scholarly journals Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 668-676 ◽  
Author(s):  
M Villa-Garcia ◽  
L Li ◽  
G Riely ◽  
PF Bray
Keyword(s):  
Transcription Start Site ◽  
Phorbol Esters ◽  
K562 Cells ◽  
Start Codon ◽  
Dependent Manner ◽  
Start Site ◽  
Transcription Start ◽  
Specific Expression ◽  
Rnase Protection ◽  
Isolation And Characterization

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5′ to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3′ intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5′ to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a “promoter-less” control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5′ beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

scholarly journals Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 668-676 ◽  
Author(s):  
M Villa-Garcia ◽  
L Li ◽  
G Riely ◽  
PF Bray
Keyword(s):  
Transcription Start Site ◽  
Phorbol Esters ◽  
K562 Cells ◽  
Start Codon ◽  
Dependent Manner ◽  
Start Site ◽  
Transcription Start ◽  
Specific Expression ◽  
Rnase Protection ◽  
Isolation And Characterization

Abstract Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5′ to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3′ intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5′ to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a “promoter-less” control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5′ beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

scholarly journals Preferential Reduction of the Thermodynamically Less Favorable Electron Acceptor, Sulfate, by a Nitrate-Reducing Strain of the Sulfate-Reducing Bacterium Desulfovibrio desulfuricans 27774

2008 ◽  
Vol 191 (3) ◽  
pp. 882-889 ◽  
Author(s):  
Angeliki Marietou ◽  
Lesley Griffiths ◽  
Jeff Cole
Keyword(s):  
Transcription Start Site ◽  
Electron Acceptor ◽  
Nitrate Reduction ◽  
Desulfovibrio Desulfuricans ◽  
Start Codon ◽  
Start Site ◽  
Transcription Start ◽  
Sulfate Reducing ◽  
Reverse Transcription Pcr ◽  
Nitrate Induction

ABSTRACT Desulfovibrio desulfuricans strain 27774 is one of a relative small group of sulfate-reducing bacteria that can also grow with nitrate as an alternative electron acceptor, but how nitrate reduction is regulated in any sulfate-reducing bacterium is controversial. Strain 27774 grew more rapidly and to higher yields of biomass with nitrate than with sulfate or nitrite as the only electron acceptor. In the presence of both sulfate and nitrate, sulfate was used preferentially, even when cultures were continuously gassed with nitrogen and carbon dioxide to prevent sulfide inhibition of nitrate reduction. The napC transcription start site was identified 112 bases upstream of the first base of the translation start codon. Transcripts initiated at the napC promoter that were extended across the napM-napA boundary were detected by reverse transcription-PCR, confirming that the six nap genes can be cotranscribed as a single operon. Real-time PCR experiments confirmed that nap operon expression is regulated at the level of mRNA transcription by at least two mechanisms: nitrate induction and sulfate repression. We speculate that three almost perfect inverted-repeat sequences located upstream of the transcription start site might be binding sites for one or more proteins of the CRP/FNR family of transcription factors that mediate nitrate induction and sulfate repression of nitrate reduction by D. desulfuricans.

scholarly journals Genomic Characterization of Human DSPG3

1999 ◽  
Vol 9 (5) ◽  
pp. 449-456
Author(s):  
Michelle Deere ◽  
Jose L. Dieguez ◽  
Sung-Joo Kim Yoon ◽  
David Hewett-Emmett ◽  
Albert de la Chapelle ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Sequence Data ◽  
Stop Codon ◽  
Genomic Structure ◽  
Start Codon ◽  
Start Site ◽  
Transcription Start ◽  
Exon 2 ◽  
Ancestral Gene ◽  
Link Type

DSPG3, the human homolog to chick PG-Lb, is a member of the small leucine-rich repeat proteoglycan (SLRP) family, including decorin, biglycan, fibromodulin, and lumican. In contrast to the tissue distribution of the other SLRPs, DSPG3 is predominantly expressed in cartilage. In this study, we have determined that the human DSPG3 gene is composed of seven exons: Exon 2 ofDSPG3 includes the start codon, exons 4–7 code for the leucine-rich repeats, exons 3 and 7 contain the potential glycosaminoglycan attachment sites, and exon 7 contains the potential N-glycosylation sites and the stop codon. We have identified two polymorphic variations, an insertion/deletion composed of 19 nucleotides in intron 1 and a tetranucleotide (TATT)n repeat in intron 5. Analysis of 1.6 kb of upstream promoter sequence ofDSPG3 reveals three TATA boxes, one of which is 20 nucleotides before the transcription start site. The transcription start site precedes the translation start site by 98 nucleotides. There are 14 potential binding sites for SOX9, a transcription factor present in cartilage, in the promoter, and in the first intron of DSPG3. We have examined the evolution of the SLRP gene family and found that gene products clustered together in the evolutionary tree are encoded by genes with similarities in genomic structure. Hence, it appears that the majority of the introns in the SLRP genes were inserted after the differentiation of the SLRP genes from an ancestral gene that was most likely composed of 2–3 exons.[The sequence data described in this paper have been submitted to GenBank under accession nos.AF031658 and U63814.]

scholarly journals Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes

1999 ◽  
Vol 181 (16) ◽  
pp. 5075-5080 ◽  
Author(s):  
Hiroshi Kinoshita ◽  
Tomohiro Tsuji ◽  
Hiroomi Ipposhi ◽  
Takuya Nihira ◽  
Yasuhiro Yamada
Keyword(s):  
Transcription Start Site ◽  
Dna Sequences ◽  
Target Genes ◽  
Start Codon ◽  
Receptor Protein ◽  
Upstream Region ◽  
Start Site ◽  
Transcription Start ◽  
S1 Nuclease ◽  
Nuclease Mapping

ABSTRACT BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barBupstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barBtranslational start codon. The BARE located on the barAupstream region (BARE-3) was found 101 to 76 bp upstream of thebarA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barBtranscription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

scholarly journals Promoter Characterization and Constitutive Expression of the Escherichia coli gcvR Gene

1998 ◽  
Vol 180 (7) ◽  
pp. 1803-1807 ◽  
Author(s):  
Angela C. Ghrist ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Transcription Start Site ◽  
Promoter Region ◽  
Start Codon ◽  
Start Site ◽  
Transcription Start ◽  
Translation Start Codon ◽  
Translation Start ◽  
Glycine Cleavage ◽  
Extension Analysis

ABSTRACT The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of thegcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstreambcp-lacZ fusion, and primer extension analysis of thebcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.

scholarly journals Location of promoter elements necessary and sufficient to direct testis-specific expression of the Hst70/Hsp70.2 gene

2004 ◽  
Vol 379 (3) ◽  
pp. 739-747 ◽  
Author(s):  
Dorota ŚCIEGLIŃSKA ◽  
Natallia VYDRA ◽  
Zdzisław KRAWCZYK ◽  
Wiesława WIDŁAK
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Gene Promoter ◽  
Specific Gene ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Specific Expression ◽  
High Level ◽  
Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 are expressed specifically in pachytene primary spermatocytes and spermatids. Here we demonstrate that a 165 bp fragment of the Hst70 gene promoter, containing the T1 transcription start site region, entire exon 1 and 42 bp 5´ region of the intron, is sufficient to drive testis-specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice with the same developmentally regulated pattern as the endogenous Hsp70.2 gene. We show further that high-level tissue-specific gene expression requires additional sequences localized upstream of the T2 transcription start site. Electrophoretic mobility-shift assay analysis revealed that only testes of juvenile rats, when Hst70 gene expression is repressed, contain proteins that specifically bind to the Oct (octamer) sequence localized directly downstream of the T1 site.

scholarly journals Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

Microbiology ◽  
2009 ◽  
Vol 155 (2) ◽  
pp. 584-593 ◽  
Author(s):  
Gang Liu ◽  
Jin Zhong ◽  
Jianqiang Ni ◽  
Meiling Chen ◽  
Haijie Xiao ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Disulfide Bridge ◽  
Start Codon ◽  
Start Site ◽  
Streptococcus Bovis ◽  
Chromosomal Dna ◽  
Knockout Mutant ◽  
Transcription Start ◽  
Translation Start ◽  
Disulfide Oxidoreductase

Bovicin HJ50 is a new lantibiotic containing a disulfide bridge produced by Streptococcus bovis HJ50; its encoding gene bovA was reported in our previous publication. To identify other genes involved in bovicin HJ50 production, DNA fragments flanking bovA were cloned and sequenced. The bovicin HJ50 biosynthesis gene locus was encoded by a 9.9 kb region of chromosomal DNA and consisted of at least nine genes in the following order: bovA, -M, -T, -E, -F, ORF1, ORF2, bovK and bovR. A thiol–disulfide oxidoreductase gene named sdb1 was located downstream of bovR. A knockout mutant of this gene retained antimicrobial activity and the molecular mass of bovicin HJ50 in the mutant was the same as that of bovicin HJ50 in S. bovis HJ50, implying that sdb1 is not involved in bovicin HJ50 production. Transcriptional analyses showed that bovA, bovM and bovT constituted an operon, and the transcription start site of the bovA promoter was located at a G residue 45 bp upstream of the translation start codon for bovA, while bovE through bovR were transcribed together and the transcription start site of the bovE promoter was located at a C residue 35 bp upstream of bovE. We also demonstrated successful heterologous expression of bovicin HJ50 in Lactococcus lactis MG1363, which lacks thiol–disulfide oxidoreductase genes; this showed that thiol–disulfide oxidoreductase genes other than sdb1 are not essential for bovicin HJ50 biosynthesis.

scholarly journals Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication.

Genetics ◽  
1992 ◽  
Vol 131 (1) ◽  
pp. 79-90 ◽  
Author(s):  
S Qian ◽  
B Varjavand ◽  
V Pirrotta
Keyword(s):  
Transcription Start Site ◽  
Binding Sites ◽  
Start Site ◽  
Transcription Start ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Directed Mutation ◽  
Sequence Deletion ◽  
Promoter Interaction

Abstract We have analyzed the eye and testis enhancers located 1 kb upstream of the transcription start site of the white gene. Both enhancers confer the corresponding tissue-specific expression on a heterologous promoter as well as on the white promoter. The eye determinant consists of multiple elements, each able to stimulate eye-specific expression. It also contains five binding sites for the zeste protein while the immediately adjacent testis element contains none. Site-directed mutation of these zeste binding sites abolishes the zeste-white interaction but does not significantly affect the eye enhancer activity, indicating that they are not important for the eye enhancer activity per se. Other zeste binding sites just upstream of the promoter are not necessary for the zeste-white interaction. We conclude that the overlap of the eye enhancer with the zeste binding sites is responsible for the zeste-white interaction and explains why this interaction affects eye but not testis expression. Sequence deletion or substitution experiments suggested that the white promoter is internal to the transcription start site; the zeste protein is not required for distant enhancer action but a 95-bp promoter-proximal sequence is essential for distant enhancer-promoter interaction. This element may serve as an anchor to stabilize formation of a loop that brings the enhancer to the vicinity of the promoter.

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