Differential expression of potato U1A spliceosomal protein genes: a rapid method for expression profiling of multigene families.
The spliceosomal protein, UIA, is a component of the U1snRNP essential to pre-mRNA splicing. From the ubiquitous nature of the splicing machinery, expression of U1A genes is expected to be constitutive. However, many plant genes are organised in multigene families that exhibit variation in expression profiles. Without detailed knowledge of the size of the U1A gene family or their degree of sequence variation, we examined the expression of the U1A genes using a novel approach. The approach was based on 5' RACE with [32P]-labelled primers and separation of products on high-resolution DNA sequencing gels to give a 'snapshot' of the expression of U1A genes. This was followed by sequencing of cloned 5' RACE products and of products re-amplified from excised bands. In combination with RT-PCR/SSCP, these analyses allowed the rapid identification of different gene transcripts and assessment of their relative expression profiles. Transcripts from four U1A genes (U1A-1 to U1A-4) were identified, of which U1A-1 and U1A-2 were expressed much more highly than U1A-3 and U1A-4. Differential expression of the two most highly expressed genes, U1A-1 and UIA-2, was observed in that only U1A-2 was expressed in flowers. Upstream sequences of U1A-1 and U1A-2 were cloned and the gene-specific promoters identified on the basis of the sequence variation defined from the 5' RACE products. The differential expression of these genes may be due to a 1.3 kb insertion less than 200 bp upstream of the U1A-1 coding sequence. This approach can be used more generally to examine expression profiles of multigene families, and in particular to refine information from EST or microarray analyses, and to isolate rapidly gene-specific promoters.
Ibrahim
A F
AF
Department of Cell & Molecular Genetics, Scottish Crop Research Institute, Dundee, UK.
Watters
J A
JA
Brown
J W
JW
D001483:Base Sequence; D015139:Blotting, Southern; D018744:DNA, Plant / Q000235:genetics; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D014644:Genetic Variation; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000235:genetics*; D014018:Tissue Distribution; D014158:Transcription, Genetic
D018744:DNA, Plant; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D005966:Glucuronidase