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M J Imperiale

Publications and source records attributed to M J Imperiale.

At least 55 records · Page 3Linked to original sources
PubMed1990

Mutational analysis of simian virus 40 small-t antigen.

Several point mutations in the simian virus 40 (SV40) small-t antigen have been analyzed for their effects on protein stability, transformation, transactivation, and binding of two cellular proteins. All mutations which affected cysteine residues in two cysteine clusters produced highly unstable small-t antigens. Four point mutations outside these clusters and one in-frame deletion mutant, dl890, produced stable proteins but reduced transformation efficiency. These were able to transactivate the EII promoter and bind the cellular proteins, suggesting that these activities are not sufficient for small-t-mediated enhancement of transformation.

Amino Acid Sequence↗
PubMed1989

Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit.

The adenovirus major late transcription unit (MLTU) encodes five colinear mRNA families, L1 through L5, each distinguished by a unique poly(A) site. Site selection is regulated during the course of infection, predominating early at the L1 site and late at the L2 through L5 sites. Two general mechanisms can be invoked to explain predominant usage of the L1 site early in infection. MLTU site selection may proceed in a first-come, first-serve manner whereby the L1 site is used most frequently because it is closest to the promoter. Alternatively, specific sequences flanking the L1 site may control predominant L1 site usage in a position-independent manner. To distinguish between these mechanisms, we constructed deletions in the L1 flanking sequences and inserted the mutated sites into either simple transcription units or mini-MLTUs encoding two poly(A) sites. The pattern of site selection for each construct was then quantitated by S1 nuclease analysis after transfection into 293 cells. The results indicated that L1 sequences upstream of AAUAAA define a novel selector element that can cause predominant L1 site usage at either position of a tandem transcription unit. The element did not significantly affect the stability or nucleocytoplasmic transport of L1 transcripts and was not required for efficient 3'-end processing in simple transcription units. Predominant L1 site usage required physical linkage of the processing signals and was independent of the major late promoter.

Adenoviridae↗
PubMed1989

Viral growth, origin binding, and p53 binding properties of simian virus 40 large T antigen transformation and replication mutants.

The viability, p53 binding, and SV40 origin binding of a series of SV40 large T antigen point mutants, which map to the amino terminal one-third of the molecule, were examined. Two mutants which yield small plaques were found to have altered kinetics of replication upon infection of permissive cells. Mutants which did not bind to the origin of replication were not able to replicate, but the reverse was not always true. Replication defective mutants which bound the SV40 origin were found; these map both inside and outside of the origin binding domain. All the transformation defective mutants bound the cellular protein, p53.

Animals↗
PubMed1988

Analysis of adenovirus type 2 L1 RNA 3'-end formation in vivo and in vitro.

Downstream sequence requirements for efficient cleavage and polyadenylation at the adenovirus type 2 L1 poly(A) site were determined in vivo in 293 cells and in vitro by using RNA precursors in HeLa cell nuclear extracts. The two cleavage sites used were found to differ in sensitivity to 3'-end deletion in vivo and in vitro.

Adenoviruses, Human↗
PubMed1987

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions.

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.

Animals↗
PubMed1986

Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants.

We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated.

Animals↗
PubMed1985

An enhancer-like element in the adenovirus E2 promoter contains sequences essential for uninduced and E1A-induced transcription.

Using a chloramphenicol acetyltransferase gene under the control of a series of adenovirus E2 promoter mutants, we have determined the sequence requirements for E1A-induced as well as uninduced expression of the E2 promoter. Fully induced expression requires 79 nucleotides of upstream sequence, and the same sequences are required for uninduced expression. A promoter containing 70 nucleotides of upstream sequence is inducible, although to a lower level than wild type, while a promoter containing 59 nucleotides of upstream sequence is not inducible. The sequences required for induction as well as for uninduced promoter activity map between 21 and 79 nucleotides upstream of the transcriptional initiation site and function in either orientation and at a distance. We suggest that E1A-mediated induction involves the effective increase in a factor utilizing this upstream site and that the factor can functionally interact with the site irrespective of its location relative to the start of transcription.

Acetyltransferases↗
PubMed1985

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.

Animals↗
PubMed1984

Requirement of a downstream sequence for generation of a poly(A) addition site.

The 3' terminus of most, if not all, eucaryotic polyadenylated mRNAs is formed as a result of endonucleolytic cleavage of a larger precursor RNA. That is, transcription does not terminate at the mRNA 3' sequence but rather proceeds through this site, terminating at some distance downstream. Using a plasmid containing the adenovirus E2A transcriptional unit, we have investigated the sequence requirement for the formation of a mature mRNA 3' terminus, focusing on the role of sequences immediately distal to the poly(A) addition site. Deletion mutants were constructed in the region distal to the poly(A) addition site and assayed by transfection into human 293 cells. The results demonstrate that 35 nucleotides distal to the site of poly(A) addition are sufficient for the formation of a mature E2 mRNA. However, removal of an additional 15 nucleotides, leaving 20 nucleotides distal to the poly(A) site, abolished the ability to produce functional E2A mRNA. The defect in the production of functional mRNA from such a mutant appears to be in the proper cleavage of the primary transcript at the poly(A) addition site. It would thus appear that sequences immediately distal to the site of poly(A) addition do not contribute to the mature mRNA but are essential for the formation of mature mRNA.

Adenoviruses, Human↗
PubMed1984

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Adenoviruses, Human↗
PubMed1984

Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation.

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

Adenoviruses, Human↗
PubMed1983

Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element.

A plasmid containing the adenovirus E2 gene, a gene normally requiring E1A-mediated induction during viral infection, is expressed very poorly upon transfection into mouse L cells. If the same plasmid is transfected into 293 cells, which constitutively express the adenovirus E1A gene, or into L cells together with a plasmid containing the E1A gene, the E2 gene is expressed at higher levels. Cotransfection of the E2 plasmid with a plasmid containing the pseudorabies virus (a herpesvirus) immediate early gene results in an even higher increase in the level of E2 expression. In addition, efficient E2 expression in the absence of trans induction was obtained by inserting E1A upstream promoter sequences at the 5' or 3' end of the E2 gene, indicating that these E1A sequences possess enhancer properties. Thus the efficient expression of the E2 gene can be obtained either by a structural change in the gene itself or by a trans-acting induction.

Adenoviruses, Human↗
PubMed1982

Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor.

Adenovirus mutants carrying a defective E1A gene, such as dl312, are unable to express any of the early viral genes upon infection of HeLa cells. However, efficient expression of the other early adenovirus genes was obtained when dl312-infected HeLa cells were coinfected with pseudorabies virus, a herpesvirus. By employing a temperature-sensitive pseudorabies mutant (tsG1) it was demonstrated that the herpesvirus function responsible for the induction of adenovirus transcription was the immediate early gene, a gene required for the activation of herpesvirus early gene expression and the maintenance of early and late herpesvirus transcription. Specifically, HeLa cells coinfected with dl312 and tsG1, when shifted to the nonpermissive temperature, lost their capacity to express the early adenovirus genes. Furthermore, activation of early adenovirus gene expression in herpesvirus coinfection occurred earlier and at a higher level than in wild-type adenovirus infection. Therefore, the herpesvirus immediate early protein not only activates the early adenovirus transcription units but apparently does so more efficiently than the adenovirus E1A gene product. Because of this fact, we argue that the activation, either by the E1A protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.

Adenoviruses, Human↗
PubMed1982

Functionally distinct helper T cells enriched under different culture conditions cooperate with different B cells.

Helper T cells selected from KLH-primed lymph node cells under different in vitro conditions were analyzed for the ability to cooperate in secondary hapten-specific antibody responses. It is shown that carrier-specific, MHC-restricted helper T cells selected in low-density cultures cooperate with equal efficiency in either PC-specific or DNP-specific responses. In the presence of both PC-specific and DNP-specific B precursors, however, such T cells are limited to participate in only one helper event in vitro. In contrast, the predominant helper T cell in a second population maintained in high-density lymph node cultures cooperates, presumably through a soluble factor, with multiple B precursors. These properties correspond to those previously described for specific and nonspecific effector functions. The experiments reported here further demonstrate that different B cells respond in vitro to carrier-specific and nonspecific helper signals.

Animals↗
PubMed1981

Clonal analysis of F1 hybrid helper T cells. I-A subregion-encoded hybrid determinants restrict the activity of keyhole limpet hemocyanin-specific helper T cells.

Clonal expansion of isolated precursors to helper T cells was induced in limiting dilution cultures of keyhole limpet hemocyanin (KLH)-primed F1 hybrid lymph node cells. Progeny of each isolated precursor was tested for helper activity by transfer to independent cultures with hapten-primed B cells of either parental or F1 hybrid origin. The major histocompatibility complex (MHC) restriction specificity of each F1 hybrid helper T clone was determined. To assess the contribution of I-A and I-E subregion-encoded genes to the expression of these restriction elements, helper T cell cultures derived from F1 hybrids between strains with recombinant H-2 haplotypes were analyzed. Parental and unique F1 hybrid MHC determinants that are encoded entirely within the I-A subregion were found to restrict the activity of KLH-specific helper T cells.

Animals↗