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N Sarver

Publications and source records attributed to N Sarver.

At least 37 records · Page 2Linked to original sources
PubMed1991

Ribozymes: a new frontier in anti-HIV strategy.

A recent addition to the arsenal of antiviral agents are small, single-stranded catalytic RNAs (ribozymes) capable of specific destruction of target RNAs, leading to their functional inactivation. These dual features--substrate specificity inherent in nucleic acid complementarity coupled with catalytic degradation of the bound substrate--are analogous to sophisticated antisense oligonucleotides having restriction enzyme properties. Ribozymes fulfill certain criteria critical to the design of effective antiviral agents in (1) having the ability to interrupt a critical step early in the virus replication cycle before the virus has extensively damaged cellular machinery; (2) having sufficient specificity to interfere with critical viral functions without affecting host processes; and (3) having activity against a variety of viral strains generated throughout the course of the infection, especially for highly mutable pathogens, such as HIV. Several reports have now demonstrated the capability of molecularly tailored ribozymes to block gene expression by specifically cleaving the cognate RNA transcript.

Antiviral Agents↗
PubMed1991

Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells.

Expression of the human immunodeficiency virus type 1 nef gene was studied by in vitro transcription-translation and by transfection into monkey COS cells. Two Nef-related peptides, of 27 and 25 kDa, were identified by immunoprecipitation with anti-Nef antibodies. The relation between these two proteins was determined by metabolically labeling transfected COS cells and by deleting the initiator methionine of nef. We found that the 25-kDa polypeptide is not a cleavage product of 27-kDa Nef but rather is initiated from an internal ATG 57 bases downstream from the Nef initiation site. Myristoylation of the 27-kDa but not of the 25-kDa Nef was demonstrated by the contranslational modification of Nef in an in vitro reticulocyte translation system. The myristoylation pattern of the two Nef polypeptides further implies that the 25-kDa polypeptide lacks the amino terminus of 27-kDa Nef. Cellular localization of the various forms of Nef was studied in transiently transfected COS cells. Myristoylation was found to be necessary for membrane association of Nef. Myristoylation-deficient 27-kDa Nef mutant and 25-kDa Nef were confined to the soluble cytoplasmic fraction of transfected cells, whereas part of the wild-type 27-kDa Nef was membrane attached.

Animals↗
PubMed1990

Ribozymes as potential anti-HIV-1 therapeutic agents.

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1990

RNA enzymes (ribozymes) as antiviral therapeutic agents.

Among the landmark discoveries of recent years are ribozymes, RNA molecules which possess enzymatic, self-cleaving activities. The concept of exploiting the ribozyme catalytic center for cleaving (inactivating) a specific RNA transcript is now emerging as a potential therapeutic or preventative strategy in human diseases, veterinary medicine and agriculture. Linked to the catalytic center of the ribozyme are RNA sequences which are complementary to, and thus serve to target the ribozyme to, a unique RNA sequence. Specific association of the ribozyme with its target via base pairing, cleavage of the RNA substrate and subsequent recycling of the ribozyme make these catalytic RNA molecules attractive as antiviral agents. Theoretically, ribozymes can be adapted for the destruction of any RNA species, whatever its origin.

Animals↗
PubMed1990

Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.

Amino Acid Sequence↗
PubMed1988

Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells.

Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35-45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF-producing cells formed small, non-progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.

Animals↗
PubMed1988

Utilization of an Epstein-Barr virus replicon as a eukaryotic expression vector.

Epstein-Barr virus (EBV) replicons which include the genetic element oriP and a functional gene for Epstein-Barr nuclear antigen (EBNA-1) can be maintained episomally in a variety of mammalian cell lines [Yates et al., Nature 313 (1985) 812-815]. We have assessed the application of an EBV replicon for foreign gene expression. Two cDNAs, human interferon-gamma (IFN-gamma) and the extracellular domain of the human epidermal growth factor receptor (EGF-Rex), cloned in an EBV replicon, were efficiently expressed and the protein was secreted into the extracellular media. Expression in human embryonic 293 cells was approximately ten-fold higher than in CV-1 cells. The expression of the human protein is dependent upon the orientation of the IFN-gamma transcriptional cassette relative to the other genetic elements within the vector.

Animals↗
PubMed1987

Optimizing gene expression in BPV-transformed cells: effects of cell type on enhancer/promoter interaction.

We have compared several combinations of enhancers and promoters in expressing the chloramphenicol acetyl transferase gene in transient assays, in mouse C127, the most widely used host cell for the bovine papilloma virus (BPV) expression vector. Of the various combinations tested, the unit comprised of the SV40 enhancer and adenovirus type 2 major late promoter (MLP) was the most active in BPV transformed C127 cells. We further demonstrate that untransformed and BPV transformed C127 cells respond differently to the various enhancer/promoter combinations tested. Moving the SV40 enhancer closer to the cap site of a complete MLP (from -414 to -107) reduced potentiation to less than half in BPV transformed cells. The level of potentiation with enhancer at either site was similar in human HeLa cells. In BPV transformed C127 cells, the SV40 enhancer and the MLP (at the -414 site) supports 4-5 fold greater levels of expression than the murine sarcoma virus (MSV) enhancer/mouse metallothionein (MT) promoter which has previously been extremely effective in BPV vectors. Our findings provide a basis for the improvement of the BPV vector system in supporting increased levels of expression of proteins of important therapeutic application.

Acetyltransferases↗
PubMed1987

Stable expression of recombinant factor VIII molecules using a bovine papillomavirus vector.

The bleeding disorder in hemophilia A results from a deficiency or abnormality of Factor VIII (FVIII), a member of the coagulation cascade. FVIII is a large glycoprotein (approximately 350,000 daltons) that is activated by a series of proteolytic cleavages. During activation, a large internal domain (B domain) is removed, resulting in an active complex comprised of the amino and carboxyl subunits of the parental molecule. Using a bovine papillomavirus expression vector system, we have established stable, genetically engineered cell lines harboring either full-length FVIII cDNA or variant FVIII cDNA (delta FVIII), the latter containing an extensive deletion in the region encoding the B domain. We demonstrate that the two recombinant FVIII molecules manifest the biological attributes of native FVIII. Relative to full-length FVIII transformants, cells harboring delta FVIII cDNA are five to eight times more efficient in expressing coagulant activity. This difference is due to a post-transcriptional event.

Bovine papillomavirus 1↗
PubMed1985

Enhancer-dependent expression of the rat preproinsulin gene in bovine papillomavirus type 1 vectors.

The effect of position in a bovine papillomavirus type 1 (BPV-1) vector on foreign gene expression was assessed with the rat preproinsulin (rI1) gene. The rI1 gene was inserted at each of the BPV-1/pML2d junctions in either transcriptional orientation in derivatives of the pdBPV-1(142-6) vector which consists of the BamHI linear genome of BPV-1 DNA cloned into pML2d. Transformed lines of C127 cells were established and assayed for rI1 gene expression. Cells containing the rI1 gene at the 3' end of the BPV-1 transforming region expressed rat proinsulin, whereas cells with the gene at the 5' end of the nontransforming region did not. Variability in the plasmid copy number or in the extent of DNA rearrangement could not account for this difference. We conclude that the expression of the rat preproinsulin gene (which is normally tissue specific for pancreatic islet cells) in C127 cells depends on the transcriptional activation afforded by viral enhancer sequences located at the 3' end of the transforming region. Intervening BPV-1 or pML2d sequences appear to block this enhancer-mediated gene activation. In agreement with enhancer-dependent activation, a rat preproinsulin gene located in a blocked position (i.e., not adjacent to the BPV-1 enhancer) could be activated by the insertion of a DNA fragment containing the simian virus 40, Moloney murine sarcoma virus, or BPV-1 enhancer element adjacent to the rI1 gene. Thus, a gene which is normally not expressed in a particular cell may be activated when placed adjacent to a viral enhancer in a BPV-1 vector.

Animals↗
PubMed1984

Localization and analysis of bovine papillomavirus type 1 transforming functions.

Bovine papillomavirus type 1 (BPV-1) or cloned BPV-1 DNA can transform susceptible rodent cells, and the viral DNA remains as a stable extrachromosomal plasmid in the transformed cells. The transforming region of the BPV-1 genome has previously been localized to a specific fragment comprising 69% of the genome, which also contains the elements sufficient for extrachromosomal plasmid maintenance. To define more precisely the viral DNA sequences which are involved in cellular transformation, we have tested the ability of defined deletion mutants of BPV-1 DNA to morphologically transform mouse C127 cells. Cells containing the mutated DNAs have been examined for anchorage independence and tumorigenicity in nude mice. Several distinct regions of the BPV-1 genome were found to influence expression of the viral transformation functions. A transcriptional regulatory region located in the noncoding region 5' to the early open reading frames is essential for transcriptional activity and transformation. A transcriptional enhancer element, located 3' to the polyadenylation site for the viral RNAs expressed in transformed cells, has previously been shown to be essential for transformation (Lusky et al., Mol. Cell. Biol., 3:1108-1122, 1983). Deletion mutants affecting the E2 open reading frame, particularly the NH2 half, are significantly impaired in their ability to transform, suggesting that the E2 gene product is an important transforming protein of BPV-1. Mutants lacking the E6 and E7 open reading frames are still able to induce transformation but at a lowered efficiency, and the transformants have altered characteristics. Mutations localized within the E1 open reading frame do not significantly affect the transforming functions but result in the integration of the viral genome in the transformed cells, implicating the E1 gene product in stable plasmid replication and maintenance.

Animals↗
PubMed1982

Transformation and replication in mouse cells of a bovine papillomavirus--pML2 plasmid vector that can be rescued in bacteria.

The unique ability of bovine papillomavirus (BPV-1) DNA to replicate as a stable, multicopy plasmid in transformed mouse cells has led to its utilization as a eukaryotic cloning vector. One limitation of the system has been the marked reduction in transformation efficiency when BPV-1 DNA or the subgenomic transforming segment of BPV-1 DNA (BPV69T) is covalently linked to pBR322 sequences. A dual host replicon consisting of BPV-1 DNA and pML2d, a deletion variant of pBR322, was constructed and shown to be highly efficient for transformation of mouse cells in vitro. The hybrid molecule replicates as a stable, unintegrated, multicopy plasmid in transformed mouse cells. The resident BPV-1-pML2d plasmid DNA was rescued in bacteria and the recovered plasmids were shown to be identical in structure and to have the same transformation efficiency as the original transforming DNA. In contrast, the transforming efficiency of BPV69T DNA is less than 1/100th that of BPV-1 DNA when the DNA is left covalently linked to pML2d. These observations indicate that, although the nontransforming region of BPV-1 (BPV31NT) DNA is not essential for transformation, it has a facilitative role in the transformation process.

Animals↗
PubMed1981

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

Animals↗