|ognizant Communication Corporation|
VOLUME 10, NUMBER 3
Gene Expression, Vol. 10, pp. 93-100
1052-2166/02 $20.00 + .00
Copyright © 2002 Cognizant Comm. Corp.
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Analysis of the Mutations in the Active Site of the RNA-Dependent RNA Polymerase of Human Parainfluenza Virus Type 3 (HPIV3)
Achut G. Malur, Neera K. Gupta, Bishnu P. De* and Amiya K. Banerjee
Department of Virology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195
The large protein (L) of the human parainfluenza virus type 3 (HPIV3) is the functional RNA-dependent RNA polymerase, which possesses highly conserved residues QGDNQ located within motif C of domain III comprising the putative polymerase active site. We have characterized the role of the QGDNQ residues as well as the residues flanking this region in the polymerase activity of the L protein by site-directed mutagenesis and examining the polymerase activity of the wild-type and mutant L proteins by an in vivo minigenome replication assay and an in vitro mRNA transcription assay. All mutations in the QGDNQ residues abolished transcription while mutations in the flanking residues gave rise to variable polymerase activities. These observations support the contention that the QGDNQ sequence is absolutely required for the polymerase activity of the HPIV3 RNA-dependent RNA polymerase.
Key words: Human parainfluenza virus type 3 (HPIV3); RNA-dependent RNA polymerase; Mutagenesis; In vitro transcription
Address correspondence to Amiya K. Banerjee, Department of Virology NN1-10, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Tel: (216) 444-0625; Fax: (216) 444-2998; E-mail: firstname.lastname@example.org
*Present address: Department of Genetic Medicine, Weill Medical College, Cornell University, 515 East 71st Street, Starr 505, New York, NY 10021.
Differential Display: A Critical Analysis
Dana R. Crawford,1 John C. Kochheiser,1 Gary P. Schools,2 Sharon L. Salmon,1 and Kelvin J. A. Davies3
1Center for Immunology and Microbial Disease and 2Center
for Pharmacology and Neuroscience, The Albany Medical College, Albany,
3Ethel Percy Andrus Gerontology Center and Division of Molecular Biology, University of Southern California, Los Angeles, CA
Differential display (DD) is a well-established analytical tool for measuring gene expression that is still popular due to its documented success and ability to identify novel genes not yet available for analysis by more powerful microarray hybridization. For a comprehensive analysis of all mRNAs in a given cell, it is statistically predicted that at least 240 different DD primer combinations are required. This prediction, however, has never been empirically tested. Using far more primer combinations than that predicted to evaluate 90% of the mRNAs in a cell, plus other modifications, we identified and confirmed the induction of five mRNAs by hydrogen peroxide in HA-1 hamster cells. However, five other known oxidant-inducible mRNAs were not identified by DD. Filter microarray hybridization did not result in the identification of any additional species modulated twofold or greater but previous two-dimensional protein gel electrophoresis identified 15 induced protein species. We conclude that the current statistical prediction for comprehensive analysis of all the mRNAs in a given cell is inaccurate, at least in our hands, and further conclude that DD is a useful but less than comprehensive method for assessing changes in mRNA levels.
Key words: Differential display; Gene expression; Hydrogen peroxide; Oxidative stress; Hamster fibroblasts
Address correspondence to Dana R. Crawford, Ph.D., Center for Immunology and Microbial Disease, The Albany Medical College, Albany, NY 12208. Tel: (518) 262-6652; Fax: (518) 262-5689; E-mail: email@example.com
Genetic Analysis of the Basis of Translation in the -1 Frame of an Unusual Non-ORF Sequence Isolated From Phage Display
Jennifer Zemsky, Wlodek Mandecki,* and Emanuel Goldman
Department of Microbiology & Molecular Genetics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103
An unusual peptide-encoding sequence, called H10, and several derivatives of this sequence were previously isolated from a random peptide library screened by phage display during drug discovery protocols. The H10 family of sequences had the unusual property of being expressed despite the absence of an open reading frame. When these sequences were fused to a reporter lacZ gene in all three frames, b-galactosidase was expressed not only from the parental non-open reading frame, consistent with the original isolations, but also from the frame -1 to the parental. This unexpected translation in a second reading frame could result from either a recoding event or from an internal translation initiation event. In order to elucidate which type of event, a genetic approach was selected to eliminate a potential downstream initiator site within the H10 sequence. This report provides strong evidence that translation in the -1 frame in this family of sequences is indeed originating from a downstream translation initiation event. Unexpectedly, the mutation eliminating the downstream initiation event in the -1 frame simultaneously elevated expression in the original non-open reading frame.
Key words: E. coli protein synthesis; Recoding; Readthrough of UGA codons; Programmed translational frameshifts; Internal initiation or reinitiation
Address correspondence to Emanuel Goldman, Department of Microbiology & Molecular Genetics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103. Tel: (973) 972-4367; Fax: (973) 972-3644; E-mail: egoldman @umdnj.edu
*Current address: PharmaSeq, Inc., 1 Deer Park Drive, Suite F, Monmouth Junction, NJ 08852.
Heterogeneity in Expression of DNA Polymerase b and DNA Repair Activity in Human Tumor Cell Lines
Nandan Bhattacharyya, Huan-Chao Chen,* Liming Wang,** and Spira Banerjee
Department of Cancer Biology/NB40, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
The 39-kDa DNA polymerase b (polb) is an essential enzyme in short-patch base excision repair pathway. A wild-type and a truncated forms of polb proteins are expressed in primary colorectal and breast adenocarcinomas and in a primary culture of renal cell carcinoma. To test whether polb has a contributory role in tumorigenicity of human tumor cell lines, we have undertaken a study to determine expression of polb in colon, breast, and prostate tumor cell lines. Unlike primary colon tumor cells, three types of polb mRNA have been identified in HCT 116, LoVo, and DLD1, colon tumor cell lines. A 111-bp-deleted polb transcript was expressed in MCF7, a breast tumor cell line, but not in primary breast tumor cells. An expression of a smaller polb transcript has been revealed in DU145, a prostate tumor cell line, whereas, a single base (T) deletion in mRNA at codon 191 was found in prostate cancer tissue. Interestingly, a wild-type polb transcript was also expressed in all tumor cell lines similar to primary tumor cells. Furthermore, the cell extract of LoVo exhibited highest gap-filling synthesis function of polb when the extract of DU145 showed lowest activity. MNNG, a DNA alkylating agent, enhanced the gap-filling synthesis activity in extracts of LoVo cell line. Furthermore, the cellular viability of LoVo and HCT116 cells is sensitive to MNNG when DU145 cells are resistant. These results demonstrate heterogeneity in polb mRNA expression, which may be a risk factor related to tumorigenic activities of tumor cell lines.
Key words: DNA polb; Human tumor cell lines; mRNA; Protein; Gap-filling synthesis; DNA binding; MNNG
Address correspondence to Sipra Banerjee, Ph.D., Department of Cancer Biology/NB40, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Tel: (216) 444 0631; Fax: (216) 445 6269; E-mail: firstname.lastname@example.org
*Present address: Department of Urology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106.
**Present address: Metro Health Medical Center, Rammelkamp Research & Education Center, 2500 Metro Health Drive, Cleveland, OH 44109.
Modulation of c-myc, max, and mad Gene Expression During Neural Differentiation of Embryonic Stem Cells by All-trans-Retinoic Acid
Suparna A. Sarkar and Raghubir P. Sharma
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602
c-Myc regulates cellular proliferation, differentiation, and apoptosis. Temporal expression of c-Myc during all-trans-retinoic acid (RA)-mediated neural differentiation in murine embryonic stem cell (ES) was investigated. Correlation to the modulation of dimerizing partners Max and Mad that may influence c-Myc signaling and transcription regulation was elucidated for the first time in these cells. In RA-treated cells, increase in c-myc mRNA was detected by reverse transcriptase polymerase chain reaction on days 11 and 14 of differentiation compared with the vehicle-treated controls. The results were further corroborated by ribonuclease protection assay (RPA). Western blots revealed an increase in c-Myc protein only on day 14 of differentiation in RA-treated cells. Increases in max and mad gene transcription detected by RPA at times of elevated c-Myc in RA-treated ES cells suggest that a transient increase in c-Myc protein expression may influence differential dimerization of Myc partners needed for signaling in the neural differentiation of ES cells.
Key words: Murine embryonic stem cells; c-Myc; Differentiation; Retinoic acid; Max; Mad
Address correspondence to Dr. Raghubir P. Sharma, Department of Physiology
and Pharmacology, College of Veterinary Medicine, The University of Georgia,
Athens, GA 30602-7389. Tel: (706) 542-2788; Fax: (706) 542-3015; E-mail: