Biomedical subjects

R Z Florkiewicz

Publications and source records attributed to R Z Florkiewicz.

25 records · Page 2Linked to original sources

PubMed1989

Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons.

Human basic fibroblast growth factor (bFGF) is an angiogenic polypeptide mitogen present in a wide variety of mesoderm- and neuroectoderm-derived tissues. bFGF cDNA and genomic clones predict a 17.8-kDa (155-amino acid) gene product based on the presence of a single putative translational initiator ATG codon. However, a bFGF protein isolated from human placenta contains two additional amino acids NH2-terminal to the predicted initiator methionine. We report here that the human cell line SK-HEP-1 coexpresses four molecular forms (17.8, 22.5, 23.1, and 24.2 kDa) of bFGF. The 17.8-kDa bFGF protein is translationally initiated at the previously predicted methionine (AUG) codon, whereas the 22.5-, 23.1-, and 24.2-kDa proteins initiate at unusual non-AUG codons. The higher molecular weight forms are colinear NH2-terminal extensions of the 18-kDa bFGF.

Animals↗

PubMed1987

An internalized amino-terminal signal sequence retains full activity in vivo but not in vitro.

Internalization of the signal sequence of the vesicular stomatitis virus glycoprotein was accomplished by extending the amino-terminal coding sequence with sequences derived from pBR322. Such constructs were then expressed in eukaryotic cells. It was found that amino-terminal extensions consisting of 20, 61, or 102 amino acids totally unrelated to any signal peptide affected neither the function nor cleavage of the signal sequence in vivo. Subsequent transport of the glycoprotein was also not affected. Although the internalized signals functioned with wild-type efficiency in vivo, membrane insertion in vitro (as determined by proteolysis protection assays), signal cleavage, and glycosylation were only achieved when the amino-terminal presequences were short.

Amino Acid Sequence↗

PubMed1986

Secretion of phytohemagglutinin by monkey COS cells.

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.

Animals↗

PubMed1985

A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

Animals↗

PubMed1984

A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH.

A stable cell line expressing a complementary DNA clone encoding the vesicular stomatitis virus glycoprotein fused and formed polykaryons at pH 5.5. The formation of polykaryons was dependent on the presence of glycoprotein anchored at the cell surface and could be prevented by incubation of cells with a monoclonal antibody to the glycoprotein. Fusion occurred at a pH 0.5 unit lower than that observed for cells infected with vesicular stomatitis virus.

Animals↗

PubMed1983

Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein.

We have characterized two stable transformed mouse cell lines (CG1 and CTG1) that express either the normal vesicular stomatitis virus glycoprotein (G) or a truncated form of the G protein (TG) that lacks the COOH-terminal anchor sequences and is secreted from the cells. These cell lines were obtained using a hybrid vector consisting of the transforming DNA fragment of bovine papilloma virus linked to a segment of the SV40 expression vector pSV2 containing cloned cDNA encoding either the normal or truncated form of the vesicular stomatitis virus G protein. Using indirect immunofluorescence we have found that greater than 95% of the cells in each line express the G protein(s), although the level of expression within the population is variable. The normal G protein expressed in these cells obtains its complex oligosaccharides in less than 30 min and is transported to the cell surface. In contrast, the TG protein obtains its complex oligosaccharides with a half-time of about 2.5 h. Immunofluorescence data show an apparent concentration of the TG protein in the rough endoplasmic reticulum. These data together suggest that transfer of this anchorless protein from the rough endoplasmic reticulum to the Golgi apparatus is the rate-limiting step in its secretion. We observed, in addition to normal G protein, two smaller G-related proteins produced in the CG1 cell line. We suggest that these proteins could result from aberrant splicing from sites within the G mRNA sequence to the downstream acceptor in the pSV2 vector.

Amino Acid Sequence↗

PubMed1980

Sequence of picornavirus RNAs containing a radioiodinated 5'-linked peptide reveals a conserved 5' sequence.

Virion RNA (vRNA) from poliovirus type 1 (PV1), poliovirus type 2 (PV2), and coxsackie virus B1 (Cox B1) were treated with proteinase K to remove all but a small peptide of the covalently attached 5' genome-linked virion protein (VPg). The peptide on these RNA molecules was then treated with Bolton-Hunter 125I reagent, which iodinates primary amine groups, in order to obtain specific 5'-terminal radioactive labeling. Sequences of 125I-labeled vRNAs were determined by using a set of base-specific RNases and a partial alkaline hydrolysis "ladder." The first 20 positions of these RNAs show a remarkable conservation of sequence. The initial 10 nucleotides are identical in PV1, PV2, and Cox B1, with the sequence VPg-pU-U-A-A-A-A-C-A-G-C. The next 10 nucleotides show a one-base difference between PV1 and PV2 and 50% homology between PV1 and Cox B1. This conserved 5' region may provide a recognition site for interaction between the viral mRNA and the host translation system.

Base Sequence↗