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At least 991 records · Page 55Linked to original sources

PubMed1993

Promoter region of the rat phospholipase C-gamma 1 gene.

We have cloned the upstream region of the rat phospholipase C-gamma 1 gene and characterized its promoter activity. The 5'-upstream sequence is highly rich in GC, enriched with CpG dinucleotides and lacks a TATA motif. This sequence also contains many putative binding sites for regulatory proteins. Primer extension and RNase protection assay demonstrated a single transcriptional start site located at 103 nucleotides upstream of the ATG start codon. The transcriptional activities of various 5'-deleted fragments, fused with chloramphenicol acetyltransferase gene, were examined after transfection into C2C12 myoblast. Multiple positive and negative regulatory sites were observed.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and sequencing of sulfated glycoprotein-2 cDNA from testis of mouse: implications of two different mRNAs of SGP-2.

We cloned a mouse homolog of sulfated glycoprotein-2 (SGP-2) cDNA by screening a mouse testicular cDNA library and its nucleotide sequence was determined. The predicted amino acid sequence of the cDNA shares 93% identity with that of rat SGP-2. The nucleotide sequences of both cDNAs show extensive homology throughout the open reading frames and 3' untranslated regions. The 5' untranslated regions, however, share homology only up to 28 bp upstream from the start codons; the rest of sequences are quite different. DNA sequence homology search to mouse SGP-2 cDNA through the EMBL/GenBank database and a recent study on the genomic organization of rat TRPM-2 gene suggest a possibility that there are at least two different SGP-2 mRNAs as a result of alternative splicing and/or different promoter usage in mouse.

Alternative Splicing↗

PubMed1994

Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene.

Prostaglandin H synthase (PGHS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. The human PGHS has two isoforms. PGHS-1 is a house keeping gene whereas PGHS-2 is an inducible gene. We reported here the isolation of the entire PGHS-2 gene and its 5'-flanking region from a human bacteriophage P1 genomic library. The gene containing 10 exons is 7.5 kb in length and located at chromosome 1. The transcriptional start site was mapped at 134 bases upstream from the ATG start codon. Nucleotide sequence of 1.8 kb promoter region contains a TATA box and a number of potential regulatory elements including CRE, NF-kappa B, Sp1 and AP2 sites. Studies of the promoter activity showed that the first 460 nucleotides of 5'-flanking region efficiently drove transcription of the luciferase reporter gene in human umbilical vein endothelial cells upon stimulation with phorbor ester.

Animals↗

PubMed1995

Genomic structure of the mouse delta opioid receptor gene.

Using mouse delta opioid receptor (DOR) cDNA sequence to probe genomic libraries in bacteriophage lambda and P1 vectors, clones traversing the entire DOR coding sequence and 5' and 3' flanking regions were isolate. Genomic sequence encoding mature DOR message, including 5' and 3' untranslated sequence, is divided by two introns of 26 kb and 3 kb, resulting in the gene occupying 32 kb of chromosomal DNA. Multiple putative transcription initiation sites were located, by RNase protection assay, in TATA-less G+C rich sequence between 390 and 140 nucleotides upstream from the ATG translation start codon. A polyadenylation site was located 1.24 kb downstream from the TGA translation stop codon. Examination of 1.3 kb of 5'flanking sequence revealed potential binding sites for several known transcription factors including: Sp1, Ap-2, NF-kappa B, NF-IL6, and NGFI-B.

Animals↗

PubMed1995

Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene.

The chromatin structure of 120kb of genomic DNA 5' to the CFTR gene has been analysed in a number of CFTR expressing and non-expressing cell types, including primary genital duct epithelial cells. Novel DNAse I hypersensitive sites have been observed at -79.5kb and -20.5kb 5' to the ATG translation start codon of the CFTR coding sequence. Neither of these sites appears to show strong correlation with CFTR expression in the cell types investigated, hence they are unlikely to reflect the sites of binding of the major CFTR tissue specific regulator(s). However, they may still play an important part in the complex series of events involved in the regulation of CFTR transcription.

Base Sequence↗

PubMed1995

Identification of an upstream region that controls the transcription of the human autocrine motility factor receptor.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

3T3 Cells↗

PubMed1996

Chromosomal localization and 5' sequence of the human protein serine/threonine phosphatase 5' gene.

Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involved in many cellular processes. Protein phosphatase 5 (PP5) is a novel member of the protein serine/threonine phosphatase family. The majority of the cDNA sequence of PP5 has been reported recently. In our study, a sequence encoding the whole open reading frame of PP5 was cloned from a human fetal brain cDNA library. The protein phosphatase cDNA sequence of our clone is longer at the 5' end than the recently published sequence. It's likely that the extended sequence contains the start codon ATG, since a translation stop codon TAG is present upstream of the ATG codon in the same open reading frame. The mRNA of the PP5 gene was detected in all the human tissues examined. The PP5 gene was localized to human chromosomal region 19q13.3.

Amino Acid Sequence↗

PubMed1996

Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro.

Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.

Base Sequence↗

PubMed1996

An Mse I RFLP in the human CTLA4 promotor.

We present a PCR-based Mse I restriction fragment length polymorphism (RFLP) in the promotor of the human CTLA4 gene at position -318 relative to the ATG start codon. In a random caucasian population including 239 individuals the allele frequency of the polymorphism was 13.4%, with a calculated heterozygosity rate of 23.2% and an observed one of 25.1%. CTLA4 and CD28 are two genes in close proximity on the long arm of chromosome 2. Both of them influence T-cell activity in an antigen-independent way. They represent candidate genes for several immunological disorders. Recently association between the CTLA4 gene and Graves' disease has been shown. The Mse I RFLP in the CTLA4 promotor might serve as a useful tool for further association studies.

Abatacept↗

PubMed1997

Sequencing, analysis and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein.

A previous paper presented data on a cDNA sequence encoding a protein associated with the AIDS related pathogen, Cryptosporidium parvum. However, the position of the start codon was uncertain, and the 5' end was continuous, lending doubt about the size and complete sequence of the final protein product. Herein we present the complete gene sequence and conclude the predicted size of the putative protein to be 16.2 kDa.

Amino Acid Sequence↗

PubMed1997

Cell cycle-dependent regulation of the mouse DNA topoisomerase IIalpha gene promoter.

Expression of DNA topoisomerase (topo) IIalpha varies through the cell cycle with its peak in G2/M. To investigate the mechanism controlling the topo IIalpha gene expression, we cloned the 5' upstream region of the mouse topo IIalpha gene. Although there was no TATA-like sequence, two GC and seven CCAAT boxes were found in the upstream region 5' distal to the major transcription start sites, which were located 137, 124, and 105 bp upstream from the ATG start codon. Luciferase vectors with the upstream sequences were constructed and transfected into HeLa cells, followed by cell cycle arrest either in G1 by treatment with mimosine, in S with thymidine, or in G2/M with colcemid. We found that the topo IIalpha gene promoter has the cell cycle-dependent activity, which is low in G1, rises in S, and peaks in G2/M. We suggest that the level of topo IIalpha mRNA is determined by the cell cycle-regulated promoter.

Animals↗

PubMed1997

Sequence of a cDNA clone encoding a novel somatolactin in goldfish, Carassius auratus.

As a first step towards the development of a sensitive ribonuclease protection assay to study the regulation of somatolactin (SL) mRNA expression in pituitary cells of goldfish, we have isolated a complementary DNA (cDNA) clone encoding precursor sequence of SL from a cDNA library prepared from goldfish pituitary poly(A)+ RNA. The 843-bp goldfish SL (gfSL) cDNA has an open reading frame of 693 nucleotides with two possible start codons of AUG. Amino acid sequence alignment revealed that gfSL has the characteristics of four conserved domains (A, B, C and D) common to all SLs with the domain B being the most conserved region among all the characterized SLs. Similar to other teleost SLs, this gfSL is similarly related but clearly distinct from growth hormone and prolactin of goldfish and other teleosts. However, unlike most other known teleost SLs which have more than 70% amino acid sequence identity to each other, the overall amino acid sequence identity of this novel gfSL with other previously characterized SLs ranges from only 36% to 51%. Moreover, this gfSL contains only six cysteine residues, rather than seven in most other SLs, in conserved positions. Northern blot analysis revealed a single gfSL mRNA transcript of approximately 1 kb in the pituitaries of both sexually regressed and maturing male and female goldfish.

Amino Acid Sequence↗

PubMed1997

Characterization of the genomic structure and promoter of the mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5'-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5'-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.

Animals↗

PubMed1997

Molecular structure of the mouse CCK-A receptor gene.

We have cloned the mouse CCK-A receptor gene (Cckar), determined its nucleotide sequence, and analyzed its expression. The receptor protein is encoded in five exons distributed over 9 kb of genomic DNA. Intron/exon borders were determined by comparing the genomic nucleotide sequence with the mouse cDNA sequence obtained by reverse transcriptase polymerase chain reaction. RNase protection analysis of Cckar transcripts revealed the presence of a splice acceptor site 200 bp upstream of the translational start codon, indicating that the promoter is associated with a non-translated exon at an upstream site. The second coding exon contains a rarely used alternative splice site that would result in the production of a truncated, 48 amino acid protein. Cckar is widely expressed in the gastrointestinal system (pancreas, gallbladder, intestine, colon and stomach), as well as in brain and kidney.

Alternative Splicing↗

PubMed1997

Gene structure for mouse glutathione reductase, including a putative mitochondrial targeting signal.

Glutathione reductase (GR) is an important component of cellular antioxidant defense functions. Although GR activities are found in mitochondria and cytoplasm, the sorting mechanisms of mammalian GR into mitochondria have not been elucidated. To identify the mouse GR gene structure, including a sequence for a potential mitochondrial targeting signal (MTS), we screened a mouse genomic library and isolated four contiguous clones that covered the entire coding region of the gene. The coding region is composed of 13 exons. Exon 1 has two in-frame start codons separated by a sequence for an arginine-rich peptide segment. Expression studies, in which Chinese hamster ovary cells were transiently transfected with a plasmid containing the first 78 bp of the mouse exon 1 attached 5' to the human GR cDNA, showed marked and selective increases in mitochondrial GR activities. The data indicate that this 78 bp sequence encodes a potential MTS for GR in mice.

Amino Acid Sequence↗

PubMed1997

Transcriptional analysis of the threonine dehydrogenase gene of Xanthomonas campestris.

The nucleotide sequence has previously been determined for the Xanthomonas campestris pv. campestris gene coding for threonine dehydrogenase (tdh). Flanking this gene are the upstream region possessing promoter activity and the downstream perfect inverted repeat having potential to form a stem-loop structure which resembles a transcription terminator. In addition, Northern blot analysis suggested the transcript of this gene to be monocistronic. In the present study, the essential region for promoter activity was narrowed down to a stretch of 57 bp which still retained 84% of the promoter activity. The first nucleotide to be transcribed is the guanosine at 30 nt upstream from the proposed tdh start codon. The putative terminator exhibited transcriptional termination activity bidirectionally in both Escherichia coli and X. campestris. These observations indicate that the transcriptional structure of X. campestris tdh is different from that of E. coli where tdh and kbl are organized into the tdh operon. Furthermore, the expression of tdh in X. campestris is repressed by leucine, a situation different from that in E. coli where leucine induces the expression of tdh operon.

Alcohol Oxidoreductases↗

PubMed1998

Expression of a novel isoform of cyclin I in human testis.

A new isoform of cyclin I, designated cyclin ITI, was cloned from the human testis cDNA-lambda gt10 library by using a approximately 0.5 kb PCR fragment that was obtained by using primers based on the published cyclin I sequence from the human brain. The cyclin ITI cDNA is 1443-bp long and has an ORF of 178 aa with the first ATG Met start codon at nt 1 and has the well-conserved sequence of cyclin box. The 5' flanking sequence of 728-bp has a high similarity (99.6%) with the 5'-intron of the human beta-polymerase gene. The nt sequence of cyclin ITI cDNA has high similarity (99.3%) with the cyclin I gene sequence. The cyclin ITI was found to have three amino acids mutations at nt 25, 172, 223, respectively, including one in the well-conserved sequence of cyclin box. Northern blot analysis indicated that the cyclin ITI is expressed in human testis at increased level compared to other tissues. The in vitro translated protein of the cyclin ITI cDNA was recognized specifically by the antibodies raised against the human sperm proteins, indicating its presence in human sperm. This new isoform may have a physiological role in spermatogenesis and/or human sperm cell function--especially in capacitation and/or acrosome reaction.

Amino Acid Sequence↗

PubMed1999

A core promoter and a frequent single-nucleotide polymorphism of the mismatch repair gene hMLH1.

The hMLH1 gene encodes a protein that is involved in the DNA mismatch repair system. The coding region of the hMLH1 gene has been known to be mutated in a subset of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Our current research characterized the promoter region of the hMLH1 gene and searched for mutations correlating to HNPCC. Utilizing the oligo-capping method, major transcription start sites of the hMLH1 gene were mapped at two locations. The core promoter region of about 180 bp was determined by the luciferase assay of serial deletion mutants. Although we did not find any pathogenic mutation in the hMLH1 promoter region by PCR-SSCP, we found a single-nucleotide polymorphism at position -93 nt from the adenine residue of the start codon. By PCR-RFLP analysis with Pvu II for this polymorphism, we detected LOH in four tumors from three patients. An easy detection of this polymorphism with PCR-RFLP and high incidence ( approximately 50%) of informative cases make this polymorphism a suitable marker for the detection of hMLH1 allelic losses.

3T3 Cells↗