Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.
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PMID:Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs. 41 5

An HLA-E-specific oligonucleotide probe was used to study the expression of HLA-E. This probe detects two HLA-E transcripts, 1.8 and 2.7 kb in size, which are present in varying ratios in all tissues and cell lines investigated. We demonstrate that alternative poly(A) site usage accounts for the differential regulation of the two HLA-E mRNA species. Sequence analysis of three cDNA clones, representing the two transcripts of HLA-E, and of an HLA-E gene encoded by cosmid cd3.14, revealed identity of gene and cDNA in the 3' untranslated region. S1 nuclease protection assays confirmed that the two HLA-E transcripts are not alternative splicing products. Introduction of cd3.14, together with human beta 2 m into the murine myeloma cell line P3X63-Ag8.653, resulted in a cell surface expression of an HLA-class I heavy chain detectable by indirect immunofluorescence whereas transfection into the human beta 2m expressing mouse L cell line, J27 was negative with regard to cell surface expression. Cell surface labeling of transfectants and immunoprecipitation with a monomorphic HLA class I-specific antibody or an antibody against human beta 2m confirmed the presence of an HLA-E H chain on the cell surface. These results indicate that the HLA-E gene codes for a class I H chain that can be expressed on the cell surface.
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PMID:The HLA-E gene encodes two differentially regulated transcripts and a cell surface protein. 140 23

We have isolated a chicken cellular myosin II heavy chain isoform cDNA clone that overlaps the published sequence for MHC-A (Shohet et al., 1989, Proc Natl Acad Sci 86, 7726-7730) and contains three canonical AAUAAA-polyadenylation signals in an additional 374 nucleotides at its 3' end. S1 nuclease protection analysis and PCR-amplification of MHC-A cDNA 3' ends have confirmed that all three of the signals are used in vivo. Differential usage of these signals without differential splicing in this region yields three messages that differ at their 3' ends but appear to encode the same protein. Comparison of the new chicken sequence with the homologous human MHC-A cDNA sequence (Saez et al., 1990, Proc Natl Acad Sci 87, 1164-1168) has revealed a number of similarities at this end of their long 3' untranslated regions (3'-UTRs). The three chicken polyadenylation signals reported here are positioned similarly to three signals evident in the human sequence. This region also contains distinct stretches of identity that are interspersed with regions of little homology. Within these regions of identity are a number of conserved sequence motifs, some of which have been demonstrated to be involved in mRNA metabolism in other systems. The pattern of mRNA sequence conservation demonstrated here suggests that the mechanisms for regulating MHC-A mRNA metabolism have been conserved between chickens and humans.
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PMID:Multiple polyadenylation signals and 3' untranslated sequences are conserved between chicken and human cellular myosin II transcripts. 168 16

cDNA sequences for both human high molecular weight (HMW) and low molecular weight (LMW) prekininogens have been isolated by molecular cloning and determined by sequence analysis. The sequence determination together with the S1 nuclease mapping and RNA blot-hybridization analyses indicate that human HMW and LMW prekininogen mRNAs share an identical sequence throughout the 5'-untranslated region and the protein-coding region up to the sequence encoding the 12 amino acids distal to the bradykinin sequence, and the two mRNAs then completely diverge from each other. The signal peptide, the heavy chain (H chain), and the bradykinin moiety, which are common between the two prekininogens, consist of 18, 362, and 9 amino acids, respectively, while the light chains (L chains) of the HMW and LMW prekininogens are composed of 255 and 38 amino acids, respectively. All 17 cysteine residues present in the human and bovine H chains are located at exactly equivalent positions, indicating that the human H chain, like the bovine counterpart, can form 8 loop structures, each connected by two adjacent cysteine residues. The L chains of human and bovine kininogens differ in the protein lengths as well as in some amino acids crucial for the processing of the kininogens by kallikrein. Based upon this finding, we have discussed the molecular basis for the different modes of processing of human and bovine HMW kininogens and for the different kinetics of contact activation reactions exhibited by the two HMW kininogens.
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PMID:Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens. 298 93

The major transplantation (or H-2) antigens in the mouse are cell-surface glycoproteins composed of a heavy chain and a light chain, the beta-2 microglobulin (beta 2m). The expression of these proteins is regulated during development. Embryonic cells at early stages of development do not express these proteins. On the other hand, these molecules are present on the surface of all adult somatic cells. We investigated whether the expression of both chains was coordinately regulated. Using specific single-stranded DNA probes in an S1 nuclease analysis, we compared the relative amounts of H-2D and beta 2m transcripts in normal tissues, in transformed cells, and during embryonic development. Our results show that (1) the steady state level of beta 2m transcripts varies from one adult organ to another, while that of H-2D transcripts stays approximately the same; (2) upon transformation, the amount of H-2D-specific mRNA increases drastically, while the beta 2m mRNA level remains constant; (3) whereas the quantity of beta 2m mRNA increases during early development, the amount of H-2D mRNA remains at a very low level. These data suggest that the regulation of H-2D and beta 2m genes are not identical and that their activation during development is not synchronous.
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PMID:Asynchronous regulation of mouse H-2D and beta-2 microglobulin RNA transcripts. 299 82

We report here a 54 base pair deletion in the CH3 exon of the alpha gene in the mouse myeloma W3129. This deletion results in a loss of 18 amino acids and a change from a glycine to a serine at position 464. The extent of the deletion was determined by sequencing a portion of CH3 cloned from a variant of W3129, and S1 nuclease protection showed the deletion pre-exists in the parental cell line. The deletion removes the donor splice site normally used in joining CH3 to the alpha membrane (MB) exon when forming MB-specific mRNA. Examination of cytoplasmic RNA by blot hybridization and S1 nuclease protection using MB-specific probes showed a complete lack of membrane mRNA in W3129 and its derivatives. An RNA transcript of unknown origin and function which includes sequences from the CH3-MB intron was seen in W3129 and in J558, an IgA, lambda myeloma with specificity for alpha (1----3) linked dextrans. We discuss the possible influence of the mutation on the W3129 protein. In contrast to the other myelomas studied in this laboratory, light chain loss variants are readily isolated from W3129 and are stable in their production of heavy chain [Dackowski and Morrison (1981) Proc. natn. Acad. Sci. U.S.A. 78, 7091-7095].
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PMID:The IgA myeloma W3129 contains a deletion in CH3 which prevents the formation of the membrane form of heavy chain mRNA. 313 59

We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the gamma heavy chain locus (S gamma). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-myc oncogene translocated to a S region depends on the stage of B-cell differentiation of the cells harboring the translocated c-myc gene and not on alterations in the structure of the translocated c-myc oncogene.
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PMID:The translocated c-myc oncogene of Raji Burkitt lymphoma cells is not expressed in human lymphoblastoid cells. 385 23

Transcriptional competence of the immunoglobulin heavy-chain locus (IgH) is established at an early stage of lymphoid cell development, leading to the appearance of RNA components, previously called C mu RNA1 or sterile-mu RNA2, which contain constant-region sequences but lack variable-region sequences. These components are of two types: those which initiate in the D region of alleles that have undergone DJH (diversity-joining region) rearrangement (D mu transcripts) and those which initiate within the JH-C mu intron (hereafter termed I mu transcripts). In pre-B and early B cells, D mu and I mu transcripts are nearly as abundant as the messenger RNA encoding mu heavy chain. The D mu transcripts are spliced into RNAs containing D, JH and C mu sequences, and in some, but not all, cases these RNAs are translated into D mu proteins. To establish whether the I mu transcripts have any translational potential and to elucidate the structure of their promoter region, we have determined their transcription initiation sites and their mode of splicing. As reported here, by using sequence analysis of cloned I mu complementary DNAs, primer extension and S1 nuclease mapping, we have found that these transcripts have remarkable 5' heterogeneity: there are more than five distinct start sites spanning a region of 44 nucleotides that is located downstream of an octanucleotide found in all variable-region promoters. Such imprecise initiation may result from the lack of a well-defined TATAA motif and the unusual proximity of the octanucleotide to the enhancer region. Approximately 700 nucleotides downstream from these initiation sites, a cryptic splice site is used to create a nontranslatable exon ('nontron') which is joined to the C mu 1 domain. The properties of the nontron may be important for the mechanism of allelic exclusion.
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PMID:C mu-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5'-nontranslatable exon. 393 61

We examine the influence of the immunoglobulin locus on the expression of the translocated c-myc oncogene in mouse plasmacytomas. The level of c-myc RNA was 30- 35-fold greater in tumor cells than in normal, quiescent B cells. Mitogen stimulation of the lymphocytes with lipopolysaccharide induced a 15-fold increase in c-myc expression per cell to a level that was similar to that in the transcription of the translocated c-myc gene involved initiation from sequences in the first c-myc intron. Abundant RNA transcripts were also found from the noncoding strand of the c-myc intron in most tumor lines. S1 nuclease mapping was used to locate the intronic sequences that are used to initiate the tumor-specific c-myc RNAs. Six different initiation sites within the intron were mapped, none of which have the TATA sequence usually associated with eucaryotic RNA polymerase II promoters. The noncoding strand transcripts were also found to initiate in the c-myc intron. Transcription of the c-myc coding strand was independent of the position of the translocation breakpoint, even when the heavy chain switch and constant regions were deleted.
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PMID:Transcriptional activation of the translocated c-myc oncogene in mouse plasmacytomas: similar RNA levels in tumor and proliferating normal cells. 632 72

FS-4 fibroblasts were found to produce 37-kDa HLA class I heavy chain in response to IFN-gamma or TNF in a time- and dose-dependent fashion, and a synergism between IFN-gamma and TNF was observed. Immunoprecipitation of IFN-gamma- or TNF-induced FS-4 cell culture supernatants by mAb A1.4 revealed an additional 33-kDa protein in association with the 37-kDa heavy chain. The 33-kDa protein appeared to be expressed in a 38-kDa form on the membrane of FS-4 cells induced by IFN-gamma or TNF, as A1.4 immunoprecipitated the 38-kDa band in association with the 44-kDa transmembrane HLA class I heavy chain. Release of the 37-kDa heavy chain could well be due to an alternative RNA splicing with the deletion of exon 5 encoding the hydrophobic transmembrane region of membrane-anchored HLA class I heavy chain. Northern blot analysis and S1 nuclease protection assay suggested the existence of HLA class I heavy chain mRNA lacking exon 5 in IFN-gamma- or TNF-induced FS-4 cells. Southern blot analysis on the products of reverse transcription-polymerase chain reaction amplification from cytoplasmic RNA confirmed induction of alternative splicing by these cytokines. Our results suggest that cytokine-induced production of soluble HLA class I molecules may play important roles in the regulation of T cell interaction with antigen-presenting cells.
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PMID:Alternative splicing of HLA class I transcripts induced by IFN-gamma and TNF in fibroblasts: release of soluble HLA class I heavy chain and an associate protein. 770 5


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