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Results for “multiple nucleotide deletion”

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At least 217 records · Page 12Linked to original sources

PubMed2006

Translocation-excision-deletion-amplification mechanism leading to nonsyntenic coamplification of MYC and ATBF1.

Despite oncogene amplification being a characteristic of many tumor types, the mechanisms leading to amplicon formation have remained largely unresolved. In this study, we used a combinatorial approach of fluorescence in situ hybridization and single-nucleotide polymorphism chip gene copy number analyses to unravel the mechanism leading to nonsyntenic coamplification of MYC and ATBF1 in SJNB-12 cells. To explain our findings, we propose a complex series of events consisting of multiple double-strand breaks, accompanied (or triggered) by the formation of a reciprocal translocation t(8;16), as well as excisions and deletions near the translocation breakpoints. This study provides evidence for a translocation-excision-deletion-amplification sequence of events rather than a breakage-fusion-bridge model, which has been more frequently proposed to explain proto-oncogene amplification. Furthermore, it illustrates the power of presently available tools for detailed analysis of the complex rearrangements that accompany amplicon formation.

Cell Line, Tumor↗

PubMed1998

Identification of the oim mutation by dye terminator chemistry combined with automated direct DNA sequencing.

The homozygous oim/oim mouse, a model of moderate-to-severe human osteogenesis imperfecta, contains a G-nucleotide deletion in the Cola-2 gene (the murine pro alpha(I) collagen gene) that results in accumulation of alpha1(I) homotrimer collagen. Although these mice have a distinctive phenotype that includes multiple fractures and deformities, genotyping is necessary to distinguish them from their wildtype (+/+) and heterozygote (oim/+) littermates. In this study, the dye primer and dye terminator chemistry methods, in combination with automated direct DNA sequencing, were compared for accuracy and ease in genotyping. A total of 82 mice from 14 litters were bred and genotyped; this resulted in 18 +/+, 35 oim/+, and 29 oim/oim mice. The dye primer and dye terminator chemistry methods worked equally well for identification of the deletion mutation and thus the genotype of all of the mice. However, the dye terminator method was found to be superior on the basis of the reduced amount of sample handling and reduced quantity of reagent required.

Animals↗

PubMed1984

Sequence analysis of ultraviolet-induced mutations in M13lacZ hybrid phage DNA.

We have studied the specificity of ultraviolet (u.v.) mutagenesis in single-stranded DNA phage by analyzing u.v.-induced forward mutations in the lac insert of M13mp2 hybrid phage. Sequence analysis of 114 lac mutants derived from u.v.-irradiated phage grown in u.v.-irradiated cells showed that ultraviolet induces mainly single-nucleotide substitutions and deletions in progeny phage DNA. A total of 74% of the single-base substitution mutations occurred at sites of adjacent pyrimidines in the single-stranded DNA, with both T----C and C----T transitions predominating in the u.v. spectrum. Single-nucleotide deletion mutations occurred preferentially in tracts of repeated pyrimidine nucleotides. Tandem, double-base substitutions did not represent a major class of u.v.-induced mutations, but nearly 10% of mutant clones contained multiple, non-tandem nucleotide changes.

Base Sequence↗

PubMed2003

Structure and promoter activity of the human glia maturation factor-gamma gene: a TATA-less, GC-rich and bidirectional promoter.

Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity.

Base Sequence↗

PubMed1999

Identification of two essential sequence elements in the nonconsensus type II PatpB-290 plastid promoter by using plastid transcription extracts from cultured tobacco BY-2 cells.

In higher plants, plastid genes are transcribed by at least two types of DNA-dependent RNA polymerases. One of them is the well-known plastid-encoded prokaryotic type of polymerase that recognizes sigma(70)-type promoters consisting of -35 and -10 consensus elements. The other recently recognized RNA polymerase has been found to be encoded entirely in the nucleus, and it recognizes a completely different set of promoters, designated previously as nonconsensus type II (NCII) promoters. Here, we report the development of an in vitro transcription system using nonphotosynthetic plastids of cultured tobacco BY-2 cells. This system preferentially and accurately initiates transcription from NCII promoters. The conditions for in vitro transcription were optimized by using the tobacco PatpB-290 promoter, which has been found to be the most highly expressed NCII promoter in vivo. Analysis of in vitro transcription initiation in a series of PatpB-290 5' deletion constructs revealed that sequences upstream of nucleotide -41 do not influence the transcriptional activity of this promoter. A 43-bp region (nucleotides -35 to +8) was further analyzed by introducing single or multiple nucleotide substitutions into two regions (box I and box II) of high sequence conservation. We report here that the ATAGAA sequence comprising box II and the -11 to +4 region (relative to transcription initiation) in box I significantly influence the activity of this NCII promoter.

Base Sequence↗

PubMed2000

Defects of intergenomic communication: where do we stand?

An expanding number of autosomal diseases has been associated with mitochondrial DNA (mtDNA) depletion and multiple deletions. These disorders have been classified as defects of intergenomic communication because mutations of the nuclear DNA are thought to disrupt the normal cross-talk that regulates the integrity and quantity of mtDNA. In 1989, autosomal dominant progressive external ophthalmoplegia with multiple deletions of mitochondrial DNA was the first of these disorders to be identified. Two years later, mtDNA depletion syndrome was initially reported in infants with severe hepatopathy or myopathy. The causes of these diseases are still unclear, but genetic linkage studies have identified three chromosomal loci for AD-PEO. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder associated with both mtDNA depletion and multiple deletions, is now known to be due to loss-of-function mutations in the gene encoding thymidine phosphorylase. Increased plasma thymidine levels in MNGIE patients suggest that imbalanced nucleoside and nucleotide pools in mitochondria may lead to impaired replication of mtDNA. Future research will certainly lead to the identification of additional genetic causes of intergenomic communication defects and will likely provide insight into the normal "dialogue" between the two genomes.

Cell Nucleus↗

PubMed1998

In vitro genetic analysis of the RNA binding site of vigilin, a multi-KH-domain protein.

The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3' untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A)nCU and UC(A)n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A)nCU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3' end of human dystrophin mRNA. RNA sequences from the 3' UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs.

Animals↗

PubMed2000

Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.

Adenosine↗

PubMed2004

Identification and characterization of human FOXK1 gene in silico.

Forkhead-box (FOX) family transcription factors are implicated in carcinogenesis and embryogenesis. Here, we identified and characterized the human FOXK1 gene by using bioinformatics. Complete coding sequence of human FOXK1 cDNA was determined by assembling CB959941 EST, AW206906 EST, and 5'-truncated FLJ16099 (AK122663.1) cDNA. FOXK1 gene, consisting of nine exons, was mapped to human chromosome 7p22.1. Mouse Foxk1 (NM_199068.1) was an aberrant cDNA with frame shifts due to multiple insertions and deletions, while mouse IMAGE6853263 (BC060238.1) was a Foxk1 cDNA with a frame shift due to two base deletions. Complete coding sequence of mouse Foxk1 cDNA was determined by inserting CA nucleotides between nucleotide position 1628 and 1629 of BC060238.1. Foxk1 gene, consisting of nine exons, was mapped to mouse chromosome 5G2. Because interleukin enhancer-binding factor 1 (ILF1) gene at human chromosome 17q25.3 was the paralog of FOXK1 gene, ILF1 gene was designated the FOXK2 gene. Xenopus BC046369.1 cDNA was Foxk2 ortholog rather than Foxk1 ortholog. Human FOXK1 (733 aa) showed 88.7% total amino-acid identity with mouse Foxk1 (719 aa), 48.7% total amino-acid identity with human FOXK2, and 47.5% total amino-acid identity with Xenopus Foxk2. Forkhead associated (FHA) domain and FOX domain were conserved among human FOXK1, FOXK2, mouse FoxK1, and Xenopus Foxk2. At least 42 FOX family genes, including FOXK1, FOXN5 (FOXR1) and FOXN6 (FOXR2), have been identified within the human genome.

Amino Acid Sequence↗

PubMed1996

Transcription of the human hepatic lipase gene is modulated by multiple negative elements in HepG2 cells.

The expression of the hepatic lipase (HL) gene is highly tissue specific. In order to identify cis-acting elements which regulate the expression of this gene in the liver, multiple deletion mutants of the 5'-flanking region of the HL gene fused to the human growth hormone gene were transfected in HepG2 cells, which normally produce HL. Transient expression assays indicated the presence of negative (at nucleotides (nt) -1576(/)-1342 and -623(/)-407) and positive (at nt -1862(/)-1576 and -50(/)-9) regulatory elements. Transfection of HeLa cells, which do not produce HL, with the same deletion constructs resulted in a similar pattern of promoter activities. However, additional negative (nt -138/-50) and positive (nt -407(/)-138) elements were found. DNase I footprint analysis of the proximal and distal HLpromoter sequences with HepG2 and HeLa cell nuclear extracts identified seven protected regions: A, nt -1540(/)-1527; B, -1505(/)-1473; C, -1467(/)-1460; D, -592(/)-577; E, -565(/)-545; F, -234(/)-220; and G, -70(/) -48. Sites A, B, C, D and E were located within regions containing negative regulatory elements. In order to determine which nuclear factor interacts with the negative elements, sites B, D and E were mutated and the effects of mutation on competition in a gel retardation assay and on promoter activity were studied. When the binding motif for AP1 in sites B, D and E was mutated, the specific DNA-protein complexes were not competed with the mutant oligonucleotides and promoter activity increased twofold. The magnitude of the increase is less than expected from the deletion analysis, and simultaneous mutations did not cause further increase in promoter activity, which suggests that other sites are involved in this negative modulation. These results suggest that the transcription of the HLgene in HepG2 cells is negatively modulated by multiple cis-acting negative elements and AP1-like nuclear factor may play some role in this modulation.

Base Sequence↗

PubMed1995

Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids.

Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been associated with missense or deletion mutations in the mtrR (multiple transferable resistance Regulator) gene of laboratory-derived strains but their prevalence in clinical isolates was heretofore unknown. Since faecal lipids provide strong selective pressure for the emergence of variants resistant to HAs (HAR), the nucleotide sequence of the mtrR gene from rectal isolates of N. gonorrhoeae, which displayed different levels of HAR, was determined. Compared to the mtrR gene possessed by the HA-sensitive strain FA19, each clinical isolate contained mutations in the coding and/or promoter regions of their mtrR gene. A missense mutation in codon 45 (Gly-45 to Asp) was the most common mutation found in the strains studied and impacted the structure of the helix-turn-helix domain of the MtrR protein thought to be important in DNA-binding activity. Two clinical isolates bearing a missense mutation in codon 45 also contained a single basepair deletion in a 13 bp inverted sequence positioned within the mtrR promoter region. Introduction of mtrR sequences amplified from the clinical isolates into strain FA19 revealed that acquisition of the single basepair deletion was correlated with high level HAR while mutations in the mtrR-coding region provided for an intermediate level of HAR.

Amino Acid Sequence↗

PubMed1997

Frequency of the CCR5 delta 32 mutant allele in HIV-1-positive patients, female sex workers, and a normal population in Taiwan.

A specific 32-nucleotide deletion mutant of the CCR5 gene (Accr5), the coreceptor gene for human immunodeficiency virus type 1 (HIV-1), can effectively suppress the transmission and pathogenesis of the virus. Individuals homozygous for the delta ccr5 allele resist primary macrophage-tropic HIV-1 infection, despite multiple high-risk sexual exposures. This gene deletion is relatively common among Caucasians but uncommon among Africans, Asians, and South Americans. We used polymerase chain reaction (PCR) technology to determine the frequency of the delta ccr5 allele in a Taiwanese population with diverse health status and social backgrounds. Subjects included 24 HIV-1-infected persons in the northern and southern parts of Taiwan; 131 HIV-1 high-risk, licensed female sex workers in the northern part of the island (21% of whom were aborigines); and 187 unrelated, healthy, HIV-1-negative individuals in southern Taiwan. PCR with primers encompassing the entire CCR5 gene was used to explore possible deletions at regions other than the 32-nucleotide area in the female sex workers. No ccr5 deletions were detected, indicating that they are rare or absent in the Taiwanese population. This finding implies that delta ccr5 is not likely to be part of the defense against the spread of HIV-1-infection in Taiwanese.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Double deletions and missense mutations in the first nucleotide-binding fold of the ATP-binding cassette transporter A1 ( ABCA1) gene in Japanese patients with Tangier disease.

Tangier disease (TD) is a rare autosomal recessive disease characterized by plasma high-density lipoprotein deficiency caused by an ATP-binding cassette transporter A1 ( ABCA1) gene mutation. We describe three different mutations in Japanese patients with TD. The first patient was homozygous for double deletions of 1221 bp between intron 12 and 14 and 19.9 kb between intron 16 and 31. The breakpoint sequence analyses suggest that it is a simultaneous event caused by double-loop formation through multiple Alu. The second patient was homozygous for a novel mutation of A3198C in exon 19, resulting in Asn935His. The third patient was homozygous for A3199G of exon 19 that leads to Asn935Ser, which is the same mutation found in German and Spanish families. Both Asn mutations involved Walker A motif of the first nucleotide-binding fold.

ATP Binding Cassette Transporter 1↗

PubMed1995

Autosomal dominant optic nerve colobomas, vesicoureteral reflux, and renal anomalies.

We describe a father and 3 sons with optic nerve colobomas, vesicoureteral reflux, and renal anomalies. The youngest son had congenital renal failure and ultimately underwent renal transplantation. The father and one son had high frequency hearing loss. There were no other affected relatives. We conclude that the association of optic nerve colobomas, renal anomalies, and vesicoureteral reflux comprises a unique autosomal dominant syndrome. Molecular investigations have determined this disorder to be associated with a single nucleotide deletion in the PAX2 gene.

Abnormalities, Multiple↗

PubMed1995

Large deletions in the 5'-untranslated region of foot-and-mouth disease virus of serotype C.

Nucleotide sequences of the 5'-untranslated region (5'-UTR), at the 3'-side of the poly C tract, have been compared for 21 isolates of foot-and-mouth disease virus (FMDV) of serotype C from Europe, South America and The Philippines. A deletion of 43 nucleotides is present in the European isolates as compared with most American isolates. A larger deletion of 86 nucleotides is present in some viruses from South America and The Philippines. These deletions include the loss of one or two pseudoknot structures predicted in this region of the 5'-UTR. In addition, multiple point mutations have allowed the derivation of a phylogenetic tree which defines a grouping of isolates very similar to that derived from the capsid gene sequences of the same viruses. The study provides evidence that deletion (or addition) events must be very frequent during evolution of FMDV type C, since viruses which are phylogenetically very closely related (they belong to the same tree branch) may differ in the presence or absence of these deletions. Implications for FMDV evolution are discussed.

Animals↗

PubMed2025

Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.

DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.

Arabidopsis↗

PubMed1991

[DNA analysis and gene diagnosis of cystic fibrosis].

Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, affecting approximately 1 in 2,000 newborns but the actual estimate varies with the geographic location. The incidence of CF in non-Caucasian populations is low. Intensive efforts using genetic linkage information ultimately led to the cloning of the CF gene prior to the identification of the gene product or its function. The gene encodes what is believed to be a transmembrane protein, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR contains two nucleotide-binding folds (NBF) which show homology to numerous transport proteins with the greatest homology to the P-glycoproteins that are encoded by the multiple drug-resistance loci. A three- base deletion resulting in the loss of phenylalanine residue (delta F508) in the tenth exon of the CFTR gene is the mutation occurring on the majority of CF chromosomes. The overall frequency of delta F508 in the present mutant CF gene pool is about 70%, but the study populations are not equally represented: there is marked variation in the population of delta F508 among different geographic populations. Recently, numerous additional, less common mutations have been found. Some mutations occur on 2-5% of the CF chromosomes. Many of these are rare 'private' mutations, occurring in individual families of all racial and ethnic backgrounds. By contrast over 80% of Western European CF mutations have been identified. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but further improvements are needed in DNA-based genetic screening for CF carrier status.

Cystic Fibrosis↗

PubMed1993

Characterization of the gene for ADP-ribosylation factor (ARF) 2, a developmentally regulated, selectively expressed member of the ARF family of approximately 20-kDa guanine nucleotide-binding proteins.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking in vivo. The six cloned mammalian ARFs can be grouped into three classes based on size and sequence identity. ARF 2 is a class I ARF, whose approximately 2.6-kilobase mRNA exhibits species and tissue selective expression and is developmentally regulated in rat brain. Here we report the sequence, structure, and functional promoter region of the bovine ARF 2 gene, which was facilitated by constructing a composite cDNA. The ARF 2 cDNA, constructed from a partial cDNA clone and polymerase chain reaction-amplified fragments from reverse-transcribed poly(A)+ RNA, was approximately 2270 base pairs (bp) (minus the poly(A) tail). In the 3'-untranslated region, there are two potential polyadenylation signals, ATTAAA and AATAAA, at positions 1064 and 2232, respectively, and two ATTTA motifs, believed to signal mRNA degradation, at positions 2115 and 2165. The ARF 2 gene, represented in three overlapping genomic clones, spans approximately 20 kilobase pairs with five exons and four introns. Consensus sequences for guanine nucleotide-binding and GTP hydrolysis are in separate exons, except for the NKXD sequence, which is divided by intron 4. There are multiple transcriptional initiation sites. Transient transfection of embryonic trachea cells with deletion constructs defined the functional promoter region to be within 400 bp upstream of the most 5' site of transcription initiation. This 400-bp region lacks a TATA-like sequence but contains six inverted CCAAT boxes, four potential Sp1-binding sites, and a potential AP-2-binding site. Although the pattern of expression of ARF 2 is unique among the ARFs, the structures of the class I ARF genes are conserved among its members and across species.

ADP-Ribosylation Factors↗