of the 42S RNA and is used as a messenger for the structural proteins of the virus. The nucleotide sequence in the noncoding 5' region of the 26S RNA (51 bases) was thus established, completing the primary structure of the 26S RNA molecule (for earlier sequence work, see Garoff et al., Proc. Natl. Acad. Sci. U.S.A. 77:6376-6380, 1980, and Garoff et al., Nature (London) 288:236-241, 1980). An examination of the nucleotide sequences upstream from the initiator codon for the structural proteins on the 42S RNA genome shows that all reading frames are effectively blocked by stop codons, which means that the nonstructural genes in the 5' end of the 42S RNA molecule do not overlap with the structural ones at the 3' end of the molecule.

Base Sequence↗
PubMed1981

cDNA clone coding for part of a mouse H-2d major histocompatibility antigen.

mRNA coding for mouse major transplantation antigens of the d haplotype was partially purified, copied into double-stranded cDNA, and cloned in Escherichia coli. Clones were selected by their ability to hybridize specifically with mRNA coding for H-2K, D, or L antigens. One of these clones, pH-2d-1, carries a 1200-base-pair insert, comprising the noncoding region, including poly(A) at the 3' end and part of the coding region. A partial sequence of the latter region showed extensive homology with the known amino acid sequences of H-2Kb,Kk, and HLA-B7 antigens. From this comparison, it appears that the coding region extends from amino acid 133 in the second domain, through the third domain, to the cytoplasmic COOH-terminal region. A stretch of 24 hydrophobic or uncharged residues, located 31 amino acids from the COOH-terminal end, could represent the segment that spans the membrane. This is followed on the cytoplasmic side of the membrane by a cluster of basic amino acids and a possible phosphorylation site on a threonine residue.

Amino Acid Sequence↗
PubMed1980

A subcloning strategy for DNA sequence analysis.

We describe here a new strategy of fragment preparation for sequencing procedures using endlabelled DNA fragments as substrates (2,3) which is directly applicable to DNA fragments cloned into the Pst I site of pBR322, or in modified form, to inserts into the BamH I or Sal I site of the same plasmid. Ordered sets of subclones of predetermined overlap are are generated. These can be sequenced directly without further strand- or fragment separation steps.

Base Sequence↗
PubMed1980

The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region.

The amino acid sequence of the capsid (C) protein was deduced from the nucleotide sequence of the C gene. This part of the viral 42S RNA genome was transcribed into double-stranded cDNA. The cDNA was cloned in the Escherichia coli chi 1776-pBR322 host-vector system and then the base sequence was determined with the technique described by Maxam and Gilbert. The amino acid sequence of the C protein shows a clustering of basic amino acids and prolines within the first 110 amino acids.

Amino Acid Sequence↗
PubMed1980

Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell.

In Semliki Forest virus (SFV)-infected cells, all structural proteins are translated from a 26S mRNA using a single initiation site. The capsid protein which is made first is released into the cytoplasm whereas the two membrane proteins, p62 (the precursor for E2 and E3) and E1, are inserted into the rough endoplasmic reticulum membrane. Based on gradient centrifugation and cross-linking studies, it can be seen that the p62 and E1 polypeptides form a complex immediately after synthesis and migrate to the plasma membrane in the form of a p62-E1 complex. The processing of p62 to E2 and E3 is first seen 25 to 30 min after a 10 min pulse of radioactive amino acids. This cleavage can be inhibited by addition of antisera specific for E1 and E3, thus supporting the view that, as in the case of the related Sindbis virus, this cleavage occurs on the external face of the plasma membrane. Proteolytic digestion of crude vesicle preparations derived from plasma membranes, combined with peptide mapping, indicate that the carboxy-terminal end of E2 spans the cell plasma membrane, there being a portion of mol. wt about 3000 located towards the cytosol.

Animals↗
PubMed1979

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin.

Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2Dd histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence H-2Kd antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2Dd and H-2Kd antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. beta 2-Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, beta-microglobulin can bind to newly synthesized H-2d glycoproteins. The mRNAs coding for beta 2-microglobulin and H-2Dd antigen could be separated on aqueous sucrose gradients.

Animals↗