Multiple mRNA isoforms of the transcription activator protein CREB

We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREBA and CREBa, of which the rat and human homologues have been previously identified. Both encode proteins with CREbinding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway

The intron-containing gene for yeast profilin (PFY) encodes a vital function

The gene coding for profilin (PFY), an actin-binding protein, occurs as a single copy in the haploid genome of Saccharomyces cerevisiae and is required for spore germination and cell viability. Displacement of one gene copy in a diploid cell by a nonfunctional allele is recessively lethal: tetrad analysis yields only two viable spores per ascus. The PFY gene maps on chromosome XV and is linked to the ADE2 marker. The primary transcript of about 1,000 bases contains an intron of 209 bases and is spliced into a messenger of about 750 bases. The intron was identified by comparison with a cDNA clone, which also revealed the 3' end of the transcript. The 5' end of the mRNA was mapped by primer elongation. The gene is transcribed constitutively and has a coding capacity for a protein of 126 amino acids. The deduced molecular weight o

Using Expressing Sequence Tags to Improve Gene Structure Annotation

Finding all gene structures is a crucial step in obtaining valuable information from genomic sequences. It is still a challenging problem, especially for vertebrate genomes, such as the human genome. Expressed Sequence Tags (ESTs) provide a tremendous resource for determining intron-exon structures. However, they are short and error prone, which prevents existing methods from exploiting EST information efficiently. This dissertation addresses three aspects of using ESTs for gene structure annotation. The first aspect is using ESTs to improve de novo gene prediction. Probability models are introduced for EST alignments to genomic sequence in exons, introns, interknit regions, splice sites and UTRs, representing the EST alignment patterns in these regions. New gene prediction systems were developed by combining the EST alignments with comparative genomics gene prediction systems, such as TWINSCAN and N-SCAN, so that they can predict gene structures more accurately where EST alignments exist without compromising their ability to predict gene structures where no EST exists. The accuracy of TWINSCAN_EST and NSCAN_EST is shown to be substantially better than any existing methods without using full-length cDNA or protein similarity information. The second aspect is using ESTs and de novo gene prediction to guide biology experiments, such as finding full ORF-containing-cDNA clones, which provide the most direct experimental evidence for gene structures. A probability model was introduced to guide experiments by summing over gene structure models consistent with EST alignments. The last aspect is a novel EST-to-genome alignment program called QPAIRAGON to improve the alignment accuracy by using EST sequencing quality values. Gene prediction accuracy can be improved by using this new EST-to-genome alignment program. It can also be used for many other bioinformatics applications, such as SNP finding and alternative splicing site prediction

A method of precise mRNA/DNA homology-based gene structure prediction

BACKGROUND: Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, micro-exons and partial gene structure predictions that span across several genomic clones. RESULTS: We present a mRNA/DNA homology based gene structure prediction tool, GIGOgene. We use a new affine gap penalty splice-enhanced global alignment algorithm running in linear memory for a high quality annotation of splice sites. Our tool includes a novel algorithm to assemble partial gene structure predictions using interval graphs. GIGOgene exhibited a sensitivity of 99.08% and a specificity of 99.98% on the Genie learning set, and demonstrated a higher quality of gene structural prediction when compared to Sim4, est2genome, Spidey, Galahad and BLAT, including when genes contained micro-exons and non-canonical splice sites. GIGOgene showed an acceptable loss of prediction quality when confronted with a noisy Genie learning set simulating ESTs. CONCLUSION: GIGOgene shows a higher quality of gene structure prediction for mRNA/DNA spliced alignment when compared to other available tools

Molecular characterisation of Ovine CD1

The CD1 molecules are a family of ß2microglobulin- associated glycoproteins with strong structural homology, but weaker sequence homology, to the MHC class I antigens. In contrast to the classical class I antigens, CD1 molecules exhibit restricted tissue expression (cortical thymocytes, dendritic cells, a subset of B cells and some intestinal epithelial cells), and are nonpolymorphic. Five CD1 genes have been identified in humans, two in the mouse and several in other mammalian species (Calabi et al, 1991). CD1 expression has also been detected by immunohistological techniques in the cow, sheep and pig.The MHC class I -like structure of CD1 and the expression on classical antigen presenting cells of the immune system has pointed to a role for CD1 in antigen presentation. Indeed, evidence has been accumulating over the past few years to support this view, with several reports suggesting that CD4 -8- T cells in particular may be able to recognise nonclassical presentational elements including MHC class lb molecules such as TLa and Qa, as well as CD1. Most recently, CD1b molecules on human monocytes have been demonstrated to restrict the response of CD4 -8- T cells to antigens derived from M. tuberculosis (Porcelli et al, 1992).Previous studies on the ovine CD1 family have involved the use of monoclonal antibodies to assess tissue expression and distribution, and biochemical analyses of the ovine CD1 antigens. However, no studies have been carried out to investigate ovine CD1 at the molecular level. Therefore, a human CD1 C a3 probe was used to screen several sheep thymocyte cDNA libraries. The HCD1 B -like clone SCD1 A25 was isolated from a foetal thymocyte library. A homologous probe comprising the a3/TM /CYT domains from this clone was derived by PCR amplification and used to identify a further three ovine clones - SCD1 B -42, SCD1 B -52 and SCD1T10. Three of the four clones are truncated at the 5' end, with sequences beginning towards the end of the al domain or the start of the a2 domain. These 5' truncation events probably reflect poor reverse transcriptase activity during library preparation. The fourth clone, SCD1 B -52, represents a transcript containing a precise a3 deletion. The PCR technique was used to amplify the missing 5' ends from two of the three truncated$equences, thus generating full length coding sequence for two of the four ovine CD1's identified.Comparison of the ovine CD1 sequences amongst themselves has shown them to be 81 -96% identical at the nucleotide level and 79 -90% identical at the amino acid level, suggesting that the four clones represent different gene products rather than allelic variants of CD1. The sheep sequences have also been analysed by comparison to the human, mouse and rabbit coding seqences. Perhaps unexpectedly, given the existence of five different human CD1 genes, all of the ovine CD1 sequences are most homologous to human CD1 B at both the nucleotide and amino acid levels. The sheep CD1 sequences also show a high percentage sequence identity to the cottontail rabbit sequence, which is itself most similar to HCD1 B.Southern blot analysis of genomic DNA digested with a variety of enzymes and probed with the homologous a3 probe has indicated the possible existence of up to seven ovine CD1 genes. Further studies are required to determine which of these genes are expressed and to identify the genes encoding the CD1 molecules recognised by the monoclonal antibodies. The significance and implications of these results are discussed and potential further experiments suggested

A method of precise mRNA/DNA homology-based gene structure prediction

Background: Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, microexons and partial gene structure predictions that span across several genomic clones. Results: We present a mRNA/DNA homology based gene structure prediction tool, GIGOgene. We use a new affine gap penalty splice-enhanced global alignment algorithm running in linear memory for a high quality annotation of splice sites. Our tool includes a novel algorithm to assemble partial gene structure predictions using interval graphs. GIGOgene exhibited a sensitivity of 99.08% and a specificity of 99.98% on the Genie learning set, and demonstrated a higher quality of gene structural prediction when compared to Sim4, est2genome, Spidey, Galahad and BLAT, including when genes contained micro-exons and non-canonical splice sites. GIGOgene showed an acceptable loss of prediction quality when confronted with a noisy Genie learning set simulating ESTs. Conclusion: GIGOgene shows a higher quality of gene structure prediction for mRNA/DNA spliced alignment when compared to other available tools