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J L Manley

Publications and source records attributed to J L Manley.

At least 163 records · Page 9Linked to original sources
PubMed1983

A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells.

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA.

Animals↗
PubMed1983

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

Avian Sarcoma Viruses↗
PubMed1982

Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression.

DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a casual link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopus laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa II site at 0.72 SV40 map units was specifically methylated with Hpa II methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end of the late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.

Animals↗
PubMed1982

Transcription initiation by RNA polymerase II is inhibited by S-adenosylhomocysteine.

Most eukaryotic mRNAs are blocked at their 5' termini by guanylylation and methylation. These "cap structures" have been shown to play important roles in increasing the stability and translatability of mRNAs. Previous in vitro and in vivo data suggest that these modifications occur extremely early in the synthesis of RNA transcripts by RNA polymerase II. Here we show that S-adenosylhomocysteine (AdoHcy), both a product and an inhibitor of transmethylation reactions, inhibits transcription initiation by RNA polymerase II, but not by RNA polymerase III, in a HeLa whole-cell lysate. AdoHcy must be present during initiation to inhibit transcription and does not affect elongation by RNA polymerase II or the stability of the resultant transcript. Furthermore, AdoHcy does not inhibit transcription by purified HeLa RNA polymerase II. These results suggest that formation of the 5'-cap structure is coupled to initiation of transcription and is consistent with a close association between the capping enzymes and RNA polymerase II at the time of initiation.

Adenoviruses, Human↗
PubMed1981

DNA sequence required for initiation of transcription in vitro from the major late promoter of adenovirus 2.

We have identified a region of the viral genome required for the initiation of transcription in vitro from the major late promoter of adenovirus 2. A fragment of the adenovirus genome containing the cap site of the major late transcripts was inserted into plasmid pBR322 and cloned. Deletions were then generated in vitro in and around the T-A-T-A-A-A-A sequence located 25-31 nucleotides (positions -25 to -31) upstream from the cap site. DNAs with these deletions were tested for their ability to initiate transcription in vitro by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Whereas removal of sequences upstream from position -47 or downstream from position -12 did not abolish transcription, deletions extending into, or beyond, the T-A-T-A-A-A-A sequence reduced transcription to less than 1/10th. Removal of the normal cap site slightly reduced, but did not abolish, transcription. These results indicate that the region of the genome upstream of the cap site, with boundaries within 15-17 nucleotides to either side of the T-A-T-A-A-A-A sequence, is required for the initiation of transcription in vitro from the major late promoter of adenovirus 2.

Adenoviruses, Human↗
PubMed1981

Base substitution in an intervening sequence of a beta+-thalassemic human globin gene.

beta globin gene fragments from a patient with homozygous beta+-thalassemia have been cloned and subjected to restriction endonuclease, nucleotide sequence, and in vitro trancription analyses. Restriction endonuclease mapping of the cloned gene fragments revealed no deletions or other rearrangements, and transcription of the thalassemic gene appeared to be normal in vitro. However, nucleotide sequence analysis of the beta+-thalassemic gene fragments permitted identification of a single base change in the body of the small intervening sequence. This nucleotide change creates a sequence much like that of the 3' splice site of the small intervening sequence. The presence of a potential anomalous splicing site as a result of this base change suggests a mechanism for defective posttranscriptional processing of beta globin mRNA precursor molecules in beta+-thalassemia.

Base Sequence↗
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

Structure and in vitro transcription of human globin genes.

The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

Base Sequence↗
PubMed1980

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

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.

Adenoviruses, Human↗
PubMed1979

RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor.

We have analyzed the RNA produced in vitro by incubating nuclei from HeLa cells infected with adenovirus serotype 2. Our results show that adenovirus-specific RNA is produced at a linear rate for up to 2.5 hr. Hybridization analysis of RNA produced in nuclei isolated 18 hr after infection indicated that transcription begins at the "late promoter" at map position 16.5. Sequence analysis of the 5' termini of the in vitro transcripts showed that this system initiates RNA chains de novo at the correct promoter and that the 5' terminus is capped.

Amanitins↗