Isolation of three testis-specific genes (TSA303, TSA806, TSA903) by a differential mRNA display method
ABSTRACT We isolated three human testis-specific genes by a differential mRNA display method. The cDNAs contained open reading frames of 1620, 453, and 333 nucleotides, encoding 540, 151, and 111 amino acids, respectively. The first of these genes, designated TSA303, encodes a novel protein homologous to TCP20, one of the subunits of the human TRiC chaperonin complex that can bind newly synthesized or unstable folding intermediates of polypeptides and assist substrate proteins in folding, assembly, and transport. The second, TSA806, encodes a novel protein containing 3.3 contiguous repeats of the cdc10/swi6 (ankyrin) motif that was originally found in products of cell cycle control genes of yeast and cell fate determination genes in Drosophila and Caenorhabditis elegans. The third gene, TSA903, encodes a protein homologous to the C-terminal region of murine uridine monophosphate kinase. Northern blot analysis confirmed that in 16 human adult tissues examined, each of these genes was expressed specifically in the testis. From the results of cDNA screening of nearly 1 million plaques, the abundance of each transcript in a preparation of total mRNA was estimated as 0.0004% (TSA303), 0.0006% (TSA806), and 0.0002% (TSA903). Our results imply that the differential display method is a powerful tool for isolation of tissue-specific genes even if they are expressed at a level as low as 1 in several hundred thousand to a million molecules of total mRNA.
SourceAvailable from: Saiful Anuar Karsani[Show abstract] [Hide abstract]
ABSTRACT: A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories - structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins - 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 - were found to correlate with the corresponding proteins' abundance. The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins.Proteome Science 01/2014; 12(1):3. DOI:10.1186/1477-5956-12-3 · 1.88 Impact Factor
Article: Differential RNA display identifies novel genes associated with decreased vitamin D receptor expression 1 The work in this paper was funded by various grants: M.D., Polish State Committee for Scientific Research Grant No. 4P05A08709; E.R., West Midlands Regional Health Authority; R.B., Medical Research Council Grant No. G9517674. 1[Show abstract] [Hide abstract]
ABSTRACT: To characterize further the function of the intracellular vitamin D receptor (VDR), we have developed stable transfectant variants of a vitamin D-responsive cell line (U937) which express either decreased or increased numbers of VDR. In this study we have analyzed changes in gene expression associated with this variable VDR expression. Initial experiments indicated that a 50% decrease in VDR levels was associated with a 2-fold increase in cell proliferation and a similar rise in c-myc mRNA expression. Further studies were carried out using differential RNA display (DD). Sequence analysis of DD products revealed two cDNAs with identity to known gene products: the catalytic sub-unit of DNA-protein kinase (DNA-PKCS), and the peroxisomal enzyme 17β-hydroxysteroid dehydrogenase type IV (17β-HSD IV). Northern analysis confirmed that expression of both mRNAs was reduced in cells with decreased numbers of VDR. Down-regulation of 17β-HSD IV mRNA expression was associated with enhanced estradiol inactivation by U937 cells, suggesting a link between estrogenic pathways and cell proliferation. Further Northern analyses indicated that there was no significant change in 17β-HSD IV or DNA-PKCS mRNA levels following treatment with 1,25(OH)2D3, although expression of both genes varied with changes in cell proliferation. These data suggest that, in addition to its established role as a hormone-dependent trans-activator, VDR may influence gene expression by ligand-independent mechanisms.Molecular and Cellular Endocrinology 07/1998; 142(1):131-139. DOI:10.1016/S0303-7207(98)00111-7 · 4.24 Impact Factor
01/2014; 35(2):130-135. DOI:10.5958/0976-0741.2014.00090.7