No abstract

Based on precedents from other retroviruses, the precursor of the human immunodeficiency virus (HIV-1) reverse transcriptase is predicted to be a polyprotein with a relative molecular mass (Mr) of 160,000 (160K) encoded by both the viral pol gene and the upstream gag gene. These two genes lie in different translational reading frames, with the 3' end of gag overlapping the 5' end of pol by 205 or 241 nucleotides. Thus, production of the gag-pol fusion protein would require either messenger RNA processing or translational frameshifting. The latter mechanism has been shown in the synthesis of the gag-pol proteins of two other retroviruses, Rous sarcoma virus (RSV) and mouse mammary tumour virus (MMTV). Here we report that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV-1 gag-pol overlap region. Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to a UUA leucine codon near the 5' end of the overlap.

Mutations in the human APC gene are linked to familial adenomatous polyposis and to the progression of sporadic colorectal and gastric tumors. To gain insight into APC function, APC-associated proteins were identified by immunoprecipitation experiments. Antibodies to APC precipitated a 95-kilodalton protein that was purified and identified by sequencing as beta-catenin, a protein that binds to the cell adhesion molecule E-cadherin. An antibody specific to beta-catenin also recognized the 95-kilodalton protein in the immunoprecipitates. These results suggest that APC is involved in cell adhesion.

The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5' direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

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Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone afactor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium. Previous studies on the expression of B-lactamase-proinsulin gene fusions in Escherichia coli showed that the /3-lactamase signal sequence is cleaved and proinsulin is secreted into the periplasm (4). We reasoned, therefore, that the leader sequences of secreted yeast proteins may allow more efficient processing and secretion of heterologous proteins by yeast.Most studies on secretion of yeast proteins have used enzymes such as invertase and acid phosphatase (2, 5, 6), which are secreted into the periplasmic space or cell wall.We have used the peptide mating pheromone a-factor, which is efficiently secreted into the medium. a-Factor is a 13-residue peptide, secreted by cells of the a mating type, that acts on cells of the opposite a mating type to promote efficient conjugation between the two cell types leading to the formation of aa diploid cells (7). Studies on the sequence of the afactor structural gene (8) and on the synthesis and processing of the a-factor peptide (9-11) have shown that a-factor is synthesized as a precursor of 165 amino acids containing an 83-residue leader and four a-factor coding regions, each preceded by a short spacer peptide. The leader and spacer amino acids appear to contain the signals necessary for proteolytic processing and secretion. A recent study (12) shows that fusion of the leader region of the a-factor precursor to invertase, another secreted yeast protein, directs its export.To investigate whether the a-factor leader sequences are sufficient to allow efficient processing and secretion, we have constructed plasmids in which the genes coding for foreign secreted proteins have been fused to the yeast a-factor gene and expressed under the control of the a-factor promoter. Several different constructions were made in which the

These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.