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Microsatellite marker development, mapping and applications in rice genetics and breeding.

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10,000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trail locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.

Breeding↗

A novel microsatellite polymorphism in the human OB gene: a highly polymorphic marker for linkage analysis.

The mouse ob gene and its human homologue OB have recently been cloned. The mutations in the ob gene are known to be associated with extreme obesity. The relationship between the human OB gene and disease, however, is largely unknown due to the lack of suitable markers within or adjacent to the OB gene. To obtain informative markers, we searched for simple tandem repeat polymorphisms in the genomic sequence of the human OB gene and identified a novel tetranucleotide repeat in the 3' flanking region. Fifteen alleles were detected in this marker with a heterozygosity of 0.85 and polymorphism information content of 0.83, indicating a highly informative nature of this marker. Two-point linkage mapping in two Centre Etude Polymorphisme Humaine (CEPH) reference families suggested that this marker is located in the interval between D7S514 and D7S530, the same interval where the OB gene is located (recombination fractions with D7S514 and D7S530 were 0.026 and 0.034, respectively). Although allele frequency distributions of this marker did not differ between 84 control subjects and 69 NIDDM patients, there was a tendency to higher body weight in control subjects with class I/class I genotype than in those without this genotype (68.8 +/- 11.1 vs 60.8 +/- 10.3 kg, p = 0.05). The highly polymorphic nature of this marker and its location in the OB gene makes this marker useful for linkage studies of the OB gene with a number of phenotypes, such as obesity, non-insulin-dependent diabetes mellitus, hypertension and the insulin resistance syndrome.

Alleles↗

Characterization of a G-quartet formation reaction promoted by the beta-subunit of the Oxytricha telomere-binding protein.

Telomeres, the ends of linear chromosomes, typically consists of tandem repeats of a simple guanine-rich sequence. Telomeric DNA is able to form intermolecular G-quartet structures. The beta-subunit of the Oxytricha telomere-binding protein acts as a molecular chaperone to promote the formation of dimers and specific higher order complexes of telomeric DNA stabilized by G-quartets; these reactions occur under physiological conditions in vitro. In the present article, we show that, at saturating protein concentrations (> or = 200 nM), beta-mediated G-quartet formation is a first-order reaction with respect to DNA concentration, with k approximately 1 h-1 at 37 degrees C. In contrast, the protein-independent reaction is a second-order reaction. The beta-subunit enhances the rate of G-quartet formation by 10(5)-10(6)-fold at a telomeric DNA concentration of 20 nM. The beta-mediated higher order complexes are identified as parallel four-stranded tetramers of telomeric DNA (G4-DNA). Poly-L-lysine also promotes formation of the tetramers, but not dimers. These DNA structures were studied by irreversible thermal melting experiments and probed by annealing to different complementary strands. Guanine residues important for structure formation were analyzed by methylation interference experiments. On the basis of these data, models for the beta-mediated structures are proposed, and possible mechanisms for the beta-mediated reaction are discussed. In addition, we found that the beta-subunit promotes the annealing of two complementary strands into a duplex, as do many other basic proteins. However, not all proteins with annealing-promoting activity are active in the formation of G-quartet structures. The activity of the telomere protein in promoting the formation of telomeric DNA structures may enable chromosome-chromosome association or the regulation of telomerase activity in vivo.

Animals↗

Large CAG/CTG repeat templates produced by PCR, usefulness for the DIRECT method of cloning genes with CAG/CTG repeat expansions.

We report here a simple method for generating large CAG/CTG repeat sequences. We have applied this method to clone the genomic sequence containing the CAG/CTG repeat and its upstream intronic sequence present in spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) by a modified DIRECT method. With these modifications we have considerably simplified the generation of the repeat probe used to screen for anomalous bands. This method will facilitate the molecular approach to other genetic disorders where expansions of repeat sequences could be involved.

Base Sequence↗

Telomeric repeats of Tetrahymena malaccensis mitochondrial DNA: a multimodal distribution that fluctuates erratically during growth.

The linear mitochondrial DNA (mtDNA) of Tetrahymena malaccensis has tandem 52-base-pair repeats at its telomeres. The mtDNA has a multimodal distribution of telomeres. Different groups in the distribution have different numbers of telomeric repeats. The standard deviation of the size of each end group is independent of the mean size of the end group. The two sides of the mtDNA have different multimodal distributions of repeats. Cloned cell lines have multimodal distributions of mtDNA telomeres distinct from that of the original cell line. The number of telomere end groups and the average size of the end groups change in an erratic fashion as the cells are passaged and do not reach a stable equilibrium distribution in 185 generations. We propose that the mean size of a telomere end group and the size distribution of an end group are independently regulated. The system controlling the average size of end groups may be defective in T. malaccensis, since a closely related species (T. thermophila) does not have a multimodal distribution of mtDNA telomeres. T. hyperangularis, which has different telomeric repeats on each side of its mtDNA, has a multimodal distribution of mtDNA telomeres on only one side, suggesting that the mechanism controlling the average number of repeats in an end group can be sequence specific. These mitochondrial telomeres provide a new example of the more general phenomenon of expansion and contraction of arrays of repeated sequences seen, for example, with simple-sequence "satellite" DNAs; however, the mitochondrial telomeres change on a very short time scale.

Animals↗

[Microsatellite marker development and applications in wheat genetics and breeding].

Microsatellites are simple, tandemly repeated one to six nucleotide sequence motifs. They are very useful as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily assayed by the polymerase chain reaction ( PCR ). In wheat, SSR markers have been applied to genetic mapping,detection of genetic diversity, identification of varieties and genotypes,gene tagging, QTL analysis, and marker-assisted selection.

English Abstract↗

[Genome organization in eukaryotes].

The review discussed problems of genome organization in the chromosomes of eukaryotes based on the data of genetical and molecular-biological studies. Particular attention is paid to the peculiarities of organization of genetic material in Drosophila. The main (euchromatic) part of the genome is considered as the totality of separate functional units (chromomere, gene loci), which have in the average a higher excess of DNA compared to the sized of the structural gene. The possible ways of internal organization of these units and the role of different types of DNA sequences in their function are discussed. The features of organization and function of the heterochromatic regions of chromosomes consisting of simple sequences of satellite DNA and tandemly repeated genes (polygenes), that code for general cellular functions (similar to the ribosomal genes) are considered. The instability of the number and even the sites of localization of polygenes in the chromosome, which can be determined by the peculiarities of their functions during differentiation of somatic cells is notes.

Animals↗

Aloe L.--a second plant family without (TTTAGGG)n telomeres.

The physical ends of chromosomes are protected and stabilised by telomeres. The sequence of telomeric DNA normally consists of a simple repeating unit that is conserved in many organisms. Most plants examined have been shown to possess Arabidopsis-type telomeres consisting of many repeat copies of the sequence 5'-TTTAGGG-3'. Using fluorescent in situ hybridisation, slot blotting and the asymmetric polymerase chain reaction we demonstrate an absence of Arabidopsis-type telomeres in the genus Aloe (family Asphodelaceae). The only other plant genera so far reported without such telomeres are Allium, Nothoscordum, and Tulbaghia (family Alliaceae). As these genera and Aloe are petaloid monocots in the Asparagales, it is suggested that an absence of Arabidopsis-type telomeres may be characteristic of this related group of plants.

Allium↗

Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA.

The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis. When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease. A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease. The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide. In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence. This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.

Animals↗

Terminal repeats in long repeat arrays are likely to reflect the early evolution of Balbiani ring genes.

Balbiani ring (BR) genes in Chironomus tentans are 35 to 40 kb (1 kb = 10(3) bases or basepairs) in length and encode secretory proteins of exceptional size. Each gene contains a large homogeneous core block consisting of approximately 100 tandemly arranged, highly homologous repeat units. The repeat unit has a constant (C) region and a subrepeat (SR) region. The various BR genes exhibit similar C regions, while the SR regions differ as to sequence, length and number of subrepeats. To study early steps in the evolution of the coding repeat arrays of the BR genes we have analyzed the 3' ends of the four BR genes in C. tentans: BR1, BR2.1, BR2.2 and BR6. In each gene the very end of the core block consists of two or three repeat unit variants; in each variant repeat the C region is linked to a Cys region, replacing the SR region. Sequence comparisons between the C regions of the closely related BR1 and BR2 genes show that during evolution the terminal repeat unit variants have to a large extent been isolated from the remainder of the core block and have probably been more conserved than the interior repeat units. Detailed analysis of the structure of the variant repeat units further supports this latter notion and suggests that the BR core blocks have evolved from an array of a simple 36 base-pair long sequence; larger, more complex repeat units containing subrepeats were gradually formed and spread in the block, mainly by homologous unequal recombination events. During this evolution the interior of the core blocks evolved as a homogeneous repetitive structure, while ancestor repeat units remained as sequence relicts in the terminal parts.

Animals↗

Sequence variation of new alleles at the short tandem repeat D19S253 locus.

This paper reports the sequences of two new alleles identified in a population database study on the short tandem repeat D19S253 locus. A Portuguese Caucasian population and a Portuguese African population were studied. Forty-four selected alleles were sequenced and 11 different alleles were found. All the sequenced alleles shown to possess a simple tetranucleotide GATA repeat region structure. The two new alleles, alleles 6 and 16, follow the simple repeat pattern. During paternity investigation casework, 1028 meiosis were analyzed and five isolated genetic incompatibilities detected. In one case, a non-detectable allele with the used set of primers could be the explanation. In the other four cases, single-step mutations could be considered. The mutation rate obtained for this locus was 3.89 x 10(-3).

Alleles↗

The contribution of slippage-like processes to genome evolution.

Simple sequences present in long (> 30 kb) sequences representative of the single-copy genome of five species (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae, E. coli, and Mycobacterium leprae) have been analyzed. A close relationship was observed between genome size and the overall level of sequence repetition. This suggested that the incorporation of simple sequences had accompanied increases of genome size during evolution. Densities of simple sequence motifs were higher in noncoding regions than in coding regions in eukaryotes but not in eubacteria. All five genomes showed very biased frequency distributions of simple sequence motifs in all species, particularly in eukaryotes where AAA and TTT predominated. Interspecific comparisons showed that noncoding sequences in eukaryotes showed highly significantly similar frequency distributions of simple sequence motifs but this was not true of coding sequences. ANOVA of the frequency distributions of simple sequence motifs indicated strong contributions from motif base composition and repeat unit length, but much of the variation remained unexplained by these parameters. The sequence composition of simple sequences therefore appears to reflect both underlying sequence biases in slippage-like processes and the action of selection. Frequency distributions of simple sequence motifs in coding sequences correlated weakly or not at all with those in noncoding sequences. Selection on coding sequences to eliminate undesirable sequences may therefore have been strong, particularly in the human lineage.

Animals↗

"Cryptic" repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes.

Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are "cryptic" repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29-40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differentially distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. Our data suggest how simple tandem repeats could evolve from cryptic repeats.

Aged↗

RAPD-based screening of genomic libraries for positional cloning.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.

Ascomycota↗

Advances in the genetics of movement disorders: implications for molecular diagnosis.

Recent developments in molecular genetics have had a profound influence on the diagnosis and classification of inherited movement disorders. Huntington's disease is caused by the expansion of an unstable trinucleotide repeat sequence. Molecular diagnosis can now be performed by a simple PCR-based assay, and the study of the effects of the repeat expansion on the function of the encoded protein will allow to elucidate the molecular pathogenesis of the disease. Wilson's disease is caused by a large number of different mutations, which complicates molecular diagnosis. Genes for a number of inherited dystonic syndromes have been mapped, one of them, the gene for dopa-responsive dystonia, has already been identified. The genetic basis of several other prevalent movement disorders, such as essential tremor and the restless-legs syndrome however, is still obscure. Current research is also directed at the identification of inherited risk-factors in genetically complex movement disorders, such as Parkinson's disease.

Atrophy↗

Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene.

Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.

Amino Acid Sequence↗

The paradox of MHC-DRB exon/intron evolution: alpha-helix and beta-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with beta-sheet codons.

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)n(ga)m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)n(ga)m structure is fixed in evolution for 7 x 10(7) years whereas ample variations exist in the tandem (gt)n and (ga)m dinucleotides and especially their "degenerated" derivatives. Phylogenetic trees for the alpha-helices and beta-pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB beta-sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast alpha-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)n(ga)m repeat is discussed with respect to these genetic exchange mechanisms during evolution.

Alleles↗