Biomedical subjects
Publications and source records attributed to B J Carter.
This study examines biopsychological responses and background practices of 182 military personnel participating in a simulated chemical defense warfare scenario. The study explores the relationship between demographic, training, biopsychological response, and termination variables. Common features of dropouts are described.
We used rep+ and rep- recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant (genr) cells containing integrated vectors. The genr cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep- vector gave high CAT expression whereas only 50% of those from the rep+ vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep+ or rep- vector expressed CAT, and several clones from the rep+ vector gave very high yields. We also analyzed integrated rep+ vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAV-CAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p40 promoter (as well as p5 and p19) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector (rep+ or rep-) but not continued expression of rep, and possibly global effects of the surrounding chromatin.
Adeno-associated virus (AAV) vectors readily express the gene for geneticin-resistance under control of the AAV p40 promoter when chromosomally integrated at low copy number in mammalian cells. We show that a truncated AAV rep gene, transcribed from the p5 and p19 promoters, mediates a negative effect on expression of geneticin-resistance in human 293 cells and a positive effect in HeLa cells. Also, we describe a novel phenotype for a mutant expressing the p19 rep gene alone which has a negative effect in 293 cells but no positive effect in HeLa cells.
We studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p40 promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p40. When the rep gene was present in cis or in trans, cat expression from p40 was decreased 3- to 10-fold, but there was a 2- to 4-fold increase in the level of p40 mRNA. Conversely, cat expression increased and the p40 mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p40 mRNA levels. We also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p5 promoter in a fashion independent of rep.
Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.
A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.
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We used a recombinant plasmid containing an adeno-associated virus (AAV) genome to construct several vectors which express the gene for chloramphenicol acetyltransferase (CAT). We transfected four different AAV-CAT vectors into human 293 (adenovirus-transformed) cells and analyzed CAT activity. We show that, for vectors using the AAV p40 and p19 promoter, the chimeric AAV-CAT transcripts began from the correct 5' position but the basal level of CAT expression depended in part on the structure of the transcript. We also examined the effects of coinfection of the cells with the helper adenovirus or cotransfection with a plasmid which expressed the adenovirus translational control RNA, VA1 RNA. Cotransfection with plasmids containing the gene for VA1 RNA resulted in elevated levels of CAT activity. VA1 RNA stimulated translation of the chimeric mRNA. However, in two cases, the VA1 RNA apparently decreased the level of mRNA. These results suggest that in addition to its function in translation, VA1 RNA acts at a second site to alter cytoplasmic accumulation of some mRNAs. Infection with adenovirus increased CAT activity several-fold by increasing the cytoplasmic levels of the chimeric AAV-CAT transcript. When the CAT gene is inserted down stream of the AAV intron, adenovirus and not VA1 RNA alone increased CAT activity by promoting accumulation of a spliced transcript.
The rep gene of the defective human parvovirus, adeno-associated virus, (AAV) mediates several trans-acting functions important to virus replication, transcription, and gene expression. At least four overlapping polypeptides are expressed from the rep gene. We have constructed a prokaryotic vector which expressed in Escherichia coli a region of AAV comprising 93% of the largest AAV rep protein. The protein expressed in E. coli, rep 78.93, was used to raise specific antibodies in rabbits. These antibodies were capable of detecting all four AAV rep proteins in human cells transfected with AAV-containing plasmids as well as new species of 47 and 35 kDa in molecular weight. These new rep proteins originate from the transcription promoter at map unit 19 in the AAV genome and may indicate use of alternate AUG codons or protein modification. The antibodies also recognized novel forms of the rep proteins expressed from mutant AAV genomes. Immunofluorescence analysis of AAV-infected human cells revealed that the rep proteins are localized primarily in the nucleus of the infected cell and have a distribution different from that of AAV capsid protein. These results demonstrate that antisera raised against an AAV rep protein synthesized in E. coli are capable of detecting wild-type AAV rep proteins in virus-infected mammalian cells. These specific antibodies should facilitate further characterization of the functionally pleiotropic viral rep proteins.
Prior genetic analysis provided evidence for trans-acting regulatory proteins (Rep) coded by the left-hand open reading frame (orf-1) of adeno-associated virus (AAV). We have used immunoblotting analysis to identify four protein products of orf-1. Antibodies elicited against an oligopeptide encoded by orf-1 were reacted with extracts of cells that were infected with AAV or transfected with AAV recombinant vectors in the presence or absence of helper adenovirus. The antibody recognized four polypeptides with apparent molecular weights of 78,000, 68,000, 52,000, and 40,000. The 78,000-dalton (78K) (Rep78) and 68K (Rep68) proteins appear to be encoded by the unspliced 4.2-kilobase (kb) and spliced 3.9-kb mRNAs, respectively, transcribed from the p5 promoter. The 52K (Rep52) and 40K (Rep40) proteins appear to be the products of the unspliced 3.6-kb and the spliced 3.3-kb mRNAs, respectively, transcribed from the p19 promoter. Rigorous identification of Rep68 as an AAV-coded protein is compromised by a cross-reacting cellular protein of similar size. All four proteins were expressed in the human cell lines 293, HeLa, HT29, and A549 infected with AAV together with adenovirus. Rep78 and Rep52 were detected at lower levels in cells infected with AAV at high multiplicity in the absence of adenovirus. Human 293 cells transfected with a recombinant AAV vector (pAV2) also expressed Rep proteins in the presence or absence of adenovirus. Mutations introduced into the Rep region of pAV2 further identified the Rep proteins. The amount of each Rep protein varied between nuclear and cytoplasmic extracts, but all four proteins accumulated during the lytic cycle of the viral infection. Other studies have indicated that the Rep proteins have independent trans-acting functions in viral DNA replication and negative and positive regulation of gene expression. Correlation of each trans-acting function with individual Rep proteins will be facilitated with the antibodies described herein.
We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.
Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.
EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.
We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.
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To investigate adrenergic control of blood vessels during aging, rats aged 6, 12, 20, and 27 months were studied using in vitro techniques. Accumulation of [3H]norepinephrine, one index of adrenergic nerve density, did not alter with age in the femoral or renal arteries or renal vein. In the femoral vein [3H]norepinephrine accumulation was greater at 6 and 27 months of age. Norepinephrine sensitivity was determined in both an innervated vessel, the femoral artery, and a non-innervated vessel, the carotid artery. In both cases, sensitivity to norepinephrine did not alter with age. In the renal and femoral arteries and veins, no significant changes in maximum responses to norepinephrine (10(-5) M), potassium chloride, or transmural nerve stimulation were seen with advancing age. Furthermore, frequency response curves (2-16 Hz, 200 pulses) did not differ with age for any of the four vessels studied, with one exception. The response to stimulation at 4 Hz of the femoral vein from 6-month-old rats was significantly larger than responses at other ages. During nerve stimulation, the renal vein exhibited rapid contractions superimposed upon the maintained contractile response. This type of rapid contraction occurred only rarely (1 out of 5) in the renal vein from 27-month-old rats. In summary, neither adrenergic nerve density as reflected by [3H]norepinephrine accumulation nor norepinephrine sensitivity decline with age. As the net effect of various components, the ability of vascular smooth muscle to respond to adrenergic nerve stimulation is also maintained during advancing age.
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