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E B Ziff

Publications and source records attributed to E B Ziff.

87 records · Page 5Linked to original sources
PubMed1985

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation.

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

Adenovirus Early Proteins↗
PubMed1984

HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism.

We report that the rate of transcription of cellular beta-tubulin genes increases during the early phase of adenovirus infection of HeLa cells, with kinetics very similar to those routinely found for viral genes. This activation depends upon adenovirus early region E1a, which encodes products that activate early virus transcription. To compare the responses of viral and cellular genes to E1a, we infected HeLa cells with dl312, a transcriptionally inactive deletion mutant that lacks a functional E1a gene. We then superinfected the cells with a helper virus, dl327, which encodes active E1a products, and measured changes in the rates of transcription of various cell and viral genes. Early region E3 of dl312 was activated 0 to 6 h postinfection and then repressed at 8 h postinfection, thus reproducing the two-step kinetics characteristic of a wild-type infection. Synthesis of beta-tubulin nuclear RNA was also transiently induced two- to six-fold, rising and falling in a manner similar to E3 transcription. An increase in helper virus multiplicity gave an increase in beta-tubulin stimulation, but dl312 alone, even at a high multiplicity of infection, gave no induction, confirming the requirement for E1a. beta-Actin nuclear RNA was actively synthesized before infection, but it was not further stimulated by the virus. Cellular beta-globin gene transcription was not stimulated by the virus, although transcription of a transfected beta-globin plasmid was induced by the virus or from a cotransfected E1a expression plasmid. We conclude that adenovirus 5 can stimulate beta-tubulin gene transcription. We discuss the significance for the viral life cycle of viral stimulation of cell genes and consider possible mechanisms in the light of the results obtained with beta-actin and beta-tubulin.

Adenoviridae↗
PubMed1982

Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomycin block of protein synthesis.

The transcription of adenovirus type 2 genes proceeds through a broad three-phase program. From 1 to 4 h postinfection six early transcription units (EIa, EIb, EII, EIII, EIV, and the promoter-proximal segment of the late transcription unit) are activated. From 4 to 6 h postinfection transcription of the early genes is depressed. After the onset of viral DNA replication at approximately 6 h postinfection, the transcript from the late promoter is antiterminated, and this transcript dominates viral RNA synthesis. The early activation period also proceeds through a series of stages; early regions EIa and EIV are activated first, followed by early region EII. We show that in the presence of anisomycin, a stringent inhibitor of protein synthesis, nuclear RNA and cytoplasmic polyadenylated RNA from regions EIa and EIV accumulate at normal rates, whereas RNAs from regions EII and EIII do not accumulate. We also show that failure to accumulate RNAs from regions EII and EIII is due to reduction of the rate of transcription by greater than 90%. We conclude that the regulation of the promoters for early regions EII and EIII is distinct from the mechanism that operates on the other early transcription units. The promoters for early regions EII and EIII diverge and lie approximately 500 nucleotides apart. We examined the structure of viral chromatin in this region early during infection by mild DNase I digestion in isolated nuclei and indirect end labeling with a DNA probe near these promoters. In control, drug-free cells where EII and EIII are transcribed and in anisomycin-treated cells where EII and EIII are not transcribed we detect the same regular DNase I pattern. We suggest that regulation of EII and EIII is not mediated through a change in gross chromatin structure, but rather by a viral effector, possibly a product of region EIa.

Adenovirus Early Proteins↗
PubMed1981

In vitro transcription of adenovirus.

A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.

Adenoviruses, Human↗
PubMed1980

Transcription and RNA processing by the DNA tumour viruses.

Messenger RNA synthesis by the DNA tumour viruses proceeds by a complex but versatile series of transcription and RNA processing steps. The major mechanistic features of this pathway are probably very similar to those used by the animal cell host itself. The viruses have, however, evolved intricate arrangements of protein coding sequences and sites for RNA initiation, polyadenylation and splicing which allow them to use their genetic information to maximum advantage.

Adenoviruses, Human↗
PubMed1980

Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families.

The major late promoter of adeovirus-2 is located at coordinate 16.45 and initiates synthesis of nuclear precursors that are processed into mRNAs which fall into five 3- co-terminal families, L1-L5. These mRNAs all share a common tripartite 5' leader with a capped terminus encoded at th RNA initiation site. We show that the coorindate 16.45 RNA initiation site is also an early promoter, and yields transcripts detectable as early as 1 hr post-infection, prior to DNA replication and the early-late switch at 6-8 hr. Polyadenylated cytoplasmic RNA from the first 3- co-terminal family, L1, is also produced from the earliest stages of infection. L1 mRNA accumulates in the cytoplasm in the presence of cycloheximide, which blocks DNA replication and the onset of the late phase. Early nuclear RNA contains the same capped 5' terminal RNAase T-1 undecanucleotide and promoter proximal oligonucleotides present in late transcripts. This implies that precisely the same transcription start site is utilized for early L1 mRNA synthesis as is used during the late stage for L1-L5 late mRNA synthesis. In contrast to the early apperance of L1 mRNA, neither L2 nor L3 mRNAs are detected until 5-6 hr post infection. We cnclude that a major event in the Ad-2 early-late switch is a novel form of control which activates L2-L5 mRNA production.

Adenoviridae↗
PubMed1978

Coincidence of the promoter and capped 5' terminus of RNA from the adenovirus 2 major late transcription unit.

During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed approximately 30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at approximately 16, 20 and 27, and protein coding sequences which map downstream. This report maps the late promoter and the capped 5' end of nuclear and cytoplasmic RNAs from this transciption unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 +/- 0.5 and contain the sequences intervening between the leader segments. We map the capped 5' terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5' terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5' end of mRNA. Residues removed from transcripts by splicing were identified. These plus caps were detected in large polyadenylated nuclear RNA, indicating that capping and polyadenylation can occur on unspliced molecules. Residues retained in the mRNA first leader contain a nine residue sequence adjacent to the cap which is complementary to the 3' end of 18S rRNA, suggesting that the first leader functions in ribosome binding. Nucleotide sequences from the promoter region are compared with cellular counterparts. Strong homologies at cap sites and splice points suggest that for the noted cases, the virus and cell share closely related mechanisms for mRNA 5' end synthesis and splicing.

Adenoviruses, Human↗
PubMedDate not supplied

Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene.

Transcription of the c-fos proto-oncogene is greatly increased within minutes of administering purified growth factors to quiescent 3T3 cells. This stimulation is the most rapid transcriptional response to peptide growth factors yet described, and implies a role for c-fos in cell-cycle control. Transformation by c-fos may result from a temporal deregulation of this control.

Actins↗