PMC:2575619 / 19873-23336 JSONTXT

{"project":"2_test","denotations":[{"id":"18593464-9750195-4200943","span":{"begin":1198,"end":1200},"obj":"9750195"}],"text":"Cloning of novel alternative splice variants coding for secreted receptor isoforms\nTo identify novel splice variants from cell surface receptor genes, we performed RT-PCR using a complex mRNA pool representing major human tissue types and tumors. We intended to identify novel splice patterns that lead to the formation of secreted receptor isoforms. To do so, we selected forward PCR primers that flank the start codon and reverse primers that are located in the transmembrane regions. The amplified PCR products were separated on agarose gels and the DNA bands were extracted, purified, and individually cloned to generate gene-specific plasmid cDNA libraries. Two hundred to 1,000 random recombinant clones within each library were screened using PCR amplification to analyze the insert sizes. Clones with subtle differences in insert sizes on agarose gel electrophoresis were selected for complete DNA sequencing. Novel splice variants were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts of sequence databases of National Center for Biotechnology Information (NCBI) using the splice variant analysis software SIM4 [47]. Only transcripts with canonical donor–acceptor splicing sites (for example, GT–AG) were considered for further analysis, so that potential PCR artifacts were excluded. We defined a novel splice variant as an alteration in splice patterns to the existing full-length transcript sequences from available sequence databases, including Geneseq and other public databases.\nA total of 60 full-length splice variants, derived from the extracellular domains of the 21 type 1 receptor genes, were confirmed to be novel – with variants from the c-Met proto-oncogene being the most diverse (Table 1). Sequences of the 60 full-length novel splice variants were deposited with GenBank (accession numbers EU826561 to EU826620; see also Additional files 1 and 2). Alignment of the cloned splice variant cDNA sequences with the corresponding genomic and known transcript sequences in available databases revealed that a total of 83 alternative splice events occurred in the 60 novel variants (Figure 1). We categorized the alternative splice events, and found that 67.5% led to intron fusion (intron sequences inserted into mature mRNA). These include novel exon insertion, exon extension, and intron retention. The remaining 32.5% of alternative splice events resulted in exon loss (a portion or whole exon was skipped). A total of 18% of the exon extensions and 50% of the exon truncations identified in this study occurred at the 5' end of the alternatively spliced exons. All of the 60 transcript variants encounter a stop codon within the extracellular regions. As a result, these variants encode soluble receptor isoforms, and were subsequently referred to as ASV.\nTable 1 Cloned alternative splice variant mRNAs Sixty novel alternative splice variants were cloned from 21 cell surface receptor genes by RT-PCR amplification followed by extensive colony screening. The number of novel alternative splice variants is presented for each receptor tested. NCBI, National Center for Biotechnology Information.\nFigure 1 Splice events categorized by type. A total of 83 alternative splicing events were identified in the 21-gene array (Table 1). The identified splicing events fell into five listed types. The splice pattern of the known transcript is depicted as type 1.\n\nD"}