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)

We have determined the structure of the human ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. This gene was assigned to human chromosome 18 at region q21.3, by fluorescent in situ hybridization. The gene contains a total of 11 exons and has a minimum size of about 45 kb. The exon/intron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to encode functional protein domains. A major site of the transcription initiation, determined by S1 nuclease mapping, was assigned to an adenine base 89 bases upstream from the adenine base of the translation initiation ATG. The promoter region contains a potential binding site for Sp1, NF-E2 and erythroid-specific transcriptional factor GATA-1, but not a typical TATAA or CCAAT sequence. Analysis of primer extension showed that the transcription starts at the same position between hepatoma HepG2 and erythroleukemia K562 cell mRNA, thereby suggesting that there can be a single transcript in erythroid and non-erythroid cells.
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PMID:Structure of the human ferrochelatase gene. Exon/intron gene organization and location of the gene to chromosome 18. 155 82

The glycoprotein (GP) IIb/IIIa heterodimer functions as a receptor for fibrinogen, von Willebrand factor, and fibronectin on activated platelets; it is dysfunctional in the bleeding diathesis Glanzmann's thrombasthenia. This receptor is a member of the integrin family, which includes homologous membrane receptors involved in a number of different cell-cell and cell-matrix adhesive interactions. Knowledge of the sequence and organization of the GPIIb and GPIIIa genes will help in understanding evolutionary relationships and functional homologies of this family of adhesion protein receptors and will facilitate analysis of molecular defects responsible for thrombasthenia. Using the GPIIb cDNA as a probe, we have isolated overlapping genomic clones encompassing the entire coding region, the 5'- and 3'-untranslated sequences, and the immediate flanking regions for the GPIIb gene. The gene spans approximately 17.2 kilobases (kb); all but approximately 2.6 kb of intronic DNA sequence has been determined. The GPIIb gene contains 30 exons whose demarcations do not correlate with previously suggested functional domains. Two intron/exon borders have the rare GC splice donor sequence instead of the consensus GT sequence. There are at least seven complete and three partial AluI sequence repeats within the intron sequences. RNase protection, S1 nuclease analysis, and primer extension studies using human erythroleukemia (HEL) cell RNA and platelet RNA map a major transcription start site 32 base pairs (bp) 5' to the beginning of the coding region; however, there are no canonical consensus TATA or CAAT boxes in the region immediately 5' to the proposed cap site. The immediate 5'-flanking sequence of rodent GPIIb demonstrates complete identity near the proposed cap site with its human counterpart, but again, no TATA or CAAT boxes are apparent.
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PMID:Organization of the gene for platelet glycoprotein IIb. 232 58

Somatic cell hybridization of mouse erythroleukemia (MEL) cells and HEL cells, a human erythroleukemia line that produces fetal (gamma) but fails to express adult (beta) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome 11-retaining HEL-MEL hybrids expressed human beta-globin, suggesting that the HEL beta-globin genes (i) are transcriptionally competent, (ii) become activated in response to a positive trans-acting element within the MEL environment, and (iii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of beta-globin gene expression. In addition to beta-globin, the primary HEL-MEL hybrids co-expressed gamma-globin; however, gamma-globin expression segregated by subcloning so that secondary and tertiary clones either expressed only beta-globin or co-expressed gamma- and beta-globin. The results of subcloning can be explained by assuming that gamma-globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL gamma-globin genes become randomly modified during the continuous proliferation of hybrids.
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PMID:Adult and fetal human globin genes are expressed following chromosomal transfer into MEL cells. 257 80

Previous studies have indicated that control and hemin-treated human erythroleukemia K-562 cells fail to produce adult-type beta-globin mRNA transcripts and to translate them into nascent beta-globin chains. Expression of the beta-globin DNA sequences in K-562 cells can occur, however, under certain conditions. To readdress this issue and to examine the possibility of whether these cells produce immature and untranslatable beta-globin RNA transcripts, we prepared total cytoplasmic RNA from control and inducer-treated cells and performed Northern blot hybridization analysis using 5' end-labeled fragments of the human beta-globin DNA rather than 3' end fragments as probes. Although hybridization of both cytoplasmic and nuclear K-562 RNA with a 32P-labeled 3' end fragment (1.6kb Bam H1 cut) coding for a large part of the first exon of beta-globin failed to detect beta-globin RNA transcripts, hybridization with a 5' end 32P-Labeled 2.0kb Bam H1 fragment (coding for the third exon and part of the second) revealed the presence of relatively small (less than 7S) RNA molecules both in nuclear and cytoplasmic fraction. S1 nuclease mapping of both cytoplasmic and nuclear RNA with the use of 5' end-labeled 2.0 kb Bam H1 fragment of human beta-globin DNA indicated protection of a small portion located 64 bp 5' upstream from the Bam H1 site of the second exon. The amount of protected portion was relatively higher in K-562 cells undergoing erythroid maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hemin increase production of beta-like globin RNA transcripts in human erythroleukemia K-562 cells. 279 52

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.
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PMID:Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes. 282 17

We used a gene-specific S1 nuclease assay to study the changes in steady-state mRNA levels of several core histone variants during the differentiation of murine erythroleukemia cells. These studies allowed us to distinguish three distinct expression classes of histone genes. The expression of the major replication-dependent class of histone genes was tightly linked to DNA synthesis. The concentrations of these transcripts decreased rapidly as cell division slowed during the process of differentiation. In contrast, the replication-independent H3.3 transcript levels were constitutively maintained throughout differentiation and were unaffected by inhibitors of DNA or protein synthesis. We also identified among the cloned histone genes used as probes a third expression class, the partially replication-dependent variants. Expression of these transcripts became transiently uncoupled from the reduced rate of DNA synthesis accompanying the early stages of differentiation. We show that their synthesis is sensitive to the DNA synthesis inhibitor hydroxyurea but that selective uncoupling from DNA synthesis of these histone mRNAs occurs at a specific stage of differentiation. We present several hypotheses to explain how this might be accomplished. The expression characteristics of the mRNAs studied coincided with those of the proteins for which they code, indicating that changes in the relative levels of the different variants is mediated at least in part by changes in mRNA levels.
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PMID:Changes in the levels of three different classes of histone mRNA during murine erythroleukemia cell differentiation. 301 84

Eukaryotic protein synthesis initiation factor 4A (eIF-4A), a 46-kDa polypeptide, is involved both in mRNA cap recognition and in the binding of mRNA to 40S ribosomal subunits. A 41-mer oligodeoxynucleotide probe was synthesized complementary to a portion of the published coding sequence of eIF-4A mRNA [Nielsen et al., Nucleic Acids Res. 13 (1985) 6867-6870] and used to screen a mouse genomic library. We have isolated and characterized a full-length clone from that library. The eIF-4A sequence is contained in eleven exons. The eleventh exon also has the 3'-nontranslated sequence and two separate polyadenylation sites. Northern-blot analysis of mouse poly(A)+RNA indicates that there are several distinct mRNA species coding for eIF-4A. Two of these contain the same coding sequence and differ only in the length of the 3'-nontranslated region. Two of the eIF-4A mRNAs are therefore likely to be the result of differential processing at the 3'-end. We have used a fragment of the genomic clone to measure the steady-state levels of eIF-4A mRNA during the induced differentiation of murine erythroleukemia cells. S1 nuclease protection experiments demonstrated that by the fourth day after induction eIF-4A mRNA declined to 25% of its steady-state level in uninduced cells. In contrast, the steady-state level of beta-globin mRNA increased dramatically during differentiation. In vitro transcription assays using nuclei isolated from uninduced and induced cells show that the rate of transcription of eIF-4A mRNA was 40% greater in differentiated cells, indicating a posttranscriptional component is involved in the regulation of the steady-state mRNA level.
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PMID:Isolation and mapping of a gene for protein synthesis initiation factor 4A and its expression during differentiation of murine erythroleukemia cells. 321 17

The protein p53 is functionally implicated in the normal regulation of cell proliferation. We have previously reported that the rate of p53 protein synthesis is reduced during the cessation of cellular proliferation which accompanies the in vitro induced differentiation of Friend-erythroleukemia cells. In this work we followed the p53 mRNA expression during the differentiation of these cells. We report on a new type of p53 mRNA with a slower electrophoretic mobility on gels, which appeared in the cytoplasmic fraction of the erythroleukemia cells between 1 to 3 days following induction of differentiation and persisted in the cells until Day 7. The larger type of p53 mRNA was found associated with polysomes, suggesting that it is translatable in cells. The difference in size between the noninduced and the differentiation-specific type of p53 mRNAs (about 200 nucleotides) was not abrogated following the deadenylation of the mRNAs, thus excluding the possibility that the altered size might result from a longer poly(A) tract. S1 nuclease mapping of the 3' termini of the p53 mRNAs revealed that the 3' ends of both p53 mRNA types were identical, suggesting that either alternative splicing or a longer 5' noncoding region could cause this heterogeneity in p53 mRNA transcripts.
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PMID:Changes in p53 mRNA expression during terminal differentiation of murine erythroleukemia cells. 331 97

In order to test whether the ontogenetic origin of human parental cells influences the expression of globin genes, we did fusions of mouse erythroleukemia (MEL) cells with fetal or adult nonerythroid cells and we assessed globin expression in hybrids. For selection of hybrid clones we used a monoclonal antibody that recognizes a human chromosome 11-linked cell surface marker. Globin expression was assessed with fluorescent antibody labeling, globin isoelectric focusing, and S1 nuclease mapping. Chromosome 11-containing hybrids from human nonerythroid cells (adult or fetal lymphoid cells; fetal fibroblasts) produced human adult (beta) but not fetal (gamma) globin chains. These results suggest that the MEL x nonerythroid cell hybrids express the adult human globin independent of whether the human cells derive from the fetal or from the adult stage of human development. Our findings, together with previous observations in MEL x fetal erythroid or MEL x HEL hybrids, show that the globin program of the parental human cell is the main determinant of the expression of fetal human globin in these hybrids.
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PMID:Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids. 346 79

The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes.
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PMID:Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation. 348 Oct 36


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