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Results for “multiple nucleotide deletion”

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PubMed1992

Isolation of the avian transforming retrovirus, AS42, carrying the v-maf oncogene and initial characterization of its gene product.

A novel avian transforming retrovirus was isolated from a chicken musculoaponeurotic fibrosarcoma. This virus (called AS42) induces tumors histopathologically indistinguishable from the original sarcoma after a long latent period when inoculated into newborn chickens. AS42 also exhibits a weak transforming activity when infected into chicken embryo fibroblasts (CEF). This virus is replication-defective and associated with a helper virus of subgroup A (called ASAV). An AS42-specific protein of about 100 kDa was immunoprecipitated from lysates of AS42-transformed CEF with antiserum directed against avian retrovirus virion proteins. Molecular analysis of the genomic structure of the AS42 virus has revealed that this 100-kDa protein represents a novel oncogene, v-maf of cellular origin, which is fused with a part of the viral gag gene (Nishizawa et al., Proc. Natl. Acad. Sci. USA 86, 7711-7715, 1989). Interestingly, some size variation was observed among the gag-maf fusion proteins found in individual clones of transformed CEF. Consistent with this observation, Southern blot analyses and nucleotide sequence determination of several independent isolates of proviral DNA indicated that this virus segregates multiple forms of deletion mutants, probably through homologous recombinations among the repetitive sequences present within the v-maf coding region.

Animals↗

PubMed1999

[Mutational analysis of the CytR protein binding site within the regulatory region of Escherichia coli udp gene].

Site-specific mutagenesis of the pentameric motif TGCAA within the regulatory region of the udp gene with coordinates -68 and -64 relative to the transcription initiation site was performed. Nine mutant promoters containing multiple nucleotide base-pair substitutions in this pentameric motif were isolated and characterized. One mutant contained a deletion of the C/G nucleotide pair in the -66 position. Isolated mutant promoters were cloned into a low-copy-number expression vector pJEL250 to determine the level of their expression, depending on the allelic state of cytR and cya genes. The level of CytR-dependent regulation of the udp gene and the ability to titrate the CytR repressor in vivo were shown to be drastically decreased in all mutant promoters isolated. On the basis of these results, it is concluded that the pentameric motif TGCAA plays a key role in binding the CytR repressor protein to the udp gene promoter.

Binding Sites↗

PubMed2002

Evidence for trafficking of Epstein-Barr virus strains between hairy leukoplakia and peripheral blood lymphocytes.

Hairy leukoplakia (HL), an epithelial lesion found on the side of the tongue in immunocompromised individuals, is characterized by high-level replication of Epstein-Barr virus (EBV) and multiple EBV strains. The source of these strains and their relationship to peripheral blood lymphocyte (PBL) strains has not previously been characterized. Using matched pairs of HL scrapings and PBL from 16 HIV-positive men, variation in EBV strain identity was characterized by detection of a 30 nucleotide deletion of the EBV latent membrane protein (LMP)-1 gene, variation in the LMP-1 repeat region and typing for Epstein-Barr nuclear antigen (EBNA)-2. Multiple EBV strains were found in both the HL and PBL specimens, but 13 of 16 (81%) patients showed evidence of strain identity for at least one strain and analysis of two patients suggested that EBV strains from HL could infect the PBL. Our data are consistent with active trafficking of EBV between these two compartments.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Specific sequence changes in multiple transcript system DYT3 are associated with X-linked dystonia parkinsonism.

X-linked dystonia parkinsonism (XDP) is an X-linked recessive adult onset movement disorder characterized by both dystonia and parkinsonism. We report delineation of the disease gene within a 300-kb interval of Xq13.1 by allelic association. Sequencing of this region in a patient revealed five disease-specific single-nucleotide changes (here referred to as DSC) and a 48-bp deletion unique to XDP. One of the DSCs is located within an exon of a not previously described multiple transcript system that is composed of at least 16 exons. There is a minimum of three different transcription start sites that encode four different transcripts. Two of these transcripts include distal portions of the TAF1 gene (TATA-box binding protein-associated factor 1) and are alternatively spliced. Three exons overlap with ING2 (a putative tumor suppressor) and with a homologue of CIS4 (cytokine-inducible SH2 protein 4), both of which are encoded by the opposite strand. Although all DSCs are located within this multiple transcript system, only DSC3 lies within an exon. This exon is used by all alternative transcripts making a pathogenic role of DSC3 in XDP likely. The multiple transcript system is therefore referred to as DYT3 (disease locus in XDP).

Adult↗

PubMed1988

Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G-rich sequence element lying between them with the characteristics of a basal promoter element.

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G-rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP-inducibility. A synthetic oligonucleotide, containing two copies of the G-rich element from the CP1 gene, will reconstitute cAMP-inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.

Amino Acid Sequence↗

PubMed1993

Mutational analysis of patients with adenomatous polyposis: identical inactivating mutations in unrelated individuals.

Samples of constitutional DNA from 60 unrelated patients with adenomatous polyposis coli (APC) were examined for mutations in the APC gene. Five inactivating mutations were observed among 12 individuals with APC; all were different from the six inactivating mutations previously reported in this panel of patients. The newly discovered mutations included single-nucleotide substitutions leading to stop codons and small deletions leading to frameshifts. Two of the mutations were observed in multiple APC families and in sporadic cases of APC; allele-specific PCR primers were designed for detecting mutations at these common sites. No missense mutations that segregated with the disease were found.

Adenomatous Polyposis Coli↗

PubMed1997

Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements.

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides -1321 to -1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

Animals↗

PubMed2003

Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.

Base Sequence↗

PubMed1989

Gap costs for multiple sequence alignment.

Standard methods for aligning pairs of biological sequences charge for the most common mutations, which are substitutions, deletions and insertions. Because a single mutation may insert or delete several nucleotides, gap costs that are not directly proportional to gap length are usually the most effective. How to extend such gap costs to alignments of three or more sequences is not immediately obvious, and a variety of approaches have been taken. This paper argues that, since gap and substitution costs together specify optimal alignments, they should be defined using a common rationale. Specifically, a new definition of gap costs for multiple alignments is proposed and compared with previous ones. Since the new definition links a multiple alignment's cost to that of its pairwise projections, it allows knowledge gained about two-sequence alignments to bear on the multiple alignment problem. Also, such linkage is a key element of recent algorithms that have rendered practical the simultaneous alignment of as many as six sequences.

Animals↗

PubMedDate not supplied

Diversification of the Ig variable region gene repertoire of synovial B lymphocytes by nucleotide insertion and deletion.

Although the changes that occur in Ig V region genes during a B lymphocyte's response to antigen usually result from point mutations, nucleotide insertion and deletion also alter gene sequence. We identified nucleotide insertions and deletions (3 to 12 bp) at a frequency of 1.34%, in Ig V gene cDNA from B lymphocytes residing in the synovial tissues of patients with rheumatoid arthritis. Because the added or lost nucleotides occurred in multiples of 3, they maintained the original reading frame and coded a potentially intact receptor. These V gene modifications were generated somatically, because they were identified in the original cDNA by HCDR3-specific polymerase chain reaction and were not found in other B cells using the same VH genes. Insertions and deletions were detected only in IgG+ and IgA+ transcripts, which exhibited 3 times more point mutations than IgM+ transcripts. In addition, they were usually found in the complementarity determining region, typical targets of somatic mutation. The occurrence of insertion/ deletion in isotype-switched cDNA with higher numbers of V gene mutations that localized to hot spots for V gene mutation suggests that these diversification events were related to the somatic hypermutation process. In support of this, an AGY hot spot motif and a short stretch of DNA similar in sequence to the inserted or deleted segments could be found next to the insertions/deletions, suggesting that these modifications arose from DNA duplication following DNA stand breaks. Thus, nucleotide insertion/deletion can lead to B-cell receptor diversification in B lymphocytes that clonally expand in synovial tissues of patients with rheumatoid arthritis.

B-Lymphocytes↗

PubMed1993

Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter.

hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

Animals↗

PubMed2003

Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.

Analysis of multiple transcripts for the Arf-specific guanine nucleotide exchange factor GBF1 identified three positions displaying small in-frame deletions and insertions. Sequencing of genomic DNA for CHO GBF1 and analysis of the human gene established that those variations were consistent with alternate splicing events. RT-PCR analysis of CHO mRNA confirmed that these small in-frame deletions occurred at significant and similar frequencies in both WT and BFA resistant CHO cells. These splice variants behaved like GBF1 in biological assays based on the observation that GBF1 is cytotoxic at high levels but will confer resistance to BFA when moderately overexpressed. Comparison of variants with larger deletions defined regions of 75 (exons 5-7) and 412 (exons 31-39) amino acid residues that were required for cell killing but were dispensable for promoting BFA resistance.

Alternative Splicing↗

PubMed1995

Packaging of multiple copies of reduced-size genomic segments by bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. Packaging of individual segments is dependent upon unique packaging sequences of about 300 nucleotides near the 5' ends of the segments. We have prepared segments L, M, and S with internal deletions that decrease their size by as much as sixfold without affecting either their packaging sequences or their 3' ends. Although packaging of genomic segments is normally very precise, with only one of each in a procapsid, these smaller segments are packaged in multiples such that the total number of nucleotides for each segment class approaches that of the normal genomic segment.

Bacteriophages↗

PubMed1981

The nucleotide sequence of the polypeptide IX gene of human adenovirus type 3.

The nucleotide sequence of the DNA segment encompassing the polypeptide IX gene of class B human adenovirus serotype 3 (Ad3) has been determined using cloned restriction fragments. There is only a single, open translational reading frame capable of specifying a protein of 138 amino acids, comparable to the Mr 12 000-13 000 of protein IX detected in virions (Wadell, 1980). The corresponding region of a closely related class B virus, Ad7, is virtually identical (Dijkema et al., 1981), but the comparable segments of class C viruses Ad2 or Ad5 are much less homologous (Aleström et al., 1980; Maat et al., 1980). There are 150 single bp changes and 19 deletion-insertions, at least one frameshift, together affecting 210 nucleotides within the 455 bp comparison positions of the protein-coding regions of Ad2 (423 bp) and Ad3 (417 bp). Each of the 19 deletion-insertions involves an integral multiple of 3 bp in phase with the open translation frame. There is no "TATA" promoter box in Ad3 DNA at the position comparable to that of Ad2. The deduced protein sequences near the amino-terminus are extensively conserved between the two classes of viruses, but the carboxy-terminal portion and the nucleotide sequences flanking the gene are much more diverged. In both classes, these N- and C-terminal regions of the inferred proteins are linked by an alanine-rich chain, an arrangement suggestive of two functional domains.

Adenoviruses, Human↗

PubMed2000

The DNA of a plant retroviroid-like element is fused to different sites in the genome of a plant pararetrovirus and shows multiple forms with sequence deletions.

Carnation small viroid-like RNA (CarSV RNA) and its homologous DNA are the two forms of a unique plant retroviroid-like system. CarSV RNA is a 275-nucleotide noninfectious viroid-like RNA, present in certain carnation plants, which can adopt hammerhead structures in both polarity strands and self-cleave accordingly. CarSV DNA is organized as a series of head-to-tail multimers forming part of extrachromosomal elements in which CarSV DNA sequences are fused to sequences of carnation etched ring virus (CERV), a plant pararetrovirus. Analysis of more than 30 CarSV-CERV DNA junctions showed that distinct regions of the viral genome seem able to interact with CarSV DNA. All these junctions were short nucleotide stretches common to both CarSV and CERV DNAs. This suggests a polymerase-driven mechanism for their origin involving an enzyme with low processivity, most likely the viral reverse transcriptase. This view was further supported by the observation that most of CarSV sequences forming part of the junctions correspond either to strong secondary structure motifs in the conformation proposed for CarSV RNA or to its self-cleavage sites, which may have facilitated polymerase jumping. Accompanying the most-abundant CarSV RNA, a series of CarSV RNAs with sequence deletions were previously characterized. Here we have identified some of their corresponding DNA forms, together with other CarSV DNA forms with deletions not found in any CarSV RNA species identified so far. Some of these CarSV DNA forms have also been detected fused to CERV sequences. The existence of these shortened CarSV DNA versions may provide a continuous input of their corresponding transcripts and explain the persistence of CarSV RNAs with defective hammerhead structures for which an RNA-RNA model of amplification seems unlikely.

Base Sequence↗

PubMed1988

Transcripts from the frameshifted MURF3 gene from Crithidia fasciculata are edited by U insertion at multiple sites.

In trypanosome mitochondria an RNA editing process is operative, which co- or post-transcriptionally alters the nucleotide sequence of transcripts by insertion and/or deletion of U residues at specific sites. To increase our understanding of the mechanism of this process we have compared the nucleotide sequence of the frameshifted mitochondrial MURF3 gene from Crithidia fasciculata to that of a large number of MURF3 cDNAs. We found cDNAs derived from transcripts edited at two different sites in the protein coding sequence: (i) at the frameshift position five extra U residues connect the two reading frames and (ii) at the 5' terminus 22 inserted Us shift a putative initiator codon out of phase. The collection also contained cDNAs that were derived from non-edited transcripts. Partially edited sequences were not found, except in one cDNA, which contained an edited frameshift site in combination with a non-edited 5' terminus. The analysis further showed that MURF3 transcripts have a 3'-terminal poly(AU) extension, which varies in sequence. The implications of these results are discussed.

Amino Acid Sequence↗

PubMed1989

Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome.

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

Animals↗

PubMed1991

Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region.

We have cloned and sequenced the 5' untranslated region of the transforming growth factor-beta 3 (TGF-beta 3) mRNA as well as the adjacent genomic sequence. S1 nuclease analysis identified a single transcription start site. We have thus determined that the 5' untranslated region is about 1.1 kb long and contains 11 open reading frames. In vitro translation of the TGF-beta 3 precursor coding sequence was markedly inhibited by the presence of the 5' untranslated region. Similarly, when the 5' untranslated region of TGF-beta 3 was introduced upstream of the coding sequence of chloramphenicol acetyltransferase, in vitro translation was inhibited. Furthermore, upon transfection into 293 cells, chloramphenicol acetyltransferase expression was inhibited by the 5' untranslated region of TGF-beta 3. The degree of translational inhibition was inversely proportional to the amount of transfected DNA. Mutation analysis implicated multiple segments of the 5' untranslated region as contributing to the inhibitory effect. Deletion of much of the 5'-most 640 nucleotides, including 8 of the 11 upstream ATGs, relieved much but not all of the inhibitory influence of the 5' untranslated region of TGF-beta 3 mRNA. The two upstream open reading frames closest to the initiator codon for the TGF-beta 3 coding sequence also decreased translational efficiency, since mutation of either ATG resulted in increased translation. Transfection results with T47-D cells, a cell line which expresses TGF-beta 3 mRNA, were similar to those obtained with the 293 cell line. Thus, TGF-beta 3 mRNA is a recent example of an expanding group of growth-related mRNAs in which the 5' untranslated region contains upstream open reading frames and other sequences which inhibit translation.

Amino Acid Sequence↗