An efficient cis-element discovery method using multiple sequence comparisons based on evolutionary relationships

The discovery of cis-element control motifs in noncoding DNA poses a difficult problem in genome analysis. Functional analysis by means of reporter constructs expressed in transgenic organisms is the most reliable method, but is by itself time-consuming and expensive. Searching noncoding DNA for known control motifs by sequence analysis is problematic, since protein binding motifs are short, in the range of 8-10 bp, and occur frequently by chance. Heretofore, the most reliable sequence analysis method has been the comparison of homologous sequence domains in related but moderately evolutionarily divergent species such as, for example, mouse and human. In such pairwise combinations, control regions are conserved because they serve a vital function and can be identified by their similar sequences. Single pairwise comparisons, however, allow the discovery of conserved sequence strings only at low resolution and without specific identity. We have investigated the possibility of using multiple sequence comparisons to correct these shortcomings. We applied this method to the Hoxc8 early enhancer region that has been previously analyzed in depth by functional methods and through its application successfully identified known protein binding cis-element motifs. Candidate protein binding sites could also be identified. This method, based on evolutionarily related sequence comparisons, should be quite useful as a prescreening step prior to functional analysis with corresponding savings in time and resources.     

Assessing wheat (Triticum aestivum) genotypes for "Yr" resistance genes using conserved regions and simple-sequence motifs

Analysis of DNA sequence variation among genotypes is useful for differentiation of wheat accessions, selection strategies and genetic development of crop plants. We screened molecular markers for yellow rust resistance genes (Yr7, Yr9, Yr15, Yr18, Yr26, and YrH52), which are in the gene-rich regions of wheat chromosomes 1B, 2B, and 7D, to investigate DNA sequence differences and repeat motifs and numbers between wheat cultivars resistant (Izgi2001, Sonmez2001, PI178383) and susceptible (Aytin98, ES14, Harmankaya99) to yellow rust. The F(2) individuals derived from the crosses were evaluated for yellow rust resistance at both the seedling and adult stages to identify DNA markers genetically linked to yellow rust resistance. The most repeated motif was found to be GA and the least repeated motif TAGA among the cultivars. When we examined DNA sequence differences (insertion, deletion and single nucleotide changes), the molecular markers Xgwm526 (Yr7) and Xgwm273 (YrH52) were found to have the most conserved regions and Yr15 (Xgwm413) the least conserved regions among the cultivars. This DNA sequence information can be used for selection of suitable parents, creating mapping populations and developing molecular markers associated with yellow rust resistance in plant breeding programs.     

New techniques for DNA sequence classification.

DNA sequence classification is the activity of determining whether or not an unlabeled sequence S belongs to an existing class C. This paper proposes two new techniques for DNA sequence classification. The first technique works by comparing the unlabeled sequence S with a group of active motifs discovered from the elements of C and by distinction with elements outside of C. The second technique generates and matches gapped fingerprints of S with elements of C. Experimental results obtained by running these algorithms on long and well conserved Alu sequences demonstrate the good performance of the presented methods compared with FASTA. When applied to less conserved and relatively short functional sites such as splice-junctions, a variation of the second technique combining fingerprinting with consensus sequence analysis gives better results than the current classifiers employing text compression and machine learning algorithms.     

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.     

The fragment transformation method to detect the protein structural motifs

To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the betabetaalpha-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the betabetaalpha-metal binding motif and the treble clef motif. The betabetaalpha-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions.     

Position-dependent motif characterization using non-negative matrix factorization

MOTIVATION: Cis-acting regulatory elements are frequently constrained by both sequence content and positioning relative to a functional site, such as a splice or polyadenylation site. We describe an approach to regulatory motif analysis based on non-negative matrix factorization (NMF). Whereas existing pattern recognition algorithms commonly focus primarily on sequence content, our method simultaneously characterizes both positioning and sequence content of putative motifs. RESULTS: Tests on artificially generated sequences show that NMF can faithfully reproduce both positioning and content of test motifs. We show how the variation of the residual sum of squares can be used to give a robust estimate of the number of motifs or patterns in a sequence set. Our analysis distinguishes multiple motifs with significant overlap in sequence content and/or positioning. Finally, we demonstrate the use of the NMF approach through characterization of biologically interesting datasets. Specifically, an analysis of mRNA 3'-processing (cleavage and polyadenylation) sites from a broad range of higher eukaryotes reveals a conserved core pattern of three elements.     

The divergence-homogenization duality in the evolution of the b1 mating type gene of Coprinus cinereus.

The A mating type locus of the fungus Coprinus cinereus is a complex, multigenic locus which regulates compatibility and subsequent sexual development. Genes within the A locus such as the b1 gene studied here exhibit extreme sequence variation. In this work, we asked how b1 alleles have evolved high levels of variation and, at the same time, conserved function. We compared sequence variation in 17 alleles characterized as belonging to seven different compatibility classes. Comparison of sequence variation between representatives of these seven classes shows that different regions of the b1 gene have been subject to varying levels of substitution, recombination, and structural/functional constraints. The N-terminal region of the encoded protein, which has been previously demonstrated to govern self/nonself recognition, exhibited hypervariability with levels of amino acid identity as low as 41%. We used a novel analysis of neutral mutations accumulating in this gene to rule out the possibility that the N-terminal region is hypermutable. In contrast, the C-terminal region displayed heterogeneous levels of variation, with functional motifs being better conserved. In fact, there is a duality in the b1 gene between variability and conservation; recombination events have homogenized the C-terminal region, while recombination events are undetectable in the N-terminal region. The ability to regulate sexual development is maintained in all of the mating compatibility alleles studied, and these data suggest that some functional motifs may tolerate high levels of substitution.     

Mutational bias in penguin microsatellite DNA

Analysis of nucleotide sequence variation at a microsatellite DNA locus revealed extensive size homoplasy of alleles in Adélie penguins (Pygoscelis adeliae). Variation in the flanking regions at this locus allowed discrimination between mechanisms proposed for length changes in microsatellite DNA alleles. We further examined the structure of alleles for the same microsatellite DNA locus across 11 additional species of penguin (Spheniscidae) by mapping allele sequences onto an independent penguin phylogeny. Our analysis indicated that the repeat motifs appear to have evolved independently on several occasions. We observed sequence instability in the region bordering the repeat tract with a transversional bias predominating. We propose that this bias results from inaccurate DNA replication owing to the sequence context of this repeat tract. Because we show that regions flanking repeat sequences exhibit this mutational bias, this cautions against the use of such regions for phylogeny reconstruction.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

Insights into recombination from population genetic variation

Patterns of genetic variation in natural populations are shaped by, and hence carry valuable information about, the underlying recombination process. In the past five years, the increasing availability of large-scale population genetic data on dense sets of markers, coupled with advances in statistical methods for extracting information from these data, have led to several important advances in our understanding of the recombination process in humans. These advances include the identification of large numbers of 'hotspots', where recombination appears to take place considerably more frequently than in the surrounding sequence, and the identification of DNA sequence motifs that are associated with the locations of these hotspots.     

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.)

The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymorphism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of alleles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic library-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic diversity was seen in GA-containing microsatellites of genomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential location of specific motifs in coding versus non-coding regions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatellite motif and the standard deviation of the molecular-weight of amplified fragments. The reliability of molecular weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed. Received: 5 May 1999 / Accepted: 16 August 1999     

Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models

The assessment of nucleotide polymorphisms in environmental samples of obligate pathogens requires DNA amplification through the polymerase chain reaction (PCR) and bacterial cloning of PCR products prior to sequencing. The drawback of this strategy is that it can give rise to false polymorphisms owing to DNA polymerase misincorporation during PCR or bacterial cloning. We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Phakopsora pachyrhizi, an obligate biotrophic fungus that causes the Asian soybean rust. Field-collected samples of P. pachyrhizi were obtained from all major soybean production areas worldwide, including Brazil and the United States. Bacterially-cloned, PCR products were obtained using a high fidelity DNA polymerase. A total of 370 ITS sequences that were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, the pattern of nucleotide polymorphisms, GC content, and the presence of conserved motifs. The sequences exhibited features of functional rRNAs. Overall, polymorphisms took place within less conserved motives, such as loops and bulges; alternatively, they gave rise to non-canonical G-U pairs within conserved regions of double stranded helices. We discuss the usefulness of structural analyses to filter out putative 'suspicious' bacterially cloned ITS sequences, thus keeping artificially-induced sequence variation to a minimum.     

Identification of the blast resistance gene Pit in rice cultivars using functional markers

DNA markers that allow for identification of resistance genes in rice germplasm have a great advantage in resistance breeding because they can assess the existence of the genes without laborious inoculation tests. Functional markers (FMs), which are designed from functional polymorphisms within the sequence of genes, are unaffected by nonfunctional allelic variation and make it possible to identify an individual gene. We previously showed that the resistance function of the rice blast resistance gene Pit in a resistant cultivar, K59, was mainly acquired by up-regulated promoter activity through the insertion of a long terminal repeat (LTR) retrotransposon upstream of Pit. Here, we developed PCR-based DNA markers derived from the LTR-retrotransposon sequence and used these markers to screen worldwide accessions of rice germplasm. We identified 5 cultivars with the LTR-retrotransposon insertion out of 68 rice accessions. The sequence and expression pattern of Pit in the five cultivars were the same as those in K59 and all showed Pit-mediated blast resistance. The results suggest that the functional Pit identified using the markers was derived from a common progenitor. Additionally, comparison of the Pit coding sequences between K59 and susceptible cultivars revealed that one nucleotide polymorphism, which caused an amino acid substitution, offered another target for a FM. These results indicate that our DNA markers should enhance prediction of Pit function and be applicable to a range of rice varieties/landraces cultivated in various regions worldwide and belonging to the temperate japonica, tropical japonica, and indica groups.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.