Characterization of the patterns of polymorphism in a "cryptic repeat" reveals a novel type of hypervariable sequence.

Alternating purine and pyrimidine repeats (RY(i)) are an abundant source of polymorphism. The subset with long tandem repeats of GT or AC (GT(i)) have been studied extensively, but cryptic RY(i) (i.e., no single tandem repeat predominates) have received little attention. The factor IX gene has a polymorphic cryptic RY(i) of 142-216 bp. Previously, there were four known polymorphic alleles, of the form AB, A2B, A2B2, and A3B2, where A = (GT)(AC)3(AT)3(GT)(AT)4 and B = A with an additional 3'' AT dinucleotide. To further characterize this locus, we examined more than 1,700 additional human chromosomes and determined the sequences of the homologous sites in orangutans and chimpanzees. The novel alleles found in humans expand the repertoire of A/B alleles to A0-4B1 and A1-3B2. The AnB2 series are abundant in Caucasians but are absent in blacks and Asians. Conversely, the A0B1 allele is common in blacks but is not found in more than 1,700 Caucasian chromosomes. The data are compatible with a model in which recombination is more frequent than polymerase slippage at this locus. In orangutans, the RY(i) is present, but the sequence is markedly different. An A/B-type of pattern was discerned in which B differs from A by an additional six (AT) dinucleotides at the 3'' end. In chimpanzees, the size of the RY(i) locus was greatly expanded, and the sequence showed a novel pattern of hypervariability in which there are many tandem repeats of the form (GT)n(AC)o(AT)p(GT)q(AT)s, where n, o, p, q, and s are different integers. The sequences of the factor IX intron 1 cryptic RY(i) in three primates provide perspective on the range of possible patterns of polymorphism. Analysis of the patterns suggests how the RY(i) can be conserved during evolution, while the precise sequence varies.     

Screening the V2R-type putative odorant receptor gene repertoire in bitterling Tanakia lanceolata

To study evolution of dinucleotide simple sequence repeats (diSSRs) we searched recently available mammalian genomes for UTR-localized diSSRs with conserved upstream flanking sequences (CFS). There were 252 reported Homo sapiens genes containing the repeats (AC)n, (GT)n, (AG)n or (CT)n in their UTRs including 22 (8.7%) with diSSR-upstream flanking sequences conserved comparing divergent mammalian lineages represented by Homo sapiens and the marsupial, Monodelphis domestica. Of these 22 genes, 19 had known functions including 18 (95%) that proved critical for mammalian nervous systems (Fishers exact test, P<0.0001). The remaining gene, Cd2ap, proved critical for development of kidney podocytes, cells that have multiple similarities to neurons. Gene functions included voltage and chloride channels, synapse-associated proteins, neurotransmitter receptors, axon and dendrite pathfinders, a NeuroD potentiator and other neuronal activities. Repeat length polymorphism was confirmed for 68% of CFS diSSRs even though these repeats were nestled among highly conserved sequences. This finding supports a hypothesis that SSR polymorphism has functional implications. A parallel study was performed on the self-complementary diSSRs (AT)n and (GC)n. When flanked by conserved sequences, the self-complementary diSSR (AT)n was also associated with genes expressed in the developing nervous system. Our findings implicate functional roles for diSSRs in nervous system development.     

Segments containing alternating purine and pyrimidine dinucleotides: patterns of polymorphism in humans and prevalence throughout phylogeny.

Tandem dinucleotide repeats of GT or AC [(GT)n/(AC)n] where n greater than or equal to 14 are highly polymorphic and other simple repeats such as (CT)n/(AG)n and (A)n(T)n are also polymorphic. The uniformity of these sequences precludes a mechanistic differentiation between recombination or polymerase slippage. Since (GT)n/(AC)n or (CT)n/(AG)n segments of desired size were not available in our gene of interest, we analyzed a 187+ bp segment in the factor IX gene with multiple short dinucleotide repeats. This sequence contains a melody of short dinucleotide repeats which includes a 142+ bp segment of alternating purines and pyrimidines. Amplification of this sequence in 167 individuals of different ethnicity and direct sequencing of 106 individuals (23 kb of sequence) failed to reveal simple variation in the number of tandem dinucleotide repeats. However, polymorphism in the 142+ alternating purine and pyrimidine segment (RY)n was detected due to the insertion of two related repeat units of 24 bp (A) and 26 bp (B). Two previously described alleles (AB, A2B2) and two novel presumptive recombinants were found (A2B, A3B2) for a total of four alleles. An analysis of (RY)n segments in GenBank revealed an extraordinary enrichment in the genome of mammals, invertebrates, and yeast and a marked reduction in bacteria. Rodent (RY)n were larger and substantially more frequent than those in primates. When a second (RY)n was examined in the exon 8 of human factor IX gene, it was polymorphic at short repeats of (GT)n/(AC)n (n = 3-6) in Western Europeans and Koreans. In addition, an (RY)n in the dystrophin gene had four polymorphic alleles involving AT and GT dinucleotides. Thus (RY)n segments appear to be abundant and highly polymorphic. The asymmetric patterns of polymorphism and the absence of simple dinucleotide variation in 23 kb of sequence are compatible with recombination or sister chromatid exchange, but not polymerase slippage. By inference, recombination should underlie the polymorphisms at (GT)n/(AC)n since they are a subset of (RY)n and they commonly occur in the context of longer (RY)n.     

Prediction of Recognition Sites for Genomic Replication of Classical Swine Fever Virus with Information Analysis

In order to explore the mechanism for the genomic replication of classical swine fever virus (CSFV), so as to make a basis for investigating its pathogenicity, an introduction of the information theory is presented in connection with the statistical mechanics, whence small-sample statistics appears naturally as a consequence of the Bayesian approach. Furthermore, a selection rule for identifying the pattern of a recognition site for an RNA-binding protein is proposed by means of the maximum entropy principle. Based on those, the information contents of 3"-untranslated regions (3"UTRs) of genomes of 20 CSFV strains and 5"-untranslated regions (5"UTRs) of genomes of 58 CSFV strains are analyzed with a computational algorithm in a reduction mode, and the 3"UTR sites of 20 strains and 5"UTR sites of 58 strains containing important motifs are extracted from the unaligned RNA sequences of unequal lengths. These sites, which have the patterns of sequence and structure similar to the putative cis elements related to the regulation of genomic replication, would be identified as the potential recognition sites in 3"UTRs and 5"UTRs for CSFV replicase responsible for classical swine fever virus genomic replication, and to some extent, this identification is supported by experimental evidence. Finally, information analysis allows a presumption to be made about the CSFV RNA replication initiation mechanism.     

The abundance of various polymorphic microsatellite motifs differs between plants and vertebrates.

The abundance of different simple sequence motifs in plants was accessed through data base searches of DNA sequences and quantitative hybridization with synthetic dinucleotide repeats. Database searches indicated that microsatellites are five times less abundant in the genomes of plants than in mammals. The most common plant repeat motif was AA/TT followed by AT/TA and CT/GA. This group comprised about 75% of all microsatellites with a length of more than 6 repeats. The GT/CA motif being the most abundant dinucleotide repeat in mammals was found to be considerably less frequent in plants. To address the question if plant simple repeat sequences are variable as in mammals, (GT)n and (CT)n microsatellites were isolated from B.napus. Five loci were investigated by PCR-analysis and amplified products were obtained for all microsatellites from B. oleracea, B.napus and B.rapa DNA, but only for one primer pair from B.nigra. Polymorphism was detected for all microsatellites.     

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats.

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.     

Short tandem repeats are associated with diverse mRNAs encoding membrane-targeted proteins

Within the genomes of multicellular organisms, short tandem repeating sequences (STRs) are ubiquitous, yet usage patterns remain obscure. The repeats (AC)n and (GU)n appear frequently in the untranslated regions (UTRs) of messenger RNAs (mRNAs). To investigate STR usage patterns, we used three approaches: (1) comparisons of individual mRNA database sequences including annotations and linked references, (2) statistical analysis of complete, UTR databases and (3) study of a large gene family, the aquaporins. Among 500 (AC)n- or (GU)n-containing mRNAs, 58 (12%) had known functions. Of these, 50 (86%) encoded proteins whose activities involved membranes or lipids, including integral membrane proteins, peripheral membrane proteins, ion channels, lipid enzymes, receptors and secreted proteins. A control sequence (AU)n also occurred in mRNAs, but only 5% encoded membrane-related functions. Investigation of all reported 3' UTR sequences, demonstrated that the STR (AC)n was 9 times more common in mRNAs encoding membrane functions than in the total UTR database (P < 0.001). Similarly, (GU)n was 8 times more common in membrane-function mRNAs than in the total database (P < 0.001). These observations suggest that (AC)n and (GU)n may be UTR signals for some mRNAs encoding membrane-targeted proteins.     

Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)n.(dG-dT)n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)n.(dG-dT)n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)n.(dG-dT)n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)n.(dG-dT)n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)n.(dG-dT)n polymorphisms regardless of the repeat sequence category.     

红原鸡全基因组中微卫星分布规律研究

本文对红原鸡Gallus gallus全基因组中微卫星数量及分布规律进行了分析,查找到l～6个碱基重复类型的微卫星序列共282728个,约占全基因组序列(1.1Gb)的0.49%,分布频率为1/3.89kb,微卫星序列的长度主要在12～70个碱基长度范围内。第1、2、3条染色体上微卫星分布频率较高,而32号染色体上无微卫星分布。不同类型微卫星中,单碱基重复类型数目最多,为184192个,占总数的65.1%;其次是四、二、三、五、六碱基重复单元序列,分别占到总数的12.8%、9.7%、7.2%、4.6%、0.8%。T、A、AT、GTTT、AAAC、G、C、ATTT、AC、GT、AAAT、ATT、AAC、AAT、GTT、AG、CT、CTTT、AAAG、GTTTT、AAACA、AAGG、CCTT是红原鸡基因组中最主要的微卫星重复类型。本研究为红原鸡微卫星标记的分离筛选、遗传多样性的研究以及不同物种微卫星的比较分析奠定了基础。     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

Survey of human and rat microsatellites

Length variations in simple sequence tandem repeats (microsatellite DNA polymorphisms) are finding increasing usage in mammalian genetics. Although every variety of short tandem repeat that has been tested has been shown to exhibit length polymorphisms, little information on the relative abundance of the different repeat motifs has been collected. In this report, summaries of GenBank searches for all possible human and rat microsatellites ranging from mononucleotide to tetranucleotide repeats are presented. In humans, the five most abundant microsatellites with total lengths for the runs of repeats of greater than or equal to 20 nucleotides contained repeat sequences of A, AC, AAAN, AAN, and AG, in order of decreasing abundance, where N is C, G, or T. These five groups comprised about 76% of all microsatellites. Many other human simple sequence repeats were found at low frequency. In the 745 kb of human genomic DNA surveyed, one microsatellite of greater than or equal to 20 nucleotides in length was found, on average, every 6 kb. Only 12% of the human microsatellites had total lengths greater than or equal to 40 nucleotides. Roughly 80% of the A, AAN, and AAAN microsatellites and 50% of the AT microsatellites, but few of the other human microsatellites, were found to be associated with interspersed, repetitive Alu elements. In rats, the five most abundant microsatellites contained AC, AG, A, AAAN, and AAGG sequences, respectively. Rat microsatellites were generally longer than human microsatellites, with 43% of the rat sequences greater than or equal to 40 nucleotides.     

Informativeness of dinucleotide repeat-based primers in fungal pathogen of rice Magnaporthe grisea

Microsatellites or simple sequence repeats (SSRs) occur ubiquitously and show complex patterns in length, motif size and sequence. Among SSRs, dinucleotide repeats occur in high abundance in fungi with shorter length as compared to other organisms. In this study, multilocus profiles obtained in Magnaporthe grisea, a model plant pathogen were evaluated. The results showed lower rate of polymorphism by (GT)(n)/(TG)(n) repeat-based primers and suggested occurrence of (GA)(n)/(AG)(n) repeats as integral repeats and (TC)(n)/(CT)(n) and (AC)(n)/(CA)(n) as non-integral repeats. Low repeat length variation was found to be correlated with less number of repeat motifs. The study provides an insight into the possibility of molecular coevolution of mobile elements and dinucleotide repeats in fungi. The study could be applied to other species for wider applications including evolutionary and population genetics.     

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3' untranslated regions (3' UTRs) of target mRNAs. In yeast, Puf3p binds the 3' UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3' UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3' UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3' UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.     

The bovine genome contains polymorphic microsatellites

Dinucleotide repeats constitute so-called microsatellites of the human and other eukaryotic genomes. Microsatellite polymorphisms can be identified through the amplification of the microsatellite DNA using the polymerase chain reaction (PCR), followed by resolution of the amplified DNA fragments on a polyacrylamide sequencing gel. We performed a preliminary sequence database search to identify bovine sequences containing (CA)n, (AC)n, (GT)n, or (TG)n blocks, with n greater than or equal to 6. This search yielded 10 sequences containing one or two of the specified repeat blocks and often additional dinucleotide repeat blocks. One of the microsatellite-containing regions has been sequenced twice from independent clones and the reported sequences showed variation in the number of repeats. PCR-amplified fragments of another sequence, the gene for steroid 21-hydroxylase, ranged from 186 to 216 nucleotides in 43 unrelated animals. The database search, as well as the hypervariable microsatellite in the bovine steroid 21-hydroxylase gene, indicates that dinucleotide blocks may be an abundant source of DNA polymorphism in cattle.     

A microsatellite polymorphism in intron 2 of human Toll-like receptor 2 gene: functional implications and racial differences

The human Toll-like receptor 2 (TLR2) mediates responses of both innate and adaptive immunity to Gram-positive bacteria, including mycobacteria. We sought functional polymorphisms in the 5'-untranslated region (UTR) of TLR2. We found a highly polymorphic (GT)n dinucleotide repeat 100 bp upstream of the TLR2 translational start site in intron 2. The numbers of GT repeats varied from 12 to 28. There were significant differences in allele distribution between African Americans and Caucasians (P=0.008) and between African Americans and Koreans (P=0.0003). The promoter activities of recombinant promoter-intron2/reporter constructs including the shortest [GT)n=12] or longest [(GT)n=28] alleles were significantly more stimulated when exposed to 200 IU ml(-1) of interferon-gamma than when exposed to 100 IU ml(-1) of GM-CSF (P<==0.03). Since TLR2 plays a critical role in the human innate immune response, this functional microsatellite polymorphism may be important in the pathogenesis of infectious and inflammatory diseases.     

Evolution of alu family repeats since the divergence of human and chimpanzee

Summary The DNA sequences of three members of the Alu family of repeated sequences located 5 to the chimpanzee 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.