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Results for “multiple nucleotide deletion”

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At least 163 records · Page 9Linked to original sources

PubMed1993

Alignment-ambiguous nucleotide sites and the exclusion of systematic data.

Molecular systematists generally rely on computer algorithms to establish the alignment of DNA sequences. However, when alignment regions are characterized by multiple insertions and deletions, these gap-filled stretches of DNA are often excised before phylogenetic reconstruction. This exclusion of systematic data is generally determined by subjective criteria. We explore a replicable methodology in which the comparison of several multiple sequence alignments can be used to eliminate regions of unstable sequence alignment. Using crocodilian and insect mitochondrial (mt) ribosomal (r) DNA as examples, we caution against the removal of sequence data prior to phylogenetic reconstruction.

Algorithms↗

PubMed2006

Region 3.2 of the sigma subunit contributes to the binding of the 3'-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation.

Region 3.2 of the RNA polymerase sigma subunit forms a loop that protrudes toward RNA polymerase active center and partially blocks RNA exit channel. To provide some insights into the functional role of this region, we studied a deletion variant of the Escherichia coli sigma(70) subunit that lacked amino acids 513-519 corresponding to the tip of the loop. The deletion had multiple effects on transcription initiation including: (i) a significant decrease in the amount of short abortive RNAs synthesized during initiation, (ii) defects in promoter escape, (iii) loss of the contacts between the sigma subunit and the nascent RNA during initiation and, finally, (iv) dramatic increase in the K(m) value for the 3'-initiating nucleotide. At the same time, the mutation did not impair promoter opening and the binding of the 5'-initiating purine nucleotide. In summary, our data demonstrate an important role of sigma region 3.2 in the binding of initiating substrates in RNA polymerase active center and in the process of promoter clearance.

Binding Sites↗

PubMed2001

Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2alpha A) is inhibited by tumor necrosis factor-alpha.

The transcription factor RUNX2 (Cbfa1/AML3/Pebp2alphaA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor alpha (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of osteoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC(50) for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6-kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide -108 upstream of the translational start site; however, localization downstream of nucleotide -108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states.

3T3 Cells↗

PubMed2000

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy.

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15-18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM.

Adolescent↗

PubMed1992

Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor.

To understand the role of mitochondria in carcinogenesis, we compared the amount of deleted mtDNAs between human hepatic tumors and surrounding cirrhotic portion of the liver of ten patients by using polymerase chain reaction (PCR). Multiple mtDNA deletions were detected in cirrhotic portion, but no deletions were detected in the tumor portion. Direct sequencing of the fragments revealed a 7,079-bp deletion (nucleotide position 8,992-16,072) involving no direct repeated sequences and a 7,436-bp deletion (position 8,649-16,084) involving a 12-bp directly repeated sequence of 5'-CATCAACAACCG-3' exists in both the ATP6 gene and the D-loop region. These mtDNA mutations could be one of the endogenous factors that induce somatic mutations in nuclear genome and etiologically contribute to human carcinogenesis.

Adenoma, Bile Duct↗

PubMed2006

Comprehensive mutation identification in an evolved bacterial cooperator and its cheating ancestor.

Precise characterization of the mutation histories of evolutionary lineages is crucial for understanding the evolutionary process, yet mutation identification has been constrained by traditional techniques. We sought to identify all accumulated mutations in an experimentally evolved lineage of the cooperative bacterium Myxococcus xanthus, which constructs fruiting bodies by a process of social multicellular development in response to starvation. This lineage had undergone two major transitions in social phenotype: from an ancestral cooperator to a socially defective cheater, and from the cheater to a competitively dominant cooperator that re-evolved social and developmental proficiency. The 9.14-Mb genome of the evolved, dominant cooperator (strain "PX") was sequenced to approximately 19-fold coverage by using recent "sequencing-by-synthesis" technology and partially sequenced (approximately 45%) by using capillary technology. The resulting data revealed 15 single-nucleotide mutations relative to the laboratory ancestor of PX after the two phases of experimental evolution but no evidence of duplications, transpositions, or multiple-base deletions. No mutations were identified by capillary sequencing beyond those found by pyrosequencing, resulting in a high probability that all mutations were discovered. Seven errors in the reference strain previously sequenced by the Sanger approach were revealed, as were five mutational differences between two distinct laboratory stocks of the reference strain. A single mutation responsible for the restoration of development in strain PX was identified, whereas 14 mutations occurred during the prior phase of experimental evolution. These results provide insight into the genetic basis of two large adaptive transitions in a social bacterium.

Adaptation, Biological↗

PubMed1998

Exon sequence is required for regulated RNA splicing of the human fibroblast growth factor receptor-1 alpha-exon.

Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the alpha-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the alpha-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5'-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for alpha-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for alpha-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the alpha-exon.

Alternative Splicing↗

PubMed1997

The human POLD1 gene. Identification of an upstream activator sequence, activation by Sp1 and Sp3, and cell cycle regulation.

The promoter of the human POLD1 gene encoding the catalytic subunit of DNA polymerase delta is G/C-rich and does not contain a TATA box. Transient transfection analysis in HeLa cells employing POLD1-luciferase chimeric plasmids revealed a core promoter region extending 328 base pairs (bp) from the major transcription initiation site. Multiple elements in this region including two 11-bp direct repeats located between nucleotide positions -92 and -22, play an important role in POLD1 promoter activity. Deletion or linker-replacement mutations of the repeats drastically reduced the promoter activity. A 70-bp DNA fragment containing the two repeats could stimulate the expression of the POLD1 or a heterologous promoter in an orientation-independent manner. DNase I footprinting and band-shift assays showed that HeLa nuclear extracts contained proteins specifically binding to the repeat sequences. Southwestern blot and UV cross-linking analyses identified Sp1 and two 85-kDa proteins that bound to the repeats. Additionally, screening of HeLa cDNA expression libraries for the sequence-specific DNA-binding protein using the 11-bp repeat sequences as the probe, identified a cDNA that corresponds to Sp3, a member of the Sp1 family. Cotransfection studies in Drosophila SL2 cells showed that both Sp1 and Sp3, but not Sp2, could activate the POLD1 promoter through the repeat sequences. The POLD1 promoter activity was induced about 4-fold at the late G1/S boundary in serum-stimulated cells. The 11-bp repeats together with an E2F-like sequence, located adjacent to the major transcription initiation site, were important for the stimulation. Taken together, this study provides a direct evidence for transcriptional regulation of the human POLD1 gene.

Base Sequence↗

PubMed1995

Functional characterisation of the genomic and antigenomic promoters of Sendai virus.

A natural Sendai virus internal deletion defective interfering (DI) RNA, previously shown to encode a truncated NP protein and previously cloned under the control of the T7 RNA polymerase promoter, was expressed from plasmid and shown to replicate in cell tissue culture when the viral proteins NP, P, and L were coexpressed from cloned genes. The efficient replication was dependent on the total length of the RNA to be a multiple of 6 nucleotides, showing that the "rule of six" applied for a DI RNA that has conserved the end sequences of the nondefective viral RNA. Compared to the copy-back H4 DI RNA, the replication efficiency of the internal deletion DI RNA was reproducibly 20-fold lower. Reciprocal exchanges between the minus-strand 3'-end primary sequences of the two DI RNAs showed that the replication efficiency of the derivatives obtained directly correlated with the origin and the extent of the primary sequence. Moreover, some of the derivatives exhibited a replication efficiency comparable to that of the copy-back DI RNA with, however, the ability to transcribe a functional mRNA similar to the internal deletion DI RNA. This indicated that the transcription ability of a viral RNA was not sufficient to explain a low replication efficiency.

Animals↗

PubMed1994

Antigen diversity in the bacterium B. hermsii through "somatic" mutations in rearranged vmp genes.

B. hermsii counters host immunity with multiphasic antigenic variation. This is conferred by interplasmidic and intraplasmidic rearrangements of vmp genes. In several independent events, activation of a silent vmp gene through intraplasmidic deletions but not interplasmidic recombinations was followed by the appearance at its 5' end of multiple mutations that were not present in the silent gene. The prevalence of mutant alleles in postwitch populations increased during infections. Differences between the silent and expressed genes were at the same nucleotides at which vmp pseudogenes differed, suggesting these were templates for postswitch gene conversions. The mechanism of this bacterium to generate diversity, namely, intramolecular deletions followed by mutations in the rearranged gene, mirrors the strategy used by vertebrate hosts to eliminate it.

Animals↗

PubMed1990

Recombination sites in cauliflower mosaic virus DNAs: implications for mechanisms of recombination.

Pairs of mutant cauliflower mosaic virus (CaMV) DNAs readily recombine in plants. Five plasmid clones of CaMV DNAs resulting from infection of turnips with pairs of mutant DNAs from DNAs resulting from infection of turnips with pairs of mutant DNAs from different isolates were obtained. Restriction analysis and nucleotide sequencing identified deletions in two cloned recombinants, VR1249 and VR244B. The sequence missing in the former was consistent with its deletion by splicing of an RNA intermediate. These DNAs were not infectious in turnips. VR1243, VR244A, and VR246 induced in turnips disease symptoms that were mixtures of those produced by the parental isolates. Junctions between sequences of the parental isolates were identified by restriction fragment analysis. Three cloned chimeras resulted from multiple recombination events. Nucleotide sequencing identified more precisely the junctions in the five cloned chimeras and in three chimeras previously characterized. Consistent with a model in which reverse transcription plays a major role in generating recombinants, six chimeras had junctions at or near the site for initiation of DNA(-) strand synthesis, three had junctions near the initiation site of 35 S RNA transcription, and one junction was found near the initiation site of 19 S mRNA transcription. Junctions were also found in regions not bearing any obvious relation to DNA (-) strand synthesis by reverse transcription, suggesting that recombination of double-stranded DNAs may also generate CaMV DNA recombinants.

Base Sequence↗

PubMed2000

Novel deletional mutation of the MEN 1 gene in a kindred with multiple endocrine neoplasia type 1.

MEN1 gene mutation in a Japanese kindred with multiple endocrine neoplasia type 1 was examined. A heterozygous deletion involving 29 base pairs in exon 10 (1606del29) was identified in the proband, and the same deletion was found in the affected family members. Most previously reported germline MEN1 gene mutations are nucleotide substitutions and small insertions/deletions, and a large deletion is rare. The hairpin structure mediated by an incomplete palindromic sequence at deletion termini is the most likely mechanism to be associated with the deletion in the present family.

Adult↗

PubMed2003

Binding of AP-2 and ETS-domain family members is associated with enhancer activity in the hypersensitive site III region of the human growth hormone/chorionic somatomammotropin locus.

The human GH gene family is specifically expressed in somatotrophs of the anterior pituitary and placental syncytiotrophoblast. Two nuclease-hypersensitive sites, HS III and HS V, are associated with a region of chromatin located 28 and 30 kb upstream of the pituitary GH gene transcription initiation site (+1) in both pituitary and placenta nuclei. A role for this region in pituitary GH gene expression has been reported, but the potential relevance to placental gene expression has not been determined. Deletion analysis of a 5.2-kb region (nucleotides - 27,568/-32,746) containing HS III to V-related sequences localized significant enhancer activity to a 574-bp HS III fragment (nucleotides -27,676/-28,249) in multiple transfected cell lines. Four nuclease-protected regions [footprints (FP) 1-4] were identified in the 574-bp fragment. FP2 and FP3 were detected with placenta cell nuclear protein, whereas FP1 and FP4 were observed with placental and nonplacental cell nuclear extract. Disruption of FP1 had no effect on heterologous promoter activity in transfected pituitary and placental cells, whereas loss of FP2 and FP3 resulted in modest increases in placental cells, reflecting the presence of repressor activity associated with these regions in vitro. In contrast, disruption of the FP4 region by mutation or deletion significantly reduced enhancer activity. As a result, 30-fold enhancer activity was localized to a 41-bp region in transfected placental tumor cells. Binding of candidate proteins, activator protein (AP)-2 (FP3) and Elk-1 (FP4), was confirmed using competition assays with specific oligonucleotides and antibodies. Moreover, these factors were associated with the hyperacetylated HS III region in human pituitary [activator protein 2 (AP-2) and Elk-1] and term placenta (AP-2) chromatin. These data implicate AP-2 and ETS-domain family members in the regulation of the GH/CS locus and raise the possibility that different complexes form in the HS III region in placenta and pituitary cells.

Acetylation↗

PubMedDate not supplied

Conformational mutations in human mitochondrial DNA.

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.

Base Sequence↗

PubMed1993

Promoter region of the rat phospholipase C-gamma 1 gene.

We have cloned the upstream region of the rat phospholipase C-gamma 1 gene and characterized its promoter activity. The 5'-upstream sequence is highly rich in GC, enriched with CpG dinucleotides and lacks a TATA motif. This sequence also contains many putative binding sites for regulatory proteins. Primer extension and RNase protection assay demonstrated a single transcriptional start site located at 103 nucleotides upstream of the ATG start codon. The transcriptional activities of various 5'-deleted fragments, fused with chloramphenicol acetyltransferase gene, were examined after transfection into C2C12 myoblast. Multiple positive and negative regulatory sites were observed.

Amino Acid Sequence↗

PubMed1999

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts.

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Animals↗

PubMed2004

A novel method of identifying genetic mutations using an electrochemical DNA array.

We describe the development of a new type of DNA array chip that utilizes electrochemical reactions and a novel method of simultaneously identifying multiple genetic mutations on an array chip. The electrochemical array (ECA) uses a threading intercalator specific to double-stranded nucleotides, ferrocenylnaphthalene diimide (FND), as the indicator. ECA does not require target labeling, and the equipment is simple, durable and less expensive. The simultaneous multiple mutation detection (SMMD) system using an ECA chip and FND utilizes an enzyme to simultaneously distinguish several genetic mutations such as single nucleotide polymorphism (SNP), insertion, deletion, translocation and short tandem repeat. We examined this SMMD system using an ECA chip, by detecting seven different mutations on the lipoprotein lipase (LPL) gene for 50 patients in a blind test. It turned out that all the results obtained were concordant with the sequencing results, demonstrating that this system is a powerful tool for clinical applications.

DNA Mutational Analysis↗

PubMed2000

Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

Aged↗