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J L Manley

Publications and source records attributed to J L Manley.

At least 145 records · Page 8Linked to original sources
PubMed1987

Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

Cell Nucleus↗
PubMed1987

Factors influencing alternative splice site utilization in vivo.

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

Antigens, Viral, Tumor↗
PubMed1986

Structure and function of the S1 nuclease-sensitive site in the adenovirus late promoter.

We analyzed an S1 nuclease-sensitive site present in supercoiled, but not linear, recombinant plasmids containing the adenovirus late promoter. S1 nicking was detected on both strands, primarily in the TATA box. Analysis of deletion mutants showed that sequences upstream of -47 and downstream of -12 are not required for S1 cutting. However, a number of different base substitution mutations in stretches of G residues upstream and/or downstream of the TATA box were sufficient to eliminate S1 cutting. When the transcriptional activities of these mutant promoters were assayed in vivo, six of seven mutants lacking the ability to form the S1-sensitive structure showed no reduction in transcriptional potential. In fact, several showed increased promoter activities. These data show that the S1 nuclease cutting site in the adenovirus late promoter has precise nucleotide sequence requirements for its formation. However, the ability of recombinant plasmids to adapt this conformation in vitro is not necessary for such plasmids to serve as templates for transcription in vivo.

Adenoviridae↗
PubMed1986

In vitro splicing of simian virus 40 early pre mRNA.

The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery.

Antigens, Viral, Tumor↗
PubMed1986

Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo.

Most eukaryotic messenger RNAs are transcribed as precursor molecules that must be processed by capping, splicing, 3' cleavage, and polyadenylylation to yield mature mRNAs. An important, unresolved issue is whether any of these reactions are linked either to transcription by RNA polymerase II or to each other. To address one aspect of this question, we constructed a chimeric gene containing an RNA polymerase III promoter (the adenovirus VAI promoter) fused to the body and 3'-flanking sequences of a protein-coding gene (the herpesvirus tk gene). Here we show that this hybrid gene was transcribed from the RNA polymerase III promoter following transfection of human 293 cells and that the transcripts produced were stable and efficiently transported to the cytoplasm. Although a significant proportion of the transcripts were prematurely terminated at specific sites within the gene, a high percentage of the full-length RNA was accurately cleaved and polyadenylylated. These results demonstrate that cleavage and polyadenylylation of mRNA precursors are not obligatorily coupled to transcription by RNA polymerase II in vivo.

Endonucleases↗
PubMed1986

Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication.

A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box-containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed.

Adenoviridae↗
PubMed1985

Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed.

Base Sequence↗
PubMed1985

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.

Base Sequence↗
PubMed1985

Control of adenovirus late promoter expression in two human cell lines.

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.

Adenovirus Early Proteins↗
PubMed1984

Generation and functional analyses for base-substitution mutants of the adenovirus 2 major late promoter.

The function of guanosine residues surrounding the TATA box of the adenovirus 2 major late promoter (MLP) in promoting efficient transcription initiation and in selecting a specific transcription start site in vitro has been examined. Multiple and single base substitutions (G----A) were generated in this region (from -63 to +25 relative to the cap site, +1) of the MLP. The promoter activities of the wild type and 21 mutants were assayed in an in vitro transcription system using whole cell extract (WCE) prepared from HeLa cells. The results suggest that the strings of G residues immediately adjacent to the TATA box are not required for full promoter activity in vitro. These G residues also appear not to be involved in the selection of a specific transcription start site by RNA polymerase II in vitro, since the identical cap site was used by wild-type and mutated MLP's. However, two G residues (-55 and -57) were identified as part of an upstream promoter element: a G----A transition at either -55 or -57 resulted in a 2.6 fold reduction in promoter activity. However, neither single nor double G----A transitions at -62 and -63 had an effect on promoter activity.

Adenoviruses, Human↗
PubMed1984

In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites.

We report that adenovirus 2 DNA sequences located between positions-66 and -51 upstream of the major late cap site (position +1) enhance transcription initiation from this promoter by up to 5- to 10-fold in HeLa whole cell lysates. This enhancing effect is template concentration-dependent and is abolished by truncation of the template immediately upstream of -66. Additionally, specific transcripts are not detected from templates truncated at +33 downstream of the cap site. These results define a minimum region of approximately 100 base pairs encompassing the transcription start site that appears to interact with the RNA polymerase II transcription complex during initiation. Analysis of the shortest runoff transcripts that can be synthesized in vitro revealed that RNAs as short as 50 nucleotides are quantitatively modified by guanylylation and methylation to cap 1 structures. In contrast, short RNAs containing guanylylated but unmethylated cap structures are not efficiently utilized as substrates by endogenous cap-methylating enzymes in the HeLa lysate. These findings, together with the observation that the synthesis of short transcripts is sensitive to the presence of the methyltransferase inhibitor S-adenosylhomocysteine, suggest that cap formation is a promoter-proximal event that occurs concomitantly with the synthesis of a nascent RNA polymerase II transcript.

Adenoviruses, Human↗
PubMed1984

Transcription of methylated eukaryotic viral genes in a soluble in vitro system.

SV40 and adenovirus-2 (Ad2) recombinant plasmids containing long segments of poly(dC-dG) cloned adjacent to transcription control regions were methylated in vitro with Hbal methylase and transcribed in a soluble in vitro system. The addition of up to 40 or more base pairs of poly(m5dC-dG) immediately upstream or downstream of promoter regions was shown to have no effect on the accuracy or efficiency of specific transcription from these promoters in vitro. Methylation at various naturally occurring C-G sequences within or near these promoters also had no effect on transcription in vitro. The significance of these results with respect to possible mechanisms whereby DNA methylation might regulate eukaryotic gene expression is discussed.

Adenoviruses, Human↗
PubMed1984

Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs.

To examine the role of small nuclear ribonucleoproteins (snRNPs) in mRNA splicing, we have injected SV40 DNA, in the presence or absence of anti-Sm or anti-(U1)RNP antibodies, into the nucleus of X. laevis oocytes, and analyzed the viral specific RNAs and proteins that were synthesized. In the absence of antibodies, the majority of the viral mRNAs were spliced, giving rise to transcripts and proteins analogous to those found in infected monkey cells. However, the relative efficiencies with which the various splice sites were utilized were different in the two cell types. When sera from systemic lupus erythematosus (SLE) patients containing anti-Sm or anti-(U1)RNP antibodies were coinjected with the viral DNA, splicing of L-strand-specific (late) mRNA was dramatically inhibited. Cleavage at both 5' and 3' splice sites was blocked, leading to an accumulation of unspliced primary transcripts. Neither the total amount of late RNA synthesized nor the formation of mature polyadenylated late mRNA 3' ends was affected. These results indicate that U1 snRNPs play a crucial role in mRNA splicing in vivo. Unexpectedly, the effects of the sera on E-strand-specific (early) viral mRNA splicing were different. All anti-Sm or -(U1)RNP sera tested had no detectable effect on the splicing of the mRNA coding for the small tumor antigen. A subset of these sera, however, inhibited large tumor antigen mRNA splicing. On the basis of these data it is suggested that different pre-mRNAs, or even different splice sites within the same pre-mRNA, have dissimilar interactions with snRNP particles in the splicing reaction.

Animals↗
PubMed1983

Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate.

Conditions have been developed which allow for the efficient, accurate, and specific polyadenylation of exogenously added mRNA precursors in a whole-cell lysate derived from HeLa cells. Precursors are prepared by in vitro transcription using linear DNA templates in a HeLa whole-cell lysate, purified, and added to another lysate. Under optimal conditions (which are quite precise with respect to several variables), 70% or more of the precursor molecules become polyadenylated, and the length of the poly(A) segment added is controlled much as it is in vivo, giving rise to 150-300-nucleotide long stretches of poly(A). Under suboptimal conditions, both the fraction of precursor RNA that becomes polyadenylated and also the length of the poly(A) segment added are reduced. The in vitro polyadenylation reaction is also remarkably specific: Only in vitro-synthesized pre-mRNAs that contain a 3' end located at or slightly downstream from the corresponding in vivo mRNA 3' end site can be efficiently polyadenylated in vitro. These results suggest that the poly(A) polymerase requires one or more protein (or RNA) factors in order to bring about accurate and specific polyadenylation in vitro, and that the poly(A) polymerase complex must interact with a specific recognition signal at the 3' end of mRNA precursors in order to catalyze subsequent polyadenylation.

Base Sequence↗
PubMed1983

A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus.

We have constructed a frameshift mutation in the simian virus 40 early region using a novel method of oligonucleotide-directed mutagenesis. The mutated DNA specifies an 84,000-dalton large tumor antigen that consists of approximately equal to 75,000 daltons encoded by the wild-type reading frame and 9,000 daltons, by the alternative reading frame (wild-type large tumor antigen is approximately equal to 82,000 daltons). The frameshifted carboxyl terminus of the protein bears a strong similarity to the same region of polyoma virus middle-sized tumor antigen. We have found that the mutant DNA is unable to replicate when introduced into permissive monkey cells and incapable of transforming nonpermissive mouse cells.

Amino Acid Sequence↗