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The human ac-tropomyosin gene hTM.m has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.Alternative splicing of pre-mRNA produces different isoforms of mRNA from a single gene, often resulting in the production of multiple protein isoforms. In many cases, the proportions of the various isoforms alter during cell differentiation or development. Mutually exclusive splicing, whereby only one of two adjacent exons is incorporated between common flanking exons, is associated particularly with transcripts which are expressed primarily in muscle cells or which exhibit muscle-specific switches in the exon selected. This pattern of splicing raises three principal problems: (i) the two alternative exons are not usually spliced to each other, and where the process appears to be regulated, (ii) one exon is preferred in most cell types but (iii) the alternative exon is incorporated in the specific tissue.This pattern of splicing has been studied most intensively in two tropomyosin gene systems: the 5'-proximal exons 2 and 3 of rat a-tropomyosin, in which exon 2 is incorporated specifically in smooth muscle (39), and the more central exons 6 and 7 (or 6A and 6B) of the 3-tropomyosin homologs in rats and chickens, in which the 3' most of the two exons is incorporated in skeletal muscle (19,24,26). The results of these studies do not address all of the issues described above, but perhaps surprisingly, it is clear that mutually exclusive splicing is not determined by a common mechanism. In the case of the rat a-tropomyosin gene, it appears that exons 2 and 3 do not splice together because the long polypyrimidine tract of exon 3 forces the branch site too close to the 5' splice site of exon 2 (39). The default exon 3 appears to be selected because both exons are in competition for splicing to exon 1, but exon 3 is preceded by a more favorable polypyrimidine tract (32); sequences near or within ...

The neural cell adhesion molecule (N-CAM) is an important mediator of calcium independent cell-cell interactions. Variations in the primary structure of the protein are due to alternative splicing of pre-mRNA in the region encoding the extracellular, trans-membrane and cytoplasmic domains. In order to identify the patterns of exon usage during development of skeletal muscle and brain of the mouse, a coupled reverse-transcriptase/polymerase chain reaction was used to identify the murine homologues of the muscle-specific domain (MSD), located between exons 12 and 13 in human N-CAM mRNA. The cDNAs produced have been cloned and sequenced, or analysed directly. The amplification reactions were shown to maintain the concentration ratios of the initial cDNAs. The results indicate that the mouse homologue to exon MSD1a is under tissue and developmental regulation that is independent of exons MSD1b and MSD1c. The inclusion of the triplet exon AAG is also regulated in a cell- and stage-specific manner, which is independent of the other alternatively spliced exons of this domain.

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A full understanding of the immune system and its responses to infection by different pathogens is important for the development of anti-parasitic vaccines. A growing number of large-scale experimental techniques, such as microarrays, are being used to gain a better understanding of the immune system. To analyse the data generated by these experiments, methods such as clustering are widely used. However, individual applications of these methods tend to analyse the experimental data without taking publicly available biological and immunological knowledge into account systematically and in an unbiased manner. To make best use of the experimental investment, to benefit from existing evidence, and to support the findings in the experimental data, available biological information should be included in the analysis in a systematic manner. In this review we present a classification of tasks that shows how experimental data produced by studies of the immune system can be placed in a broader biological context. Taking into account available evidence, the classification can be used to identify different ways of analysing the experimental data systematically. We have used the classification to identify alternative ways of analysing microarray data, and illustrate its application using studies of immune responses in mice to infection with the intestinal nematode parasites Trichuris muris and Heligmosomoides polygyrus.