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Only a small fraction of large genomes such as that of the human contains the functional regions such as the exons, promoters, and polyA sites. A platform technique for selective enrichment of functional genomic regions will enable several next-generation sequencing applications that include the discovery of causal mutations for disease and drug response. Here, we describe a powerful platform technique, termed "functional genomic fingerprinting"(FGF), for the multiplexed genomewide isolation and analysis of targeted regions such as the exome, promoterome, or exon splice enhancers. The technique employs a fixed part of a uniquely designed Fixed-Randomized primer, while the randomized part contains all the possible sequence permutations. The Fixed-Randomized primers bind with full sequence complementarity at multiple sites where the fixed sequence (such as the splice signals) occurs within the genome, and multiplex amplify many regions bounded by the fixed sequences (e.g., exons). Notably, validation of this technique using cardiac myosin binding protein-C (MYBPC3) gene as an example strongly supports the application and efficacy of this method. Further, assisted by genomewide computational analyses of such sequences, the FGF technique may provide a unique platform for high-throughput sample production and analysis of targeted genomic regions by the next-generation sequencing techniques, with powerful applications in discovering disease and drug response genes.

We have developed AspAlt-a web-based comparative analytical platform for exploring the variations in alternative transcription (AT) events and alternative splicing (AS) events in eukaryotes. AspAlt provides integrated access to 2.1 million AT-AS annotations from 1,58,876 multi-isoform genes and has the following user-friendly analytical features:(1) advanced graphical display to visualize and analyze AT-AS events in 46 eukaryotic genomes;(2) compare and identify the differences in AT-AS patterns among a group of genes specified by the user or among homologous gene groups;(3) inter-database comparative viewer to analyze the differences in the AT-AS annotations for the same gene among Ensembl, RefSeq and AceView databases;(4) dynamically classify and generate graphical plots of AT-AS events from mRNA annotations submitted by the user; and (5) download genomic AT-AS annotations of 46 eukaryotes in XML and tab-delimited formats. The AspAlt resource is available at http://66.170.16.154/AspAlt.

We have developed ExDom, a unique database for the comparative analysis of the exon-intron structures of 96 680 protein domains from seven eukaryotic organisms (Homo sapiens, Mus musculus, Bos taurus, Rattus norvegicus, Danio rerio, Gallus gallus and Arabidopsis thaliana). ExDom provides integrated access to exon-domain data through a sophisticated web interface which has the following analytical capabilities:(i) intergenomic and intragenomic comparative analysis of exon-intron structure of domains;(ii) color-coded graphical display of the domain architecture of proteins correlated with their corresponding exon-intron structures;(iii) graphical analysis of multiple sequence alignments of amino acid and coding nucleotide sequences of homologous protein domains from seven organisms;(iv) comparative graphical display of exon distributions within the tertiary structures of protein domains; and (v) visualization of exon-intron structures of alternative transcripts of a gene correlated to variations in the domain architecture of corresponding protein isoforms. These novel analytical features are highly suited for detailed investigations on the exon-intron structure of domains and make ExDom a powerful tool for exploring several key questions concerning the function, origin and evolution of genes and proteins. ExDom database is freely accessible at: http://66.170.16.154/ExDom/.

The mechanism by which protein-coding portions of eukaryotic genes came to be separated by long non-coding stretches of DNA, and the purpose for this perplexing arrangement, have remained unresolved fundamental biological problems for three decades. We report here a plausible solution to this problem based on analysis of open reading frame (ORF) length constraints in the genomes of nine diverse species. If primordial nucleic acid sequences were random in sequence, functional proteins that are innately long would not be encoded due to the frequent occurrence of stop codons. The best possible way that a long protein-coding sequence could have been derived was by evolving a split-structure from the random DNA (or RNA) sequence. Results of the systematic analyses of nine complete genome sequences presented here suggests that perhaps the major underlying structural features of split-genes have evolved due to the indigenous occurrence of split protein-coding genes in primordial random nucleotide sequence. The results also suggest that intron-rich genes containing short exons may have been the original form of genes intrinsically occurring in random DNA, and that intron-poor genes containing long exons were perhaps derived from the original intron-rich genes.

MOTIVATION: Despite increased availability of genome annotation data, a comprehensive resource for in-depth analysis of splice signal distributions and alternative splicing (AS) patterns in eukaryote genomes is still lacking. To meet this need, we have developed EuSplice-a unique splice-centric database which provides reliable splice signal and AS information for 23 eukaryotes. RESULTS: The EuSplice database contains 95,822 AS events and 2.1 million splice signals associated with over 270,000 protein-coding genes. The intuitive, user-friendly EuSplice web interface has powerful data mining and graphics capabilities for inter-genomic comparative analysis of splice signals, putative cryptic splice sites and AS events. Moreover, the seamless integration of splicing data to extensive gene-specific annotations such as homolog annotations, functional information, mutations and sequence details makes EuSplice a powerful one-stop information resource for investigating the molecular mechanisms of complex splicing events, disease associations, and the evolution of splicing in eukaryotes. AVAILABILITY: http://66.170.16.154/EuSplice SUPPLEMENTARY INFORMATION: Supplementary tables and figures included.

Antibodies specific for the modified nucleoside N6-(delta 2-isopentenyl) adenosine (i6A) were employed to identify the tRNAs containing i6A from an unfractionated tRNA mixture by a nitrocellulose filter binding assay. When radioactive aminoacyl-tRNAs were incubated with i6A-specific antibodies and filtered through nitrocellulose membrane filters, the tRNAs possessing i6A (tRNAtyr and tRNAser) remained on the filters. tRNAarg and tRNAlys which do not contain i6A showed no binding. This finding will be useful as a very simple and rapid assay of such RNAs under a variety of conditions. Purification of i6A containing tRNAs from an unfractionated tRNA mixture was achieved by affinity chromatography of the tRNAs on an i6A antibody-Sepharose column. Nonspecific binding of tRNAs to the column was avoided by the use of purified antibodies.

Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes. We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328. Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom. The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure. Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes. The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.