ognizant Communication Corporation



Gene Expression, Vol. 14, pp. 59-69
1052-2166/07 $90.00 + .00
E-ISSN 1555-3884
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Stress-Induced Expression of a Novel Variant of Human Fumarate Hydratase (FH)

Heli J. Lehtonen,1* Sanna K. Ylisaukko-Oja,1* Maija Kiuru,1* Auli Karhu,1 Rainer Lehtonen,1 Sakari Vanharanta,1 Anu Jalanko,2 Lauri A. Aaltonen,1 and Virpi Launonen1

1Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
2National Public Health Institute, Department of Molecular Medicine, Biomedicum Helsinki, Helsinki, Finland

Fumarate hydratase (FH) is an enzyme of the mitochondrial tricarboxylic acid cycle (TCAC). Here we report the characterization of a novel FH variant (FHv) that contains an alternative exon 1b, thus lacking the mitochondrial signal sequence. Distinct from mitochondrial FH, FHv localized to cytosol and nucleus and lacked FH enzyme activity. FHv was expressed ubiquitously in human fetal and adult tissues. Heat shock and prolonged hypoxia increased FHv expression in a cell line (HTB115) by nine- and fourfold, respectively. These results suggest that FHv has an alternative function outside the TCAC related to cellular stress response.

Key words: Tricarboxylic acid cycle (TCAC); Fumarate hydratase (FH); Alternative exon; Hereditary leiomyomatosis and renal cell cancer (HLRCC); Hypoxia; Heat shock; Stress

Address correspondence to Virpi Launonen, Department of Medical Genetics, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland. Tel: INT +358-9-1911; Fax: INT +358-9-19125105; E-mail: virpi.launonen@helsinki.fi

*These authors contributed equally to this work.

Gene Expression, Vol. 14, pp. 71-81
1052-2166/07 $90.00 + .00
E-ISSN 1555-3884
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Mouse and Human La Proteins Differ in Kinase Substrate Activity and Activation Mechanism for tRNA Processing

Jung-Min Park,* Robert V. Intine,** and Richard J. Maraia

Intramural Research Program, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

The La protein interacts with a variety of small RNAs as well as certain growth-associated mRNAs such as Mdm2 mRNA. Human La (hLa) phosphoprotein is so highly conserved that it can replace the tRNA processing function of the fission yeast La protein in vivo. We used this system, which is based on tRNA-mediated suppression (TMS) of ade6-704 in S. pombe, to compare the activities of mouse and human La proteins. Prior studies indicate that hLa is activated by phosphorylation of serine-366 by protein kinase CK2, neutralizing a negative effect of a short basic motif (SBM). First, we report the sequence mapping of the UGA stop codon that requires suppressor tRNA for TMS, to an unexpected site in S. pombe ade6-704. Next, we show that, unlike hLa, native mLa is unexpectedly inactive for TMS, although its intrinsic activity is revealed by deletion of its SBM. We then show that mLa is not phosphorylated by CK2, accounting for the mechanistic difference between mLa and hLa. We found a PKA/PKG target sequence in mLa (S199) that is not present in hLa, and show that PKA/PKG efficiently phosphorylates mLa S199 in vitro. A noteworthy conclusion that comes from this work is that this fission yeast system can be used to gain insight into differences in control mechanisms used by La proteins of different mammalian species. Finally, RNA binding assays indicate that while mutation of mLa S199 has little effect on pre-tRNA binding, it substantially decreases binding to a probe derived from Mdm2 mRNA. In closing, we note that species-specific signaling through La may be relevant to the La-dependent Mdm2 pathways of p53 metabolism and cancer progression in mice and humans.

Key words: tRNA processing; ade6-704; tRNA-mediated suppression; CK2; Protein kinase A; La motif; Mdm2; C5 RNA

Address correspondence to Richard J. Maraia, Intramural Research Program, National Institute of Child Health and Human Development, National Institutes of Health, 31 Center Drive, Room 2A25, Bethesda, MD 20892-2426, USA. Tel: 301-402-3567; Fax: 301-480-6863; E-mail: maraiar@mail.nih.gov

*Present address: The Korean Intellectual Property Office, Government Complex-Deajeon, Dunsan-dong, Seo-gu, DeaJeon Metropolitan City 302-701.

**Present address: Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, 3333 Green Bay Rd., North Chicago, IL 60064, USA.

Gene Expression, Vol. 14, pp. 83-100
1052-2166/07 $90.00 + .00
E-ISSN 1555-3884
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Disruption of ZAS3 in Mice Alters NF-kB and AP-1 DNA Binding and T-Cell Development

Carl E. Allen,1,2 John Richards,3 Natarajan Muthusamy,4 Herbert Auer,1 Yang Liu,3 Michael L. Robinson,1 John A. Barnard,1 and Lai-Chu Wu2,4,5

1Department of Pediatrics and Center for Cell and Developmental Biology, Columbus Children's Research Institute, Columbus, OH 43205, USA
22Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, OH 43210, USA
3Department of Pathology, The Ohio State University, Columbus, OH 43210, USA
4Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA
5Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA

The large zinc finger proteins, ZAS, regulate the transcription of a variety of genes involved in cell growth, development, and metastasis. They also function in the signal transduction of the TGF-b and TNF-a pathways. However, the endogenous protein of a representative member, ZAS3, is rapidly degraded in primary lymphocytes, which limits the determination of its physiological function in vitro. Therefore, we have generated mice with targeted disruption of ZAS3. Oligonucleotide-based microarray analyses revealed subtle but consistent differences in the expression of genes, many of which are associated with receptor or signal transduction activities between ZAS3+/+ and ZAS3-/- thymi. Gel mobility shift assays showed altered DNA binding activities of NF-kB and AP-1 proteins in ZAS3-deficient tissues, including the thymus. Lymphocyte analysis suggested a subtle but broad function of ZAS3 in regulating T-cell development and activation. In CD3+ ZAS3-/- thymocytes, the CD4/CD8 ratio was decreased and CD69 expression was decreased. In peripheral CD4+ ZAS3-/- lymphocytes we observed an increased number of memory T cells.

Key words: Transcription; Signal transduction; Gene regulation; Zinc finger proteins; T-cell development

Address correspondence to Lai-Chu Wu, Department of Internal Medicine, The Ohio State University, 480 Medical Center Drive, Columbus, OH 43210, USA. Tel: 614-293-3042; Fax: 614-293-5631; E-mail: laichu.wu@osumc.edu

Gene Expression, Vol. 14, pp. 101-115
1052-2166/07 $90.00 + .00
E-ISSN 1555-3884
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Stem Cell Gene Expression Changes Induced Specifically by Mutated K-ras

Feijun Luo,1 Rifat Hamoudi,1 David G. Brooks,1 Charles E. Patek,2 and Mark J. Arends1

1Department of Pathology, Addenbrooke's Hospital, Hills Road, University of Cambridge, Cambridge, CB2 2QQ, UK
2Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK

K-Ras proteins transduce signals from membrane-bound receptors via multiple downstream effector pathways and thereby regulate fundamental stem cell processes that affect neoplasia, including proliferation, apoptosis, and differentiation, but their contribution to tumourigenesis is unclear. Because cancers develop from stem cells, we set out to determine the characteristic changes in gene expression brought about by mutated K-ras (without interference from normal K-ras) in otherwise normal stem cells. cDNA microarrays were used to analyze gene expression profiles comparing wild-type murine embryonic stem (ES) cells with K-rasVal12 expressing ES cells (previously made null for both endogenous K-ras alleles and transfected with K-rasVal12, with valine for glycine at codon 12). K-rasVal12 was expressed at 1.2-fold normal K-ras levels and produced transcripts for both activated K-Ras4A and 4B isoforms. The array expression data were confirmed by real-time quantitative PCR analysis of selected genes expressed both in the K-rasVal12 expressing ES cells (R = 0.91 with array data) and in the normal intestinal tissues of K-rasVal12 transgenic mice (R = 0.91 with array data). Changes in gene expression were correlated with the effects of K-rasVal12 expression on ES cells of enhancing self-renewal in an undifferentiated state, increasing susceptibility to DNA damage-induced apoptosis, and increased proliferation. These expression data may explain, at least in part, some neoplasia-related aspects of the phenotypic changes brought about in this ES cell line by mutated K-ras, in that upregulation of cell growth-related proteins and DNA-associated proteins is consistent with increased proliferation; upregulation of certain apoptosis-related proteins is consistent with a greater susceptibility to DNA damage-induced apoptosis; and downregulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with a less differentiated phenotype.

Key words: K-ras; Gene expression; Stem cell; Apoptosis; Differentiation

Address correspondence to Dr. Mark J. Arends, Department of Pathology, University of Cambridge, Box 235, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK. Tel: 01223 217813; Fax: 01223 216980; E-mail: mja40@cam.ac.uk

Gene Expression, Vol. 14, pp. 117-127
1052-2166/07 $90.00 + .00
E-ISSN 1555-3884
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

EST-Based Identification of Genes Expressed in Perch (Perca fluviatilis, L.)

Federica Rossi, Valentina Chini, Anna Giulia Cattaneo, Giovanni Bernardini, Genciana Terova, Marco Saroglia, and Rosalba Gornati

Dipartimento di Biotecnologie e Scienze Molecolari, Università dell'Insubria, I-21100 Varese, Italy

Perch are promising species for freshwater aquaculture and, differently from other fish, have not yet been domesticated through artificial selection; therefore, they show a wide genetic variability that is undesirable for aquaculture. In addition to the more traditional methods of aquatic biotechnology, the most recently developed molecular biological techniques can augment the overall efficiency of aquaculture. To help these new molecular techniques find their place in the everyday management of fish farming, we should make an effort to reduce the gap in genomic resources that separates farming species from "model organisms." We performed single-pass sequencing on 1237 randomly selected clones from a perch liver cDNA expression library, 350 clones of a brain-minus-liver, and 639 clones of a liver-minus-brain subtraction library. The sequences were deposited in the NCBI Expressed Sequence Tags database (www.ncbi.nlm.nih.gov/projects/dbEST). In the three libraries we identified 108, 46, and 104 genes, respectively. EST cataloguing and profiling of perch will provide a basis for functional genomic research in this species, but will also promote studies in comparative and environmental genomics, for identifying polymorphic markers that are useful, for example, to survey the disease resistance of fish and for discovering of new molecular markers of exposure. Using these genomic resources, micro- and macroarrays can be produced that will give immediate and practical benefits in the field of aquaculture, allowing early diagnosis of the fish conditions and helping in the generation of new mechanistic data on the nature of fish responses to different farming conditions.

Key words: cDNA library; Hepcidin; Complement C3; Aquaculture; Marine biotechnology; Transcriptome analysis

Address correspondence to Rosalba Gornati, DBSM, Università dell'Insubria, 3 Via Dunant, I-21100 Varese, Italy. Tel: ++390332421444; Fax: ++390332421500; E-mail: rosalba.gornati@uninsubria.it