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At least 19 recordsLinked to original sources
PubMed1996

Instability of repeated dinucleotides in bacteriophage T7 genomes.

The ligase gene of bacteriophage T7 was interrupted with an insert of synthetic DNA that included a series of dinucleotide repeats which altered the reading frame of the gene and prevented the phage from growing on a host deficient in Escherichia coli ligase. The insert was designed so that gain of an additional copy of the repeat would restore the reading frame and produce ligase positive T7. It was found that a pair of nucleotides was gained at a frequency of 1.4 x 10(-3) and a dinucleotide was lost from this sequence at a frequency of 1 x 10(-4). The same measurements were made using T7 with a DNA polymerase without the 3' --> 5' exonuclease function. This mutant DNA polymerase lacks proofreading edit function and is more processive than its wild-type counterpart. Phage without the edit function gained or lost a dinucleotide repeat significantly more frequently than wild-type T7. Thus, the frequency of two base frameshifting is very high during DNA replication. Proofreading activity attenuates the frequency of two base frameshifts. Inactivation of the 3' --> 5' exonuclease of T7 DNA polymerase had essentially no effect on the frequency of deletion of one member of a pair of 10 bp tandem repeats.

Bacteriophage T7↗
PubMed2004

Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates.

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.

5' Flanking Region↗
PubMed2002

Identification of a soybean protein that interacts with GAGA element dinucleotide repeat DNA.

Dinucleotide repeat DNA with the pattern (GA)(n)/(TC)(n), so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)(n)/(CT)(n) DNA. Soybean GBP was dissimilar from the GAGA factor of Drosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)(n)/(CT)(n). GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum. In addition, Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants.

Amino Acid Sequence↗
PubMed1991

Characterization of a large deletion associated with a polymorphic block of repeated dinucleotides in the type III procollagen gene (COL3A1) of a patient with Ehlers-Danlos syndrome type IV.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant condition characterized by extreme fragility of skin, blood vessels, intestine, gravid uterus, and lungs. The phenotype is accounted for by mutations affecting the integrity and/or synthesis of the precursor procollagen molecules of type III collagen. In this article, we report the elucidation of the molecular defect in an EDS IV patient whose type III collagen was previously found to be structurally abnormal. We utilized PCR in a two-step process involving first the localization of the mutation in the mRNA and then the characterization of the defect in the gene. The results established the patient's heterozygosity for a genomic deletion of about 7.5 kb which eliminates 1,026 nucleotides of coding sequences in the message. The mutation arose as a result of an exon-to-intron recombination. The deleted segment extends from the 13th nucleotide of exon 9 to within a DNA sequence of intron 24, which is composed of a series of dinucleotide repeats. Using PCR, we tested the polymorphic nature of this DNA element on several unrelated individuals. Analysis of amplified genomic products of 45 chromosomes recognized at least four distinct allelic forms that display frequencies ranging from 5% to 61%. Mendelian segregation of three of the four alleles was established by the same method in a 3-generation family.

Alleles↗
PubMed1996

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers.

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.

Amelogenin↗
PubMed1993

A study of the origin of 'shadow bands' seen when typing dinucleotide repeat polymorphisms by the PCR.

Dinucleotide repeat polymorphisms ('microsatellites') are usually typed by resolving the products of PCR amplification on denaturing acrylamide gels. With this methodology, an allele consists not of a single fragment, but rather of a ladder of fragments, typically separated by intervals of 2nt. Mechanisms that have been invoked to explain the generation of these 'shadow bands' include slipped strand mispairing occurring during the PCR and artefactual 'recombination' caused by out-of-register annealing of truncated PCR products. The D11S527 locus contains the microsatellite sequence (GT)n(CTGT)m. By performing direct sequencing of PCR products derived from individuals homozygous at D11S527, we show that these products vary in length due solely to variations in the length of the dinucleotide repeat tract. These results rule out PCR recombination and support slipped strand mispairing as the major mechanism for generation of shadow bands.

Alleles↗
PubMed1993

Dinucleotide repeat polymorphisms isolated by the polymerase chain reaction.

DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem dinucleotide repeat sequences and limiting dilution of a target genomic library enable amplification by polymerase chain reaction (PCR) of single-target molecules containing dinucleotide repeats. Amplified material was sequenced by the PCR direct method and by the resultant sequences used to design locus-specific primers. This study identified and characterized four anonymous dinucleotide repeat sequences, three of which exhibited polymorphism. Although developed for dinucleotide repeats, the technique is universally applicable to repeat DNA elements of a size usually analyzed by PCR. The technique is comparatively rapid, eliminates library screening and its associated manipulations, and compares favorably with existing methods for the recovery of repetitive DNAs.

Base Sequence↗
PubMed1999

Transcriptional regulation of the human type I collagen alpha2 (COL1A2) gene by the combination of two dinucleotide repeats.

Human type I collagen alpha2 (COL1A2) gene has two dinucleotide repeats: one in the 5'-flanking region of the gene is composed of poly(dC-dA) and poly(dC-dG), while the other in the first intron consists of poly(dG-dT). In this study, we show that transcription of the COL1A2 gene is regulated by these repeats. Luciferase reporter gene assay indicated that the transcriptional activity of the COL1A2 gene was enhanced by the co-presence of both repeats, but not by either repeat alone. Analysis of the polymorphism in the two repeat regions indicated that both sequences have a variation in their repetition number, thus showing that these dinucleotide repeats constitute microsatellites. A study using constructs containing various combinations of the repeat alleles showed differences in their transcriptional activities. The results, however, showed that the stimulation rate of luciferase activity was not linear with the repetitive number of the repeats either in the 5' flanking region or in the first intron of the gene and that the stimulation was provided by the combination of these polymorphic repetitive sequences. These observations indicated that the dinucleotide repeats have an enhancing activity on transcription of the COL1A2 gene and that the variation in the number of repetitions may partly be responsible for the difference in the transcriptional activity of the gene.

Alleles↗
PubMed2004

A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing.

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a CFTR minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen in vivo. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.

Base Sequence↗
PubMed2004

Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle.

Polymorphic dinucleotide repeat loci ('microsatellite markers') are found in varying abundance throughout the genomes of most organisms. They have been extensively used for genetic studies, but conventional techniques used for their genotyping require sophisticated equipment. Microplate array diagonal gel electrophoresis (MADGE) has previously been extended to economical high-throughput genotyping of trinucleotide and tetranucleotide microsatellite amplicons. However, the capability of this technique to resolve the alleles of dinucleotide repeat loci has not been explored previously. Here we show that a modified microsatellite-MADGE approach can provide sufficient resolution for dinucleotide repeat typing. This enables economical and convenient set up for analysis of single markers in many samples in parallel, suitable, for example, for population association studies.

Dinucleotide Repeats↗
PubMed2000

Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro.

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.

DNA Polymerase I↗
PubMed2002

Typing dinucleotide repeats under nondenaturing conditions with single-base resolution and high sizing precision.

Dinucleotide repeats are genetic markers that are useful for many purposes, including genetic epidemiology, population genetics, and genetic diagnostics. The accuracy of analyses based on dinucleotide repeat polymorphisms is highly dependent on the success achieved in minimizing genotyping errors. Genotyping errors in dinucleotide repeat typing may arise for various reasons, including polymerase chain reaction (PCR) processing errors and the use of unsuitable electrophoretic conditions for resolving amplification products (i.e., lack of single-base resolution and inadequate precision in allele sizing). We have recently described a nondenaturing electrophoretic system useful for detecting PCR processing errors that lead to misidentification of heterozygotes as homozygotes in (AC)n repeat typing. Here, we show that this system also allows resolution of (AC)n repeats in native conditions with single-base resolution and high sizing precision, on the basis of an analysis of seven human (AC)n repeats ranging in size from 72 to 217 bp. This PAGE system is thus also useful for reducing the likelihood both of allele misidentification due to the absence of single-base resolution and of inaccuracies in allele sizing due to anomalous electrophoretic migrations among the alleles within an (AC)n repeat.

Alleles↗
PubMed2006

Studies of DNA dumbbells VIII. Melting analysis of DNA dumbbells with dinucleotide repeat stem sequences.

Melting curves and circular dichroism spectra were measured for a number of DNA dumbbell and linear molecules containing dinucleotide repeat sequences of different lengths. To study effects of different sequences on the melting and spectroscopic properties, six DNA dumbbells whose stems contain the central sequences (AA)(10), (AC)(10), (AG)(10), (AT)(10), (GC)(10), and (GG)(10) were prepared. These represent the minimal set of 10 possible dinucleotide repeats. To study effects of dinucleotide repeat length, dumbbells with the central sequences (AG)(n), n = 5 and 20, were prepared. Control molecules, dumbbells with a random central sequence, (RN)(n), n = 5, 10, and 20, were also prepared. The central sequence of each dumbbell was flanked on both sides by the same 12 base pairs and T(4) end-loops. Melting curves were measured by optical absorbance and differential scanning calorimetry in solvents containing 25, 55, 85, and 115 mM Na(+). CD spectra were collected from 20 to 45 degrees C and [Na(+)] from 25 to 115 mM. The spectral database did not reveal any apparent temperature dependence in the pretransition region. Analysis of the melting thermodynamics evaluated as a function of Na(+) provided a means for quantitatively estimating the counterion release with melting for the different sequences. Results show a very definite sequence dependence, indicating the salt-dependent properties of duplex DNA are also sequence dependent. Linear DNA molecules containing the (AG)(n) and (RN)(n), sequences, n = 5, 10, 20, and 30, were also prepared and studied. The linear DNA molecules had the exact sequences of the dumbbell stems. That is, the central repeat sequence in each linear duplex was flanked on both sides by the same 12-bp sequence. Melting and CD studies were also performed on the linear DNA molecules. Comparison of results obtained for the same sequences in dumbbell and linear molecular environments reveals several interesting features of the interplay between sequence-dependent structural variability, sequence length, and the unconstrained (linear) or constrained (dumbbell) molecular environments.

Base Composition↗
PubMed1999

Mutated gene-specific phenotypes of dinucleotide repeat instability in human colorectal carcinoma cell lines deficient in DNA mismatch repair.

Mutations in DNA mismatch repair (MMR) genes in hereditary non-polyposis colon cancer (HNPCC) patients revealed the importance of MMR deficiency as a risk for carcinogenesis. Since diverse mutations occur in several MMR genes, the instability of repeat sequences dispersed in the genome, which are also governed by the MMR system, is a well used marker. However, the relationship between repeat sequence instability and MMR gene mutation in human cells has not been well defined mainly because precise systems to analyse repeat sequences have not been available. Using our newly developed system, we analysed alteration of dinucleotide repeats in human cell lines which harbour mutations in MMR genes. Among 24 subclones of DLD-1 cells (hMSH6-) only one had a dinucleotide repeat alteration in only one microsatellite locus, while LoVo cells (hMSH2-/hMSH6-) exhibited marked dinucleotide repeat instability (DRI). HCT116 cells, a hMLH1-mutant, showed an ultimate DRI phenotype. Interestingly, SW48 cells lacking hMLH1 expression also demonstrated DRI, albeit the extent of diversity being significantly lower than HCT116. These data suggest that the DRI phenotype in human cells is highly dependent on mutated MMR genes and on forms of mutation. The results of DRI analyses used to detect MMR-deficiency should be interpreted with caution.

Adaptor Proteins, Signal Transducing↗
PubMed1981

Right-handed alternating DNA conformation: poly(dA-dT) adopts the same dinucleotide repeat with cesium, tetraalkylammonium, and 3 alpha, 5 beta, 17 beta-dipyrrolidinium steroid dimethiodide cations in aqueous solution.

We demonstrate that poly(dA-dT) can adopt two conformations in solution, with the relative proportions dependent on the nature and concentration of the counter ion and cationic ligands. The synthetic DNA exhibits a dinucleotide repeat conformation on addition of CsF and Me4NCl at molar concentrations, with the NMR spectral changes reflecting a common conformational change at one glycosidic torsion angle and one phosphodiester linkage. We also observe the same dinucleotide repeat in the neighbor-exclusion 3 alpha, 17 beta-dipyrrolidin-1'-yl-5 beta- delta 9,11-androstene dimethiodide (3 alpha, 5 beta, 17 beta-dipyrandenium) complex, with the steroid diammonium ligand binding in the groove of the stacked poly(dA-dT) duplex and the complex stabilized through the interaction of one of the charged ends with the backbone phosphate. We demonstrate further that 3 alpha, 5 beta, 17 beta-dipyrandenium bound to poly(dA-dT) at low binding ratios induces a switch to the dinucleotide repeat conformation at adjacent steroid-free duplex regions. This observation contrasts with a previous demonstration that the diastereoisomeric 3 beta, 5 alpha, 17 beta-dipyrandium binds to poly(dA-dT) by partial insertion between unstacked tilted base pairs. The NMR parameters rule out a left-handed alternating DNA structure (Z DNA) for the observed poly(dA-dT) dinucleotide repeat conformation, but right-handed alternating DNA models are under consideration. The facile interconversion of poly(dA-dT) between two conformations, one of which exhibits a dinucleotide repeat and can be induced by ligand binding, may provide a mechanism for the recognition of specific nucleic acid sequences by DNA-binding proteins.

Androstenes↗
PubMed2001

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences.

The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.

Dinucleotide Repeats↗
PubMed2005

Dinucleotide repeats negatively modulate the promoter activity of Cyr61 and is unstable in hepatocellular carcinoma patients.

Cyr61 is a secreted, cysteine-rich, heparin-binding protein that mediates diverse functions including extracellular matrix formation, differentiation, cell proliferation, adhesion, migration, survival, as well as angiogenesis and tumorigenesis. In this study, we found that Cyr61 gene expression is significantly downregulated in the tumors of hepatocellular carcinoma (HCC) patients. To elucidate its mechanism of gene regulation, we examined the promoter of Cyr61 which contains two long stretches of repeats, each comprising d(CA) dinucleotide repeats downstream of HNF3beta- and ATF-binding sites. We hypothesized that the d(CA) repeats may play an important role in regulating Cyr61 promoter activity and performed promoter reporter assays to examine this. We found that a greater number of d(CA) repeats resulted in significantly lower promoter activity of the Cyr61 gene in the KB3-1 and HepG2 cell lines, but not in the MCF-7 cell line. In addition, the d(CA) repeats, but not other random sequences, were found to be important for Cyr61 promoter activity. We further demonstrate that the ATF- and HNF3beta-binding sites upstream the d(CA) repeats positively and negatively modulate Cyr61 promoter activity, respectively. An examination of the d(CA) dinucleotide patterns in the Cyr61 promoter in HCC patients revealed that approximately 32% of these patients exhibited either loss of heterozygosity or somatic mosaicism in either the tumors, adjacent normal liver tissues or both.

Angiogenesis Inducing Agents↗
PubMed1992

Improved predictive test for MEN2, using flanking dinucleotide repeats and RFLPs.

Gene(s) for the autosomal dominant endocrine cancer syndromes, multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B (MEN2B), and familial medullary thyroid carcinoma (MTC1) all map to the pericentromeric region of chromosome 10. Predictive testing for the inheritance of mutant alleles in individuals at risk for these disorders has been limited by the availability of highly informative and closely linked flanking markers. We describe the development of eight new markers, including two PCR-based dinucleotide repeat polymorphisms and six RFLPs that flank the disease loci. One of the dinucleotide repeat markers (sJRH-1) derives from the RBP3 locus on 10q11.2 and has a PIC of .88. The other dinucleotide repeat (sTCL-1) defines a new locus, D10S176, that maps by in situ hybridization to 10p11.2 and has a PIC of .68. We have constructed a new genetic linkage map of the pericentromeric region of chromosome 10, on the basis of 13 polymorphisms at six loci, which places the MEN2A locus between the dinucleotide repeat markers, with odds of 5,750:1 over the next most likely position. Using this set of markers, predictive genetic testing of 130 at-risk individuals from six families segregating MEN2A revealed that 95% were jointly informative with flanking markers, representing a significant improvement in genetic testing capabilities.

Base Sequence↗