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Results for “Repetitive Sequences, Nucleic Acid”

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PubMed1993

Identification of multiple repeat sequences and transcription-related elements within introns 4, 8 and 9 of human Raf-1.

Oncogenic activation of the human c-raf-1 protein (Raf-1) occurs by truncation of its aminoterminus (regulatory domain) and retention of the carboxyterminus (kinase domain). To gain insight into the structure of human raf-1 genomic DNA corresponding to the regulatory domain of Raf-1, we analyzed the nucleotide sequences of introns 4, 8, and 9. Very high frequencies of the direct and indirect repeats, and A/T nucleotides were observed within introns 4, 8, and 9; and the DNA sequences, 5'-TTAGTCA-3' and 5'-TGATTCA-3', like the DNA recognition site of transcription factor AP-1, were identified within intron 4 of raf-1 in human normal and tumor DNAs. In addition, several transcriptional elements (CAAT box, TATA sequences, eukaryotic transcription initiation sites, and cellular transcriptional elements) were found within introns 4, 8, and 9 of human raf-1. These data suggest: 1) the high frequency of intron-specific repeats is a possible recombinational mechanism for derivation of truncated oncogenic forms of Raf-1; and 2) the multiple transcriptional elements within introns 4, 8, and 9 may have important implications in the regulation of raf-1 transcription and, therefore, its role in cell growth and proliferation.

Base Sequence↗

PubMed1993

The serum unresponsive Rous sarcoma virus promoter sustains a high serum response factor-dependent transcription in vitro.

CArG boxes are cis-regulatory elements which are represented both in serum responsive and unresponsive promoters. Here we show that the RSV Long Terminal Repeat contains two CArG boxes, which were efficiently recognised by purified Serum Response Factor, although they remained unresponsive to serum in transient transfection assays. However, RSV CArG boxes were as efficient as c-fos Serum Response Element in mediating a Serum Response Factor-dependent transcription in vitro. Thus, the fact that a CArG box is able to bind Serum Response Factor in an active form is insufficient for serum responsiveness in vivo.

3T3 Cells↗

PubMed1994

Gene organization and 5'-flanking region sequence of conglutinin: a C-type mammalian lectin containing a collagen-like domain.

Bovine conglutinin is a Ca(2+)-dependent serum lectin specific for N-acetylglucosamine. In the present study, we report the exon-intron organization and the sequence of 5'-regulatory region of the conglutinin gene. The coding region of conglutinin mRNA, which encoded the signal peptide and the sequence of the mature protein of 351 amino acids, consisted of seven exons. Exon III encoded the signal peptide, a cysteine-rich NH2-terminal region and six repeats of a Gly-X-Y triplet with one irregular triplet. The remaining long collagen-like domain (49 Gly-X-Y repeats with one irregular triplet) was encoded by four separate exons (Exon IV-VII). Exon VIII encoded the neck region and Exon IX encoded the carbohydrate-recognition domain. The overall exon-intron organization of conglutinin was very similar to that of mannan (mannose)-binding proteins. However, the presence of 108 bp exons in the gene encoding the collagen-like domain was a characteristic of conglutinin. In the promoter region of the gene, several putative consensus sequences that may be involved in the expression of conglutinin were identified.

Amino Acid Sequence↗

PubMed1995

Multi-strand binding of nuclear factors to a repressor of mouse mammary tumor virus transcription can be distinguished kinetically.

NRE1 is a DNA sequence element in the long terminal repeat of mouse mammary tumor virus that represses viral transcription in mature T cells. In addition to double-stranded binding activity, factors in Jurkat T cell nuclear extracts bind specifically to each of the two single-strands of NRE1. Here we show that binding to the three forms of NRE1 can be distinguished kinetically. The on rates for double, upper and lower-strand NRE1 binding were 1.5, 3, and 11 min, respectively. Binding was extremely stable with off-rates varying from 30 and 60 min for double and upper-strand binding to 12 h for lower-strand binding. In addition, a truncated form of NRE1 that is only bound as a double-strand was observed to have an on rate of binding of 4 min and an off rate of 4 h.

Base Sequence↗

PubMed1995

A regulatory element in the 5'UTR directs cell-specific expression of the mouse alpha 4 gene.

Transfection experiments showed that the mouse alpha 4 promoter contains a downstream element in its 5'UTR which is essential for efficient promoter activity. DNaseI footprinting and electrophoretic mobility shift assays (EMSA) demonstrated that the region from nt +113 to +148 can bind a cell type-specific factor (MIII-3) present in the alpha 4m expressing cell line L1210 but not in the non-expressing cell lines A9 and LMTK. Two consensus SP1 sites in this 5'UTR were recognised in L1210, A9 and LMTK, and with extracts from A9 and LMTK, an AP2-like protein was shown to bind a downstream AP2 site. Thus both ubiquitous and cell type-specific factors regulate the expression of alpha 4m.

Animals↗

PubMed1995

The human bradykinin B2 receptor gene: full length cDNA, genomic organization and identification of the regulatory region.

Hybridization of Northern blots with a probe corresponding to the coding region of the bradykinin B2 receptor revealed a single transcript of approximately 4.0 kb. Using the same probe, we detected a 2.6 kb human cDNA clone that overlapped 103 bp with the 3' end of the known coding region and contained a classical polyadenylation site and a polyA tail. The gene for the human B2 receptor was isolated from a human placenta genomic library. Analysis of several genomic lambda clones indicated that the B2 receptor gene is organized in three exons. Genomic Southern analysis revealed the B2 receptor to be encoded by a single copy gene. In situ hybridization showed that the gene is located on chromosome 14q32. By testing different putative regulatory fragments in a luciferase reporter assay, a gene activating function of the 5' upstream region of exon 1 was demonstrated.

Animals↗

PubMed1995

A novel sequence found at the 3' terminus of hepatitis C virus genome.

The 3' end region of positive-strand RNA-virus genomes is implicated in the initiation of genomic replication. We analyzed the extreme 3' end of the hepatitis C virus (HCV) genome by primer extension of the 5' end region of the antigenomic strand RNA found in infected liver. We discovered a novel sequence present downstream of the poly(U) stretch that was previously considered to be the 3' end structure of the HCV genome. The novel sequence was 98 nucleotides long and had no significant homology with any known sequences, viral or nonviral. The discovery of this novel tail on the HCV genome should contribute to the study of HCV replication.

Base Sequence↗

PubMed1996

Human CFTR gene sequences in regions flanking exon 10: a simple repeat sequence polymorphism in intron 9.

A 2,908-bp segment of genomic DNA containing exon 10 and flanking intron regions of the human cystic fibrosis transmembrane conductance regulator gene was sequenced. A 30-bp sequence discrepancy and three missing nucleotides were detected when compared to a previously published 831-bp sequence. In the 30-bp region of sequence discrepancy, only a primer based on the new sequence information presented in this study gave products from polymerase chain reaction amplification of cellular DNA and a plasmid DNA encompassing the exon 10 region of CFTR. A 4-bp (TAAA) simple repeat sequence was also identified in intron 9 region. This repeat is dimorphic with nine (TAAA)9 or eleven (TAAA)11 copies on different chromosomes. Eleven repeats were exclusively associated with chromosomes carrying the delta508 mutation. Both 9 and 11 repeats were detected in non-delta508 chromosomes.

Base Sequence↗

PubMed1996

Genomic structure of the human bradykinin B1 receptor gene and preliminary characterization of its regulatory regions.

We report on the isolation of the human BK B1 receptor gene from a human lung fibroblast genomic library. The gene is present as a single copy, spans more than 10 kilobases and includes three exons interrupted by two introns. The relative position of the exons was mapped using RNase protection assay, and genomic DNA and cDNA sequence matches. Exon and intron-exon boundaries, and 5', 3' flanking regions were sequenced. While the 5' untranslated region is distributed on all three exons, the coding region is located entirely on the third exon. The exon-intron junction sequences are highly conserved. Primer extension analysis mapped the transcriptional initiation site 21 base pairs downstream of a TATA sequence. Two Aluc sequences and two distinct functional promoters were found in the human BK B1 receptor gene.

Base Sequence↗

PubMed1997

Cloning and characterization of the 5'-flanking region of the human dopamine D4 receptor gene.

Dopamine D4 receptor (DRD4) has received attention in terms of pathogenesis of schizophrenia and association with human personalities. We isolated the human DRD4 gene containing the 5'-flanking region and determined its sequence. Analysis of the DRD4 transcripts by 5'-RACE (5'-rapid amplification of cDNA ends) revealed a region of the transcription initiation located between -501 and -436 relative to the first nucleotide of the initiation codon. There is a CpG island spanning from -900 to +500 but no TATA or CAAT boxes in the 5'-flanking region. Functional analysis of the 5'-flanking region of the DRD4 gene by a transient expression method revealed the presence of a negative modulator between -770 and -679. The region between -591 and -123 gave the highest transcriptional activity in IMR32 (neuroblastoma) and Y-79 (retinoblastoma) cells but not in HeLa cells, suggesting that this housekeeping gene-like promoter regulates the cell-type specific gene expression.

Base Sequence↗

PubMed1999

Structural and functional characterization of the 5'-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene.

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.

Alleles↗

PubMed1997

Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene.

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation of HpArs gene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms.

Amino Acid Sequence↗

PubMed1993

Relationship between scaffold-attached regions, sequences replicating autonomously in yeast, and a chromosomal replication origin in the Drosophila rDNA.

The potential relationship between anchorage of Drosophila rDNA to a nuclear substructure and replication mechanisms was studied. First, two scaffold-attached regions (SARs) were identified, in the internally transcribed spacer and in the region spanning both the intergenic spacer (IGS) and the externally transcribed spacer (ETS), respectively. These SARs define two possible loops containing the sequence coding for the 18S rRNA and part of that coding for the 28S rRNA, respectively. They also bind yeast scaffolds. Then, the presence of sequences able to promote extrachromosomal replication in yeast (ARSs) was tested. The identified ARSs comap with SARs. The tight relationship between SARs and ARSs was further investigated in the IGS-ETS region which contains a chromosomal replication origin. The topological correlation observed between SARs, ARSs, and a chromosomal replication origin suggests the physical association of the replication origin to the nuclear substructure.

Animals↗

PubMed1994

The human EPRS locus (formerly the QARS locus): a gene encoding a class I and a class II aminoacyl-tRNA synthetase.

Glutamyl-tRNA synthetase and prolyl-tRNA synthetase belong to different classes of aminoacyl-tRNA synthetases that are thought to have evolved along independent evolutionary pathways. However, both enzymes are on one polypeptide chain encoded by a single human gene, the EPRS locus, which is transcribed as one long mRNA. We report the structure of the human EPRS gene, which consists of 29 exons spread over at least 90 kb of genomic DNA. The exons, encoding the glutamyl-specific and the prolyl-specific parts of the enzyme, are each clustered in 10-kb sections located at opposite ends of the gene. These two exon clusters are separated by a long intervening DNA section with a number of exons, encoding functions that may be involved in the organization of the mammalian multienzyme synthetase complex. The upstream gene region shows structural features of a regulated gene, and preliminary experiments suggest that the gene is expressed at specific times in growth-stimulated cultured cells. We have localized the gene to the distal long arm of human chromosome 1 and to a corresponding site in mouse chromosome 1.

Amino Acid Sequence↗

PubMed1994

The human MCP-3 gene (SCYA7): cloning, sequence analysis, and assignment to the C-C chemokine gene cluster on chromosome 17q11.2-q12.

Monocyte chemotactic proteins (MCPs) are chemokines involved in macrophage recruitment during inflammation and cancer. A full-size MCP-3 cDNA was used to isolate the functional human MCP-3 gene. Based on restriction analysis, subclones were selected and the MCP-3 gene sequence was completed. In addition to a dense region with direct and inverted repeats and palindromic sequences, a double microsatellite (CA)n-(GA)n' was found at the 5'-end of the MCP-3 gene, and an RFLP was detected. The gene was regionally mapped by fluorescence in situ hybridization to human chromosome 17, subbands q11.2-q12. This site contains the MCP-subset of C-C chemokines and can be distinguished from the syntenic MIP-1 alpha locus. SCYA7 was assigned as the locus symbol of the MCP-3 gene. Double-labeling experiments confirmed the regional assignment of the MCP-3 gene close to the ERBB2 locus on human chromosome 17.

Amino Acid Sequence↗

PubMed1995

A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome.

The rapid and reproducible identification of new cellular DNA sequences adjacent to known sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully identified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome.

Base Sequence↗

PubMed1995

Sequence of human tryptophan 2,3-dioxygenase (TDO2): presence of a glucocorticoid response-like element composed of a GTT repeat and an intronic CCCCT repeat.

Abnormalities in serotonin levels have been implicated in a wide range of psychiatric disorders. Tryptophan 2,3-dioxygenase is the rate-limiting enzyme in the catabolism of tryptophan, the precursor of serotonin. As such it is a potential major candidate gene in psychiatric genetics. The regulatory, intron, and exon regions of the human TDO2 gene have been sequenced. Twelve exons were identified. The amino acid sequence of the enzyme was 88% homologous to that of the rat. Compared to the rat, the regulatory region of the human TDO2 gene had an insertion of approximately 1064 bp of random DNA beginning at -293 bp and extending to -1357 bp. This displaced the glucocorticoid response element (GRE) occurring at -1174 bp in the rat to -1500 in the human. The proximal GRE at -419 in the rat was missing in the human. However, within the DNA insert there was a GRE-like microsatellite region containing multiple GTT repeats plus additional GT(n) sequences. This could produce several staggered regions of the sequence TGTTGTnnnTGTTGT similar to a GRE consensus sequence of TGTTCAnnnTGTTCT. The intron regions 5' and 3' to each exon were sequenced This showed a HIS --> Val mutation polymorphism in exon 7. Three introns, 1,5, and 6, were completely sequenced and examined for polymorphism. This identified two polymorphisms consisting of G -- >T and G --> A mutations 2 bp apart in intron 6. The 3' end of intron 5 showed an extensive CCCT pentanucleotide repeat that was markedly polymorphic. These polymorphisms allow the TDO2 gene to be examined for a possible role in psychiatric disorders.

Amino Acid Sequence↗

PubMed1996

The complete sequence of the host cell factor 1 (HCFC1) gene and its promoter: a role for YY1 transcription factor in the regulation of its expression.

We report here the complete sequence of the Host Cell Factor (HCFC1) gene, including two kilobases of the 5'-flanking region and 5.9 kb of the first intron. The upstream and 5'-untranslated regions contain several putative transcriptional factor binding sites and a 17-nt-long repeated element (SiSa element) present in six regularly spaced copies, of which five are perfectly identical, while the sixth has a transition substitution (CT for TC) at nucleotides 13 and 14. Four copies are contained in the flanking region, the fifth is at the beginning of the mRNA (position +9), and the sixth is at position 195 of the mRNA. This 17-bp element contains at its 5' side an octamer sequence known to bind the Yin/Yang 1 (YY1) transcription factor; another YY1 binding octamer is present at the end of the first intron. The promoter also contains several Sp1 binding sites, some of which are located very close to SiSa elements. We demonstrate that YY1 binds to the 5' half of the SiSa element, whose 3' region binds in gel shift experiments an additional, as yet unidentified nuclear factor. Therefore the YY1 binding site in HCFC1 overlaps the site of a second factor, as has been described in several YY1-site-containing promoters. This suggests that HCFC1 expression might be regulated by the reciprocal interaction of several transcription factors.

Base Sequence↗