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 examined the genomic organization and the transcription unit for the human link protein gene from genomic clones and RNA prepared from human cartilage over a wide age range. Five exons cover the gene which is greater than 60 kbp. Primer extension and S1 nuclease protection analysis revealed transcription initiation to be 315 bases upstream from the translation initiation codon in RNA derived from cartilage samples ranging from fetal to 53 years of age. The first exon size therefore is 289 bp and examination of the 5' flanking sequence indicated a lack of a TATA box in close proximity to the transcription start, although a TATAA-like motif (TCTAA) was present at -75 bp. Such a sequence at a similar distance can serve as a promoter in the chicken link protein gene. The large first exon of 289 bp is similar to that of the chicken but contrasts with that described previously for human (96 bp) and rat (62 bp). We also analysed human link protein mRNA by PCR for the presence of an alternatively spliced exon that is present in rat mRNA in low abundance, but could not detect such transcripts. Equine and porcine mRNA contained this spliced form but the results suggested that this was expressed as a rare transcript.
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PMID:Human link protein gene: structure and transcription pattern in chondrocytes. 794 59

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.
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PMID:Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control. 827 54

Neuroblastoma, the most common malignant solid cancer of children, has an ability to differentiate in vitro and in vivo. This biological property has a significant influence upon the prognosis of patients with neuroblastomas. Neuronal cells express three alternatively spliced forms of c-src mRNA (nonneuronal c-src, neuronal c-srcN1, and neuronal c-srcN2), which are found at different levels in adult and fetal human brain tissue. In this study, the transcriptional levels of the three c-src mRNAs were examined in relation to the neural differentiation in eight human neuroblastoma cell lines and two clonal sublines and in seven primary neuroblastoma tissues by S1 nuclease protection assays. Neuronal c-srcN1 mRNA was expressed at high levels in neuroblastoma cell lines with the ability to differentiate but not in the cell lines lacking the capacity to mature in response to chemical inducers irrespective of N-myc gene amplification and overexpression. In terminally differentiated neuroblastoma cells, the expression of neuronal c-srcN2 mRNA, which was barely detectable at a steady-state level in the uninduced cells, increased to significant levels. Infantile neuroblastomas identified by mass screening tests expressed both neuronal c-srcN1 and c-srcN2 mRNAs at levels almost identical to that found in human brain tissue, but terminally differentiated neuroblastoma cells, neuroblastomas from older children identified based on clinical symptoms, did not. These results suggest that neuronal c-src expression and the ability of neuroblastomas to differentiate in vitro and in vivo may be correlated.
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PMID:Expression of alternatively spliced src messenger RNAs related to neuronal differentiation in human neuroblastomas. 831 27

The synthesis of estrogens from androgens is catalyzed by a microsomal cytochrome P450 termed aromatase (P450arom). The expression of this enzyme is highly regulated in both a developmental and cell-type specific fashion. We have chosen to examine the molecular basis of aromatase gene regulation by studying two models of aromatase expression: the Sebright bantam chicken and the R2C rat Leydig tumor cell line. In the first model, affected (Sebright) chickens express aromatase in many extragonadal tissues, while normal Leghorn chickens express aromatase only in the ovary and hypothalamus. Our studies have demonstrated that in normal chickens the site of transcription initiation is located approx. 147 nucleotides upstream of the initiator methionine. While Sebright animals also express aromatase mRNA initiated at an analogous initiation site in the ovary, a distinctive species of aromatase mRNA is also detected and is present in ovary and extragonadal tissues. This mRNA contains an identical coding sequence, but contains an alternatively spliced 5' noncoding exon that is derived from a distinctive promoter. The second model, the R2C Leydig tumor cell line, provides ample contrast. This cell line expresses high basal levels of aromatase (150-200 pmol/h/mg protein) that is suppressed with administration of 8 bromo cAMP or forskolin but the activity is not altered by glucocorticoids or epidermal growth factor treatment. Despite this distinctive pattern of regulation, at least three species of aromatase mRNA are detected in Northern blots, each of which is also detected in rat ovary. Primer extension and S1 nuclease assays indicate that both granulosa cells and R2C cells utilize a promoter that is located approx. 97 nucleotides upstream of the initiator methionine. These studies suggest that the "ovarian" promoter is evolutionarily conserved in both rats and chickens. These results further imply that the genetic mechanisms controlling the diversity of aromatase expression among tissues and among different species are likely to fall into two groups: those that employ distinctive promoters and alternative splicing and those that effect different patterns of regulation through a common ("ovarian") promoter.
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PMID:Diverse mechanisms of control of aromatase gene expression. 847 47

The murine Mac-2 gene is composed of six exons dispersed over 10.5 kilobases. S1 nuclease mapping showed multiple transcription initiation sites, clustered within a 30-base pair region. Sequence analysis revealed that a consensus initiator sequence is located in this area which lacks a TATA motif. The untranslated first exon contains an alternative splice donor site, confirming the existence of two cDNA species with the potential to encode proteins differing at their NH2 termini. In vitro expression and translocation experiments demonstrate that both of the alternatively spliced variants of Mac-2 encode proteins which lack a functional signal peptide. Subcellular fractionation studies indicate that most of the Mac-2 protein is present in the cytosol. These results support the view that Mac-2 is exported from the cell by an unusual mechanism which does not depend on the presence of a signal peptide.
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PMID:Structure of the murine Mac-2 gene. Splice variants encode proteins lacking functional signal peptides. 850 79

We report here the isolation and characterization of the human gene for the beta or muscle-specific isoform of the glycolytic enzyme enolase. The nucleotide sequence analysis revealed structural features, such as organization as 11 coding exons, the first exon consisting of an untranslated sequence and hence resembling sequences of the other two members of the gene family, the alpha and gamma enolase genes. The beta enolase locus spans about 6 kbp genomic DNA. Sequences matching the consensus sequence for muscle-specific regulatory factors are present in the 5'-flanking region and within the first intron. A combination of primer extension, S1 nuclease protection and RNA-sequencing experiments indicates that the gene has a unique transcriptional start site, 26 bp downstream of a TATA-like box; the differential usage of two donor sites within the untranslated exon I generates two alternatively spliced transcripts. The existence of the two mRNA, differing from one another in the presence or absence of a 42-nucleotide fragment in the leader sequence, was confirmed by cloning the corresponding cDNA using the rapid amplification of cDNA ends strategy. Secondary-structure predictions indicated that the leader sequences of the spliced forms could form hairpin structures with different free energies of formation, suggesting translational control.
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PMID:Structural features of the human gene for muscle-specific enolase. Differential splicing in the 5'-untranslated sequence generates two forms of mRNA. 851 87

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipper protein that has been shown to play a central role in the functional activity of c-Myc as a transcriptional activator. Max potentiates the binding of Myc-Max heterodimers through its basic region to its specific E-box Myc site (EMS), enabling c-Myc to transactivate effectively. In addition to the alternatively spliced exon a, several naturally occurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been detected. Using Western blot (immunoblot) and immunoprecipitation analysis, we have identified a variant form of Max protein (16 to 17 kDa), termed dMax, in detergent nuclear extracts of murine B-lymphoma cells, normal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing revealed that dMax contains a deletion spanning the basic region and helix 1 and the loop of the helix-loop-helix region, presumably as a result of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells. The dMax protein, prepared via in vitro transcription and translation, associated with bacterially synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-synthesized dMax failed to bind EMS DNA, presumably because of the absence of the basic region. Coexpression of dMax inhibited EMS-mediated transactivation by c-Myc. Thus dMax, which can interact with c-Myc, appears to function as a dominant negative regulator, providing an additional level of regulation to the transactivation potential of c-Myc.
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PMID:Variant Max protein, derived by alternative splicing, associates with c-Myc in vivo and inhibits transactivation. 852 35

We have cloned the full-length cDNA encoding the rat homolog of type I neurofibromin isoform, a protein product of a gene linked to neurofibromatosis type 1. Rat type I neurofibromin isoform is composed of 2,820 amino acid residues and shares about 98.5% amino acid identity with the human counterpart. By S1 nuclease mapping analysis of the alternatively spliced neurofibromin mRNAs in adult rat tissues, we showed that type I isoform mRNA was predominantly expressed in the brain, pituitary, and testis, while type II mRNA, carrying a 63-nucleotide insertion in the region coding for the domain related to GTPase-activating protein, was predominantly expressed in most other tissues, such as heart, kidney, and ovary. Furthermore, type II mRNA is predominant in the testis at age 1 week, while type I mRNA becomes predominant at 3 weeks and is subsequently expressed at higher levels, as seen in the adult testis. In contrast, type I mRNA is the predominant form in the brain throughout the postnatal development. Thus, the relative expression levels of type I and type II mRNAs may be specific to the tissues and to the developmental stage of certain tissues.
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PMID:Differential tissue-specific expression of neurofibromin isoform mRNAs in rat. 898 75

Expression of syntrophin genes, encoding members of the dystrophin-associated protein complex, was studied in the mouse brain. In the hippocampal formation there is distinctive co-localization of specific syntrophins with certain dystrophin isoforms in neurons, e.g. alpha1-syntrophin with the C-dystrophin in CA regions and beta2-syntrophin with the G-dystrophin in the dentate gyrus. Expression of the alpha1-syntrophin is predominant in CA regions and the olfactory bulb and it is also present in the cerebral cortex and the dentate gyrus. The beta2-syntrophin mRNA is most abundant in the dentate gyrus and is also evident in the pituitary, the cerebral cortex and in Ammon's horn and in traces in the caudate putamen. The choroid plexus was labelled by both alpha1- and beta2-syntrophin-specific probes. The expression of syntrophins in the brain correlates with expression of dystrophins and dystroglycan. There are brain areas such as the cerebral cortex where several different syntrophins and dystrophins are expressed together. Syntrophin expression co-localizes with utrophin in the choroid plexus and caudate putamen. Finally, no syntrophin was detected in the cerebellar Purkinje cells where the specific dystrophin isoform (P-type) is present. This specific distribution of syntrophins in the brain is particularly interesting, as muscle syntrophin interacts with neuronal nitric oxide synthase. This may suggest that the dystrophin-associated protein complex may be involved in synaptic organisation and signal transduction machinery in both muscle and neurons. The dystrophin isoform, with exons 71-74 spliced out and hence lacking syntrophin binding sites, had been believed to be predominant in the brain, but our analyses using in situ hybridization, S1 nuclease protection and the semi-quantitative polymerase chain reaction revealed that this alternatively spliced mRNA is a minor, low abundance form in the brain.
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PMID:Differential expression of syntrophins and analysis of alternatively spliced dystrophin transcripts in the mouse brain. 918 49

Fatty acid transport protein (FATP) was identified by expression cloning strategies (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436) and shown by transfection analysis to catalyze the transfer of long-chain fatty acids across the plasma membrane of cells. It is expressed highly in tissues exhibiting rapid fatty acid metabolism such as skeletal muscle, heart, and adipose. FATP mRNA levels are down-regulated by insulin in cultured 3T3-L1 adipocytes and up-regulated by nutrient depletion in murine adipose tissue (Man, M. Z., Hui, T. Y., Schaffer, J. E., Lodish, H. F., and Bernlohr, D. A. (1996) Mol. Endocrinol. 10, 1021-1028). To determine the molecular mechanism of insulin regulation of FATP transcription, we have isolated the murine FATP gene and its 5'-flanking sequences. The FATP gene spans approximately 16 kilobases and contains 13 exons, of which exon 2 is alternatively spliced. S1 nuclease and RNase protection assays revealed the presence of multiple transcription start sites; the DNA sequence upstream of the predominant transcription start sites lacks a typical TATA box. By transient transfection assays in 3T3-L1 adipocytes, the inhibitory action of insulin on FATP transcription was localized to a cis-acting element with the sequence 5'-TGTTTTC-3' from -1347 to -1353. This sequence is very similar to the insulin response sequence found in the regulatory region of other genes negatively regulated by insulin such as those encoding phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and insulin-like growth factor-binding protein 1. Fluorescence in situ hybridization analysis revealed that the murine FATP gene is localized to chromosome 8, band 8B3.3. Interestingly, this region of chromosome 8 contains a cluster of three other genes important for fatty acid homeostasis, lipoprotein lipase, the mitochondrial uncoupling protein 1 (UCP1) and sterol regulatory element-binding protein 1. These results characterize the murine FATP gene and its insulin responsiveness as well as present a framework for future studies of its role in lipid metabolism, obesity, and type II diabetes mellitus.
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PMID:Characterization of the murine fatty acid transport protein gene and its insulin response sequence. 976 71


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