Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.
 ...
PMID:Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts. 20 13

The presence of viral-like sequences in the RNA of various types of leukemic cells was investigated by hybridizing cellular poly(A)-containing RNA with cDNA synthesized in an endogenous system of purified Moloney murine sarcoma virus [M-MSV-(MLV)]. Poly(A)-RNA-cDNA hybrids were detected by assaying their resistance to S1 nuclease. Hybrids were found in 22 out of the 46 leukemias that were tested. None of the controls, including material obtained from buffy coats, bone marrow cells, and a continuous human cell line, was positive. Positive cases were found in all the different categories of leukemias with the exception of chronic myelogenous leukemias. There was no definite corelation between the category of leukemia and positivity. A few cases contained a very high proportion of poly(A)-RNA-cDNA hybrid.
 ...
PMID:Search for nucleic acid sequences complementary to a murine oncornaviral genome in poly(A)-rich RNA of human leukemic cells. 106 Oct 78

Phosphoribosylpyrophosphate (PP-Rib-P) synthetase (EC 2.7.6.1) subunit I gene (PRPS1) is constitutively expressed in various tissues (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989) Biochim. Biophys. Acta 1007, 203-208). We report here the exon-intron organization and the transcription promoter sequence of rat PRPS1 gene. This gene has 22 kilobases and is split into 7 exons ranging in size from 99 to 251 base pairs (bp), except for exon 7 (1008 bp). A putative PP-Rib-P binding site is encoded in exon 5. The exon-intron boundaries are similar to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG initiation codon. In the upstream region from the transcriptional start sites, we observed a TATA-like sequence (TAATTTAAT) at nucleotides -28, a CCAAT element (AGCCAATC) at nucleotides -80, and three GC boxes (putative Sp1-binding sites) at nucleotides -103, -43, and -10. A comparison of the promoter region for PRPS1 with those of other housekeeping genes revealed a homology resembling that of the beta-actin gene.
 ...
PMID:Structure of the rat PRPS1 gene encoding phosphoribosylpyrophosphate synthetase subunit I. 215 94

We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells. 298 39

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs. 298 40

We examined the molecular basis for phenotypic reversion in cells infected with a transformation mutant of murine sarcoma virus, MuSVts110. In MuSVts110-infected NRK cells (6m2 cells), the manifestation of the transformed phenotype at 33 degrees C and the normal phenotype at 39 degrees C is governed by thermosensitive splicing of the MuSVts110 primary transcript, a 4.0-kilobase (kb) RNA which contains the gag and mos genes joined out of frame. At 33 degrees C, selectively, the 4.0-kb RNA is processed to a spliced 3.5-kb RNA in which the gag and mos genes are rejoined in a continuous open reading frame, thus allowing synthesis of the P85gag-mos-transforming protein. In contrast, the MuSVts110 revertant cell lines (designated 54-5A4 and 204-3) appear transformed at all growth temperatures from 33 to 39 degrees C and express a P100gag-mos-transforming protein from an apparently unprocessed 4.0-kb viral RNA. In the current study we established both by S1 nuclease analysis and primer extension sequencing that the revertant 54-5A4 and 204-3 4.0-kb viral RNAs suffered a 5-base deletion at the intron-exon border of the 3' splice site. The effect of this deletion is twofold. First, because of the damage to the 3' splice site, the revertant viral 4.0-kb RNAs cannot be processed to the spliced 3.5-kb RNA and, consequently, cannot be translated to P85gag-mos. Second, the 5-base deletion excises an in-frame stop codon positioned at the intron-exon border in the parental RNA and restores the original mos gene reading frame. The net effect is to produce a continuous open reading frame from the gag, alternate mos, and authentic mos gene reading frames which are fused together in the revertant 4.0-kb RNA. This continuous open reading frame can be translated into the P100gag-mos-transforming protein at any growth temperature.
 ...
PMID:Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110. 300 54

Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.
 ...
PMID:Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells. 303 49

A number of deletion mutants were isolated, including 5', 3', and internal deletions in the 5'-flanking region of the human cellular oncogene related to the Harvey sarcoma virus (c-H-ras), and their transforming activities were examined in NIH 3T3 cells. DNA sequences which could not be detected without losing transforming activity were localized to a relatively short stretch upstream of the region which showed homology to the 5'-flanking region of v-H-ras oncogene. S1 nuclease analysis indicated that there were two clusters of mRNA start sites at positions that were about 1,371 and 1,298 base pairs upstream of the first coding ATG. The minimum region required for promoter function was estimated to be a 51-base-pair-long (or less) DNA segment. The promoter was GC rich (78%) and did not contain the consensus sequences that are usually observed in PolII-directed promoters but contained a GC box within which one of the mRNA start sites was included. In addition, two sets of positive and negative elements seemed to be located between the promoter and the protein-coding region, which appeared to influence positively and negatively, respectively, the efficiency of transformation with the c-H-ras oncogene.
 ...
PMID:Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5'-flanking region by focus formation assay. 367 Mar

Valuable information about proto-oncogenes and their physiological function has been obtained by studying their expression in normal cells. However, expression of the c-mos gene, the cellular homologue of the transforming gene of Moloney murine sarcoma virus, has not been detected in normal mouse cells or tissues. The conservation of the c-mos open reading frame strongly indicates that the gene must function during some portion of the animal life cycle, and other lines of evidence suggested to us that the c-mos proto-oncogene may be expressed at very low levels in normal tissues. We have used a sensitive S1 nuclease assay to screen RNA preparations from mouse tissues and describe here the detection of c-mos-related transcripts especially in mouse embryos, testes and ovaries. The transcripts found in testis RNA are estimated to be approximately 1.7 kilobases (kb) long by Northern analysis. S1 analysis demonstrated that the entire mos open reading frame is present. In contrast, we detect approximately 1.4-kb transcripts in ovary RNA and at least two major transcripts, approximately 2.3 and approximately 1.3 kb, in embryo RNA. The latter transcripts have in common sequences of at least 1 kb, representing most of the c-mos open reading frame. The variation in size of the mos transcript in different tissues suggests a novel regulatory mechanism for the expression of this proto-oncogene.
 ...
PMID:Expression of c-mos proto-oncogene transcripts in mouse tissues. 400 Feb 80

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.
 ...
PMID:Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes. 609 86


1 2 Next >>