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PubMed2004

Protection against MUC1 expressing mouse tumours by intra-muscular injection of MUC1 cDNA requires functional CD8+ and CD4+ T cells but does not require the MUC1 tandem repeat domain.

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background.

Animals↗
PubMed2002

Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi/Angelman syndrome region.

A human imprinted domain at 14q32 contains two co-expressed and reciprocally imprinted genes, DLK1 and GTL2, which are expressed from the paternally and maternally inherited alleles, respectively. We have previously shown that another imprinted locus, on human 15q11-q13, contains a large number of tandemly repeated C/D small nucleolar RNA genes (or C/D snoRNAs) only expressed from the paternal allele. Here we show that the region downstream from the GTL2 gene is also characterized by a transcription unit spanning many repeated intron-encoded C/D snoRNA genes, most of them arranged into two tandem arrays of 31 and 9 copies. Intriguingly, these snoRNAs depart from previously reported rRNA or snRNA methylation guides by their tissue-specific expression and by their lack of complementarity to rRNA or snRNA within their sequences. Analysis of the orthologous region in the mouse shows that the previously reported maternally expressed Rian gene, located downstream of Gtl2 on the distal 12 chromosome, encodes at least nine C/D snoRNAs. Through a systematic search in rodents, we could identify other C/D snoRNA genes in this domain. All snoRNAs identified on mouse distal 12 are brain-specific and only expressed from the maternally inherited allele. The human imprinted 14q32 domain therefore shares common genomic features with the imprinted 15q11-q13 loci. This link between tandemly repeated C/D snoRNA genes and genomic imprinting suggests a role for these snoRNAs and/or their host non-coding RNA genes in the evolution and/or mechanism of the epigenetic imprinting process.

Angelman Syndrome↗
PubMed1992

Isolation of human minisatellite loci detected by synthetic tandem repeat probes: direct comparison with cloned DNA fingerprinting probes.

As a direct comparison with cloned 'DNA fingerprinting' probes, we present the results of screening an ordered array Charomid library for hypervariable human loci using synthetic tandem repeat (STR) probes. By recording the coordinates of positive hybridization signals, the subset of clones within the library detected by each STR probe can be defined, and directly compared with the set of clones detected by naturally occurring (cloned) DNA fingerprinting probes. The STR probes vary in the efficiency of detection of polymorphic minisatellite loci; among the more efficient probes, there is a strong overlap with the sets of clones detected by the DNA fingerprinting probes. Four new polymorphic loci were detected by one or more of the STR probes but not by any of the naturally occurring repeats. Sequence comparisons with the probe(s) used to detect the locus suggest that a relatively poor match, for example 10 out of 14 bases in a limited region of each repeat, is sufficient for the positive detection of tandem repeats in a clone in this type of library screening by hybridization. These results not only provide a detailed evaluation of the usefulness of STR probes in the isolation of highly variable loci, but also suggest strategies for the use of these multi-locus probes in screening libraries for clones from hypervariable loci.

Base Sequence↗
PubMed2004

An isothermal amplification system based on the tandem-repeated DNA probe.

The hybridization methods and polymerase-based amplification methods are usually employed to detect pathogens and gene expressions quantitatively in clinical practices nowadays. However, the sensitiveness of the former and the specificity of the latter are not yet satisfied. To solve this problem, some promising comprehensive methods have been developed recently. Here we reported a new comprehensive method, a tandem repeated DNA probe-based amplification (TRPBA) system. To establish the TRPBA, TR48, an artificial tandem repeated DNA probe with 48 repeats of a 50 base pair unit was constructed. It could be efficiently amplified by Bst DNA polymerase at 61 degrees C for only 1 h. The products were analyzed either by direct gel electrophoresis or by gel electrophoresis after the digestion with restriction endonuclease HincII. The sensitiveness was as few as 100 copies per test, which was comparable with PCR-based techniques. The TR48 splicing with the DNA fragment of hepatitis B virus used as probe could successfully develop TRPBA to detect hepatitis B virus DNA. The TRPBA can be used in the future to detect many other genes or microorganisms simply by splicing TR48 with their DNA fragments. Thus, TRPBA might be useful because of its sensitiveness and simpleness.

DNA Probes↗
PubMed2006

Analysis of tandem repeats found in 44 prokaryotic genomes.

Genomes contain various types of repetitive sequences. They may be used as probes for seeking genome rearrangements because they are rather free from the natural selection if they are located in the intergenic regions. In this study, we searched for tandem repeats (TRs) in 44 prokaryotic genomes by the color-coding method and sought the signs of genome rearrangements by detailed analysis of the detected TRs. We found 13,542 tandem repeats from 44 prokaryotic genomes in total ranging from several tens to one thousand per genome. The results of statistical analysis show that TRs tend to exist on high base composition bias regions in some genomes. Moreover, we recognized the characteristic distribution patterns of equivalent TR-pairs in 12 genomes, which are expected to indicate the occurrence of whole-genome duplication (WGD) on the genomes. It is demonstrated that TRs could indeed be used for seeking genome rearrangements. Although it has not been made clear at this time whether or not WGD had occurred in prokaryotic genomes, the results of the analyses of equivalent TR-pairs in this study are thought to be evidences of WGD in these genomes.

Base Sequence↗
PubMed2002

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.

The Ki-67 antigen, pKi-67, is one of the most commonly used markers of proliferating cells. The protein can only be detected in dividing cells (G(1)-, S-, G(2)-, and M-phase) but not in quiescent cells (G(0)). The standard antibody to detect pKi-67 is MIB-1, which detects the so-called 'Ki-67 motif' FKELF in 9 of the protein's 16 tandem repeats. To investigate the function of these repeats we expressed three of them in an inducible gene expression system in HeLa cells. Surprisingly, addition of a nuclear localization sequence led to a complete absence of signal in the nuclei of MIB-1-stained cells. At the same time antibodies directed against different epitopes of pKi-67 did not fail to detect the protein. We conclude that the overexpression of the 'Ki-67 motif', which is present in the repeats, can lead to inability of MIB-1 to detect its antigen as demonstrated in adenocarcinoma tissue samples. Thereafter, in order to prevent the underestimation of Ki-67 proliferation indices in MIB-1-labeled preparations, additional antibodies (for example, MIB-21) should be used. Additionally, we could show in a mammalian two-hybrid assay that recombinant pKi-67 repeats are capable of self-associating with endogenous pKi-67. Speculating that the tandem repeats are intimately involved in its protein-protein interactions, this offers new insights in how access to these repeats is regulated by pKi-67 itself.

Amino Acid Motifs↗
PubMed2005

Evaluation of the discriminatory power of variable number tandem repeat (VNTR) typing of Mycobacterium bovis strains.

The discriminatory power of variable number tandem repeat (VNTR) typing based on 16 known loci (12 MIRUs, 3 ETRs and VNTR 3232) was assessed for Mycobacterium bovis strains collected sequentially at the slaughterhouse of N'Djaména, Chad. Of 67 M. bovis strains analyzed, 67% were clustered. In this study, VNTR typing was highly discriminative with an overall allelic diversity (h(oa)) of 0.922. We defined five loci (ETR A, B, C and MIRU 26, 27) as highly (h>0.25), two loci (MIRU 4, and VNTR 3232) as moderately (0.11<h<0.25) and three loci (MIRU 16, 20, 31) as poorly (0.01<h<0.11) discriminative. Six loci (MIRU 2, 10, 23, 24, 39, and 40) showed no polymorphism at all. VNTR typing of the five highly discriminative loci (h=0.917) proved to be most appropriate for first line typing of M. bovis strains of Chad and superior than spoligotyping (h(sp)=0.789). In contrast to Mycobacterium tuberculosis strains, a consensus on VNTR loci needs to be found for M. bovis strains. The selection of a generally agreed set of VNTR loci for molecular discrimination of M. bovis in different geographical settings is discussed.

Alleles↗
PubMed1999

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus.

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.

Alleles↗
PubMed2026

The first complete mitochondrial genome of Strigea falconis (Digenea: Strigeidae) reveals six tandemly repeated trnE-containing units and provides mt evidence for the non-monophyly of the family Strigeidae.

BACKGROUND: Phylogenetic relationships among members in the order Diplostomida remain contentious, with mitochondrial (mt) and nuclear genomic data often yielding conflicting topologies. A major limitation is the availability of only a few mt genomes from the type genus Strigea, hindering a robust test of the monophyly of the family Strigeidae and the order Diplostomida. RESULTS: The mt genome of S. falconis was completely sequenced for the first time, which was a circular molecule of 16,872&#xa0;bp in length, encoding the typical set of 36 mt genes and six duplicate tRNA-Glu genes. Notably, there were seven identical and consecutive tandem repeat units each consist of a 169&#xa0;bp non-coding region followed by a trnE gene in the newly assembled genome. Phylogenomic analyses based on concatenated predicted amino acid sequences of 12 proteins robustly placed S. falconis in the same clade as Apharyngostrigea pipientis. Crucially, the family Strigeidae was not recovered as monophyletic. Instead, two species within Strigeidae, Cardiocephaloides medioconiger and Cotylurus marcogliesei, clustered with representatives of Diplostomidae, providing mt evidence for the paraphyly of Strigeidae under the current sampling. CONCLUSIONS: The newly sequenced mt genome of S. falconis reveals a previously unreported six-copy tandem repeat of trnE-containing units among currently available diplostomoid mt genomes. Phylogenetic analyses based on mt protein-coding genes provide additional mt evidence that the family Strigeidae was not recovered as monophyletic under the present taxon sampling. However, because mt genomes represent a single maternally inherited linkage group, broader taxon sampling, independent nuclear phylogenomic data, and explicit sensitivity analyses will be required to confirm these relationships and guide any formal systematic revision.

Animals↗
PubMed1992

Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.

In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.

Amino Acid Sequence↗
PubMed2005

Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli.

Human beta-defensin-2 (hBD2) is a cysteine-rich cationic antimicrobial peptide with low molecular weight that exhibits a broad range of antimicrobial activity. To improve the expression level of hBD2 in Escherichia coli, tandem repeats of mature hBD2 gene were constructed and expressed as fusion proteins (TrxA-nmhBD2, n = 1, 2, 4, 8) by constructing the vectors of pET32-nsmhBD2 (n = 1, 2, 4, 8). The results showed that the tandem repeats of mhBD2 gene were highly expressed in our constructed system. Comparing the expression levels of soluble mhBD2, BL21(DE3)/pET32-2smhBD2 was selected as an ideal recombinant strain for mature hBD2 production. Under the optimized conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the maximum expression level of soluble mature hBD2 (0.76 g/l) with the highest percentage of fusion protein in soluble proteins (62.2%) was obtained in the present work, which was the highest yield of hBD2 reported so far.

Biotechnology↗
PubMed2000

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats.

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.

Alleles↗
PubMed1996

Improved conditions for the analysis of large variable number of tandemly repeated (VNTR) unit polymorphisms.

Large variable number of tandemly repeated (VNTR) unit polymorphisms were identified by amplification of genomic DNA with appropriate primers, followed by agarose gel electrophoresis and ethidium bromide staining. However, when genomic DNA was extracted using paramagnetic beads, we found that the efficiency of polymerase chain reaction amplification of VNTR's was affected by the purity of the solid phase-eluted DNA. Here we report that VNTR's located in the human interleukin-1 receptor antagonist and interleukin-4 genes are clearly and reliably amplified only when more stringent conditions are used in order to obtain purer DNA eluates from DNA-bead complexes. In addition, polyacrylamide gel electrophoresis followed by silver staining is superior to ethidium bromide staining of agarose gels in visualizing the different allelic bands of heterozygous carriers.

DNA↗
PubMed1996

Analysis of a short tandem repeat locus on chromosome 19 (D19S253).

A tetranucleotide tandem repeat locus on chromosome 19 (D19S253) was analysed. PCR products were detected by denaturing polyacrylamide gels with fluorescent-based technology. This study has confirmed a polymorphism with 9 alleles ranging from 209 to 241 bp with a simple repeat structure arranged from 7 to 15 repeats. Family studies confirmed mendelian inheritance of alleles. The efficiency on DNA extracted from bloodstains and cigarette butts has been evaluated. The protocol has shown sensitivity and reproducibility.

Alleles↗
PubMed1992

Continuous linkage map of human chromosome 14 short tandem repeat polymorphisms.

Nine moderately to highly informative short tandem repeat polymorphisms were assigned to chromosome 14 using somatic cell hybrids and were mapped using linkage analysis. The nine markers formed a continuous linkage map covering almost the entire long arm from 14q11.2 to q32. The markers filled a large gap within previously reported linkage maps for this chromosome. Best order of the new loci from q11.2 to q32 was D14S50, D14S54, D14S49, D14S47, D14S52, D14S53, D14S55, D14S48, and D14S51. The order shown for all adjacent pairs of loci was very strongly favored with the exception of loci pair D14S55 and D14S48, for which the order was moderately favored. Map lengths for the nine loci were 142 cM in females and 72 cM in males. Female recombination frequencies exceeded male recombination frequencies in the middle and distal portions of the map.

Alleles↗
PubMed1990

Minisatellite linkage maps in the mouse by cross-hybridization with human probes containing tandem repeats.

Tests of 29 human variable number of tandem repeat probes in inbred mouse lines showed that 80% (23/29) cross-hybridize, and 48% (14/29) produce multiple band, minisatellite polymorphisms (fingerprint patterns). Mini-satellite-type polymorphisms detected by 11 probes were characterized in eight different strains; on average, 240 polymorphic differences were detected between pairs of strains. Reproducible fingerprint patterns permit the study of the segregation of the minisatellite polymorphisms in experimentally designed crosses. As an example, we constructed primary minisatellite genetic linkage maps containing 346 polymorphic bands, distributed in 101 groups of closely linked systems, from genotypes on a recombinant inbred panel (C57BL/6J X DBA/2J); 38 of the groups were assigned by linkage to 15 autosomal chromosomes. The minisatellite genetic maps of C57BL/6J and DBA/2J can be applied in other linkage studies involving these strains.

Animals↗
PubMed1997

Multiplex systems for the amplification of short tandem repeat loci: evaluation of laser fluorescence detection.

Short tandem repeat (STR) loci are ideal markers for personal identification and for genomic mapping. Two fluorescent multiplex systems, each designed for simultaneous PCR amplification of four polymorphic STR loci (HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31, and HUMF13A01, HUMFESFPS, HUMBFXIII and HUMLIPOL), were evaluated on three laser fluorescence detection instruments. Concordant DNA typing results were obtained with all three detection methods. These fluorescent multiplex STR systems offer an accurate, reproducible and versatile method of DNA profiling that is well-suited for forensic identity testing and other genetic analyses.

Alleles↗