exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Bioinformatic analysis of exon repetition, exon scrambling and trans-splicing in humans

MOTIVATION: Using bioinformatic approaches we aimed to characterize poorly understood abnormalities in splicing known as exon scrambling, exon repetition and trans-splicing. RESULTS: We developed a software package that allows large-scale comparison of all human expressed sequence tags (EST) sequences to the entire set of human gene sequences. Among 5,992,495 EST sequences, 401 cases of exon repetition and 416 cases of exon scrambling were found. The vast majority of identified ESTs contain fragments rather than full-length repeated or scrambled exons. Their structures suggest that the scrambled or repeated exon fragments may have arisen in the process of cDNA cloning and not from splicing abnormalities. Nevertheless, we found 11 cases of full-length exon repetition showing that this phenomenon is real yet very rare. In searching for examples of trans-splicing, we looked only at reproducible events where at least two independent ESTs represent the same putative trans-splicing event. We found 15 ESTs representing five types of putative trans-splicing. However, all 15 cases were derived from human malignant tissues and could have resulted from genomic rearrangements. Our results provide support for a very rare but physiological occurrence of exon repetition, but suggest that apparent exon scrambling and trans-splicing result, respectively, from in vitro artifact and gene-level abnormalities. AVAILABILITY: Exon-Intron Database (EID) is available at http://www.meduohio.edu/bioinfo/eid. Programs are available at http://www.meduohio.edu/bioinfo/software.html. The Laboratory website is available at http://www.meduohio.edu/medicine/fedorov Supplementary information: Supplementary file is available at http://www.meduohio.edu/bioinfo/software.html.     

Exon 26-coded polypeptide: An isolated hydrophobic domain of human tropoelastin able to self-assemble in vitro

Hydrophobic domains of human tropoelastin are able to aggregate in a variegated manner. Some aggregates have typical features of the whole protein while others show peculiar self-assembling profiles. Among these hydrophobic domains, an important role in the self-assembling properties of tropoelastin in vitro could be assigned to the peptide encoded by exon 26 of the human tropoelastin gene, that, although unstructured in solution, has great tendency to self-assemble in an ordered manner. The present report describes the aggregation properties of this hydrophobic domain of human tropoelastin analysed by different ultra-structural approaches. Transmission electron microscopy shows that the peptide is able to form different aggregation entities from short rods to very long and flexible fibers, depending on the temperature and on the incubation time. At a microm scale, very long fibers as well as fractal aggregation patterns were observed. Data show that the isolated domain encoded by exon 26 of the tropoelastin gene is able to aggregate in a manner very similar to the whole tropoelastin protein. The aggregation properties are due to the peculiar sequence of EX26, and not to its amino acid composition, as evidenced by the supramolecular analysis of a scrambled sequence of exon 26-coded domain of human tropoelastin, showing a quite different aggregation patterns. These findings confirm that specific sequences can play a driving role in the aggregation process of tropoelastin molecule, at least in vitro, and indicate exon 26-encoded domain among these sequences.     

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

Combinatorial control of a neuron-specific exon.

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.     

Cloning and characterization of an alpha 1-antitrypsin like gene 12 KB downstream of the genuine alpha 1-antitrypsin gene

Cosmid clones containing alpha 1-antitrypsin (alpha 1AT) gene sequences were observed to contain alpha 1AT-like sequences approximately 12 kb downstream of the authentic alpha 1AT gene. Restriction mapping suggested the alpha 1AT-like gene lacks promoter sequences. Cosmid clones from one library contained a truncated alpha 1AT-like gene with a deletion encompassing 1745 bp, including the whole exon IV and part of exon V. Sequencing of exon II of this truncated gene revealed a nucleotide homology of 76% but included critical mutations in the start codon (ATG - greater than ATA) and the 3' exon-intron junction. These results strongly suggest that the truncated alpha 1AT-like gene is a pseudogene, which is present at a frequency of 0.30 in the Dutch population.     

Rudimentary phosvitin domain in a minor chicken vitellogenin gene

We have determined the nucleotide sequence and the derived amino acid sequence of the phosphoprotein-encoding region of the chicken vitellogenin III gene. The sequence of this minor vitellogenin could be aligned with exon 22 up to exon 27 of the previously sequenced major vitellogenin II gene (van het Schip et al., 1987). The exon 23 and 25 sequences are rich in serine codons (26% and 41%, respectively), and this region encodes at least one of the small egg yolk phosphoproteins. The major egg yolk phosphoprotein, phosvitin, is encoded by the analogous region in vitellogenin II. Comparison of the vitellogenin II and vitellogenin III sequences shows a great reduction in the size of the putative exon 23 of the latter (321 base pairs as opposed to 690). The number of serine codons is also drastically reduced from 124 in exon 23 of the vitellogenin II gene to 28 in vitellogenin III. The grouping of synonymous serine codons, as has hitherto been observed in sequenced vitellogenin phosphoproteins, has been maintained in vitellogenin III. A putative asparagine-linked N-glycosylation site which was conserved in the chicken vitellogenin II and the Xenopus laevis vitellogenin A2 gene, at the beginning of exon 23, is also present in vitellogenin III. The two chicken vitellogenins show a low conservation in the phosphoprotein-encoding region (average 33%, at the protein level) compared to that in the peripheral sequences (58% identity), which indicates that it is a rapidly evolving domain of the vertebrate vitellogenin gene.     

Internal organization of the major adult alpha- and beta-globin genes of X. laevis

We describe the isolation of two recombinant lambda phages, each containing genomic DNA fragments encoding both the major adult alpha- and beta-globin mRNAs of X. laevis. The DNA fragment in the two clones have restriction maps which indicate that they are each derived from a different member of the pair of alleles present in the heterozygote used as the source of DNA for cloning. The characterization of these two clones by restriction mapping, R looping and DNA sequencing shows that the alpha 1- and beta 1-globin genes lie in the orientation separated by 7.7 kb of DNA. There are two introns in the alpha 1-globin gene and two in the beta 1-globin gene, and they interrupt the genes at exactly the same positions as the introns found in all known mammalian alpha- and beta-globin genes. The exon sequences proximal to the introns show a much higher degree of homology with mammalian sequences than the sequences distal to intron/exon junctions, and the introns in the beta 1-globin gene of X. laevis are very similar in length to the corresponding introns in the beta-globin genes of several mammals and the chicken.