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Results for “Simple repeated sequences”

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PubMed1999

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Case-Control Studies↗

PubMed1999

Somatic mutation of the PTEN/MMAC1 gene in breast cancers with microsatellite instability.

The extent to which microsatellite instability (MI) contributes to the etiology of breast cancer has not been established in any large-scale studies. We examined 528 samples of tumor DNA from patients with primary breast cancer for MI, using 14 polymorphic CA-repeat markers. The frequency of MI in these tumors was unexpectedly low (10/528, 1.9%). The ten MI+ tumors were analyzed for mutations in five potential target genes that contain simple repeat sequences (TGFBIIR, IGF2R, hMSH6, BAX and PTEN/MMAC1). A somatic insertion of an extra adenine in the (A)6 region at codon 321-323 (exon 8) of the PTEN/MMAC1 gene, leading to a frame-shift, was identified in one tumor. This observation represented the first documented instance of PTEN/MMAC1 alteration in a MI+ primary breast cancer.

Breast Neoplasms↗

PubMed2001

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype.

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.

Amino Acid Sequence↗

PubMed2001

DNA profiling of disputed chilli samples (Capsicum annum) using ISSR-PCR and FISSR-PCR marker assays.

A case of marketing of spurious seeds of chilli, Capsicum annum in the brand name of an elite variety referred to us from an Indian court of law, for identification is described here. The highly reproducible molecular marker assays, inter simple sequence repeat polymerase chain reaction [ISSR-PCR] and FISSR-PCR (for fluorescent ISSR-PCR) were used for differentiating the four disputed chilli samples. A total number of 17 ISSR anchored primers, which included nine di-, and eight tri-nucleotide primers were used for the analysis. The ISSR-PCR products were separated on a 2% agarose gel. A total of 212 and 288 bands were resolved by seven di- and eight tri-nucleotide primers, respectively, with an average of 30 bands per primer. Five out of nine dinucleotide primers and four out of eight trinucleotide primers could unambiguously differentiate all the four disputed chilli samples. The sensitivity and informativeness of the ISSR-PCR assay were further enhanced by the use of FISSR-PCR technique. The FISSR-PCR assay revealed a total number of 566 bands using three tri- and one di-nucleotide primers with an average of 141 bands per primer. These four primers could reliably distinguish all the four disputed samples unambiguously. In developing countries like India, violation of Plant Breeder's Rights is a major concern of law. The present report is, therefore, a step to protect the Plant Breeder's Rights by making use of reliable and modern DNA technologies.

Breeding↗

PubMed2006

Construction of a microsatellite linkage map with two sequenced rice varieties.

Based on the successful development of new microsatellite markers from the data of two whole-sequenced rice varieties, japonica variety Nipponbare and indica variety 9311, an F2 population of 90 lines, which was derived from a single cross between Nipponbare and 9311, was applied to construct a genetic linkage framework map. The map covered 2 455.7 cM of total genomic length, and consisted of 152 simple sequence repeats (SSRs) loci including 46 pairs of new SSR primers developed by our research institute. The average genetic distance between two markers was 16.16 cM. In addition, markers RM345 and RM494, which have not been mapped on the Temnykh's map et al. (2001) were anchored on the sixth chromosome of this map. We compared this research with maps of Temnykh et al.(2001) and LAN et al. (2003) regarding the aspects of type and size of population, type and quantity of markers, and the marker arrangement order on chromosome, etc. Results indicated that the similarity of marker linear alignment was 93.81% between this map and T-map. Finally, the important significance of using sequenced rice varieties to construct linkage map was also discussed.

Chromosome Mapping↗

PubMed2006

Analysis of microsatellites in citrus unigenes.

Simple sequence repeats (SSRs) were investigated in the unigene sequences from expressed sequence tags (EST) of sweet orange (Citrus sinensis osbeck), trifoliate orange (Poncirus trifoliata Raf.) and other citrus species and cultivars. A total of 37 802 citrus unigene sequences corresponding to 23.29 Mb were searched, resulting in the identification of 8,218 SSRs. Among them there were 4,913 (59.8%) mono-, 1,419 (17.3%) di-, 1,709 (20.8%) tri-, 114 (1.39%) tetra-, 23 (0.28%) penta- and 40 (0.49%) hexa-nucleotide SSRs. The estimated frequency of SSRs was approximately 1/2.8 kb, which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes. The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR. The overall average length of SSRs was 20.9 bp. The frequencies of different SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) were very similar between sweet orange and trifoliate orange. The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange, followed by trimeric repeats. The adenine rich repeats such as A/T, AG, AT, AAG AAAT, AAAG, AAAT, AAAAG, AAAAT etc. were predominant in each type of SSRs (mono-, di-, tri-, tetra-, and penta-), whereas the C/G, CG, CCG repeats were less abundant. Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange. The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.

Base Sequence↗

PubMed2006

QTL analysis of popping fold and the consistency of QTLs under two environments in popcorn.

Popping fold (PF) is the most important quality trait in popcorn. In this study, a total of 259 F2:3 families, derived from the cross between a dent corn inbred Dan232 and a popcorn inbred N04, were evaluated for their popping folds in replicated experiments under two environments. Of 613 simple sequence repeat (SSR) primer pairs screened, 183 pairs were selected to construct a genetic linkage map with the genetic distance of 1,762.2 cM (centimorgan) and on average 9.63 cM every marker. Quantative trait loci (QTL) were identified, and their genetic effects were estimated using CIM (composite interval mapping) method. The interactions among QTLs detected were calculated using MIM (multiple interval mapping) method. In all, 22 QTLs were detected, and only 5 of them were common under two environments. Contribution to phenotypic variation of a single QTL varied from 3.07% to 12.84%, and total contributions of all QTLs under two environments were 66.46% and 51.90%, respectively. Three QTLs (qPF-6-1, qPF-8-1 and qPF-1-3) with more than 10% contributions were observed. The additive effects were larger than dominant effects for most QTLs. The amount of QTLs showing additive, partially dominant, dominant and over-dominant effects were 4, 5, 0, 2 in spring sowing and 2, 5, 2, 2 in summer sowing, respectively. There were only 2.60% pairs of QTLs or maker intervals expressing AA, DA or DD interactions.

Environment↗

PubMed2006

Genetic diversity of source germplasm of Upland cotton in China as determined by SSR marker analysis.

The genetic diversity of 43 sources of Upland cotton germplasm with different parental origins, breeding periods, and ecological growing areas in China were studied on the basis of simple sequence repeat (SSR) markers. A total of 130 gene alleles with 80% polymorphism were detected from 36 SSR primers. The number of alleles per primer ranged from two to eight with an average of 3.6. The polymorphism information content (PIC) range was 0.278-0.865, with an average of 0.62. The average genotype diversity index (H') was 1.102, the highest was 2.039 and the lowest was 0.451. The average coefficient of the genetic similarity of SSR markers among source germplasm was 0.610, ranging from 0.409 to 0.865. These indicated that the genetic diversity at the genomic level of the selected source germplasm was rich, and was representative of the diversity of the germplasms, in general. The diversity at the genome level of the base germplasm from the second and third breeding periods was decreased compared to that of the first period, indicating that the cotton genetic background in China became narrow gradually. The diversity of SSR markers among the base germplasm from early maturity cotton growing areas in the north was higher than those from the Huanghe and Yangtze growing areas. The molecular marker genetic similarity index of the domestic varieties was higher than that in the introduced varieties, which indicates that the genetic diversity in domestic cultivars was lower than that in the introduced varieties. This study gives an overview of the genetic diversity of the cotton germplasm base in China, and provides a guide for breeders to develop new cultivars efficiently.

China↗

PubMed2000

The Tetrahymena p80/p95 complex is required for proper telomere length maintenance and micronuclear genome stability.

The telomerase enzyme adds simple sequence repeats to chromosome ends. Telomerases share two essential subunits, telomerase RNA and telomerase reverse transcriptase, that associate with species-specific proteins of predominantly unknown functions. The Tetrahymena p80/p95 complex can coimmunopurify active telomerase from cell extract, and recombinant p80/p95 can interact directly with telomerase RNA and single-stranded telomeric DNA in vitro. Here, we test the functions of p80/p95 in vivo. Surprisingly, telomerase RNA accumulation and telomerase activity in cell extract are unaffected by loss of the genes encoding p80/p95. However, in the absence of p80/p95, telomeres become elongated in both macronuclei and micronuclei. Micronuclear chromosome maintenance is also compromised. These findings suggest that p80/p95 functions to maintain appropriate telomere length and micronuclear genomic stability but does so in a manner different than previously anticipated.

Animals↗

PubMed2002

+1 Proteins and aging.

Molecular misreading is an expression used to describe errors in RNA that lead to the translation of mutated proteins. We have shown that dinucleotide deletions (delta GA, delta GU) are introduced in simple sequence repeats (e.g. GAGAG) of mRNA. If the resulting mutant transcripts escape RNA quality control systems, they are translated into +1 proteins. If functional domains are located downstream of the frameshift site, the result will be a protein with either a partial or complete loss of function. A clear example is ubiquitin(+1) (UBB(+1)), which has lost its capacity to ubiquitinate, i.e. tagging proteins destined for proteasomal degradation. This is an important step in regulating the degradation of misfolded proteins and transcription factors. In fact, UBB(+1) seems to block the proteasome. UBB(+1) and other proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD), which suggests a causal relationship. We have hypothesized that quality control mechanisms for both transcripts and proteins work less efficiently during aging. In this manner +1 proteins may become manifest and contribute to age-related diseases.

Aging↗

PubMed2002

Spontaneous and bleomycin-induced genomic alterations in the progeny of Drosophila treated males depends on the Msh2 status. DNA fingerprinting analysis.

Deficiency in DNA mismatch repair (MMR) confers instability of simple repeated sequences and increases susceptibility to cancer. Some of the MMR genes are also implicated in other repair and cellular processes related to DNA damage response. Supposedly, lack of their function can lead to a global genomic instability, besides microsatellite instability (MSI). To study the spontaneous and induced genomic instability in germ cells, related to the Msh2 status, DNA alterations in the progeny of individual crosses of Drosophila deficient in one or two copies of the Msh2 gene, were analysed by the arbitrarily primed polymerase chain reaction (AP-PCR). The results indicate that the progeny of homozygous parents for the normal Msh2 allele (+/+) presents a significantly lower frequency of genomic alterations than those from heterozygous (+/-) or mutant homozygous (-/-) parents. In addition, the DNA damage transmitted to the progeny, after the adult parental males were exposed to bleomycin, indicates that whereas the induction of mutations related to MSI depends on the lack of the Msh2 function, the induction of other mutational events may require at least one functional Msh2 allele. Thus, the results obtained with heterozygous individuals may have special relevance for cancer development since they show that a disrupted Msh2 allele is enough to generate genomic instability in germ cells, increasing the genomic damage in the progeny of heterozygous individuals. This effect is enhanced by mutagenic stress, such as occurs after bleomycin exposure.

Animals↗

PubMed2004

Analysis on frequency and density of microsatellites in coding sequences of several eukaryotic genomes.

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in the investigated species, followed by trimeric, dimeric, tetrameric, monomeric and pentameric repeats. Furthermore, the A-rich repeats are predominant in each type of SSRs, whereas G-rich repeats are rare in the coding regions.

Animals↗

PubMed2001

Genetic variability and molecular identification of Brazilian Biomphalaria species (Mollusca: Planorbidae).

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

Animals↗

PubMed2000

A microsatellite marker reveals population heterogeneity within human and animal genotypes of cryptosporidium parvum.

Isolates of the protozoan parasite Cryptosporidium parvum have been differentiated into 2 genotypes: genotype 'H', which is associated only with human infections, and genotype 'C', which is associated with both human and animal infections. To date, the analysis of polymorphisms of genes and of the small subunit ribosomal DNA have revealed no heterogeneity within the 2 genotypes. In the present study, a locus containing simple sequence repeats (microsatellites) was PCR amplified and sequenced from 94 C. parvum isolates, which were collected from humans (immunocompetent and immunocompromized individuals, outbreak and single cases) and from several animal hosts in 3 continents. The analysis revealed that genotype 'H' can be further differentiated into 2 subgenotypes, and genotype 'C' can be further differentiated into 4 subgenotypes. The 6 subgenotypes differ in terms of expansions/contractions of the microsatellite repeats and by point mutations. Some subgenotypes showed a wide geographical distribution, whereas others were restricted to specific regions. Therefore, microsatellites are informative markers for more defined studies on the epidemiology, the transmission routes, and the population structure of this parasite.

Animals↗

PubMed2003

Genetic structure of Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota).

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR-RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy-Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.

Alleles↗

PubMed2003

Mycelial incompatibility operative in pairings between single basidiospore isolates of Helicobasidium mompa.

Helicobasidium mompa is a clampless basidiomycete and binucleate both in secondary and primary hyphae. Single basidiospore isolates were paired in sibling and non-sibling combinations. We did not observe any alterations in colony morphology, such as tuft formation, but mycelial incompatibility, as indicated by the presence of a dark demarcation line between colonies, occurred in a unifactorial manner. Techniques using DNA molecular markers, such as internal transcribed spacer-restriction fragment-length polymorphism (ITS-RFLP), inter-simple-sequence-repeat polymerase chain reaction (PCR), and universally primed PCR, failed to identify nuclear migration to opposite single basidiospore isolates in all but one of the 92 pairings. These results imply that single basidiospore isolates of H. mompa may be incompetent to mate under laboratory conditions and that a single mycelial incompatibility factor operates in single basidiospore isolates.

Basidiomycota↗

PubMed2003

Distribution of Amanita spp. genotypes under eastern Australian sclerophyll vegetation.

Basidiomes of Amanita alboverrucosa, A. ochrophylla, and A. pyramidifera were collected from native mixed sclerophyll forest sites and of A. conicoverrucosa and A. punctata from planted stands of Eucalyptus maculata in New South Wales, Australia. DNA was extracted from stipe tissue and subjected to inter-simple sequence repeat (ISSR) PCR analysis conducted using the primers (GTG)5 and (GACA)4 in order to determine genotype distribution at each site. Two to nine genotypes of one of the species were identified at each field site. Genotypes of A. ochrophylla, A. conicoverrucosa and A. punctata were spread over areas of ca 10-60 m diam, suggesting vegetative spread via large below-ground mycelial genets. In contrast, genotypes of A. alboverrucosa were more spatially restricted, suggesting recent establishment via basidiospores and more limited below-ground vegetative spread. Two groups of A. pyramidifera basidiomes that were separated by ca 600 m were found to be of the same genotype. While this might reflect long distance spread of below-ground mycelium in this taxon, the proximity of the basidiomes to a roadway makes movement of vegetative basidiome tissue via vehicular activity and subsequent establishment equally plausible.

Amanita↗

PubMed2005

SSR-based detection of genetic variability in the charcoal root rot pathogen Macrophomina phaseolina.

Macrophomina phaseolina, the causal agent of charcoal root or collar rot, is an important plant pathogen especially in soybean and cotton. Single primers of simple sequence repeats (SSR) or microsatellite markers have been used for the characterization of genetic variability of different populations of M. phaseolina obtained from soybean and cotton grown in India and the USA. Genetic similarity between isolates was calculated, and cluster analysis was used to generate a dendrogram showing relationships between isolates collected from the two hosts. Forty isolates could be clustered into three major groups corresponding to their hosts and geographical region. The wide distribution of microsatellites in M. phaseolina genome was assessed by agarose gel electrophoresis of the PCR products generated by direct amplification of inter SSR regions DNA. This is the first report of the use of microsatellite markers to characterize the charcoal root rot pathogen. The SSR fingerprints (0.25-3.5 kb) generated using DNA from different populations of M. phaseolina of two hosts indicated that these repeats are interspersed within the genome of this pathogen. The variability found within closely related isolates of M. phaseolina indicated that such microsatellites are useful in population studies and represents a step towards identification of potential isolate diagnostic markers specific to soybean and cotton.

Ascomycota↗