Scaffold/Matrix Attachment Regions and intrinsic DNA curvature.

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs.

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates.

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.

Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology.

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.

Heteroduplex mobility assay for detection of new avian influenza virus variants.

Highly pathogenic avian influenza (HPAI) in poultry causes high morbidity and mortality, and it is a List A disease of the Office International des Epizooties. An outbreak of HPAI in commercial poultry not only causes direct disease losses but often results in trade restrictions for the affected country. Because HPAI viruses can mutate from H5 and H7 low pathogenic avian influenza viruses, it is necessary to monitor and control even the low pathogenic form of the virus. We report a practical approach for screening large numbers of isolates that uses amplification by reverse transcriptase-polymerase chain reaction of a segment of the hemagglutinin (HA) gene (536-560 bp) of H7 avian influenza viruses followed by the heteroduplex mobility assay (HMA). The HMA test compares the amplified polymerase chain reaction product from unknown samples with reference isolates, which allows the identification of new variants. The HMA test results were compared with sequence analysis of the isolates used in the study. On the basis of the HMA, we could identify several new variant viruses present in the live bird markets in the northeastern United States. New strains gave a distinct pattern of bands in the gels in accordance with the different heteroduplexes formed when their HA region amplification products were incubated together with the same amplification product of a reference strain. These differences correlate with phylogenetic analysis from sequence data.

Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.

BACKGROUND: The identification of somatic mutations in tissues is often difficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We found that the identification of gsp mutation was not possible by direct sequencing and present a new approach that improves the identification of gsp somatic mutations. MATERIAL/METHODS: Genomic DNA was extracted from frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplified by PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR product did not reveal mutations, probably due to the small amount of mutant alleles. To improve this amount, the PCR products were re-amplified using as template the excised products of the mutant homoduplex and heteroduplex bands obtained at the first DGGE. RESULTS: This approach resulted in the enhancing of the mutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Direct sequencing of the second round PCR clearly identified the mutation R201C in the ovarian Leydig cell tumor. CONCLUSIONS: We have demonstrated a relatively rapid, convenient and reliable method to improve gsp somatic mutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutant homoduplex bands disclosed in a first DGGE followed by direct sequencing.

Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomonas aeruginosa MutS by preventing protein oligomerization.

MutS, a component of the mismatch repair system begins the DNA reparation process by recognizing base/base mismatches or small insertion/deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa and analysed the biochemical properties of the encoded protein. Complementation of the hypermutator phenotype of a P. aeruginosa mutS mutant strain indicated that the isolated gene was functional. When purified MutS was incubated at 37 degrees C in the absence of ligands, a rapid inactivation of the oligonucleotide binding capability and ATPase activity occurred. However, the presence of ATP, ADP or heteroduplex oligonucleotides, but not homoduplex oligonucleotides, prevented the protein from being inactivated. The analysis of the protein by native PAGE indicated that the active conformation state correlates with the presence of MutS dimer. Analysis by gel-filtration chromatography showed that the inactive protein formed by incubation at 37 degrees C in the absence of ligands corresponds to the formation of a high molecular mass oligomer. The kinetic analysis of the oligomer formation showed that the extent of the reaction was markedly dependent on the temperature and the presence of MutS ligands. However, the protein inactivation apparently occurred before the maximum extent of MutS oligomerization. Further analysis of the MutS oligomers by electron microscopy showed the presence of regular structures consisting of four subunits, with each subunit probably representing a MutS homodimer. It is concluded that MutS possesses an intrinsic propensity to form oligomeric structures and that the presence of physiological ligands, such as nucleotides or heteroduplex DNA, but not homoduplex DNA, plays an important role in keeping the protein in an active conformation by preventing protein oligomerization.

Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species.

AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA. METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized. We tested 11 type cultures of Saccharomyces, 27 different cultures of S. cerevisiae and four other ascomycetic genera. CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S. cerevisiae identification. HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.

Population screening of F508del (DeltaF508), the most frequent mutation in the CFTR gene associated with cystic fibrosis in Argentina.

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. The disease can be caused by one of the more than 900 different mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. However, the deletion of the phe508-codon is the most prevalent mutation observed. Our aim was to perform a screening for this mutation (DeltaF508, or F508del) in the population of Mendoza, Argentina. For the screening, 1,000 blood samples were obtained from CF asymptomatic individuals and combined into 100 pools each containing 10 different blood samples. Pools containing at least one F508del carrier were detected by heteroduplex formation during the PCR amplification of exon 10. The PCR was designed to introduce a recognition site for a restriction enzyme that confirmed the presence of the deletion F508del in the positive pools. The results with this simple method indicate a frequency of carriers in the Mendoza population of 2.1% (1.3%-3.2, 95% confidence limits). The observed frequency of carriers is similar to that reported for European populations. Hum Mutat 18:167, 2001.

A rapid method for detecting and distinguishing clinically important yeasts by heteroduplex mobility assays (HMAs).

Heteroduplex Mobility Assays (HMAs) of 700 bp amplified products of a 17S rDNA region were used to identify and differentiate seven yeast species of clinical importance Candida albicans, Torulopsis (Candida) glabrata, Candida tropicalis, Candida parapsilosis, Candida (Pichia) guilliermondii and Hansenula (Pichia) anomala. Distance of heteroduplex migration (dHE) was found to be negatively correlated to the number of nucleotide differences between amplified DNA sequences.

HIV-1 subtyping by heteroduplex mobility assay in patients with bleeding disorders

O vírus da imunodeficiência humana do tipo 1 estabelece uma infecçäo presistente que na maioria dos casos evolui para a Síndrome de Imunodeficiência humana do tipo 1 tem sido geneticamente classificado em maior ou em outros grupos. Ogrupo maior é subdivido em nove subtipos baseados em evidências seqüênciais. A infecçäo pelo vírus da imunodeficiência humana do tipo 1 foi transmitida aos hemofílicos principalmente pelos concentrados de coagulaçäo no final da década de 70 e meados da de 80 e a síndrome da imunodeficiência adquirida tornou-se a principal causa de morbidade e morte entre estes pacientes. O Objetivo deste estudo foi o de determinar os subtipos do vírus da imunodeficiência humana em oito pacientes soropositivos com moléstias hemorrágicas de Belo Horizonte, Brasil, utilizando o ensaio de mobilidade heteroduplex, um método näo seqüêncial, que tem a propriedade de separar os portadores do vírus. Realizamos a reaçäo da cadeia de polimerase seguida pelo ensaio de mobilidade heteroduplex e obtivemos em todos os oito pacientes a confirmaçäo que os mesmos pertenciam ao subtipo B do vírus da imunodeficiência humana que é a mais prevalente nos Estados Unidos, Europa e Brasil. Os pacientes hemofílicos provavelmente foram infectados de concentrados provenientes da Europa, Estados Unidos, Säo Paulo e Rio de Janeiro (Brasil), utilizados em período anterior ao do conhecimento do vírus da imunodeficiência humana adquirida, e do uso de plasmas e concentrados näo testados, ou inativados, para o mesmo. O ensaio da mobilidade de heteroduplex e amplificaçäo do ácido desoxiribonucleico pela reaçäo de cadeia da polimerase provaram ser um modo rápido de subtipar o vírus da imunodeficiência humana adquirida em indivíduos infectados por transfusäo. O teste pode ser útil como rastreamento e detecçäo da origem e procedência do vírus da imunodeficiência adquirida.

Molecular epidemiology of HIV-1 in Brazil: high prevalence of HIV-1 subtype B and identification of an HIV-1 subtype D infection in the city of Rio de Janeiro, Brazil. Evandro Chagas Hospital AIDS Clinical Research Group.

HIV-1-positive individuals were recruited from January 1993 to December 1996 from several cohorts receiving follow-up in the city of Rio de Janeiro, Brazil, to evaluate HIV-1 genetic variability and the potential association with modes of transmission. HIV-1 subtyping was carried out using the heteroduplex mobility assay (HMA), and those samples corresponding to the typical Brazilian subtype B variant were further identified based on the Fok I restriction fragment length polymorphism (RFLP). DNA sequencing was performed to evaluate one case of subtype D infection. From the 131 HIV-1-positive individuals analyzed, 106 (80.9%) could be identified as infected by subtype B and 20 (15.3%) by subtype F. One of the samples (0.8%) was classified as subtype D. DNA samples from 4 patients (3.0%) did not yield polymerase chain reaction (PCR)-amplified products to be typed. Based on the Fok I RFLP, 39 of the 106 subtype B samples (37%) were identified as corresponding to the typical Brazilian subtype B variant containing the GWGR motif at the tip of the V3 loop. No statistically significant association could be detected between HIV-I subtypes and modes of transmission, exposure categories, or gender. This is the first reported case of HIV-1 subtype D infection in Brazil.

Frameshift mutations in the type VII collagen gene (COL7A1) in five Mexican cousins with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). In this study, we assessed the molecular basis of recessive DEB in five affected individuals from two Mexican families. Both fathers of the affected children were first cousins. Genomic DNA was extracted from peripheral blood samples and assessed for COL7A1 mutations by polymerase chain reaction (PCR) amplification, heteroduplex analysis and direct automated sequencing of PCR products displaying heteroduplex bandshifts. In one family, we identified a homozygous 1 bp insertion of a G nucleotide in exon 19 of COL7A1, designated 2470insG, in three affected sisters. This mutation causes a frameshift and a premature termination codon on both alleles 178 bp downstream from the insertion; both parents were shown to be heterozygous carriers of this mutation. In the second family, the father of the other two affected children was also found to be a heterozygous carrier of this frameshift mutation. In addition, his unrelated partner was shown to be a heterozygous carrier of a different COL7A1 frameshift mutation, an insertion of a T nucleotide in exon 32, designated 3948insT. This mutation also results in a premature termination codon, 126 bp downstream from the insertion. Both affected children were compound heterozygotes for the 2470insG/3948insT mutations in COL7A1. Overall, these molecular findings offer a genetic explanation for the skin fragility in these related Mexican patients with recessive DEB. Immediate benefits from elucidation of the mutations include assessment of carrier status in other members of the family and the feasibility of DNA-based prenatal testing in subsequent pregnancies.

PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity.

Analysis of the 16S rRNA genes retrieved directly from different environments has proven to be a powerful tool that has greatly expanded our knowledge of microbial diversity and phylogeny. It is shown here that sequence similarity between 80 and 100% among 16S rDNAs can be estimated by the electrophoretic migration of their heteroduplexes. This was measured by hybridization and electrophoresis in polyacrylamide gels of the product obtained after PCR amplification of almost the entire 16S rRNA gene from different bacterial species. These heteroduplexes were also observed after amplification of samples containing DNA from two or more bacterial species and a procedure was applied to identify reliably heteroduplexes among the amplification products. The electrophoretic migration of the heteroduplexes observed after PCR was used to detect the presence of 16S rDNAs with different sequences in DNA extracted from both a mixture of two bacterial species and samples containing a natural bacterial community.

HIV-1 detection and subtyping by PCR and heteroduplex mobility assay in blood donors: can these tests help to elucidate conflicting serological results?

Testing blood donors for the human immunodeficiency viruses (HIV-1 and 2), requires serological tests that are frequently inconclusive. Peripheral blood mononuclear cells of 50 blood donors with the screening test (ELISA) reactive to HIV-1, but with indeterminate results in the first Western Blot (WB) performed, were submitted to the polymerase chain reaction (PCR) and heteroduplex mobility assay (HMA), a nonsequencing method that can distinguish between HIV-1 subtypes (A to I). PCR amplification of HIV-1 env gene regions has been obtained in 12 (24.0%) samples. HMA of the amplified DNA showed that 12 belonged to HIV-1 B subtype and one to F subtype. PCR testing of the amplified DNA helped to elucidate doubtful serological results and HMA proved to be a relatively simple and rapid way of subtyping HIV-1 for epidemiological purposes.

Simultaneous detection of size and sequence polymorphisms in the transcribed trinucleotide repeat D2S196E (EST00493).

Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12-15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G-->A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.

Heteroduplex mobility assays (HMAs) and analogous sequence analysis of a cytochrome b region indicate phylogenetic relationships of selected callitrichids.

Heteroduplex mobility assays (HMAs) of a cytochrome b region were used for estimating genetic distances and phylogenetic relationships between some selected neotropical primates of the families Callitrichidae (marmosets and tamarins) and Cebidae [capuchin monkey, (Cebus apella)], and man (Homo sapiens). HMA distances were found to be strongly correlated to analogous estimates derived from DNA sequence data. Phylogenetic trees obtained by HMAs and sequence analyses showed similar topologies with almost identical intraspecific, intrageneric, and intergeneric relationships. The applicability of HMAs is assessed relative to different levels of molecular and organismal diversity.

Distribution of HIV-1 subtypes seen in an AIDS clinic in Sao Paulo City, Brazil.

OBJECTIVE: To determine the distribution of HIV-1 subtypes in Sao Paulo, Brazil. METHODS: Samples were obtained from 80 consecutive HIV-1-infected individuals attending the Immunodeficiency Clinic at the University of Sao Paulo in 1993. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque gradient and a portion was used for routine CD4 counts; the remainder were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. Samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset of these were also sequenced through the C2-V3 region of env. RESULTS: A total 69 of 80 samples yielded env polymerase chain reaction product enabling subtype determination; samples that did not amplify were those with low DNA yields. Among 12 injecting drug users (IDU) or sexual partners of IDU, four were typed as clade F and eight as clade B. Forty-three homosexual men or female sexual partners of bisexual men were typed as clade B. The 14 additional cases without known risk factors were typed as clade B. CONCLUSION: These data suggest that subtype F is related to injecting drug use in Brazil.

PIP: Serum samples from 80 consecutive HIV-1-infected individuals presenting to the Immunodeficiency Clinic at the University of Sao Paulo in 1993 were analyzed to determine the distribution of HIV-1 subtypes in the city. Peripheral blood mononuclear cells (PBMC) were separated using Ficoll-Hypaque gradient, a portion was used for routine CD4 counts, and the rest were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. The samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset was also sequenced through the C2-V3 region of env. 69 samples yielded env polymerase chain reaction product enabling subtype determination. The samples which did not amplify had low DNA yields. Among 12 IV-drug users or their sex partners, four were typed as clade F and eight as clade B. 43 homosexual men or female sex partners of bisexual men were typed as clade B. The 14 additional cases with no known risk factor were typed as clade B.

PCR haplotypes for the human Y chromosome based on alphoid satellite DNA variants and heteroduplex analysis.

We have developed a system for revealing informative and useful haplotypes for the human Y chromosome using PCR. Variant alphoid satellite DNA subunits were amplified and analysed by digestion with HindIII to score a restriction site polymorphism, or on polyacrylamide gels to reveal 13 heteroduplex haplotypes. Heteroduplexes are double-stranded DNA molecules containing mismatches; the haplotype is the combination of alleles on the same chromosome. Structural studies showed that the heteroduplexes analysed here were formed from loci at the left (short arm) and right (long arm) edges of the centromeric alphoid array which differed by a 4-bp insertion/deletion and several point mutations. Consequently, many haplotypes may have arisen only once and are useful for evolutionary studies.

Identification of RB1 germline mutations in Argentinian families with sporadic bilateral retinoblastoma.

Hereditary predisposition to retinoblastoma is caused by germline mutations in the RB1 gene. Most of these mutations occur de novo and differ from one patient to another. DNA samples from 10 families with a child presenting sporadic bilateral retinoblastoma have been analysed for the causative mutation. Using intragenic DNA polymorphisms we detected large deletions in two patients. Heteroduplex and DNA sequence analysis of PCR products from each exon and the promoter region showed small mutations in four patients: a C to T transition in exon 18; 1 bp and 2 bp deletion in exons 20 and 19 respectively; and a 4 bp insertion in exon 7. All these mutations are likely to result in premature termination of transcription. In one of these families, an unaffected carrier was detected. This emphasises the importance of detection of the causative mutation for predictive diagnosis in families with sporadic bilateral retinoblastoma.