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Nine multiplex schizophrenia families were genotyped with seven highly polymorphic microsatellite loci used in the construction of an index map of chromosome 21. Assuming either autosomal dominant or recessive transmission, evidence of linkage was not found. In addition, the nonparametric sib-pair test did not yield significant evidence of linkage.
BACKGROUND: Cultivated strawberry (Fragaria x ananassa) represents one of the most valued fruit crops in the United States. Despite its economic importance, the octoploid genome presents a formidable barrier to efficient study of genome structure and molecular mechanisms that underlie agriculturally-relevant traits. Many potentially fruitful research avenues, especially large-scale gene expression surveys and development of molecular genetic markers have been limited by a lack of sequence information in public databases. As a first step to remedy this discrepancy a cDNA library has been developed from salicylate-treated, whole-plant tissues and over 1800 expressed sequence tags (EST's) have been sequenced and analyzed. RESULTS: A putative unigene set of 1304 sequences--133 contigs and 1171 singlets--has been developed, and the transcripts have been functionally annotated. Homology searches indicate that 89.5% of sequences share significant similarity to known/putative proteins or Rosaceae ESTs. The ESTs have been functionally characterized and genes relevant to specific physiological processes of economic importance have been identified. A set of tools useful for SSR development and mapping is presented. CONCLUSION: Sequences derived from this effort may be used to speed gene discovery efforts in Fragaria and the Rosaceae in general and also open avenues of comparative mapping. This report represents a first step in expanding molecular-genetic analyses in strawberry and demonstrates how computational tools can be used to optimally mine a large body of useful information from a relatively small data set.
We recently demonstrated that insulin growth factor-I (IGF-I) cosegregates with bone mineral density (BMD) in progenitor crosses of two inbred strains of mice. Additionally, we reported that men with idiopathic osteoporosis (IOM) have low serum IGF-I levels, which can be related to BMD and bone turnover. In this study, we considered the possibility that serum IGF-I levels are influenced by molecular genetic variation in the IGF-I structural gene, and that a polymorphic microsatellite (CA repeat) in this locus can be used as a genetic marker for such comparisons. We studied 171 men and women, classified according to the trial in which they were participating. First, in 25 Caucasian men with IOM we noted a very high frequency (64%) of homozygosity for the most common allele (192 bp) in a dinucleotide repeat 1 kb upstream from the transcription start site of the IGF-I gene. This compared with a frequency of only 32% in healthy populations (both men and women) (P < 0.004). Next, we determined that for 116 healthy Caucasian men and women the 192/192 genotype was associated with lower serum IGF-I levels than all other genotypes (192/192: 129 +/- 7 ng/mL vs. others: 154 +/- 7 ng/mL, P = 0.03). We also noted that subjects possessing one 194-bp allele exhibited serum IGF-I levels 25% higher than those homozygous for 192 bp (192/192), (P < 0.005) even after correction for age and sex. Similarly, for men with the 192/192 genotype, serum IGF-I concentrations were lower than any other genotype (145 +/- 10 ng/mL vs. 183 +/- 9 ng/ml P < 0.02). In conclusion, low serum IGF-I levels found in men with IOM are associated with homozygosity for a specific allele of the IGF-I microsatellite (192/192), and individual variation in serum IGF-I levels is influenced by genetic factors and may be specifically influenced by variation at the IGF-I structural locus. Further family and pedigree studies are needed to characterize the relationship of bone mass acquisition to the IGF-I genotype.
Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n > or = 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.
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This report describes a new polymorphism, in intron 3 of the p53 gene, which consists of a single repeat of 16 nucleotides, absent in the published wild-type p53 gene sequence. In the Caucasian population tested (n = 82), 28% of individuals were heterozygotes for this polymorphism. Using PCR-based analysis, we were able to demonstrate p53 allelic losses in three of six breast tumors from heterozygote patients tested.
The identification of the gene Pp523, conferring downy mildew resistance to adult plants of broccoli (Brassica oleracea convar. italica), led to the construction of a genetic map that included this resistance locus, 301 amplified fragment length polymorphisms, 55 random amplified polymorphic DNAs, 46 inter-simple sequence repeats, three simple sequence repeats, four other PCR markers and a flower colour locus, all gathered into nine major linkage groups. Nineteen additional molecular markers were clustered into one group of four markers, one group of three markers and six pairs of markers. The map spans over 731.9 cM, corresponding to 89.5% of the 818 cM estimated to be the total genome length. A significant number of the mapped markers, 19.3%, showed distorted segregation. The average distance between mapped adjacent markers is 1.64 cM, which places this map among the densest published to date for this species. Using bulked segregant analysis, we identified a group of molecular markers flanking and closely linked in coupling to the resistance gene and included these in the map. Two markers linked in coupling, OPK17_980 and AT.CTA_133/134, are located at 3.1 cM and 3.6 cM, respectively, at each side from the resistance gene. These markers can be used for marker-assisted selection in breeding programs aiming at the introgression of this gene in susceptible B. oleracea genotypes. The fine mapping of the genomic region surrounding the Pp523 resistance gene is currently being carried out, a basic condition for its isolation via positional cloning.
A new linkage map of Cucumis melo, derived from the F2 progeny of a cross between PI 414723 and C. melo 'TopMark' is presented. The map spans a total of 1421 cM and includes 179 points consisting of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), simple sequence repeats (SSRs), and restriction fragment length polymorphism (RFLP) markers. The map also includes an aphid resistance trait (Vat) and the sex type gene, andromonoecious (a), the two of which are important in resistance breeding and the control of hybrid seed production, as well as a seed-color gene, Wt-2. Most RFLPs represent sequence-characterized cDNA probes from C. melo and Cucumis sativus. These include resistance gene homologues and genes involved in various aspects of plant development and metabolism. A sub-set of our SSR and RFLP markers were also mapped, as part of this study, on additional mapping populations that were published for this species. This provides important reference points ("anchors"), enabling us to identify several linkage groups with respect to other melon maps.
Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multi-chromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)n (8 Mb), (AAGAG)n (7 Mb) and (AATAT)n (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin.
Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.
Three Drosophila melanogaster satellite DNAs (1.672, 1.686, and 1.705 g/ml in CsCl), each containing a simple sequence repeated in tandem, were cloned in pBR322 as small fragments about 500 base pairs long. This precaution minimized deletions, since inserts of the same size as the fragments used for cloning were recovered in a stable form. A homogeneous tandem array of one sequence type usually extended the length of the insert. Eleven distinct repeat sequences were discovered, but only one sequence was predominant in each satellite preparation. The remaining classes were minor in amount. The repeat unit lengths were restricted to 5, 7, or 10 base pairs, with sequences closely related. Each sequence conforms to the expression (RRN)m(RN)n, where R is A or G. The multiplicity of simple repeated sequences revealed despite the small sample size suggests that numerous repeat sequences reside in heterochromatin and that particular rules apply to the structure of the repeating sequence.
A total of 568 new simple sequence repeat (SSR)-based markers for barley have been developed from a combination of database sequences and small insert genomic libraries enriched for a range of short simple sequence repeats. Analysis of the SSRs on 16 barley cultivars revealed variable levels of informativeness but no obvious correlation was found with SSR repeat length, motif type, or map position. Of the 568 SSRs developed, 242 were genetically mapped, 216 with 37 previously published SSRs in a single doubled-haploid population derived from the F(1) of an interspecific cross between the cultivar Lina and Hordeum spontaneum Canada Park and 26 SSRs in two other mapping populations. A total of 27 SSRs amplified multiple loci. Centromeric clustering of markers was observed in the main mapping population; however, the clustering severity was reduced in intraspecific crosses, supporting the notion that the observed marker distribution was largely a genetical effect. The mapped SSRs provide a framework for rapidly assigning chromosomal designations and polarity in future mapping programs in barley and a convenient alternative to RFLP for aligning information derived from different populations. A list of the 242 primer pairs that amplify mapped SSRs from total barley genomic DNA is presented.
The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.
The dipteran Chironomus tentans has complex tandemly repeated 350-bp DNA sequences at or near the chromosome ends. As in Drosophila melanogaster, short simple repeats with cytosines and guanines in different strands have never been observed. We were therefore interested in learning whether the Chironomus repeats could have evolved from simple sequence telomeric DNA, which might suggest that they constitute a functional equivalent. We screened for repeat units with evolutionarily ancient features within the tandem arrays and recovered two clones with a less-evolved structure. Sequence analysis reveals that the present-day 350-bp unit probably evolved from a simpler 165-bp unit through the acquisition of transposed sequences. The 165-bp unit contains DNA with a highly biased distribution of cytosine and guanine between the two strands, although with the ratios inverted in two minor parts of the repeat. It is largely built up of short degenerate subrepeats for which most of the sequence can be reconstructed. The consensus for the subrepeat sequence is similar to the simple telomeric repeat sequences of several kinds of eukaryotes. We propose that the present-day unit has evolved from telomeric, simple sequence, asymmetric DNA from which it has retained some original sequence features and possibly functions.
Microsatellites, also called simple sequence repeats (SSR), are simple, tandemly repeated DNA sequences with a repeat length of a few base pairs,and are very ideally used as molecular markers because of their abundance, high level of polymorphism, co-dominance and ease of assay with the polymerase chain reaction (PCR) by selecting primers as the conserved DNA sequences flanking the SSRs,as well as better stability. The experiments showed that SSRs are randomly distributed throughout the barley genome,and there are 3-18 alleles at a single SSR locus,up to 37 alleles/locus. SSR markers have being widely applied in the construction of molecular genetic map, the study of genetic diversity,the identification of germplasm, gene mapping for important traits and molecular marker-assisted selection. Meanwhile,most of markers are strongly clustered around the centromeric regions of all seven linkage groups. As a result of the clustering,genome coverage with SSRs remains incomplete with an obvious lack of markers on the long arms of chromosomes 1H and 5H and short arm of chromosome 6H. Therefore,it is very potential and necessary to further develop SSR markers in barley.
The lengths of simple repeat sequences are generally unstable or polymorphic; that is, they are highly variable with respect to the numbers of tandem repeats. To determine the structural features that cause such variability, we examined a minisatellite DNA isolated from the yellow fin sea bream Acanthopagrus latus. Electrophoresis, CD spectra, and calorimetric analyses of oligodeoxyribonucleotides comprised of the minisatellite repeat unit suggest a stable secondary structure is formed within the repeat unit.