Showing papers by "Phillip A. Sharp published in 1980"

DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract

Abstract: We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.

Transcription maps of adenovirus.

Abstract: Publisher Summary This chapter discusses the techniques for the transcription mapping of adenovirus. All true early mRNAs and proteins can be found in adenovirus 2 (Ad2) transformed cell lines. However, only a subset of these early gene products is required to establish a transformed state. With the transition from early to late phase of infection several new mRNAs appear in the cytoplasm that are transcribed from the same regions of the viral DNA as the early mRNAs, but which have novel sizes and sequence content. Two methods have been developed to map spliced Ad2 mRNAs that include (1) the nuclease gel electrophoresis procedure and (2) electron microscopy of RNA-DNA hybrids. The strengths and limitations of these two techniques are reviewed in the chapter. A summary transcription map of Ad2 is also presented.

Expression of Early and Late Simian Virus 40 Transcripts in the Absence of Protein Synthesis

Abstract: We examined the synthesis of early and late simian virus 40 (SV40) mRNA9s in SV40-infected cells treated with two kinds of protein synthesis inhibitors. SV40 stimulated the synthesis of mRNA9s for both large and small tumor antigens in cells pretreated with the drug emetine before the addition of virus. Emetine is a stringent inhibitor of protein synthesis and, thus, protein factors necessary for transcription and processing of these mRNA9s probably preexist in the cell. Surprisingly, infection of cells pretreated with the protein synthesis inhibitor cycloheximide stimulated the synthesis of about 10-fold-higher levels of early viral mRNA9s than did comparable infections of nontreated cells. This amplification of early viral mRNA steady-state levels is probably not due to inhibition of synthesis of the early A gene product since the same degree of drug-specific amplification was seen in SV40 tsA -infected cells that were cultured at the nonpermissive temperature. However, the most interesting effect of cycloheximide addition on viral mRNA synthesis was its stimulation of the appearance of late mRNA9s in the cytoplasm of cells at early periods of infection. The synthesis of late mRNA9s does not appear to require the A gene product as late RNAs can be found in the cytoplasm of cells infected with SV40 tsA mutants which have been maintained at 41°C and continuously cultured in the presence of cycloheximide. Images

Regulation of adenovirus mRNA synthesis

Abstract: The lytic cycle of adenovirus is a tightly regulated sequence of stages. When this regulation is studied at the level of mRNA production, the most significant step in controlling gene expression is initiation of transcription. Thus in preceding from one stage of expression to another, viral factors seem to turn on transcription of new sets of genes. At the moment, it is thought that viral mRNA synthesis involves initiation of transcription at ten different promoter sites. It is likely that in some manner the frequency of an initiation of transcription at nine of these sites is affected by one or more viral gene products. With the recent development of soluble in vitro transcription systems that respond to exogenously added DNA, it should be possible to begin to study regulation of gene expression at this stage of transcription. At present, these systems yield the paradoxical observation that extracts prepared from uninfected human cells more efficiently recognize the late promoter as compared to the early promoter of adenovirus. As more is learned about regulation of synthesis of viral mRNAs, examples will surely be found where RNA processing and RNA turnover play a critical role in determining the level of mRNAs. Such cases are more likely to appear in the balancing of synthesis of different mRNAs derived from one transcriptional unit. Few experiments have been directed to this possibility and the study of adenovirus molecular biology is only now entering the age of maturity where these experiments are feasible.