Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks ( family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata ( Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata ( the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleoticle distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Alternative polyadenylation and splicing of mRNAs transcribed from the human Sin1 gene

Limited but significant sequence similarity has been observed between an uncharacterized human protein, SIN1, and the S. pombe SIN1, Dictyostelium RIP3 and S. cerevisiae AVO1 proteins. The human Sin1 gene has been automatically predicted (MAPKAP1; GenBank accession number NM_024117); however, this sequence appears to be incomplete. In this study, we have cloned and characterized the full-length human Sin1 mRNA and identified a highly conserved domain that defines the family of SIN1 orthologues, members of which are widely distributed in the fungal and metazoan kingdoms. We demonstrate that Sin1 transcripts can use alternative polyadenylation signals and describe a number of Sin1 splice variants that potentially encode functionally different isoforms. (C) 2004 Elsevier B.V. All rights reserved.

ASD: the Alternative Splicing Database

Alternative splicing is widespread in mammalian gene expression, and variant splice patterns are often specific to different stages of development, particular tissues or a disease state. There is a need to systematically collect data on alternatively spliced exons, introns and splice isoforms, and to annotate this data. The Alternative Splicing Database consortium has been addressing this need, and is committed to maintaining and developing a value-added database of alternative splice events, and of experimentally verified regulatory mechanisms that mediate splice variants. In this paper we present two of the products from this project: namely, a database of computationally delineated alternative splice events as seen in alignments of EST/cDNA sequences with genome sequences, and a database of alternatively spliced exons collected from literature. The reported splice events are from nine different organisms and are annotated for various biological features including expression states and cross-species conservation. The data are presented on our ASD web pages (http://www.ebi.ac.uk/asd).

Understanding alternative splicing: Towards a cellular code

In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

Ultraconserved elements in insect genomes: A highly conserved intronic sequence implicated in the control of homothorax mRNA splicing

Recently, we identified a large number of ultraconserved (uc) sequences in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Here, we used similar methods to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to Occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pairfed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs, that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity. (C) 1999 Elsevier Science B.V. All rights reserved.