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The use of simple sequence repeats as polymorphic genetic markers has considerably facilitated the construction of reference maps of the human chromosomes and mapping of Mendelian genetic diseases. These markers display major advantages over other systems and will be widely used for linkage mapping purposes in the forthcoming years.
Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.
The development of dense genetic maps of mammalian chromosomes is facilitated when chromosome-specific libraries are used as a source of genetic markers. To saturate the genetic maps of mouse chromosomes 4 and 6, we have made use of fluorescent-activated chromosome sorting to purify a 4:6 Robertsonian chromosome from a cell line harboring the Rb(4:6)2Bnr translocation. After staining with chromomycin A3 and Hoechst 33528, this chromosome was separated from the other mouse chromosomes. DNA was isolated from the fraction containing the Robertsonian chromosome and subcloned into the insertion vector lambda gt10, generating a library with 4.6 x 10(5) independent phage. A total of 19 single-copy sequences were used to type the progeny of a C57BL/6J x Mus spretus backcross that had previously been typed for loci on chromosomes 4 and 6. Approximately 70% of the clones in the library mapped to either chromosome 4 or 6 as assessed by genetic mapping and by use of a somatic cell hybrid panel. Simple sequence repeats have also been isolated from this library. Further characterization of these microsatellites should accelerate efforts to map mouse chromosomes 4 and 6 using PCR. In addition, flow sorting of Robertsonian chromosomes suggests a general approach for making chromosome-specific libraries in mouse.
Twenty-six (CA)n polymorphic microsatellites were isolated from a flow-sorted chromosome 20 library. To reduce the number of sequencing gels, these microsatellites were genotyped on 15 CEPH families using a procedure derived from the multiplex sequencing technique of G. M. Church and S. Kieffer-Higgins (1988, Science 240:185-188). A primary map with a strongly supported order was constructed with 15 (CA)n markers. Regional localizations for the 11 other microsatellites were obtained with regard to the primary map. The 26 loci span approximately 160 cM. Regional localizations for a set of index markers (D20S4, D20S6, D20S16, and D20S19) and for other markers from the CEPH Public Database (D20S5, D20S15, D20S17, and D20S18) have also been determined. The total map spans a genetic distance of approximately 195 cM extending from the (CA)n marker IP20M7 on 20p to D20S19 on 20q. The density of microsatellite markers is markedly higher in the pericentromeric region, with an average distance of 3 to 4 cM between adjacent markers over a distance of 43 cM. Two-thirds of these randomly isolated microsatellites are clustered in that region between D20S5 and D20S16 representing approximately one-fourth of the linkage map. This might suggest a nonrandom distribution of (CA)n simple sequence repeats on this chromosome.
COL5A1, the gene for the alpha 1 chain of type V collagen, has been considered a candidate gene for certain diseases based on chromosomal location and/or disease phenotype. We have employed 3'-untranslated region RFLPs to exclude COL5A1 as a candidate gene in families with tuberous sclerosis 1, Ehlers-Danlos syndrome type II, and nail-patella syndrome. In addition, we describe a polymorphic simple sequence repeat (SSR) within a COL5A1 intron. This SSR is used to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber disease) and to add COL5A1 to the existing map of "index" markers of chromosome 9 by evaluation of the COL5A1 locus on the CEPH 40-family reference pedigree set. This genetic mapping places COL5A1 between markers D9S66 and D9S67.
We report the chromosomal localization and a simple sequence repeat (SSR) in the cytosolic phospholipase A2 (cPLA2) gene in both human and rat. A (CA)18 repeat in the promoter of the rat gene was determined to exhibit length polymorphism when analyzed using the polymerase chain reaction (PCR) in 19 different inbred rat strains. Genotyping for this marker in 234 F2 progeny of a SHR x BN intercross mapped the gene to rat chromosome 13. Using a PCR strategy, a fragment of the promoter for the human gene was isolated, and a (CA)18 repeat was identified. Since this marker displayed a low heterozygosity index, we also identified a mononucleotide repeat in the promoter for cPLA2 that displayed a polymorphism information content value of 0.76. The human gene was mapped using fluorescence in situ hybridization (FISH) to chromosome 1q25. Of interest, the gene encoding the enzyme prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), which acts on the arachidonic acid product of cPLA2, was previously localized to this same chromosomal region, raising the possibility of coordinate regulation. Identification of intragenic markers may facilitate studies of polymorphic variants of these genes as candidates for disorders in which perturbations of the eicosanoid cascade may play a role.
A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling. Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation hybridization, with a 245-kb yeast artificial chromosome clone containing the FLI1 gene, and by directed "walking" techniques. Cosmid contigs were constructed by individual clone fingerprinting using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm. The relative orientation and spacing of cosmid contigs with respect to the chromosome was determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping. Each cosmid clone in the contig was subjected to "one-pass" end sequencing, and the resulting ordered sequence fragments represent approximately 5% of the complete DNA sequence, making the entire region accessible by PCR amplification. The sequence samples were analyzed for putative exons, repetitive DNAs, and simple sequence repeats using a variety of computer algorithms. Based upon the computer predictions, Southern and Northern blot experiments led to the independent identification and localization of the FLI1 gene as well as a previously unknown gene located in this region of chromosome 11q24. This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps and provides a tool for further structural and functional analysis of the genome.
Mismatch-repair systems have been identified in organisms ranging from Escherichia coli to humans. They can repair almost all DNA base pair mismatches as well as small insertion/deletion mismatches. Molecular and biochemical analyses have shown that the core components of eukaryotic mismatch-repair systems are highly homologous to their bacterial counterparts. In humans, defects in four mismatch-repair genes have been linked both to hereditary non-polyposis colorectal cancer and to spontaneous cancers that exhibit rearrangements in DNA containing simple repeat sequences.
The use of simple sequence repeats as polymorphic genetic markers has significantly facilitated the construction of reference maps of the human chromosomes and the mapping of mendelian genetic diseases. These markers display major advantages over other systems, including restriction fragment length polymorphisms and satellites, but also have some limitations.
BACKGROUND: In 1989 the National Institute of Mental Health began a collaborative effort to identify genes for bipolar disorder. The first 97 pedigrees showed evidence of linkage to chromosomes 1, 6, 7, 10, 16, and 22 (Nurnberger et al 1997). An additional 56 bipolar families have been genotyped, and the combined sample of 153 pedigrees studied. METHODS: Three hierarchical affection status models were analyzed with 513 simple sequence repeat markers; 298 were common across all pedigrees. The primary analysis was a nonparametric genome-wide scan. We performed conditional analyses based on epistasis or heterogeneity for five regions. RESULTS: One region, on 16p13, was significant at the genome-wide p <.05 level. Four additional chromosomal regions (20p12, 11p15, 6q24, and 10p12) showed nominally significant linkage findings (p </=.01). Conditional analysis assuming epistasis identified a significant increase in linkage at four regions. Families linked to 6q24 showed a significant increase in nonparametric logarithms of the odds (NPL) scores at 5q11 and 7q21. Epistasis also was observed between 20p12 and 13q21, and 16p13 and 9q21. CONCLUSIONS: The findings are presented in rank order of nominal significance. Several of these regions have been previously implicated in independent studies of either bipolar disorder or schizophrenia. The strongest finding is at 16p13 at D16S748 with an NPL of 3.3, there is evidence of epistasis between this locus and 9q21. Application of conditional analyses is potentially useful in larger sample collections to identify susceptibility genes of modest influence that may not be identified in a genome-wide scan aimed to identify single gene effects.
Ulcerative colitis (UC) is an inflammatory disease of the colon that is associated with increased risk of colorectal cancer associated with genomic instability. We have previously demonstrated that genomic instability is present in UC patients with colonic neoplasia, and hypothesized that the chromosomal alterations may be taking place in regions that are susceptible to mutation or that provide a growth advantage to a cell undergoing neoplastic transformation. In this study, we used two polymerase chain reaction (PCR)-based DNA fingerprinting techniques (arbitrarily primed PCR and inter-simple-sequence-repeat PCR) to study the process of genomic instability. The two techniques of DNA fingerprinting cross-validate the instability observed in these studies. We analyzed the molecular basis of 10 commonly altered DNA bands obtained from DNA fingerprints of biopsies from various histologic grades of UC patients with dysplasia or cancer (UC Progressors). We determined that the band changes in the fingerprint truly represent changes in DNA sequence, and that the fingerprinting provides highly reproducible results. Furthermore, our investigation revealed that 40% of alterations involve repetitive sequences. Two frequently deleted sequences in 6q27 and 2q14 were studied further because they were frequently abnormal in the dysplastic and nondysplastic tissue of UC Progressors. The losses from 6q27 and 2q14 were confirmed by loss of heterozygosity and real-time PCR analysis. Both of these regions in chromosomes 6 and 2 are surrounded by highly repetitive and mobile LINE-1 elements, possibly making the region susceptible to mutational change. These regions were affected (lost) in UC Progressors but not in UC patients who were neoplasia free. Loss of heterozygosity at 6q27 has been described in ovarian and other cancers, while the 2q14 region has been implicated in prostate and sporadic colon cancers. Both regions are likely to contain tumor-suppressor genes. In conclusion, the genomic instability in UC Progressors can occur in regions that are susceptible to change and are locations of putative tumor-suppressor genes.
Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-beta signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-beta RII mutations were more common among nonsmokers.
Only a small portion of the total RNA transcribed in human cells becomes mature mRNA and constitutes the human transcriptome, which is context-dependent and varies with development, physiology and pathology. A small fraction of different repetitive sequences, which make up more than half of the human genome, is retained in mature transcripts and shapes their function. Among them are short interspersed elements (SINEs), of which Alu sequences are most frequent, and simple sequence repeats, which come in many varieties. In this review, we have focused on the structural and functional role of Alu elements and trinucleotide repeats in transcripts.
Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloë festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource. They permit the interrogation of 3806 Neotyphodium genes (Nchip microarray), and 4195 Neotyphodium and 920 Epichloë genes (EndoChip microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass-symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloë was performed.
Eight Y chromosome short tandem repeats (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385I/II) were used to assess haplotype distribution in non-selected, unrelated Bantu males living in Central Africa [N. Mathias, M. Bayes, C. Tyler-Smith, Highly informative compound haplotypes for the human Y chromosome, Hum. Mol. Genet. 3 (1994) 115-123; L. Roewer, J. Arnemann, N.K. Spurr, K.H. Grzeschik, J.T. Epplen, Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts, Hum. Genet. 89 (1992) 389-394; P. De Knijff, M. Kayser, A. Caglia, D. Corach, N. Fretwel, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccini, M. Prinz, C. Schmitt, P. M. Schneider, R. Szibor, J. Teifel-Greding, G. Weishold, L. Rower, Chromosome Y microsatellites: population genetic and evolutionary aspects, Int. J. Legal Med. 110 (1997) 134-149; M. Kayser, A. Caglia, D. Corach, N. Fretwel, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccini, M. Prinz, C. Schmitt, P. M. Schneider, R. Szibor, J. Teifel-Greding, G. Weishold, P. de Knijff, L. Rower, Evaluation of Y chromosome STRs: a multicenter study, Int. J. Legal Med. 110 (1997) 125-133, 141-149]. One hundred and sixty-five full haplotypes were obtained from Bantu males. The most common haplotype (DYS19-15, DYS389I-13, DYS389II-30, DYS390-21, DYS391-10, DYS392-11, DYS393-13, DYS385I/II-15,17) was shared by 5.5% of individuals. In the Bantu population in Central Africa, the haplotype diversity and the discrimination capacity of Y-STR may be estimated at 99.14% and 0.5333, respectively.
Necrophagous fly is the most common insect evidence collected during a death investigation. We analyzed the DNA polymorphism among the five forensically important fly species, namely, Lucilia sericata, Aldrichina grahami, Chrysomya megacephala, Parasarcophaga crassipalpis and Musca domestica using inter simple sequence repeat (ISSR) method. Nine ISSR primers selected from 18 primers could amplify 105 clear and stable bands, of which 95 bands were polymorphic. Some primers produced completely different band pattern in different species, indicating that they can be used to identify these species. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific ISSR fragments into the sequence-characterized amplified region (SCAR) markers that can be used for the molecular diagnosis of the five species.
Identification and typing of spoilage and pathogenic microorganisms have become major objectives over the past decade in microbiology. In food, strain typing is necessary to ensure food safety and for linking cases of foodborne infections to suspected items. Recent advances in molecular biology have resulted in the development of numerous DNA-based methods for discrimination among bacterial strains. Here, we present the use of Simple Sequence Repeats (SSR, or Microsatellites) for bacterial typing. SSRs are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. Computer-based screen of the complete genomic DNA sequences of various prokaryotes showed the existence of tens of thousands well distributed SSR tracts. Mono Nucleotides Repeats (MNRs) are the majority of SSR tracts in bacteria, therefore selected MNR loci were analyzed for variation among strains belonging to three bacterial species: Escherichia coli, Listeria monocytogenes and Vibrio cholerae. High levels of polymorphism in the number of repeats was observed. The finding that most of the MNR tracts are variable in bacterial genomes, but stable at the strain level, allows the use of MNRs for bacterial strains identification. The variation in MNR tracts enables the separation between virulent and non-virulent strain groups and further discriminates among bacterial isolates, in the three tested bacterial species. The uncovered MNR polymorphism is important as a genome-wide source of variation, both in practical applications (e.g. rapid strain identification) and in evolutionary studies. This multi-locus MNR strategy could be applied for high throughput bacterial typing by assigning an "identity number" for each strain based on MNR variations. The developed typing technology should include the fingerprint database for large bacterial strain collections and a high throughput scanner. This accurate and rapid tool can have a major role in decreasing the incidences of food-related outbreaks and will contribute to limit epidemics.
Three different DNA fingerprinting techniques, the mobile genetic element (MGE)-PCR, simple sequence repeat (SSR)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to define a large set of genetic markers to study genetic similarity within and among Trypanosoma brucei, Trypanosoma equiperdum and Trypanosoma evansi strains (n=18) from China, Africa and South America and to investigate their genetic relationships. Using the three fingerprinting techniques, >890 bands (ranging in size from 0.2 to 2kb) were defined for all 18 strains of Trypanosoma. Within each of the strains, 39-59 bands were defined. The similarity coefficients between strains ranged from approximately 41 to 94%, with a mean of 65%. There was more genetic similarity among strains within T. evansi (mean of approximately 79%) compared with T. equiperdum ( approximately 65%) and T. brucei ( approximately 59%). The similarity coefficient data were used to construct the dendrogram, which revealed that (irrespective of species) the majority of strains from China and South America grouped together to the exclusion of those from Africa. The exceptions were a T. brucei strain from Africa and a T. equiperdum strain of unknown origin. Hence, employing data sets generated using the three different fingerprinting methods, it was not possible to unequivocally distinguish among T. brucei, T. evansi and T. equiperdum, although there was a tendency for T. evansi strains to group together to the exclusion of T. brucei. The findings provide support for the hypothesis that T. evansi originated from a mutated form of T. equiperdum and stimulate further investigations of the genetic make-up and evolution of members of the subgenus Trypanozoon.