Biomedical subjects

Y Ramanathan

Publications and source records attributed to Y Ramanathan.

7 recordsLinked to original sources

PubMed2006

Squamous cell carcinoma related oncogene/DCUN1D1 is highly conserved and activated by amplification in squamous cell carcinomas.

Chromosomal amplification at 3q is common to multiple human cancers, but has a specific predilection for squamous cell carcinomas (SCC) of mucosal origin. We identified and characterized a novel oncogene, SCC-related oncogene (SCCRO), which is amplified along the 3q26.3 region in human SCC. Amplification and overexpression of SCCRO in these tumors correlate with poor clinical outcome. The importance of SCCRO amplification in malignant transformation is established by the apoptotic response to short hairpin RNA against SCCRO, exclusively in cancer cell lines carrying SCCRO amplification. The oncogenic potential of SCCRO is underscored by its ability to transform fibroblasts (NIH-3T3 cells) in vitro and in vivo. We show that SCCRO regulates Gli1--a key regulator of the hedgehog (HH) pathway. Collectively, these data suggest that SCCRO is a novel component of the HH signaling pathway involved in the malignant transformation of squamous cell lineage.

Animals↗

PubMed2003

Functional cloning of genes encoding dsRNA binding proteins.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

Cloning, Molecular↗

PubMed2002

EZ-Retrieve: a web-server for batch retrieval of coordinate-specified human DNA sequences and underscoring putative transcription factor-binding sites.

The availability of a draft human genome sequence and ability to monitor the transcription of thousands of genes with DNA microarrays has necessitated the need for new computational tools that can analyze cis-regulatory elements controlling genes that display similar expression patterns. We have developed a tool designated EZ-Retrieve that can: (i) retrieve any particular region of human genome sequence from the NCBI database and (ii) analyze retrieved sequences for putative transcription factor-binding sites (TFBSs) as they appear on the TRANSFAC database. The tool is web-based, user-friendly and offers both batch sequence retrieval and batch TFBS prediction. A major application of EZ-Retrieve is the analysis of co-expressed genes that are highlighted as expression clusters in DNA microarray experiments.

Animals↗

PubMed2002

Functional cloning, sorting, and expression profiling of nucleic acid-binding proteins.

A major challenge in the post-sequencing era is to elucidate the activity and biological function of genes that reside in the human genome. An important subset includes genes that encode proteins that regulate gene expression or maintain the structural integrity of the genome. Using a novel oligonucleotide-binding substrate as bait, we show the feasibility of a modified functional expression-cloning strategy to identify human cDNAs that encode a spectrum of nucleic acid-binding proteins (NBPs). Approximately 170 cDNAs were identified from screening phage libraries derived from a human colorectal adenocarcinoma cell line and from noncancerous fetal lung tissue. Sequence analysis confirmed that virtually every clone contained a known DNA- or RNA-binding motif. We also report on a complementary sorting strategy that, in the absence of subcloning and protein purification, can distinguish different classes of NBPs according to their particular binding properties. To extend our functional annotation of NBPs, we have used GeneChip expression profiling of 14 different breast-derived cell lines to examine the relative transcriptional activity of genes identified in our screen and cluster analysis to discover other genes that have similar expression patterns. Finally, we present strategies to analyze the upstream regulatory region of each gene within a cluster group and select unique combinations of transcription factor binding sites that may be responsible for dictating the observed synexpression.

Adenocarcinoma↗

PubMed2001

Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences.

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.

Amino Acid Sequence↗

PubMed1999

Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate.

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.

Amino Acid Sequence↗

PubMed1997

Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

Amino Acid Sequence↗