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PubMed2005

Sister chromatids are often incompletely aligned in meristematic and endopolyploid interphase nuclei of Arabidopsis thaliana.

We analyzed whether sister chromatids are continuously aligned in meristematic and endopolyploid Arabidopsis interphase nuclei by studying sister-chromatid alignment at various chromosomal positions. FISH with individual BACs to flow-sorted 4C root and leaf nuclei frequently yielded more than two hybridization signals, indicating incomplete or absent sister-chromatid alignment. Up to 100% of 8C, 16C, and 32C nuclei showed no sister-chromatid alignment at defined positions. Simultaneous FISH with BACs from different chromosomal positions revealed more frequent sister-chromatid alignment in terminal than in midarm positions. Centromeric positions were mainly aligned up to a ploidy level of 16C but became separated or dispersed in 32C nuclei. DNA hypomethylation (of the whole genome) and transcriptional activity (at FWA gene position) did not impair sister-chromatid alignment. Only 6.1% of 4C leaf nuclei showed sister-chromatid separation of the entire chromosome 1 top arm territories. Homozygous transgenic tandem repeat (lac operator) arrays showing somatic homologous pairing more often than average euchromatic loci did not promote an increased frequency of sister-chromatid alignment. The high frequency of separated sister-chromatid arm positions in > or =4C nuclei suggests that sister-chromatid cohesion is variable, dynamic, and not obligatory along the entire chromosome arm in meristematic and differentiated Arabidopsis nuclei.

Arabidopsis↗
PubMed1997

Zooming in on the human-mouse comparative map: genome conservation re-examined on a high-resolution scale.

Over the past decade, conservation of genetic linkage groups has been shown in mammals and used to great advantage, fueling significant exchanges of gene mapping and functional information especially between the genomes of humans and mice. As human physical maps increase in resolution from chromosome bands to nucleotide sequence, comparative alignments of mouse and human regions have revealed striking similarities and surprising differences between the genomes of these two best-mapped mammalian species. Whereas, at present, very few mouse and human regions have been compared on the physical level, existing studies provide intriguing insights to genome evolution, including the observation of recent duplications and deletions of genes that may play significant roles in defining some of the biological differences between the two species. Although high-resolution conserved marker-based maps are currently available only for human and mouse, a variety of new methods and resources are speeding the development of comparative maps of additional organisms. These advances mark the first step toward establishment of the human genome as a reference map for vertebrate species, providing evolutionary and functional annotation to human sequence and vast new resources for genetic analysis of a variety of commercially, medically, and ecologically important animal models.

Animals↗
PubMed2005

Haspin: a mitotic histone kinase required for metaphase chromosome alignment.

The fidelity of chromosome segregation during cell division is critical to maintain genomic stability and to prevent cancer and birth defects. A key set of kinases that regulates this process has been identified and characterized over the last few years, including the Aurora, Polo and Nek families. Recently we proposed that a little-studied kinase known as haspin is a new member of this important group. During mitosis haspin is phosphorylated, associates with the chromosomes, centrosomes and spindle, and is responsible for phosphorylation of histone H3 at threonine-3. Depletion of haspin using RNA interference prevents normal alignment of chromosomes at metaphase, suggesting that haspin plays a crucial role in chromosome segregation. Here we discuss possible mechanisms of haspin action and the function of histone phosphorylation in mitosis. We also outline some of the questions raised by these new findings and consider what role haspin might play in cancer.

Amino Acid Sequence↗
PubMed2006

Genome-wide analysis of cyclin family in rice (Oryza Sativa L.).

The cyclins together with highly conserved cyclin-dependent kinases regulate cell cycle progression in plants. Although extensive and systematic study on cell cycle mechanisms and cyclin functions in yeasts and animals has been carried out, only a small number of plant cyclins have been characterized and classified functionally and phylogenetically. We identified several types of cyclin genes in the rice genome and characterized them by phylogenetic, tandem and segmental duplications analyses. Our results indicated that there were at least 49 predicted rice cyclin genes in the rice genome, and they were distributed on 12 chromosomes. Of these cyclins, one possessed only cyclin_C domain and no cyclin_N domain, and the remaining 48 cyclins with cyclin_N domains were classified as nine types based on evolutionary relationships. Eight of these nine types were common between rice and Arabidopsis, whereas only one, known as F-type cyclins, was unique to rice. No homologues of the F-type cyclins in plants could be retrieved from the public databases, and reverse transcription-PCR analysis supported an existence of the F-type cyclin genes. Sequence alignment suggested that the cyclin genes in the rice genome experienced a mass of gene tandem and segmental duplications occurred on seven chromosomes related to the origins of new cyclin genes. Our study provided an opportunity to facilitate assessment and classification of new members, serving as a guide for further functional elucidation of rice cyclins.

Arabidopsis Proteins↗
PubMed2002

Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology.

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.

Amino Acid Sequence↗
PubMed1998

Highly specific protein sequence motifs for genome analysis.

We present a method for discovering conserved sequence motifs from families of aligned protein sequences. The method has been implemented as a computer program called EMOTIF (http://motif. stanford.edu/emotif). Given an aligned set of protein sequences, EMOTIF generates a set of motifs with a wide range of specificities and sensitivities. EMOTIF also can generate motifs that describe possible subfamilies of a protein superfamily. A disjunction of such motifs often can represent the entire superfamily with high specificity and sensitivity. We have used EMOTIF to generate sets of motifs from all 7,000 protein alignments in the BLOCKS and PRINTS databases. The resulting database, called IDENTIFY (http://motif. stanford.edu/identify), contains more than 50,000 motifs. For each alignment, the database contains several motifs having a probability of matching a false positive that range from 10(-10) to 10(-5). Highly specific motifs are well suited for searching entire proteomes, while generating very few false predictions. IDENTIFY assigns biological functions to 25-30% of all proteins encoded by the Saccharomyces cerevisiae genome and by several bacterial genomes. In particular, IDENTIFY assigned functions to 172 of proteins of unknown function in the yeast genome.

Amino Acid Sequence↗
PubMed2026

Exploring differences across pangenome-graph representations using Escherichia coli O157:H7 as a model.

Pangenome graphs are increasingly used to represent population-scale bacterial diversity, yet construction methods span fundamentally different representation paradigms whose outputs and sensitivities to assembly quality remain poorly quantified. We systematically reviewed microbial pangenome graph tools and benchmarked seven representative methods spanning gene-cluster, compacted coloured de Bruijn graph, one hybrid approach and one multiple sequence alignment method. Using a repeat-rich Escherichia coli O157:H7 dataset with complete genomes and matched short-read data, we constructed graphs from identical inputs and observed orders-of-magnitude differences in graph size and fragmentation, indicating that global topology is driven by representation strategy. Varying completeness composition revealed that assembly fragmentation is a first-order determinant of graph structure: gene-cluster graphs contracted as draft assemblies replaced complete genomes, whereas compacted coloured de Bruijn graphs expanded, with distinct degree-prevalence fingerprints across tools. In contrast, the multiple sequence alignment method could not be evaluated across fragmented inputs because it did not run reliably on draft-assembly datasets. Computational cost mirrored these shifts and depended strongly on completeness composition, including a pronounced runtime penalty for one compacted coloured de Bruijn graph implementation on all-draft inputs. Finally, analysis of Shiga toxin loci showed that pangenome-level reconciliation by gene-cluster-based tools does not reliably correct assembly artefacts at challenging multi-copy genes and that performance varies by locus. Together, these findings show that pangenome graphs are representation-dependent models of bacterial diversity, and that, in this repeat-rich O157:H7 benchmark dataset, assembly completeness is a primary determinant of their topology, scalability, and locus-level accuracy.

Escherichia coli O157↗
PubMed2006

GeMprospector--online design of cross-species genetic marker candidates in legumes and grasses.

The web program GeMprospector (URL: http://cgi-www.daimi.au.dk/cgi-chili/GeMprospector/main) allows users to automatically design large sets of cross-species genetic marker candidates targeting either legumes or grasses. The user uploads a collection of ESTs from one or more legume or grass species, and they are compared with a database of clusters of homologous EST and genomic sequences from other legumes or grasses, respectively. Multiple sequence alignments between submitted ESTs and their homologues in the appropriate database form the basis of automated PCR primer design in conserved exons such that each primer set amplifies an intron. The only user input is a collection of ESTs, not necessarily from more than one species, and GeMprospector can boost the potential of such an EST collection by combining it with a large database to produce cross-species genetic marker candidates for legumes or grasses.

Base Sequence↗
PubMed2001

Novel sigmaF-dependent genes of Escherichia coli found using a specified promoter consensus.

Availability of whole genome information opens new bioinformatics approaches to study global regulation. We developed a program, named ScanProm, that allows to search a genome database for promoter consensus elements. The program uses a multiple alignment of previously identified components of a regulon as an input and generates a consensus profile. The profile is then optimized by adjusting the cutoff value for position-specific similarity assessment and used for a genome scan to search for unknown members of the regulon. The candidates obtained are scored by their similarity to the consensus profile. The ScanProm program was applied to search for novel members of the class III flagellar regulon of Escherichia coli. The search template included the previously defined 4 bp (-35) and 8 bp (-10) promoter elements, presumably recognized by the flagellar-specific sigmaF, with additional 4 bp at the 3' of the -35 consensus. The majority of highly scoring candidates obtained from the whole genome sequence scan were known class III genes, although several new genes were also identified. We tested 10 novel highly scoring candidate class III genes by cloning their promoter fragments into a fusion vector designed to monitor the transcriptional activity with lacZ. Two of these genes, b2737(ygbK) and ppdAB, were found to be dependent on FlhDC, the master regulator of the flagellar genes. The regulation of these genes by sigmaF was further confirmed by comparing their expression in the wild-type and fliA backgrounds. An overproduction or inactivation of these genes did not exhibit any notable phenotypes in motility or chemotaxis.

Base Sequence↗
PubMed2004

Space-conserving optimal DNA-protein alignment.

DNA-protein alignment algorithms can be used to discover coding sequences in a genomic sequence, if the corresponding protein derivatives are known. They can also be used to identify potential coding sequences of a newly sequenced genome, by using proteins from related species. Previously known algorithms either solve a simplified formulation, or sacrifice optimality to achieve practical implementation. In this paper, we present a comprehensive formulation of the DNA-protein alignment problem, and an algorithm to compute the optimal alignment in O(mn) time using only four tables of size (m + 1) x (n + 1), where m and n are the lengths of the DNA and protein sequences, respectively. We also developed a Protein and DNA Alignment program PanDA that implements the proposed solution. Experimental results indicate that our algorithm produces high quality alignments.

Algorithms↗
PubMed2005

Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses.

Sequences corresponding to the 7.7K open-reading frame (ORF) of the E3 region of subspecies B1 adenoviruses (Ads) were compared with prototype strains of Ad3, Ad7, Ad16, Ad21, and Ad50 and field isolates representing a variety of genome restriction types of Ad3 and Ad7 to better assess the extent of genetic variation in this intriguing region of the viral genome encoding a product whose function is still unknown. Alignment of 55 species B1 Ad sequences revealed a marked polymorphism in the 7.7K ORF and allowed the identification of eight distinct sequence profiles (SPs) characterized by (1) deletions that retain or change the reading frame, (2) single-base mutations (SBMs) that change the start codon (ATG to ATT or ATC), and (3) other SBMs. mRNAs of expected size for the observed sequence polymorphisms were identified by RT-PCR from DNAse I-treated total RNA extracts of infected cells. Predicted proteins ranged from 0 to 94 amino acids corresponding to molecular masses of 0-11 K. Together with the hypervariable regions of the hexon gene, the E3 7.7K ORF appears to be another area of the Ad genome in which genetic diversity may be generated by illegitimate recombination.

Adenovirus E3 Proteins↗
PubMed2006

Strong regional biases in nucleotide substitution in the chicken genome.

Interspersed repeats have emerged as a valuable tool for studying neutral patterns of molecular evolution. Here we analyze variation in the rate and pattern of nucleotide substitution across all autosomes in the chicken genome by comparing the present-day CR1 repeat sequences with their ancestral copies and reconstructing nucleotide substitutions with a maximum likelihood model. The results shed light on the origin and evolution of large-scale heterogeneity in GC content found in the genomes of birds and mammals--the isochore structure. In contrast to mammals, where GC content is becoming homogenized, heterogeneity in GC content is being reinforced in the chicken genome. This is also supported by patterns of substitution inferred from alignments of introns in chicken, turkey, and quail. Analysis of individual substitution frequencies is consistent with the biased gene conversion (BGC) model of isochore evolution, and it is likely that patterns of evolution in the chicken genome closely resemble those in the ancestral amniote genome, when it is inferred that isochores originated. Microchromosomes and distal regions of macrochromosomes are found to have elevated substitution rates and a more GC-biased pattern of nucleotide substitution. This can largely be accounted for by a strong correlation between GC content and the rate and pattern of substitution. The results suggest that an interaction between increased mutability at CpG motifs and fixation biases due to BGC could explain increased levels of divergence in GC-rich regions.

Alu Elements↗
PubMed1995

Cloning and chromosomal localization of a pseudogene corresponding to a mRNA for a soluble IL-6 receptor.

A polymerase chain reaction assay (Lust J.A. et al. (1992). Cytokine 4:96-100) was used to detect a mRNA coding for a soluble IL-6 receptor in human hepatoma cells. In addition to the expected amplification product, we found a sequence (SR4) which could be aligned to it with 78% identity. After cloning and sequencing a genomic 2.5-2.7 kB EcoRI fragment containing SR4, this sequence turned out to be part of a pseudogene corresponding to the transmembrane domain deleted soluble IL-6 receptor. Screening of a panel of interspecies hybrids revealed that it maps to chromosome 9.

Antigens, CD↗
PubMed1989

Physical mapping of the Myxococcus xanthus genome by random cloning in yeast artificial chromosomes.

Random segments of Myxococcus xanthus DNA were cloned in yeast artificial chromosomes (YACs) to construct a physical map of the genome. EcoRI restriction maps of 409 YAC clones with inserts averaging 111 kilobase pairs (kb) were determined. Comparison to the map of a 300-kb region of M. xanthus obtained from clones in Escherichia coli indicates that segments of DNA cloned in YACs are stably maintained in yeast and that their sequences accurately reflect the structure of the Myxococcus genome. The 409 YAC inserts were ordered within 60 map segments (contigs) by aligning their EcoRI restriction maps and by hybridization with 18 gene-specific DNA probes. These 60 map segments may represent the entire Myxococcus genome and could be used to organize its genetic information. This study illustrates the utility of YACs for cloning large segments of DNA and for reliable long-range genomic mapping.

Chromosome Mapping↗
PubMed2005

Analysis of Acorus calamus chloroplast genome and its phylogenetic implications.

Determining the phylogenetic relationships among the major lines of angiosperms is a long-standing problem, yet the uncertainty as to the phylogenetic affinity of these lines persists. While a number of studies have suggested that the ANITA (Amborella-Nymphaeales-Illiciales-Trimeniales-Aristolochiales) grade is basal within angiosperms, studies of complete chloroplast genome sequences also suggested an alternative tree, wherein the line leading to the grasses branches first among the angiosperms. To improve taxon sampling in the existing chloroplast genome data, we sequenced the chloroplast genome of the monocot Acorus calamus. We generated a concatenated alignment (89,436 positions for 15 taxa), encompassing almost all sequences usable for phylogeny reconstruction within spermatophytes. The data still contain support for both the ANITA-basal and grasses-basal hypotheses. Using simulations we can show that were the ANITA-basal hypothesis true, parsimony (and distance-based methods with many models) would be expected to fail to recover it. The self-evident explanation for this failure appears to be a long-branch attraction (LBA) between the clade of grasses and the out-group. However, this LBA cannot explain the discrepancies observed between tree topology recovered using the maximum likelihood (ML) method and the topologies recovered using the parsimony and distance-based methods when grasses are deleted. Furthermore, the fact that neither maximum parsimony nor distance methods consistently recover the ML tree, when according to the simulations they would be expected to, when the out-group (Pinus) is deleted, suggests that either the generating tree is not correct or the best symmetric model is misspecified (or both). We demonstrate that the tree recovered under ML is extremely sensitive to model specification and that the best symmetric model is misspecified. Hence, we remain agnostic regarding phylogenetic relationships among basal angiosperm lineages.

Acorus↗
PubMed2004

A new approach that allows identification of intron-split peptides from mass spectrometric data in genomic databases.

We present a new approach that allows the identification of intron-split peptides from mass spectrometric data in genomic databases. Our algorithm uses small regions of peptide sequence information which are automatically deduced from de novo amino acid sequence predictions together with the molecular mass information of the precursor ion. The sequence predictions are based on selected collision-induced mass spectrometric fragmentation spectra. Fragments of the predicted amino acid sequence are aligned with each of the six frames of the translated genome and the precursor mass information is used to assemble the corresponding tryptic peptides using the sequence as a matrix. Hereby, intron-split peptides can be gathered and in turn verified by mass spectrometric data interpretation tools such as Sequest.

Algorithms↗
PubMed1992

Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA-directed oligonucleotide probes.

Examination of a number of previously published aligned Borrelia 16S rRNA sequences revealed the presence of regions which could serve as oligonucleotide probe targets for both species-specific identification of Borrelia burgdorferi and distinction between genomic groups. Total cellular RNA isolated from Borrelia cultures was used in slot blot analysis. Radiolabeled oligonucleotides designed to hybridize to specific 16S rRNA targets were used as probes. These probes allowed for both species-specific identification and genomic group typing of B. burgdorferi.

Bacterial Typing Techniques↗
PubMed2002

Estimation of genetic distances from human and mouse introns.

BACKGROUND: Using genetic distances measured from exons, it has been observed that the mutation rate is not constant along mammalian chromosomes. Exons constitute only 1% of the human genome, however, and thus they cannot provide a complete picture of the mutational variation in the genome. RESULTS: I calculated genetic distances between 504 human introns and their orthologous mouse counterparts from a set of 63 pairs of human and mouse genes scattered through the genome using a recently developed method that can extract reliably aligned regions from the introns in an objective manner. I found a significant correlation between the genetic distance measured in the conserved intron segments and the synonymous and nonsynonymous distances measured in the corresponding coding exons, indicating that genes with fast-evolving exons tend to have fast-evolving introns, and vice versa. CONCLUSIONS: These results indicate that introns, which extend over almost a quarter of the human genome, contain useful information for fully understanding the mutational dynamics of human and mouse genomes. This work also supports the idea that there is a mutational force that fluctuates nonrandomly along the genome, and shows for the first time that this force affects the introns and the synonymous and nonsynonymous positions in the exons of the genes simultaneously.

Animals↗