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Results for “Repetitive Sequences, Nucleic Acid”

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PubMed1987

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome.

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP does not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.

Binding Sites↗

PubMed1990

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence.

In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 bp duplication in the 5' region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 and A+ strain, an A. nidulans strain with a mutation in the 5' region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 bp sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.

Amidohydrolases↗

PubMed1992

Definition of microsatellite size variants for Tnfa and Hsp70 in autoimmune and nonautoimmune mouse strains.

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor alpha (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouse Tnfa gene. The TNF allele found in the NZW strain is distinct from those of all other H-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of the Tnfa gene and the 3' untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of the H-2 complex, centromeric to the Tnfa gene. The NOD and NZW strains carry unique HSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.

Animals↗

PubMed1994

Effects of insertion of multiple AP-1 binding sites into the U3 region of the long terminal repeat of feline immunodeficiency virus.

An oligonucleotide containing multiple AP-1 binding sites was introduced into the regulatory sequence in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV). Chloramphenicol acetyltransferase assay revealed that basal promoter activity of the mutated LTR was higher than that of the wild-type LTR in Crandell feline kidney (CRFK) cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the clone was transfected into CRFK cells. The virus production of the mutant in the cells was as high as that of the wild-type when determined by the reverse transcriptase activity assay. The growth of the mutant virus obtained from the transfected CRFK cells was examined in feline T lymphoblastoid cell lines (MYA-1 and FeL-039 cells) and primary feline peripheral blood mononuclear cells (fPBMCs). The growth was delayed when compared with that of the wild-type virus in all the cells used. Upon examination by polymerase chain reaction, the length of the LTR of the mutant virus was shortened in both MYA-1 cells and fPBMCs. Sequence analysis revealed that the insertion was completely deleted 39 days after infection in the MYA-1 cells.

Animals↗

PubMed1995

Nucleotide sequence of a 1446 base pair SalI fragment and structure of a novel early gene of Leucania separata nuclear polyhedrosis virus.

A 1446 bp SalI fragment of LsNPV was sequenced by the silver staining method, and two large open reading frames (ORFs, ORF1 and ORF2) were found, both contain typical characteristics of the 5' regulatory elements of baculovirus early genes. ORF1 is 345 bp long with the capacity to encode a putative protein of 114 amino acid residues with MW about 13 kDa and was designated p13 gene, ORF2 comprises 248 bp from the 3' end of the fragment. In the untranslated region (UTR) of ORF1, a 33 bp mini cistron (ORF3), a core recognition sequence (CGTCG) for many bHLHzip transcription factors and a late promoter sequence TTAAG are present. In the UTR of ORF2, two host transcription factor binding elements (CACGTG and GATA motif) and two CGT motifs were found. Some regular leucine zipper-like structures, designated leucine trans-conformation structure and LVT repeat, were found near the N-terminus and the middle of p13 protein. The leucine trans-conformation structure that is near the N-terminus consists of 4 leucines and 7 other amino acids between every two leucines, and every leucine is located at a conformation shift point of the predicted secondary structure of the p13 protein. In LVT repeat, L-6aa-V-6aa-T-6aa is repeated once. The functions of those structures remain unclear, and the two ORFs, not found in the genome of Autographa californica nuclear polyhedrosis virus, are possibly two new genes.

Amino Acid Sequence↗

PubMed1994

Structure and function of the long terminal repeat of the chimpanzee foamy virus isolates (SFV-6).

The complete long terminal repeat (LTR) nucleotide sequence of the chimpanzee foamy virus isolate SFV-6 was determined. Its 1761-bp size makes it the longest LTR reported to date among all retroviruses. Since the length of its LTR is similar to that of other simian isolates while its sequence homology is closer to that of HFV, SFV-6 genetic structure appears to be intermediate between simian and human foamy viruses. Transient expression assays demonstrate that SFV-6 encodes a transactivator of viral gene expression directed either by its own LTR or by heterologous promoters like HFV and HIV-1 LTRs. Our data also provide evidence for cross-transactivation between SFV-6 and HFV.

Animals↗

PubMed1995

Transcriptional regulation of the neuronal L-type calcium channel alpha 1D subunit gene.

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

Alprostadil↗

PubMed1997

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM.

Hexokinase II (HKII) plays an important role in facilitating glucose uptake by skeletal muscle, heart, and adipose tissue in response to insulin. We have cloned and sequenced the proximal promoter region of the human HKII gene, determined the transcription start sites and screened the 2.0 kb of the proximal 5' flanking region for variants in non-insulin-dependent diabetic patients and control subjects. We found three variants in this region, one in the 5' untranslated region (G-->C at +217) and two in the promoter region (T-->G at -1043 and G-->A at -1159). The allele frequencies of these variants did not differ between the diabetic and control subjects and these variants are not associated with insulin resistance. Various segments of the human HKII promoter were tested for driving expression of the luciferase reporter gene. The proximal 500 bp and 400 bp of the promoter were sufficient to drive maximal activity in adipocyte (3T3F442A) and myocyte (C2C12F3) cell lines, respectively. This region of the promoter is GC-rich and contains eight consensus binding sites for the transcription factor Sp-1, five for AP-2, two putative response elements for each of insulin and cyclic AMP. The proximal 175 bases of the promoter retained only 7-15% of maximal activity. Sequence elements located between positions -304 and -215 accounted for approximately 80% of the basal HKII promoter. In addition, the region between -215 and -184 contains a negative regulatory element for expression in 3T3F442A but not in C2C12F3 cells.

Adipocytes↗

PubMed2005

Palindrome content of the yeast Saccharomyces cerevisiae genome.

Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2-12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4-12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.

DNA, Intergenic↗

PubMed2003

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3.

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

Conserved Sequence↗

PubMed1998

P element sequences can compensate for a deletion of the yellow regulatory region in Drosophila melanogaster.

The effects of interactions between P element and yellow regulatory sequences on the control of yellow expression were studied. The y mutations used in the analysis lack a segment of upstream sequence that extends from position -146 bp to -70 bp, relative to the transcription start site of the yellow gene. This sequence has been found to be necessary for the function of the yellow promoter. The insertion of one or two P element copies at position -69 bp compensates for the deletion in the regulatory region and restores yellow expression. After mobilization of the P element, new phenotypes were selected and molecularly characterized. Two regions in the 5' part of the P element, from 23 bp to 71 bp and from 82 bp to 108 bp, can each partially compensate for the yellow deletion. In addition, deletion derivatives of the P element were themselves able to activate yellow transcription. All such P elements retain at least 108 bp of sequence at the 5' end and 15-17 bp at the 3' end. Thus, the region of the P element from 23 bp to 108 bp contains cis-regulatory elements that can influence the transcription of neighboring genes.

Animals↗

PubMed1998

The roles of vif and ORF-A genes and AP-1 binding site in in vivo replication of feline immunodeficiency virus.

To examine the in vivo roles of auxiliary genes and regulatory elements of feline immunodeficiency virus (FIV), the provirus load in various tissues of cats infected with each of the mutant viruses (delta vif, delta ORF-A and delta AP-1) was studied. Although all mutant viruses could infect various tissues, provirus loads in various tissues especially those in cats infected with delta vif virus were lower than those with the wild-type virus. Our results indicate the significance of vif and ORF-A genes and AP-1 binding site of FIV for efficient viral replication and full pathogenicity in cats.

Animals↗

PubMed1989

Characterization of the prostate-specific antigen gene: a novel human kallikrein-like gene.

Using Prostate-specific Antigen cDNA fragments as hybridization probes a clone containing the information for the gene encoding Prostate-specific Antigen was isolated form a human genomic DNA library. The complete gene (about 6 kb) was sequenced and shown to be composed of four introns and five exons. Two major transcription initiation sites were found. The sequence of the promoter region revealed the presence of various well known transcription regulatory elements including a TATA box. A high percentage of homology was found between the Prostate-specific Antigen gene and the hGK-1 gene (82%). This homology extended into the promoter region. Two previously described variant Prostate-specific Antigen cDNAs can now be explained by intron retention and alternative splicing of the primary transcript.

Amino Acid Sequence↗

PubMed1990

Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway.

An expression vector containing three copies of the AP-1 binding element (TRE) upstream of a thymidine kinase promotor which controlled the expression of the chloramphenicol acetyl transferase (CAT) gene was transiently transfected into vascular smooth muscle (VSM) cells and a human hepatocarcinoma cell line, Hep G2. Twelve hours of angiotensin (Ang) II exposure stimulated significantly CAT expression by 3.4 fold and 2.7 fold in Hep G2 and VSM cells, respectively. AngII had no effect on CAT expression of a control vector. This AngII-induced stimulation was attenuated significantly by an AngII receptor antagonist, Sar1 Ile8 AngII, and abolished completely by a PKC inhibitor, staurosporine. Our data suggest that the TRE plays a crucial role in AngII-induced gene expression that is mediated by PKC. We concluded that TRE is one of the AngII-responsive elements.

Alkaloids↗

PubMed1992

Ear3/COUP-TF binds most tightly to a response element with tandem repeat separated by one nucleotide.

Ear3/COUP-TF belongs to the steroid/thyroid hormone receptor superfamily of nuclear transcription factors. Previous studies have identified six target response elements to which the factors of the family bind with various affinities. They are palindromic pair of hexameric half site (Thyroid hormone Response Element; TRE, Estrogen Response Element; ERE, Glucocorticoid Response Element; GRE) or directly repeated half sites [Direct Repeat (DR) 3, DR4, DR5]. In this report, these six elements and three additional putative elements, DR0, DR1, and DR2, which are directly repeated half sites separated by 0, 1 and 2 nucleotides, respectively, were tested for binding to Ear3/COUP-TF protein employing gel retardation method. Among the palindromic series of response elements, TRE showed the highest affinity, ERE showed second highest, and no binding was observed for GRE. Among six direct repeat response elements, DR1 showed the highest affinity. Comparison of binding affinities between DR1 and TRE revealed that the DR1 has the highest affinity of all the nine response elements tested. The consensus sequence of the DR1 element resembles well the COUP sequence found in the upstream promoter of chicken ovalbumin gene, which has been shown to serve as a natural response element for Ear3/COUP-TF. Retinoid X receptor (RXR), another member of the receptor family, reportedly utilizes DR1 sequence as its response element, suggesting that Ear3/COUP-TF could regulate the expression of the same subsets of genes as RXR does.

Base Sequence↗

PubMed1987

12-O-tetradecanoylphorbol-13-acetate induces the enhancer function of human T-cell leukemia virus type I.

Phorbol esters were employed in studies on the molecular mechanism of the induction of expression of human T-cell leukemia virus type I (HTLV-I) by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Experiments using the chloramphenicol acetyltransferase (CAT) system showed that CAT expression directed by the long terminal repeat (LTR) of HTLV-I was induced by TPA, but not by 4 alpha-phorbol-12,13-didecanoate, which is not an activator of protein kinase C, and that like other known enhancers, irrespective of its position and orientation, a 230-bp fragment in the U3 region of the HTLV-I LTR confers susceptibility to induction by TPA.

Deltaretrovirus↗

PubMed1990

Nuclear factor-1 (NF-1) binds to multiple sites within the transcriptional enhancer of Moloney murine leukemia virus.

The transcriptional enhancer of the Moloney Murine Leukemia virus (Mo-MuLV) is comprised of a 75-bp direct repeat, each of which contains multiple binding sites for transcription factors. The occupancy of these sites determines the tissue specificity of expression and disease tropism of the virus. The identification of proteins that bind to this enhancer is therefore required in order to understand the molecular basis of this viral-host interaction. Analysis of the nucleic acid sequence of the Mo-MuLV has identified 4 potential binding sites for the transcription factor NF-1. Evidence is presented using DNAase I protection analysis that NF-1 binds to these 4 sites within the enhancer. The potential role of NF-1 binding in tissue specific expression of Mo-MuLV is discussed.

Base Sequence↗

PubMed1991

Demonstration of a transcription element in vitro between the capping site and translation initiation site of the mouse myelin basic protein gene.

A transcription element was identified, by in vitro analyses, just downstream from the capping site of the mouse myelin basic protein (MBP) gene. Deletion of this element caused a dramatic drop of transcription efficiency in mouse brain, rat liver and HeLa cell nuclear extracts, regardless of the form of DNA being closed circular or linear form. DNase I footprint analysis demonstrated the presence of a ubiquitous trans-acting factor for this region. This element functioned even when it is located in the normal direction downstream from the adenovirus major late promoter. Mutation analysis suggested that an essential part of the downstream element was located between +25 and +45.

Animals↗