scholarly journals Cloning and characterization of the human osteopontin gene and its promoter

1994 ◽  
Vol 303 (1) ◽  
pp. 255-262 ◽  
Author(s):  
N Hijiya ◽  
M Setoguchi ◽  
K Matsuura ◽  
Y Higuchi ◽  
S Akizuki ◽  
...  
Keyword(s):  
Human Monocyte ◽  
Mouse Gene ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Major Band ◽  
Cis Acting ◽  
Consensus Sequences ◽  
Shift Analysis ◽  
Intron Structure ◽  
The Difference

We isolated the human osteopontin (hOP) gene and the 5′ upstream region, and analysed its exon-intron structure and potential regulatory sequences of the promoter region in comparison with those of the mouse and porcine gene. The coding sequence is split into 7 exons which are similar to those of the mouse gene, although the hOP gene is longer than the mouse gene. The difference in length is mainly due to variations in intron 3, which is approximately 2.7-fold longer than that of the mouse OP gene. The 5′ upstream region of the hOP, which is highly conserved up to nucleotide -250, contains a number of potential cis regulatory consensus sequences. A series of sequentially 5′-deleted chimeric clones was tested for the ability to stimulate chloramphenicol acetyltransferase (CAT). Initial CAT analysis demonstrated that nucleotides at positions -474 to -270, -124 to -80, and -55 to -39 contained cis-acting enhancing sequences in a human monocyte cell line, SCC-3, although the -124 to -80 region was much more active than other regions. Deletion of the sequences between -474 and -270 localized this cis region to the sequence at positions -439 to -410, whereas the deletion between -124 to -80 localized the regions to -124 to -115, and -94 to -80. Gel-shift analysis using as probes synthesized double-stranded DNA corresponding to the 10 and 15 bp region at positions -124 to -115 and -94 to -80 respectively revealed that each probe formed a major band complexed with nuclear proteins prepared from SCC-3 cells.

Protein:DNA interactions at chromosomal loop attachment sites

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 503-509 ◽  
Author(s):  
Veronica C. Blasquez ◽  
Ann O. Sperry ◽  
Peter N. Cockerill ◽  
William T. Garrard
Keyword(s):  
Nuclear Matrix ◽  
Binding Sites ◽  
Topoisomerase Ii ◽  
Regulatory Sequences ◽  
Immunoglobulin Gene ◽  
Sequence Motifs ◽  
Cis Acting ◽  
Consensus Sequences ◽  
Dna And Rna ◽  
Multiple Binding Sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (> 10 000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse κ immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.Key words: MARs, chromatin loops, topoisomerase II, nuclear matrix.

Gene regulation in the white–opaque transition ofCandida albicans

1995 ◽  
Vol 73 (S1) ◽  
pp. 1049-1057 ◽  
Author(s):  
David R. Soll ◽  
Thyagarajan Srikantha ◽  
Brian Morrow ◽  
Anand Chandrasekhar ◽  
Klaus Schröppel ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Phenotypic Switching ◽  
Upstream Region ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Promoter Regions ◽  
Coordinate Regulation ◽  
Cis Acting ◽  
Ofcandida Albicans ◽  
Differential Transcription

Most strains of Candida albicans switch frequently and reversibly among a number of different phenotypes distinguishable by colony morphology. Previous experiments indicated that switching involved differential gene expression. Using the white–opaque transition as a model switching system, we have cloned two opaque-specific genes, PEP1 and OP4, and one white specific gene, WH11. Differential transcription of these genes suggested that switching involves the coordinate regulation of batteries of unlinked phase-specific genes. It has been demonstrated that the frequency of integration at phase specific loci is a function of the transcriptional state of the phase-specific genes. In addition, a functional dissection of the 5′-upstream region of the WH11 gene has identified two major domains containing cis-acting regulatory sequences that are involved in phase-specific transcription. Gel retardation experiments provide evidence for white phase-specific trans-acting factors which form complexes with both domains. The regulation of the switching event is discussed. Key words: Candida albicans, phenotypic switching, white–opaque transition, phase-specific genes, integrative transformation, promoter regions, WH11 gene.

scholarly journals Activator-independent gene expression in Neurospora crassa

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 417-423
Author(s):  
Wayne K Versaw ◽  
Robert L Metzenberg
Keyword(s):  
Gene Expression ◽  
Neurospora Crassa ◽  
Chromosomal Rearrangement ◽  
Position Effect ◽  
Upstream Region ◽  
Phosphorus Metabolism ◽  
Position Effects ◽  
Cis Acting ◽  
Independent Gene ◽  
Acting Mechanism

Abstract A transgenic position effect that causes activator-independent gene expression has been described previously for three Neurospora crassa phosphate-repressible genes. We report analogous findings for two additional positively regulated genes, qa-2  + and ars-1  +, indicating that such position effects are not limited to genes involved in phosphorus metabolism. In addition, we have characterized a number of mutants that display activator-independent gene expression. Each of these mutants contains a chromosomal rearrangement with one breakpoint located in the 5’-upstream region of the affected gene. This suggests that the rearrangements are associated with activator-independent gene expression and that these cis-acting mutations may represent a position effect similar to that responsible for rendering some transgenes independent of their transcriptional activators. We suggest that positively regulated genes in N.  crassa are normally held in a transcriptionally repressed state by a cis-acting mechanism until specifically activated. Disruption of this cis-acting mechanism, either by random integration of a gene by transformation or by chromosomal rearrangement, renders these genes independent or partly independent of the transcriptional activator on which they normally depend.

Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (8) ◽  
pp. 3517-3523
Author(s):  
D P McDonnell ◽  
J W Pike ◽  
D J Drutz ◽  
T R Butt ◽  
B W O'Malley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin D ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Transcription Unit ◽  
Proximal Promoter ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Osteocalcin Gene ◽  
Definition Of

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Role for mRNA localization in translational activation but not spatial restriction of nanos RNA

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 659-669 ◽  
Author(s):  
S.E. Bergsten ◽  
E.R. Gavis
Keyword(s):  
Translational Control ◽  
Rna Localization ◽  
Early Embryo ◽  
Posterior Pole ◽  
Translational Repression ◽  
Control Element ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Body Patterning ◽  
Localization Signal

Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3′ untranslated region (3′UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3′UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3′UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.

Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression

2000 ◽  
Vol 113 (18) ◽  
pp. 3173-3185 ◽  
Author(s):  
M.K. Duncan ◽  
Z. Kozmik ◽  
K. Cveklova ◽  
J. Piatigorsky ◽  
A. Cvekl
Keyword(s):  
Transgenic Mice ◽  
Binding Sites ◽  
Fiber Cell ◽  
Regulatory Sequences ◽  
Pax6 Gene ◽  
Fiber Cells ◽  
Nuclear Extracts ◽  
Shift Analysis ◽  
Beta 1 Integrin ◽  
Gel Shift

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120(ctn) as well as large aggregates of (alpha)5(beta)1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human (α)5- and (beta)1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human (alpha)5 and (beta)1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.

Regulated expression of a chimeric histone gene introduced into mouse fibroblasts

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

scholarly journals Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

1991 ◽  
Vol 11 (2) ◽  
pp. 1099-1106 ◽  
Author(s):  
F P Lemaigre ◽  
S M Durviaux ◽  
G G Rousseau
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Gene Encoding ◽  
Protein Binding Sites ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

scholarly journals Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

1989 ◽  
Vol 9 (7) ◽  
pp. 2928-2933 ◽  
Author(s):  
B W Howell ◽  
M Lagacé ◽  
G C Shore
Keyword(s):  
In Vitro Transcription ◽  
Proximal Promoter ◽  
Upstream Region ◽  
Carbamyl Phosphate ◽  
Cis Acting ◽  
Carbamyl Phosphate Synthetase ◽  
Recognition Element ◽  
Cis Element ◽  
Nuclear Extracts

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

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