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P Lebowitz

Publications and source records attributed to P Lebowitz.

At least 37 records · Page 2Linked to original sources
PubMed1982

Initiation and regulation of simian virus 40 early transcription in vitro.

We have studied initiation and regulation of early transcription of simian virus (SV40) DNA in vitro by eucaryotic RNA polymerase II, using both a crude HeLa cell extract and a partially purified calf thymus polymerase supplemented with a HeLa cell S100 fraction. Analysis of initiation sites by primer-directed cDNA synthesis and sequencing of cDNA's has revealed that early transcription is initiated at a multiplicity of sites corresponding to the 5' termini of early viral mRNA's. The pattern of in vitro initiation closely resembles the pattern of 5' termini of early mRNA's late in the lytic cycle, with principal initiations between residues 5184 to 5194, upstream from the early Hogness-Goldberg (TATA) sequence, and at residue 5123, well downstream from this sequence. In vitro transcription is initiated to a lesser extent at sites between residues 5150 and 5155, the principal 5' termini of early mRNA's in transformed cells and early in lytic infection, located 21 to 26 nucleotides downstream from the TATA sequence. Initiation occurs at identical sites and with similar efficiencies on form I and linearized DNA templates. There are minor differences in the efficiency of initiation at specific sites by the two transcriptional systems. Studies using a DNA template cleaved just downstream from the TATA sequence and a second template cleaved through a pair of 72-base-pair tandem repeats starting 87 nucleotides upstream from the TATA sequence have revealed that neither the TATA sequence nor the repeats are essential for early transcription in vitro. However, removal of the TATA and upstream sequences shifts initiation of transcription principally to the residue 5123 site. Comparison of the relative efficiencies of transcription on intact wild-type DNA, the two cleaved DNAs, and DNA from a deletion mutant suggests that all or most of the sequences constituting an early promoter lie within the genomic region 60-70 to 140 nucleotides upstream from the principal 5' termini of the early mRNA's.

Base Sequence↗
PubMed1982

Altered utilization of splice sites and 5' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.

Gene Expression Regulation↗
PubMed1981

Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs.

The 5' termini of the principal early mRNAs produced in cells transformed by wild-type simian virus 40 lie 21-25 nucleotides downstream from an A-T-T-T-A-T sequence on the DNA template. The 5' termini of early mRNAs produced by five origin-defective mutants containing deletions downstream from the A-T-T-T-A-T sequence and one viable mutant dl892 with a deletion starting 15 nucleotides upstream from this sequence were determined by a method involving synthesis, separation, and determination of the sequences of DNAs complementary to 5' termini. Mutant dl892 produced early mRNAs with the same principal 5' termini as wild-type virus; the origin-defective mutants produced mRNAs with principal 5' termini shifted downstream by a distance equivalent to the length of the deleted DNA segment. These data suggest that a DNA sequence of 29 nucleotides, which includes the A-T-T-T-A-T sequence, contains a component(s) of a promoter for early transcription. This component functions in positioning the 5' ends of the principal early mRNAs 21-25 nucleotides downstream from the A-T-T-T-A-T sequence and acts independently of these downstream sequences.

Base Sequence↗
PubMed1981

Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40.

dl-805 is a viable deletion mutant of simian virus 40 lacking 187 of the 202 nucleotides coding for the principal leader of the wild-type viral late mRNAs. The structures of the mutant late 16S and 19S RNAs and proteins were examined with the following principal findings. (i) The vast majority, if not all, of the 19S RNAs are unspliced over their entire length. These RNAs contain intervening sequences and serve as mRNAs. The vast majority, if not all, of the 16S RNAs, however, retain the characteristic wild-type splice. (ii) Mutant 16S and 19S RNAs do not terminate at residue 243 (located six nucleotides upstream from the 5' end of the deleted DNA segment), the principal 5' terminus of the wild-type late mRNAs; rather, they begin at a series of sites, mostly upstream, used infrequently or not at all by wild-type virus. (iii) Despite the presence of altered late mRNAs, dl-805 late proteins have electrophoretic mobilities similar to those of wild-type late proteins, and mutant virions contain the same relative quantities of late proteins as wild-type virions.

Base Sequence↗
PubMed1981

Simian virus 40 early mRNA's contain multiple 5' termini upstream and downstream from a Hogness-Goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large T antigen.

We have used primer-directed synthesis, separation, and sequencing of cDNA's to identify and localize the 5' termini of simian virus 40 early mRNA's. We have examined polyadenylated RNAs obtained from whole cytoplasm and polysomes of two transformed lines and from the cytoplasm of infected cells early and late in the lytic cycle, and we have attempted to correlate the results of our cDNA analyses with recent analyses of early cap structures. We have found that early mRNA's from transformed cells have three principal 5' termini, at residues 5,150, 5,154, and 5,155, with terminal transcribed sequences of CU, GC, and GG, respectively. These termini lie 21 to 26 nucleotides downstream from the early Hogness-Goldberg sequence. Transformed cell early mRNA's also contain a series of less abundant 5' termini that are copied from DNA sequences as far as 80 nucleotides downstream and a minimum of 70 to 75 nucleotides upstream from the Hogness-Goldberg sequence. The templates for the upstream 5' termini and the late simian virus 40 mRNA's overlap by a minimum of 60 to 65 nucleotides. Early mRNA's isolated from cells early in infection contain the same three principal 5' termini and downstream minor 5' termini as transformed cell mRNA's, but they lack 5' termini upstream from the Hogness-Goldberg sequence. With the onset of the late lytic phase, there is a progressive decreases in the utilization of the three principal 5' termini and additional downstream 5' termini and a progressive increase in the utilization of four major termini at residues 5,190 to 5,194, which are 10 to 15 nucleotides upstream from the Hogness-Goldberg sequence. With the onset of the late lytic phase, there is a progressive decrease in the utilization of the three principal 5' termini and additional downstream 5' termini and a progressive increase in the utilization of four major termini at residues 5,190 to 5,194, which are 10 to 15 nucleotides upstream from the Hogness-Goldberg sequence. This shift is evident in cells infected with a tsA mutant at the permissive temperature, but is aborted by growth at or shift-up to a restrictive temperature. Thus, this shift is mediated by the gene A product, large T antigen. We present two models, which are mutually exclusive, to account for the role of T antigen in the early-late shift. One involves transcription late in infection on a new DNA template synthesized during DNA replication. The second involves inhibition of initiation of early transcription at residues 5,150 to 5,155 and other downstream sites and a shift of transcription initiation principally to the upstream sites as a result of the binding of T antigen to two sites on simian virus 40 DNA downstream from the Hogness-Goldberg sequence.

Animals↗
PubMed1979

Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells.

We have studied the structure of polyadenylated virus-specific cytoplasmic mRNA's in mouse and human cells transformed by simian virus 40 and in monkey cells infected with simian virus 40 in the presence of cytosine arabinoside by means of reverse transcriptase-catalyzed complementary DNA synthesis and complementary DNA sequencing. Abundant mRNA species containing splices from residues 4490 to 4557 (0.533 to 0.546 map units [m.u.]) and 4490 to 4837 (0.533 to 0.600 m.u.) were identified in both transformed and infected cells. Two principal reverse transcriptase stops were observed at the 5' termini of these mRNA's, both occurring with approximately equal frequency. The most distal of these stops was localized at residues 5152 to 5154 (0.660 m.u.), and the second was at residues 5147 to 5148 (0.659 m.u.). Several additional minor stops, between approximately 0.62 and 0.65 m.u., were also found on complementary DNA copied from transformed cell mRNA; in contrast, only one additional stop was present on complementary DNA copied from early lytic mRNA. These data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional 5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's. Transformed cell mRNA was also found to contain a single 3' terminus at positions 2504 and 2505 (0.153 m.u.); termini lying beyond this site were not detected.

Animals↗
PubMed1978

The 5'-terminal leader sequence of late 16 S mRNA from cells infected with simian virus 40.

The 16 S mRNA that directs the synthesis of VP1, the major structural protein of Simian virus 40 (SV40), is composed of a leader sequence of 203 nucleotides joined to a transcript that contains all the codons for VP1. The leader is transcribed from DNA between 0.723 and 0.762 map units while the VP1 coding transcript is copied from DNA from 0.94 to 0.17 map units. The leader sequence joins the transcript of the coding region at a position 42 nucleotides upstream from the initiator codon for VP1 by a 3':5'-phosphodiester linkage. Longer leader sequences also occur in a portion of SV40 late mRNA.

Base Sequence↗
PubMed1978

Gaps and duplicated sequences in the leaders of SV40 16S RNA.

In order to investigate the 5' terminal structural heterogeneity of the 16S size class of SV40 late RNA, we have bound an SV40 DNA fragment labeled at its 5' termini with P32 to the .939-.945 map unit region of late lytic cytoplasmic polyadenylated RNA, used reverse transcriptase to prepare cDNA copies of the 5' termini of this RNA, separated the cDNA products on an 8% polyacrylamide-7 M urea gel and subjected these products to nucleic acid sequence analysis. A number of discrete cDNAs were obtained. Analysis of these cDNAs has suggested the presence of three categories of 16S species all containing the same body extending from residues 1381-2592 (.939-.170 m.u.) but differring in the structure of their leader segments. Members of the first category contain leaders which are colinear with SV40 DNA, have a common 3' terminus at residue 444 and extend varying distances in a 5' direction. The most abundant 16S species contains a leader of 203 nucleotides and is a member of this group. RNAs of the second category contain leaders with an internal gap between residues 211-352. The single RNA comprising the third category contains a leader with a tandem repetition of nucleotides 351-443 at the 3' terminus of its leader.

Base Sequence↗
PubMed1977

Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP.

Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.

Adenosine Triphosphate↗
PubMed1975

Simian virus 40 DNA segment of the adenovirus 7-simian virus 40 hybrid, e46, and its transcription during permissive infection of monkey kidney cells.

Nucleic acid hybridization methodology has been used to investigate the span of the simian virus 40 (SV40) DNA segment in the adenovirus 7-SV40 hybrid, E46(+), and the extent of its transcription in lytically infected monkey kidney cells. The SV40 segment of E46(+) comprises approximately 62% of the SV40 genome; it originates in the proximal region of Hin-G (the G fragment derived by cleavage of intact SV40 DNA with Haemophilus influenzae restriction endonuclease), extends sequentially through approximately 80% of this fragment, all of fragments Hin-B, -I, -H, and -A, and terminates approximately 70% of the distance through Hin-C. During E46(+) lytic infection of permissive cells, the vast majority of stable cytoplasmic SV40-specific RNA is transcribed from the minus (E) strand of the fragments Hin-A, -H, -I, and -B, comprising the early template region. Transcripts of the minus strand of the Hin-G and -C fragments are detected in much lower concentrations, especially in the total lytic cellular RNA, whereas RNA complementary to the plus (L) strand is not detected. The transcriptional pattern of the SV40 segment within E46(+) is thus very similar to that in a number of transformed cell lines and in some respects to the transcriptional pattern in a series of nondefective adenovirus 2-SV40 hybrid viruses. These results suggest a common transcriptional mechanism for integrated SV40 DNA.

Journal Article↗
PubMed1974

A colinear map relating the simian virus 40 (SV40) DNA segments of six adenovirus-SV40 hybrids to the DNA fragments produced by restriction endonuclease cleavage of SV40 DNA.

The simian virus 40 (SV40) DNA segments present in a series of adenovirus-SV40 hybrids have been mapped with respect to the sites of cleavage of SV40 DNA by restriction endonucleases. Two approaches have been used. First, nucleic acid hybridizations were performed between equimolar quantities of the denatured DNAs of SV40 and each hybrid virus and the radiolabeled transcripts of 11 DNA fragments obtained by cleavage of SV40 DNA by restriction endonuclease from Hemophilus influenzae. Secondly, selected fragments of SV40 DNA produced by the H. influenzae or H. parainfluenzae restriction endonucleases were used to form heteroduplex DNA molecules with adenovirus and adenovirus-SV40 hybrid DNA, which were then analyzed by electron microscopy. The two sets of data were consistent and have permitted alignment of the map of the SV40 segments of the hybrid viruses with the H. influenzae and H. parainfluenzae cleavage maps of SV40. Since cells infected with some of the hybrid viruses contain one or more SV40-specific antigens, the genetic determinants of these antigens could be localized on the cleavage map.

Adenoviridae↗