Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter

2002 ◽  
Vol 282 (5) ◽  
pp. F898-F909 ◽  
Author(s):  
Christie P. Thomas ◽  
Randy W. Loftus ◽  
Kang Z. Liu ◽  
Omar A. Itani
Keyword(s):  
Genomic Organization ◽  
Human Kidney ◽  
Β Subunit ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Flanking Region ◽  
Rapid Amplification ◽  
Flanking Regions

The mRNA for the β-subunit of the epithelial Na+ channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.

Molecular cloning and characterization of the yellowtail GH gene and its promoter: a consensus sequence for teleost and avian Pit-1/GHF-1 binding sites

1996 ◽  
Vol 16 (1) ◽  
pp. 63-72 ◽  
Author(s):  
T Ohkubo ◽  
M Araki ◽  
M Tanaka ◽  
S Sudo ◽  
K Nakashima
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Transcription Start ◽  
Dominant Form ◽  
Nuclear Extracts ◽  
Gh Gene ◽  
Seriola Quinqueradiata ◽  
Gel Shift Assay ◽  
Flanking Region

ABSTRACT The gene and 5′ flanking promoter region for yellowtail (Seriola quinqueradiata) GH (yGH) have been cloned, sequenced and characterized. The yGH gene spans approximately 4·6 kb and consists of six exons and five introns, as has been observed with rainbow trout, Atlantic salmon and tilapia GH genes. This result suggests that the structure of six exons and five introns is a dominant form in fish GH genes. A typical TATA box exists 26 bp upstream from the transcription start site, and Pit-1/GHF-1 (Pit-1) binding site-homologous regions were found in the promoter region of the yGH gene. In a gel shift assay, however, a single shifted band was detected with the fragments containing a region from −128 to −90 of the yGH 5′ flanking region when they were incubated with yellowtail pituitary nuclear extracts. The bound fragments contained an octamer base sequence similar, but not identical, to mammalian consensus Pit-1 binding element. A consensus octamer sequence is also proposed in this report for the binding of teleost and avian Pit-1 transcription factors.

scholarly journals Purification and Molecular Characterization of a Tripeptidase (PepT) from Lactobacillus helveticus

2000 ◽  
Vol 66 (2) ◽  
pp. 794-800 ◽  
Author(s):  
Kirsi Savijoki ◽  
Airi Palva
Keyword(s):  
Maximum Activity ◽  
Lactobacillus Helveticus ◽  
Open Reading Frames ◽  
Transcription Start ◽  
Sequence Analyses ◽  
Repeat Structure ◽  
Flanking Regions ◽  
Dairy Starter ◽  
Reading Frames

ABSTRACT A tripeptidase (PepT) from a thermophilic dairy starter strain ofLactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (Km , 2.6 mM;V max, 80.2 μmol · min−1 · μg−1). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing theLactobacillus PepT from its counterpart in mesophilicLactococcus lactis. No activity against tetrapeptides or amino acid p-nitroanilide derivatives was observed. ThepepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis. The mRNA analyses indicated thatpepT conforms a novel operon structure with an ORF1 located upstream. Several putative −35/−10 regions preceded the operon, but only one transcription start site located downstream of the first putative −10 region was identified. An inverted repeat structure with ΔG of −64.8 kJ/mol was found downstream of the PepT-encoding region.

scholarly journals Isolation and characterization of the monkey UGT2B30 gene that encodes a uridine diphosphate-glucuronosyltransferase enzyme active on mineralocorticoid, glucocorticoid, androgen and oestrogen hormones

2002 ◽  
Vol 365 (1) ◽  
pp. 213-222 ◽  
Author(s):  
Caroline GIRARD ◽  
Olivier BARBIER ◽  
David TURGEON ◽  
Alain BÉLANGER
Keyword(s):  
Cynomolgus Monkey ◽  
Genomic Organization ◽  
Human Kidney ◽  
Relative Activity ◽  
Pcr Analysis ◽  
Uridine Diphosphate ◽  
Lipophilic Compounds ◽  
Isolation And Characterization ◽  
Hormonal Steroids

The present study reports the genomic organization and the characterization of a novel cynomolgus monkey UDP-glucuronosyltransferase (UGT) enzyme, UGT2B30. UGT enzymes are microsomal proteins that catalyse the transfer of the glucuronosyl group from UDP-glucuronic acid (UDPGA) to a wide variety of lipophilic compounds, namely hormonal steroids. The 15kb UGT2B30 gene amplified by PCR showed a genomic organization similar to those encoding UGT2B human enzymes. The cDNA encoding UGT2B30 was isolated from a cynomolgus monkey prostate cDNA library, and the deduced amino acid sequence showed an identity of 94% with UGT2B19, a monkey isoform previously characterized. Stable expression of UGT2B30 protein in human kidney 293 (HK293) cells was assessed by Western-blot analysis and its conjugating activity was screened using 39 potential substrates. The UGT2B30 enzyme is active on many compounds of different classes, including testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol, androsterone, oestradiol, tetrahydroaldosterone and tetrahydrocortisone, with glucuronidation efficiencies (Vmax/Km ratios) ranging from 0.6 to 8.8μl·min−1·mg of protein−1. Reverse-transcriptase-PCR analysis revealed that the UGT2B30 transcript is expressed in several tissues, including prostate, testis, mammary gland, kidney, adrenals and intestine. The relative activity of UGT2B30 in comparison with other simian UGT2B isoforms, as well as its large variety of substrates, strongly suggest that this enzyme is essential to inactivation of several steroids.

scholarly journals Organization of the human β-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis

1997 ◽  
Vol 321 (2) ◽  
pp. 465-474 ◽  
Author(s):  
Betty YIP ◽  
Shi-Hao CHEN ◽  
Hans MULDER ◽  
Jo W. M. HÖPPENER ◽  
Harry SCHACHTER
Keyword(s):  
Genomic Dna ◽  
Transient Transfection ◽  
Transcription Start ◽  
Exon 2 ◽  
Transcription Start Sites ◽  
Exon 1 ◽  
Dna Fragment ◽  
Flanking Region ◽  
Ribonuclease Protection ◽  
I Gene

UDP-GlcNAc:α-3-d-mannoside α-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-T I) is a medial-Golgi enzyme which catalyses the first step in the conversion of oligomannose-type to N-acetyl-lactosamine- and hybrid-type N-glycans and is essential for normal embryogenesis in the mouse. Previous work indicated the presence of at least two exons in the human GlcNAc-T I gene MGAT1, exon 2 containing part of the 5ƀ untranslated region and the complete coding and 3ƀ untranslated regions, and exon 1 with the remainder of the 5ƀ untranslated region. We now report the cloning and sequencing of a human genomic DNA fragment containing exon 1, which is between 5.6 and 15 kb upstream of exon 2. Transient transfection, ribonuclease protection and reverse transcriptase-mediated PCR indicated the absence of transcription start sites in intron 1 between exons 1 and 2. Northern analysis, ribonuclease protection, primer extension analysis and rapid amplification of 5ƀ-cDNA ends showed that there are multiple transcription start sites for exon 1 compatible with the expression by several human cell lines and tissues of two transcripts, a broad band ranging in size from 2.7 to 3.0 kb and a sharper band at 3.1 kb. The 5ƀ flanking region of exon 1 has a GC content of 81% and has no canonical TATA or CCAAT boxes but contains potential binding sites for transcription factors Sp1, GC-binding factor and epidermal growth factor receptor-specific transcription factor. Chloramphenicol acetyltransferase (CAT) expression was observed on transient transfection into HeLa cells of a fusion construct containing the gene for CAT and a genomic DNA fragment from the 5ƀ flanking region of exon 1. It is concluded that MGAT1 is a typical housekeeping gene although there is, in addition, tissue-specific expression of the larger 3.1 kb transcript.

scholarly journals Cloning, sequencing and characterization of the 5′-flanking region of the human collagenase-3 gene

1997 ◽  
Vol 323 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Ginette TARDIF ◽  
Jean-Pierre PELLETIER ◽  
Martine DUPUIS ◽  
John E. HAMBOR ◽  
Johanne MARTEL-PELLETIER
Keyword(s):  
Pathological Process ◽  
Polyoma Virus ◽  
Start Codon ◽  
Activator Protein 1 ◽  
Matrix Metalloprotease ◽  
Transcription Start ◽  
Hormone Response Elements ◽  
Activator Protein ◽  
Flanking Region

Collagenase-3 (matrix metalloprotease-13) is a recently discovered human collagenase produced in normal articular cartilage chondrocytes and thought to be involved in the pathological process of osteoarthritis. We have sequenced and characterized 1.6 kb of the human collagenase-3 gene 5´-flanking region. The transcription start site was located 22 bp upstream from the ATG start codon. Sequence analysis of the 5´-flanking region revealed the presence of the consensus recognition sites for the TATA and CCAAT DNA-binding proteins, activator protein-1 and E26 transformation specific/polyoma virus enhancer, as well as three core motifs of hormone response elements. Transient transfection assays demonstrated that a small fragment of 133 bp, containing the activator protein-1 and E26 transformation specific/polyoma virus enhancer sites promoted transcription in normal and osteoarthritic human chondrocytes with significantly higher activity than the original 1.6 kb fragment. Nucleotide sequence comparison of the promoter region of human collagenase-3 revealed a stronger similarity to the mouse collagenase-1 promoter than to the human collagenase-1 promoter.

scholarly journals Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Ilaria Laudadio ◽  
Sara Formichetti ◽  
Silvia Gioiosa ◽  
Filippos Klironomos ◽  
Nikolaus Rajewsky ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Transcription Termination ◽  
Dna Integrity ◽  
Transcription Start ◽  
Cycle Progression ◽  
Primary Tumors ◽  
Transcription Start Sites ◽  
Generation Sequencing ◽  
Comprehensive Study

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5′ end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3′ end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.

Characterization of POU5F1 (OCT4) gene and its promoter in buffalo ESC-like cells identifies multiple transcription start sites and expression of four pseudogenes

Gene ◽  
2012 ◽  
Vol 491 (2) ◽  
pp. 165-172 ◽  
Author(s):  
Natwar Singh ◽  
Aman George ◽  
Ruchi Sharma ◽  
Suresh K. Singla ◽  
Prabhat Palta ◽  
...  
Keyword(s):  
Transcription Start ◽  
Transcription Start Sites
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