Heteroduplex analysis of cloned rat endogenous replication-defective (30 S) retrovirus and Harvey murine sarcoma virus.
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Animals↗
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Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata. III. Heteroduplex analysis of the C. luciliae minicircles.
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Animals↗
Heteroduplex analysis of tra delta f' plasmids and the mechanism of their formation.
Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.
Chromosome Mapping↗
Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: heteroduplex analysis of limiting dilution PCRs.
We have developed a simple, robust, highly-sensitive assay for identifying gene mutations in clinical samples. We applied this assay to detect p53 and p16 mutations in pancreatic juice obtained from patients undergoing evaluation and treatment of pancreatic disease. The assay strategy involves PCR amplifying DNA at limiting dilution (LD-PCR) followed by screening PCR products for mutations using temperature gradient capillary electrophoresis (TGCE). Compared to conventional TGCE, TGCE after LD-PCR significantly increased the number of detectable mutations in pancreatic duct juice. Using LD-PCR, mutations in p53 and/or p16 were found in the pancreatic juice of 12 of 20 individuals with pancreatic cancer compared to only 1 of 8 patients with chronic pancreatitis, 0 of 8 individuals without evidence of pancreatic disease (p<0.02). We conclude that limiting dilution PCR is an effective strategy for improving the detection of mutations in clinical samples and when applied to pancreatic juice to detect mutations of p53 and/or p16, it can help distinguish patients with pancreatic cancer from those without evidence of pancreatic neoplasia.
Diagnosis, Differential↗
A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae).
A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.
Animals↗
The clinical and molecular genetic approach to Duchenne and Becker muscular dystrophy: an updated protocol.
Detection of large rearrangements in the dystrophin gene in Duchenne and Becker muscular dystrophy is possible in about 65-70% of patients by Southern blotting or multiplex PCR. Subsequently, carrier detection is possible by assessing the intensity of relevant bands, but preferably by a non-quantitative test method. Detection of microlesions in Duchenne and Becker muscular dystrophy is currently under way. Single strand conformational analysis, heteroduplex analysis, and the protein truncation test are mostly used for this purpose. In this paper we review the available methods for detection of large and small mutations in patients and in carriers and propose a systematic approach for genetic analysis and genetic counselling of DMD and BMD families, including prenatal and preimplantation diagnosis.
DNA Mutational Analysis↗
Structure of the gene encoding the beta-subunit of chicken prolyl 4-hydroxylase.
Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of alpha 2 beta 2. In addition to the tetrameric prolyl 4-hydroxylase (alpha 2 beta 2), the free beta-subunit is also found inside cells. Recently it was shown that the beta-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the beta-subunit of chicken prolyl 4-hydroxylase. The cDNA of beta-subunit was used to screen a chicken genomic DNA library constructed with the lambda EMBL-3 vector. Two clones, lambda gCPH beta-22 and beta-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the beta-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the alpha 1(I) collagen coding gene (COL1A1). These elements may interact with nuclear factors and play important roles in expression regulation of the beta-subunit gene as has been described in COL1A1.
Amino Acid Sequence↗
Manifestations and linkage analysis in X-linked autoimmunity-immunodeficiency syndrome.
The clinical findings of a kindred with an X-linked disorder are characterized by autoimmune polyendocrinopathy, enteropathy with villous atrophy, chronic dermatitis, and variable immunodeficiency. Linkage analysis was performed on 20 members of the affected kindred to determine the location of the responsible locus. Informative recombinations limited the region to an approximate 20 cM interval bordered by DXS1055 and DXS1196/DXS1050. Multipoint analysis generated a lod score >3 for the region contained between DXS8024 and DXS8031. The candidate region includes the Wiskott-Aldrich syndrome (WAS) locus. Evaluation of the Wiskott-Aldrich syndrome protein gene by single strand conformational analysis, heteroduplex analysis, and direct sequencing of the 12 exons in an affected male and two carrier females revealed no abnormalities. We conclude that this kindred has an X-linked disorder, distinct from WAS, that results in autoimmunity and variable immunodeficiency. The responsible locus maps to the pericentromeric region Xp11.23 to Xq21.1.
Autoimmunity↗
Improved survival in women with BRCA-associated ovarian carcinoma.
BACKGROUND: The objective of this study was to determine the clinical characteristics, treatment response, and frequency of p53 overexpression in Ashkenazi Jewish women with hereditary ovarian carcinoma. METHODS: Seventy-one Jewish women with epithelial ovarian carcinoma (EOC) were tested for the three BRCA founder mutations using single-strand conformation polymorphism analysis, heteroduplex analysis, and protein truncation testing. Clinical and histopathologic data were reviewed retrospectively. In vitro chemoresistance was analyzed in 32 patients. Mutations of p53 were studied using immunohistochemical detection of p53 overexpression. RESULTS: Thirty-four of 71 Jewish patients with EOC (48%) had germline BRCA mutations (BRCA heterozygotes), including 22 BRCA1 mutations and 12 BRCA2 mutations. BRCA heterozygotes were younger compared with Jewish patients who had EOC without mutations (sporadic carcinoma; 50 years vs. 59 years, respectively; P = 0.01). BRCA1 heterozygotes were younger compared with BRCA2 heterozygotes (48 years vs. 57 years, respectively; P = 0.01). Histopathologic tumor features were similar; however, tumors with low malignant potential were seen only in women with sporadic carcinoma. Both groups had equivalent rates of surgical cytoreduction and similar median follow-up (72 months). BRCA heterozygotes had higher response rates to primary therapy compared with patients who had sporadic disease (P = 0.01). In vitro chemoresistance predicted tumor response to platinum chemotherapy correctly in BRCA heterozygotes (P = 0.0096). BRCA heterozygotes with advance-stage disease had improved survival compared with patients who had advanced stage sporadic carcinoma (91 months vs. 54 months, respectively; P = 0.046) and had a longer disease free interval (49 months vs. 19 months, respectively; P = 0.16). p53 overexpression was common in BRCA heterozygotes (80%). CONCLUSIONS: BRCA1 heterozygotes developed EOC at a younger age compared with BRCA2 heterozygotes and women who had sporadic ovarian carcinoma. BRCA heterozygotes had a better response to platinum chemotherapy compared with women who had sporadic disease, which may have contributed to their improved prognosis.
Adult↗
Cloning and characterization of putative genes that specify sensitivity to neoplastic transformation by tumor promoters.
Two putative genes (termed pro 1 and pro 2) specifying sensitivity to induction of neoplastic transformation by TPA in mouse epidermal JB6 cells were cloned by sib selection from a size-selected genomic library of clonal cells sensitive to promotion of transformation. By restriction analysis, heteroduplex analysis, direct hybridization, and sequencing, the putative genes are different from and have no homology to known oncogenes. Both genes are independently and equally active as total DNA in the transfection assay. The transformation-promoting potential of these putative genes does not appear to result from gene amplification or detectable rearrangements, suggesting that small structural changes might confer the promoting activity. The mouse pro sequences are also found in monkey and human DNAs. The pro-1 sequence is homologous to middle repetitive elements in the mouse genome, namely the BAM 5 and B1 repeats. The sequence of pro-1 was determined and suggests that it contains the signals to be transcribed by RNA polymerase II and to encode a protein of 7.1 kDa.
Amino Acid Sequence↗
Deletion screening by fluorescence in situ hybridization in Rett syndrome patients.
Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found to be a cause of Rett syndrome (RTT). Mutation screening was based on various techniques including denaturing gradient gel electrophoresis, single-strand conformation polymorphism analysis, heteroduplex analysis, DNA sequencing and recently Southern Blot analysis. Mutation detection was achieved in 80% of typical RTT with a high prevalence of recurrent mutations. In order to provide further insights into the spectrum of MECP2 rearrangements in patients without any point mutation or small deletion/insertion in the coding region MECP2 gene, we screened 25 classical RTT females using fluorescence in situ hybridization analysis. No deletion were found in our group, suggesting that MECP2 gross rearrangements are a rare cause of Rett syndrome.
Chromosomal Proteins, Non-Histone↗
APC mutation analysis by chemical cleavage of mismatch and a protein truncation assay in familial adenomatous polyposis.
Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) who have been included in studies reported in the literature. In order to determine the true frequency of detectable APC mutations, we set out to search exhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be linked to 5q markers. Chemical cleavage of mismatch analysis was employed as the initial screening technique. Mutations were confirmed by direct DNA sequencing and shown to generate a premature stop codon by an in vitro protein synthesis assay. Mutations resulting in a premature stop codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intragenic APC markers with a lodscore of 1.69 at Zmax = 0.0, further analysis of DNA, RNA and chromosome spreads from the proband failed to detect any abnormality. This was despite employing single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, reverse transcription-polymerase chain reaction (RT-PCR) analysis for splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (FISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the numerous other techniques employed could detect the mutation in the remaining kindred. This study shows the value of screening the APC gene using a combination of chemical cleavage of mismatch analysis and an in vitro protein truncation test.
Adenomatous Polyposis Coli↗
Somatic mutation detection in human biomonitoring.
Somatic cell gene mutation arising in vivo may be considered to be a biomarker for genotoxicity. Assays detecting mutations of the haemoglobin and glycophorin A genes in red blood cells and of the hypoxanthine-guanine phosphoribosyltransferase and human leucocyte antigenes in T-lymphocytes are available in humans. This MiniReview describes these assays and their application to studies of individuals exposed to genotoxic agents. Moreover, with the implementation of techniques of molecular biology mutation spectra can now be defined in addition to the quantitation of in vivo mutant frequencies. We describe current screening methods for unknown mutations, including the denaturing gradient gel electrophoresis, single strand conformation polymorphism analysis, heteroduplex analysis, chemical modification techniques and enzymatic cleavage methods. The advantage of mutation detection as a biomarker is that it integrates exposure and sensitivity in one measurement. With the analysis of mutation spectra it may thus be possible to identify the causative genotoxic agent.
Environmental Monitoring↗
A polymorphism of the CC16 gene is associated with an increased risk of asthma.
Several quantitative traits associated with the asthma phenotype have been linked to markers on chromosome 11q13, although the gene responsible has yet to be well established. The gene for Clara cell secretory protein (CC16) is an ideal candidate for involvement in an inherited predisposition to asthma because of its chromosomal location, the role of the CC16 protein in controlling airway inflammation, and differences in levels of the protein between asthmatics and healthy controls. All three CC16 exons were screened in an unselected population of 266 subjects from 76 families and a cohort of 52 severely asthmatic children. A combination of single strand conformational polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, and restriction digestion was used. Mutation detection methods identified an adenine to guanine substitution in the CC16 gene at position 38 (A38G) downstream from the transcription initiation site within the non-coding region of exon 1. In the unselected population, 43.6% were homozygous for the polymorphic sequence (38GG) and 46.2% were heterozygous (38AG). All the asthmatic and unaffected children from both populations were selected for an unmatched case control analysis consisting of 67 asthmatic and 46 unaffected subjects. Those homozygous for the published sequence (38AA) had a 6.9-fold increased risk of developing asthma (p=0.049) and heterozygotes (38AG) a 4.2-fold increased risk (p=0.028). Modelling of genotype as a continuous covariate indicated evidence of a significant linear trend across the three genotypes (odds ratio=2.84 per unit increase in genotype code, p=0.018). These associations were independent of age, gender, and tobacco smoke exposure. These data and the known anti-inflammatory role of CC16 in the respiratory tract suggest that alteration to the gene at position 38 may contribute to asthma.
Asthma↗
Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.
Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo- and heteroduplexes resulting from denaturation (at 94 degrees C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (>30 degrees C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4 degrees C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4 degrees C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of Vgamma member primers instead of Vgamma family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.
Gene Rearrangement, T-Lymphocyte↗
Genotyping of enteric adenoviruses by using single-stranded conformation polymorphism analysis and heteroduplex mobility assay.
Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results.
Adenovirus Infections, Human↗
A novel PAX6 gene mutation in an Indian aniridia patient.
PURPOSE: A mutation in the PAX6 gene is thought to be the genetic cause of aniridia. Here we search for PAX6 gene mutations in Indian aniridia patients. METHODS: We amplified the coding exons of the PAX6 gene from the genomic DNA of 15 unrelated aniridia patients using polymerase chain reaction technology. We then performed single-strand conformation polymorphism analysis and heteroduplex analysis to search for sequence variants. RESULTS: Sequencing of shifted bands in two patients revealed PAX6 gene mutations. One of these was a novel mutation, 1180insA, located in exon 10 at the start of the PST domain. The other mutation, 1080C->T (R240X), located in exon 9 within the homeodomain, and is another example of the most commonly reported PAX6 mutation. CONCLUSIONS: Although PAX6 gene mutations and polymorphisms have been reported from various ethnic groups, we report for the first time the identification of PAX6 gene mutations in Indian aniridia patients.
Adolescent↗
Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.
BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was <1% of polyclonal B-cells. INTERPRETATION AND CONCLUSIONS: Heteroduplex analysis of PCR amplified products is a simple and quick alternative for detecting monoclonally rearranged Ig genes in MM. This can be applied for diagnosis of B cell LPD and as a previous step in MRD strategies.
B-Lymphocytes↗