PARENT SESSION
Gene Regulation & Function

EXPRESSION PROFILE OF X-LINKED GENES IN BOVINE CELL CULTURES.

Farazmand, Ali¹, Stranzinger, Gerald², Basrur, Parvati¹, King, Allan¹, ^{1 2}

ABSTRACT- The X-inactive specific transcript (XIST), which is expressed exclusively from the inactive X chromosome, is a key factor in regulating X chromosome inactivation in female mammals. To test whether XIST expression is proportional to the number of the inactive X chromosomes in bovine cells, using a semi-quantitative approach the expression level of XIST was compared among normal male (XY) and female (XX) cell cultures and two aneuploid cell strains (XXX and XXY). Also, the expression profile of the X-linked genes, hypoxanthine phosphorybosil transferase (HPRT), glucose 6-phosphate dehydrogenase (G6PD), Z finger protein locus on X (ZFX), and "selected mouse cDNA on X" (SMCX), were examined. Total RNA, extracted from cells were subjected to reverse transcription (RT)-PCR using Quantum RNAż 18S Internal Standards (Ambion) according to the manufacturer instructions. For each gene six runs of PCR were carried out using cDNA samples of cells. The optical density (OD) values of the gene specific bands relative to that of the 18S (co-amplified in the same reaction as internal control) were compared to test the expression level of each gene in different cell cultures. Analysis of the data (using ANOVA) indicated that the expression of XIST is significantly (P < 0.05) different in XY and XXX compared to that in XX and XXY cell cultures, being the highest in XXX and the least in XY cells. No significant differences were observed between XX and XXY cells (with only one inactive X chromosome). Comparison of the expression level of other X-linked genes examined in this study revealed that while expression levels of G6PD, HPRT, and SMCX are comparable in all four cell types, the expression level of ZFX is significantly higher in XXX cell strain compared to that in other cells. These data indicate that while G6PD, HPRT, and SMCX are subject to inactivation, ZFX escapes inactivation in bovine cells. Expression of XIST in XY cells, although at the lowest rate compared to other cell cultures tested in this study, is unexpected as there is no need for X chromosome inactivation in normal male cells. The elevated level of XIST expression in XXX cells compared to cell cultures carrying two X chromosomes (XX and XXY) is keeping with its role in in the X chromosome inactivation and can be attributed to the presence of two active XIST loci in XXX cells. (Funded by NSERC).

KEY WORDS: X-inactive specific transcript (XIST) , X chromosome inactivation, bovine X-linked genes, bovine cell cultures