Biomedical subjects

C Afshari

Publications and source records attributed to C Afshari.

3 recordsLinked to original sources

PubMed2001

Assessing gene significance from cDNA microarray expression data via mixed models.

The determination of a list of differentially expressed genes is a basic objective in many cDNA microarray experiments. We present a statistical approach that allows direct control over the percentage of false positives in such a list and, under certain reasonable assumptions, improves on existing methods with respect to the percentage of false negatives. The method accommodates a wide variety of experimental designs and can simultaneously assess significant differences between multiple types of biological samples. Two interconnected mixed linear models are central to the method and provide a flexible means to properly account for variability both across and within genes. The mixed model also provides a convenient framework for evaluating the statistical power of any particular experimental design and thus enables a researcher to a priori select an appropriate number of replicates. We also suggest some basic graphics for visualizing lists of significant genes. Analyses of published experiments studying human cancer and yeast cells illustrate the results.

Computational Biology↗

PubMed1999

Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene.

Activation of telomerase is one of the rate-limiting steps in human cell immortalization and carcinogenesis Human telomerase is composed of at least two protein subunits and an RNA component. Regulation of expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), is suggested as the major determinant of the enzymatic activity. We report here the cloning and characterization of the 5'-regulatory region of the hTERT gene. The highly GC-rich content of the 5' end of the hTERT cDNA spans to the 5'-flanking region and intron 1, making a CpG island. A 1.7-kb DNA fragment encompassing the hTERT gene promoter was placed upstream of the luciferase reporter gene and transiently transfected into human cell lines of fibroblastic and epithelial origins that differed in their expression of the endogenous hTERT gene. Endogenous hTERT-expressing cells, but not nonexpressing cells, showed high levels of luciferase activity, suggesting that the regulation of hTERT gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments revealed that a 59-bp region (-208 to -150) is required for the maximal promoter activity. The region contains a potential Myc oncoprotein binding site (E-box), and cotransfection of a c-myc expression plasmid markedly enhanced the promoter activity, suggesting a role of the Myc protein in telomerase activation. Identification of the regulatory regions of the hTERT promoter sequence will be essential in understanding the molecular mechanisms of positive and negative regulation of telomerase.

Base Sequence↗

PubMed1998

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3' end target sequence.

Unique, small sequences (sequence tag sites) have been identified at the 3' ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3' unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3' hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3' end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

Animals↗