Identification And Function Investigation Of An Alternative Splicing For Human GIDRP88

Gene coding for growth inhibition and differentiation related protein 88 (GIDRP88) was obtained by using suppression subtractive hybridization (SSH) and other techniqueswhen the mechanism of an anti-tumour drug had been studied. It is suggested that this gene is related to the tumour differentiation, cell growth and cell cycle. The previous results also showed that there should be an alternative splicing in GIDRP88.Human GIDRP88A encoding a protein associated with cell growth and differentiation has been cloned and characterized. It is a short form of GIDRP88 whose function is unknown.The total RNA was first prepared from human breast cancers MDA-MB-231 and reverse-transcribed into ss-cDNA. Two DNA fragments were obtained when ss-DNA was amplified by using PCR with a pair of primers whose sequences were based on GIDRP88 gene.The full length of short fragment was then cloned using 3'-RACE and 5'-RACE, and named GIDRP88A.GIDRP88A is 2416bp in length and encodes a peptide with 226 amino acid residues. Sequence analysis studies showed that two alternative splicing forms are in frame. GIDRP88A cDNA is lkb shorter than GIDRP88A owing to lacking exon 6 and a part of exon 5. One more base A was inserted at the deletion site of exon 5, which results in a stop codon appeared in advance. GIDRP88A is 566aa shorter than GIDRP88 at the C terminus. Moreover, there are two repeats related with the function of GIDRP88 at the C terminus.Consequently, GIDRP1&A is lack of part of function, but retains motifs correlated with cell growth and differentiation. In addition, the tissue-specific expression of GIDRP&&A was shown in a number of human normal and tumour tissues by semi-quantity RT-PCR. The results demonstrated that GIDRPUA is predominately expressed in normal tissues, but at very low level in tumour tissues. However, GIDRP%% appears to be expressed abundantly in both normal and tumour tissues, suggesting that the possible difference exists between GIDRP%%A and GIDRPSS in the function.Subcellular localization,^ vitro translation.transient infection.prokaryotic expression of GIDRP1&A ORF in Escherichia coli and growth inhibition effect of the purified expression products against tumour cell MDA-MB-231 are studied. Human GIDRP88A has widespread subcellular expression, primarily in nuclei. The results of in vitro translation and transient infection display that the ORF of GIDRP%%A is consistent with bioinformatics analysis. This gene expresses a protein with molecular weight of 27K.D. The activity of the protein expressed in Escherichia coli is similar to the native one.The results indicate that prokaryotic expression GIDRP88A inhibits the growth of MDA-MB-231 cells.