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PubMed1989

Heat-shock protein 83 of Leishmania mexicana amazonensis is an abundant cytoplasmic protein with a tandemly repeated genomic arrangement.

The 83-kDa heat-shock protein HSP83 is a highly abundant protein in Leishmania amastigotes and promastigotes exposed to elevated temperature. Antibodies against this protein were obtained by immunization with a synthetic peptide derived from a conserved region. These antibodies recognized both the denatured and the native form of the molecule and were used for immunofluorescence analysis. These experiments, together with analysis by cell fractionation, show that HSP83 is distributed in the cytoplasm of Leishmania parasites. The gene for HSP83 in Leishmania mexicana amazonensis has been cloned from a genomic library, and molecular characterization shows it is present in several copies of 4-kb repeats arranged in tandem.

Animals↗
PubMed2003

[Analysis of short tandem repeat polymorphism in a female patient with Duchenne muscular dystrophy].

OBJECTIVE: To understand the mechanism of Duchenne/Becker muscular dystrophy (DMD) in a female patient. METHODS: A multiplex PCR (mPCR) protocol was applied to detect the dystrophin gene of the female DMD patient and her family members, whose haplotypes were analyzed in light of short tandem repeat polymorphism (STR) of five microsatellite markers (located in 5' terminus and introns 44, 45, 49, and 50). RESULTS: No deletion was detected in the affected female patient and her affected son. Examination of the female DMD patient's STR haplotypes identified in the female patient and her affected son the same haplotype inherited from her unaffected mother, who was a likely germinal mosaicism. The female patient has different haplotype from her affected son and her mother as a deletion was identified in the intron 45 of the affected son, and the female patient and her mother were probably heterozygous for this deletion. CONCLUSION: STR haplotype may not be responsible for the pathogenesis of DMD in the female patient and her affected son.

Female↗
PubMed1992

Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli.

It has been shown that tandems of rare arginine codons AGG have a strong inhibitory effect on translation of mRNA in E. coli [5]. This has been explained by the rate-limiting interaction of these codons with the less abundant tRNA(AGG) [6]. In this study tandemly repeated AGG triplets were introduced into the chloramphenicol acetyltransferase (CAT) gene either upstream of the initiation ATG codon or downstream of it (both in frame and out of frame) and the expression of the modified genes was investigated. We report that the addition of AGG clusters resulted in a substantial inhibitory effect on CAT gene expression independently of their localization in mRNA. This inhibitory effect is explained by a competition of the tandem AGGAGG with the natural Shine-Dalgarno (SD) sequence (consensus AAGGAGGU) for the 3'-end of the 16S small ribosomal RNA (rRNA).

Amino Acid Sequence↗
PubMed1999

Gene deletion and carrier detection in the family of Becker muscular dystrophy by short tandem repeat sequence polymorphism.

OBJECTIVE: To type haplotypes among the patients, carriers and normal offspring in a family of males with Becker muscular dystrophy in one generation by allelic fragment length polymorphism analysis. METHODS: Deletion analysis of the patients were performed using multiplex polymerase chain reaction (PCR) of amplification with 9 dystrophin exon primers. Intragenic short tandem repeat (STR) sequence (STR44, STR45, STR49 and STR50) were amplified by PCR to analyse allelic fragment length polymorphisms in the members of the family. RESULTS: The deletions of exons 17, 19 and 45, as well as deletions of allelic fragments at the loci of STR44 and STR45 were determined in the patients. Hemizygosity at those two loci were detected and carrier status ascertained in the mother of the patients. The normal haplotypes were typed in the sister of the patients. CONCLUSION: The method of STR sequence polymorphism analysis can determine haplotypes at normal status or at risk status. It would be used in prenatal diagnosis and carrier detection in the families of Duchenne and Becker muscular dystrophy.

Adolescent↗
PubMed1996

Human antibodies against the polymorphic epithelial mucin in ovarian cancer patients recognise a novel sequence in the tandem repeat region.

The humoral immune response to the polymorphic epithelial mucin (PEM) was studied by characterising the reactivity of human antibodies generated by EBV-immortalised B-cells from tumour-draining lymph nodes of ovarian cancer patients. All the human antibodies, selected in ELISA for their reactivity to the protein tandem core repeat sequence, reacted with PEM-expressing tumour cells. Aberrant glycosylation of the peptide core of the PEM molecule in cancer cells leads to the exposure of peptide epitopes that can be considered tumour specific. The epitope mapping of six human antibodies revealed that only one of them contained the PDTR sequence, shown to be the immunodominant epitope in the mouse. Four of the six human antibodies recognised a novel common immunogenic sequence (APPAH) in the tandem repeats. The binding of these human antibodies did not appear to be modulated by the length of the carbohydrate side chains, as shown by O-glycosylation inhibition studies. These results indicate that distinct sequences within the tandem repeat of PEM are target for a humoral immune response in humans. The presence of antibodies directed against different epitopes within the same antigenic region may modulate the antigen presentation process and the ongoing immune response. This data may help in clarifying the mechanisms of the immune response to PEM in cancer patients for the development of PEM-based immunotherapy.

Antibodies, Neoplasm↗
PubMed2001

Influence of the variable number of tandem repeats located in the promoter region of the thiopurine methyltransferase gene on enzymatic activity.

BACKGROUND: The genetic polymorphism of thiopurine methyltransferase (TPMT) activity has a significant relevance in the clinical outcome of patients receiving thiopurine drugs as immunosupressive or anticancer therapies. Apart from several open reading frame mutations unequivocally associated with decreased TPMT activity, a variable number of tandem repeats (VNTR), located within the 5' untranslated region, was recently reported as also affecting gene expression. AIMS AND METHODS: We have characterized both molecularly, by polymerase chain reaction-based techniques, and enzymatically, with an HPLC-based method, a sample of 143 Portuguese Caucasian individuals with the main objective of deepening the study of the TPMT genotype/phenotype relationship. Because two different repeated elements (A and B) do contribute to the overall VNTR variation, we set out to analyze their combined and individual effects on TPMT activity. RESULTS: Allele VNTR*6 was found to be consistently associated with decreased levels of TPMT activity, supporting previous reports that the VNTR does affect levels of TPMT activity, although moderately and in a way not yet clearly defined. Furthermore, individual analysis of the A and B variations suggested that three B repeats was the likely motif influencing gene expression toward decreased transcription. CONCLUSIONS: Our hypothesis, which emphasizes the number of particular motifs within the VNTR internal structure more than the undiscriminated number of repeats as the potential causative factor affecting TPMT activity, needs further support from future studies, namely, from enlarged population samples. However, the knowledge of the VNTR acting mechanism will represent an important step toward fully understanding how the phenotypic variability at the TPMT trait is modulated.

Alleles↗
PubMed2006

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population.

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.

Female↗
PubMed2005

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis.

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.

Amino Acid Sequence↗
PubMed2002

A tandemly repeated thyroglobulin core promoter has potential to enhance efficacy for tissue-specific gene therapy for thyroid carcinomas.

Recombinant adenoviruses, carrying herpes simplex virus thymidine kinase (HSVtk) genes, were developed to evaluate the possibility of tissue-specific gene therapy for thyroid carcinomas. The HSVtk gene was driven by a minimal thyroglobulin (TG) promoter (AdTGtk) and a tandemly repeated minimal TG promoter (Ad2 x TGtk) to obtain thyroid-specific cell killing ability. The transduction of HSVtk genes by infection with Ad2 x TGtk followed by ganciclovir (GCV) treatment showed more powerful cytotoxicity for TG-producing FRTL5 cells, a rat normal thyroid cell line, and FTC-133 cells, a human follicular thyroid carcinoma cell line, than when infected with AdTGtk in vitro. The cell killing ability of Ad2 x TGtk was 10- to 30-fold higher than that of AdTGtk and similar to that of AdCMVtk, which carries HSVtk under the control of CMV promoter. Whereas after treatment with adenovirus/GCV to non-TG-producing cell lines (undifferentiated thyroid carcinoma cell lines and carcinoma cell lines from other tissues), Ad2 x TGtk and AdTGtk needed more than 100-fold concentrated GCV to reach IC(50) compared to AdCMVtk. We confirmed the enhanced efficacy of Ad2 x TGtk for tissue-specific cytotoxicity in vivo. After adenovirus/GCV treatment for FTC-133 tumor-bearing nude mice, Ad2 x TGtk enhanced tumor growth inhibition and survival rates compared to AdTGtk. Tumor growth inhibition and survival rates by Ad2 x TGtk were similar to that by AdCMVtk. Moreover, any toxic effect for rat normal tissues was not revealed after intravenous injections with Ad2 x TGtk and intraperitoneal administrations with GCV in vivo, whereas severe liver damages were observed after treatment with AdCMVtk/GCV. These data indicate a beneficial effect of Ad2 x TGtk for tissue-specific gene therapy for TG-producing thyroid carcinomas without toxicity for normal tissues.

Adenoviridae↗
PubMed2006

Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

The molecular typing methods used so far for Mycobacterium ulcerans isolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity among M. ulcerans isolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1. Three different genotypes were found in Ghana, demonstrating for the first time the genetic diversity of M. ulcerans in an African country. While the ST1/MIRU 1 allele combination BD/BAA seems to dominate in Africa, it was only rarely found in isolates from Ghana, where the combination BD/B was dominant and observed in all districts studied. A third variant genotype (C/BAA) was found only in the Amansie-West district. The results indicate that new genetic variants of M. ulcerans emerged and spread within Ghana and support the potential of VNTR-based typing for genotyping of M. ulcerans.

Africa↗
PubMed2004

Evaluation of allelic alterations in short tandem repeats in different kinds of solid tumors--possible pitfalls in forensic casework.

Archival pathology specimens are nowadays a frequently used source in forensic identification or paternity testing, if no other material is available. A greater part of this archived material, however, consists of solid tumors known for aberrations in coding and non-coding regions of the genome. Therefore, alterations of short tandem repeats (STRs) used in forensic casework are also possible. In our study of 118 solid tumors, 46 lymph node metastases, and 16 distant metastases with the AmpFlSTR trade mark Profiler Plus PCR amplification kit comprising nine STR loci, we detected four kinds of changes between normal and tumor tissue: partial loss of one allele (pLOH), complete loss of one allele (LOH), occurrence of an additional allele and occurrence of a new allele instead of that found in normal tissue. Twenty-two percent of the tumor lesions displayed pLOH, but only in 14% one allele was completely lost. New alleles could be demonstrated in 18% of tumors, and in 8% the new allele in the tumor tissue replaced the one found in normal tissue. The changes were distributed over all nine STRs, but the STRs mostly affected were FGA, D3S1558, D18S51 and D21S11. The occurrence of new alleles in the tetra-nucleotide repeats correlated mainly with microsatellite instability in di-nucleotide and mono-nucleotide repeats. The occurrence of new alleles was most frequent in primary tumors of colon carcinomas and HNSCC metastases. In melanomas, only loss of alleles could be found. Our results demonstrate that the use of tumor tissue in forensic identification and paternity testing is questionable, especially if only tumors with known microsatellite instability are available.

Alleles↗
PubMed2004

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography.

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms.

Allelic Imbalance↗
PubMed2005

Von Willebrand gene tracking by single-tube automated fluorescent analysis of four short tandem repeat polymorphisms.

Molecular diagnosis of von Willebrand disease (VWD) has been hampered by the large size and complex genomic characteristics of the gene involved. For this reason, indirect methods using intragenic polymorphic markers described along the von Willebrand factor (VWF) gene are valuable tools for gene monitoring and linkage analysis. Several studies have demonstrated the four commonly utilized short tandem repeats (STRs), three located in intron 40 and one in the promoter region of the VWF gene, to be highly informative for this task. Our objective was t o develop a rapid, automated method to simultaneously analyze these four STRs for VWF gene tracking. Amplification of the four loci is achieved in a single multiplex fluorescent PCR which is then analyzed in the same run by capillary electrophoresis. Data processing with GeneScan and Genotyper software has simplified management and tabulation of the resulting haplotypes. Analysis of the VWF gene in DNA from 102 individuals (204 chromosomes) revealed that the three STRs within intron 40 showed significant linkage disequilibrium against each other but not against the VWP locus. Moreover, the combination of the four markers offers a high heterozygosity rate (>99%) that improves tracing VWF gene inheritance. In conclusion, the automated fluorescent capillary electrophoresis method presented here is an extremely rapid, simple and highly informative technique for association studies between VWD and the VWF gene in addition to genetic counseling and prenatal diagnosis by precise linkage analysis in VWD-affected families.

Alleles↗
PubMed1999

The use of mitochondrial DNA and short tandem repeat typing in the identification of air crash victims.

In February 1998, a civilian air plane crashed into a remote mountainside in the Philippines, killing all 104 passengers and crew. The victims were subjected to severe environmental insult, preventing conventional identification methods in most cases. As part of the identification process, samples were subjected to a combination of mitochondrial DNA (mtDNA) and short tandem repeat (STR) DNA profiling. The DNA extracted from the victims' remains was in all cases highly degraded. However, profiling using mtDNA was still successful with 95% of the victims' samples; this compared to a 50% success rate using three STR loci. The use of mtDNA and STR profiling enabled 187 human fragments from the crash site to be placed into 80 distinct groups; when combined with postmortem data, the samples could be further separated into 95 distinct groups, thereby assisting in the identification process.

Accidents, Aviation↗
PubMed2001

Perspectives on the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for short tandem repeat genotyping in the post-genome era.

The recent completion of the first rough draft of the human genome has provided fundamental information regarding our genetic make-up; however, the post-genome era will certainly require a host of new technologies to address complex biological questions. In particular, a rapid and accurate approach to characterize genetic markers, including short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) is demanded. STRs are the most informative of the two polymorphisms owing to their remarkable variability and even dispersity throughout eukaryotic genomes. Mass spectrometry is rapidly becoming a significant method in DNA analysis and has high probability of revolutionizing the way in which scientists probe the human genome. It is our responsibility as biomolecular mass spectrometrists to understand the issues in genetic analysis and the capabilities of mass spectrometry so that we may fulfill our role in developing a rapid, reliable technology to answer specific biological questions. This perspective is intended to familiarize the mass spectrometry community with modern genomics and to report on the current state of mass spectrometry, specifically electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, for characterization of STRs.

Alleles↗
PubMed1998

Short tandem repeat polymorphic markers for the rat genome from marker-selected libraries.

In an effort to generate a genome-wide set of high-quality polymorphic markers for the rat, we used the marker-selection method, which has already been proven useful for the development of markers, especially for the human genome. Small-insert (300-900 bp) rat genomic libraries were constructed with an estimated complexity of three genome equivalents and enriched for short tandem repeat sequences (STRs). The enriched libraries were found to contain 45% (CA)n and 27% (GATA)n, representing at least a 50-fold enrichment over unselected small insert genomic libraries. A subset of 2160 STR-containing clones, primarily of the (GATA)n class of repeats, were sequenced. PCR primers flanking the repeats were synthesized from some of the sequences from the (CA)n and (GATA)n classes of STRs and tested for polymorphism in a panel of eight inbred rat strains. This strategy yielded 147 polymorphic markers, which mapped with high odds to all chromosomes by linkage in three F2 populations. The integration of these STR markers with other rat genetic markers and mapping reagents will facilitate the mapping of disease genes in the rat and the identification of loci associated with complex mammalian phenotypes.

Animals↗
PubMed2002

X Chromosomal short tandem repeat polymorphisms near the phosphoglycerate kinase gene in men with chronic prostatitis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11-13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11-13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.

Alleles↗
PubMed1997

Optimization of a hexaplex DNA amplification from short tandem repeat and amelogenin loci.

An automated DNA profiling system based on the multiplex amplification of highly polymorphic short tandem repeat (STR) markers and the amelogenin locus was developed. Five STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplifications, including HUMD1S103, HUMTH01, HUMD21S11, HUMD18S51, and HUMFIBRA. One primer for each locus was labeled with a fluorescent dye (fluorescein) which allows detection on the single wavelength ALF DNA Sequencer (Pharmacia Biotech). As part of the detailed evaluation of the suitability of the hexaplex system for routine forensic use, the effect of variation in amplification parameters on the efficiency of the system was examined. Polymerase chain reaction amplification conditions were optimized to provide specific, robust amplification of forensic samples.

Autoanalysis↗