ognizant Communication Corporation

GENE EXPRESSION

ABSTRACTS
VOLUME 11, NUMBER 1

Gene Expression, Vol. 11, pp. 1-12
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Copyright © 2003 Cognizant Comm. Corp.
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Embryonic Activation and Developmental Expression of the Murine Prion Protein Gene

G. Miele,1 A. R. Alejo Blanco,1 H. Baybutt,2 S. Horvat,3 J. Manson,2 and M. Clinton1

1Department of Gene Expression & Development, Roslin Institute, Roslin, Midlothian, Scotland, EH25 9PS, UK
2BBSRC Institute for Animal Health Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, Scotland, EH9 3JF, UK
3Biotechnical Faculty, Zootechnical Department, University of Ljubljana, Slovenia

While it is well established that cellular prion protein (PrPC) expression is required for the development of transmissible spongiform encephalopathies (TSEs), the physiological function of PrPC has yet to be determined. A number of studies have examined PrP expression in different tissues and in the later stages of embryonic development. However, the relative levels of expression of PrP RNA and protein in tissues outside the central nervous system (CNS) is not well documented and the exact point of transcriptional activation of PrP during embryogenesis is unknown. We have studied PrP mRNA expression in murine embryos and both mRNA and protein expression in a variety of adult tissues. PrP RNA was detected at different levels in all tissues tested while PrPC protein was detectable in all adult tissues tested with the exception of kidney and liver. RNA and protein levels were also assessed at four points during postnatal brain development and levels of both were seen to increase with development. We also established that, during embryogenesis, induction of PrP RNA expression occurs between E8.5 and E9, during the period of transition from anaerobic to aerobic metabolism. Preliminary experiments investigating the effects of superoxide radicals on PrP expression in cultured neuroblastoma and astrocyte cells support the suggestion that PrPC forms part of a cellular antioxidant defense mechanism.

Key words: Prion protein expression; Oxidative stress; Superoxide radicals; Whole-mount in situ hybridization; Prion protein function

Address correspondence to M. Clinton, Department of Gene Expression & Development, Roslin Institute, Roslin, Midlothian, Scotland, EH25 9PS, UK. Tel: +44 131 527 4216; Fax: +44 131 440 0434; E-mail: michael.clinton@bbsrc.ac.uk




Gene Expression, Vol. 11, pp. 13-21
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Copyright © 2003 Cognizant Comm. Corp.
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Modulation of Splicing Events in Histone Deacetylase 3 by Various Extracellular and Signal Transduction Pathways

S. G. Gray,1* A. H. Iglesias,2 B. T. Teh,1 and F. Dangond2

1Van Andel Research Institute, Laboratory for Cancer Research, 333 Bostwick NE, Grand Rapids, MI 49503
2Laboratory of Transcriptional and Immune Regulation, Center for Neurologic Diseases, Brigham and Women's Hospital Laboratories, 65 Landsdowne Street, Cambridge, MA 02139

Within the context of the chromatin environment histone deacetylases are important transcriptional regulators. Three classes of human histone deacetylases have currently been identified on the basis of their similarity to yeast proteins. The class I enzymes contain four members: HDACs 1-3 and HDAC8. Of these, HDAC3 is known to generate transcript variants with altered amino-terminal regions. Here we describe the identification of a novel splice variant of HDAC3, in which exon 3 is alternatively spliced from the messenger RNA transcript. We show that this human HDAC3 splice transcript is upregulated by treatments with histone deacetylase inhibitors. We also demonstrate evidence of splicing events in murine HDAC3 as a response to various signals, including switching between splice transcript isoforms following treatments with kinase inhibitors or by osmotic shock. In contrast, such switching events were not observed in human cells. These results indicate that differential pathways in mouse and human may control the regulation of HDAC3, and that splice variants may play important roles in responding to exogenous stimuli that act via signal transduction pathways.

Key words: Alternative splicing; Histone deacetylase; Signaling

Address correspondence to F. Dangond, Laboratory of Transcriptional and Immune Regulation, Center for Neurologic Diseases, Brigham and Women's Hospital Laboratories, 65 Landsdowne Street, 3rd Floor, Cambridge, MA 02139. Tel: (617) 768-8591; Fax: (617) 768-8595; E-mail: fdangond@rics.bwh.harvard.edu

*Present address: Receptor Biology Laboratory, Novo Nordisk, Hagedorn Research Institute, Niels Steensens Vej 6, DK 2820 Gentofte, Denmark.




Gene Expression, Vol. 11, pp. 23-34
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Absolute Quantitation of Normal and ROS-Induced Patterns of Gene Expression: An In Vivo Real-Time PCR Study in Mice

María-José Prieto-Álamo, Juan-Manuel Cabrera-Luque, and Carmen Pueyo

Departamento de Bioquímica y Biología Molecular, Campus de Rabanales edificio C-6, Carretera Madrid-Cádiz Km 396-a, Universidad de Córdoba, 14071-Córdoba, Spain

Most studies using real-time PCR are taken semiquantitatively and assume a steady level of expression for the so-called housekeeping genes. By absolute real-time PCR we demonstrate that the transcript amounts of two of the most popular internal controls (coding GAPDH and b-actin) fluctuate dramatically across diverse mouse or human tissues. This raises the question about the inaccuracy of these genes as quantitative references in tissue-specific mRNA profiling. Target genes chosen for absolute real-time PCR analysis are involved in DNA repair, regulation of gene expression, and oxidative stress response. Hence, they code for 8-oxoG-DNA glycosylase/AP-lyase, major AP-endonuclease, and heme oxygenase-1. Quantitations reported: i) determine mouse-to-mouse variability in basal gene expression, ii) establish organ- and embryo-associated differences in mouse, iii) compare mouse and human tissue-specific profiles, iv) examine the time course (30-240 min) expression in liver and lung of mice treated with paraquat (superoxide generator) at 30 mg kg-1 (one half LD50 value), and v) explore the utility of absolute real-time PCR in field studies with genetically diverse mice. We conclusively establish that real-time PCR is a highly sensitive and reproducible technique for absolute quantitation of transcript levels in vivo and propose its use to quantitate gene expression modulation under mild physiological exposures and for field epidemiological studies.

Key words: Absolute real-time PCR; In vivo gene expression; Mice; GAPDH; ACTB; OGG1; APE1; HO-1; Paraquat; Oxidative stress

Address correspondence to Carmen Pueyo, Departamento de Bioquímica y Biología Molecular, Campus de Rabanales edificio C-6, Carretera Madrid-Cádiz Km 396-a, Universidad de Córdoba, 14071-Córdoba, Spain. Tel: +34 957 218695; Fax: +34 957 218688; E-mail: bb1pucuc@uco.es




Gene Expression, Vol. 11, pp. 35-46
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Differential Gene Expression in Human Glioma Cells: Correlation With Presence or Absence of DNA-Dependent Protein Kinase

Rong Ai, Ana Sandoval, and Paul Labhart

Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121

The human glioma cell line M059J is deficient in DNA-dependent protein kinase (DNA-PK) due to a frame-shift mutation in PRKDC, the gene for its catalytic subunit, while cell line M059K, isolated from the same malignant tumor, has normal DNA-PK activity. DNA-PK is required for double-strand DNA break repair, and its absence is responsible for increased radiosensitivity of M059J. We show that transcripts of several melanoma antigen subfamily A (MAGE-A) genes, the expression of which is restricted to tumor and germ-line cells, are present in M059K, but that their expression is strongly downregulated in M059J. Normal levels of MAGE-A expression are restored in the PRKDC-complemented cell line M059J/Fus1, suggesting that the presence of DNA-PK is required for MAGE-A gene transcription. We also show that the MAGE-A1 promoter is methylated in M059J, while the promoter is demethylated in M059K and M059J/Fus1. Other genes, including all three major histocompatibility class I (HLA) genes, BENE, and an unnamed gene related to CNIL (CORNICHON-like), display an opposite expression profile (i.e., they are upregulated in the DNA-PK-deficient cell line, but show low levels of expression in both M059K and in the PRKDC-complemented cell line). For these genes, differential expression does not correlate with DNA methylation in upstream promoter sequences. Our results suggest that the presence of DNA-PK can exert effects on gene expression by various mechanisms and pathways, thus affecting overall cell physiology even in the absence of DNA damage.

Key words: Malignant glioma cell lines; DNA-dependent protein kinase; Melanoma-associated antigen; DNA methylation; RNA fingerprinting

Address correspondence to Paul Labhart, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. Tel: (858) 455-3857; Fax: (858) 455-3804; E-mail: plabhart@tpims.org




Gene Expression, Vol. 11, pp. 47-53
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Strain-Specific Differences in the Expression and Activity of Ogg1 in the CNS

Diana I. Mosquera,1* Todd Stedeford,1,2* Fernando Cardozo-Pelaez,3 and Juan Sanchez-Ramos1,4

1Department of Neurology, College of Medicine, University of South Florida, Tampa, FL 33612
2Polish Academy of Sciences, Sowinskiego 5, 44-121 Gliwice, Poland
3Center for Environmental Health Sciences, Department of Pharmaceutical Sciences, University of Montana, Missoula, MT 59801
4Research Services, James A. Haley Veterans' Hospital, Tampa, FL 33612

The expression and activity of 8-oxoguanosine DNA-glycosylase (Ogg1), a key enzyme responsible for the clearance of the oxidized DNA base 8-hydroxy-2´-deoxyguanosine (oxo8dG), was determined in the cerebellum (CB) and the caudate and the putamen (CP) of male Balb/c, ICR, and C57BL/J mice. There was no significant difference in the protein expression of Ogg1 in the CB or CP. The activity of Ogg1 was not significantly different in the CB; however, in the CP of ICR mice, the activity of Ogg1 was 34% and 31% lower than Balb/c and C57BL/J, respectively. In contrast, the levels of oxo8dG in the CB and CP of C57BL/J mice were nearly twice as high as the values in both regions of Balb/c and ICR mice. The activity of superoxide dismutases (SOD) appeared to account for the differences in the levels of oxo8dG in the C57BL/J strain. Total SOD in the C57BL/J strain was two- and fourfold higher in the CB and CP, respectively, versus the other strains. These results suggest that the enhanced vulnerability of the C57BL/J strain to neurotoxicants may not be due to a decreased capacity for DNA repair, but rather, the significantly higher activity of SODs, which may cause these pathways to become more readily saturated.

Key words: DNA damage; DNA repair; Neurodegenerative disease; Striatum

Address correspondence to Juan Sanchez-Ramos, Ph.D., M.D., Department of Neurology MDC 55, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel: (813) 974-6022; Fax: (813) 974-7200; E-mail: jsramos@hsc.usf.edu

*These authors contributed equally to this work.