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The accumulation of information resulting from recent advances in genome sequencing and bioinformatics has allowed comparison of gene sequences across numerous species. However, sequence comparisons have revealed a significant number of uncharacterized genes, including many “orphan” genes. Orphan genes are putative open reading frames (ORFs) that have no resemblance to known protein coding sequences. Since there are no identified homologs to these orphan genes in other species, it is unknown whether these hypothetical sequences are actually expressed. The authors used the Access RT-PCR System(a) (Cat.# A1280) to analyze 25 strictly orphan genes from E. coli. They tested two growth conditions, exponential growth and stationary phase. Of 25 orphan genes tested, 19 transcripts were identified with two found to be expressed in only one of the two growth conditions.
Alimi, J.P., Poirot, O., Lopez, F., and Claverie, J.M.* (2000) Genome Res. 10, 959-966.
Structural and Genetic Information Laboratory, Marseille, France.
*To whom correspondence should be addressed.
Studies of the Y chromosome continue to identify putative protein coding genes. One of these genes, RBM (RNA Binding Motif), encodes a germ cell-restricted nuclear protein. Y chromosome RBM genes are found in all mammals and are implicated in spermatogenesis, although the function of these genes is not currently known. In germ cells, the RNA-binding motif protein (RBMp) is distributed throughout the nucleoplasm, and in distinct nuclear structures that also contain pre-mRNA splicing factors. This result suggests that the RBMp may interact with components of the splicing machinery. The authors have used several different techniques for assessing protein-protein interactions between RBMp and the SR family of splicing factors. At least two SR family proteins directly interact with RBMp, based on two-hybrid analysis and GST pulldown assays. Splicing assays done in vitro suggest that the RBMp is a cell-type-specific regulator of alternative pre-mRNA splicing. The TNT® Quick Coupled Transcription/Translation System (Cat.# L2080) was used to produce protein for GST pulldown assays. The transcription/translation templates were produced by PCR, and the amplification primers used were designed to add codons for additional methionine residues at the 3’ end of the coding region, to increase the 35S labeling of the translated proteins.
Elliott, D.J.1*, Bourgeois, C.F.2, Klink, A.1, Stévenin, J.2, and Cooke, H.J.1 (2000) Proc. Natl. Acad. Sci. USA 97, 5717-5722.
1Medical Research Council Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, 2Institut De Génétique et de Biologie Moléculaire et Cellulaire, Université Louis Pasteur, Illkirch, CU de Strasbourg, France.
*To whom correspondence should be addressed. E-mail: davide@hgu.mrc.ac.uk
Hox genes code for transcription factors that contain a highly conserved homeodomain. The homeodomain is a DNA binding motif consisting of three alpha helices and an N-terminal arm. In vitro, homeodomain proteins tend to recognize very similar DNA sequences. Some evidence exists that the N-terminal arm of the SCR (sex combs reduced) homeodomain helps to determine the specificity of DNA binding in vivo. The mechanism of the specificity is unclear, but this paper has looked at changes in phosphorylation of the N-terminal arm of SCR, which are vital to the proper function of SCR in developing Drosophila embryos. They have shown that the SCR homeodomain interacts with the Drosophila homolog of Phosphatase 2A (dPP2A, B´) using a yeast two hybrid system. Promega’s Serine-Threonine phosphatase assay system was used to monitor dephosphorylation by the catalytic subunit of PP2A of two phosphopeptides derived from the SCR homeodomain. Putative PKA and PKC phosphorylation sites in the N-terminal arm were tested for phosphorylation using purified PKA and PKC enzymes on biotinylated substrates, showing that the N-terminal arm is a target for PKA phosphorylation. Wildtype and two mutant proteins mimicking the active dephosphorylated state (SCR(AA)) or the inactive phosphorylated state (SCR(DD)) were expressed using the TNT® Quick Coupled Transcription/Translation System (Cat.# L2080). Gel shift assays using these proteins showed that both the wildtype SCR protein and the SCR(AA) protein were able to bind to a BS2 HOX binding site, but that the SCR(DD) protein was unable to bind. Overall, these studies suggest that the binding and function of the SCR protein depend on interactions with the dPP2A,B´ protein.
Berry, M. and Gehring, W.* (2000) EMBO J. 19, 2946-2957.
Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
*To whom correspondence should be addressed. E-mail: walter.gehring@unibas.ch
When cytochrome C is released from mitochondria, an Apaf-1/caspase-9 apoptosome is formed which induces the apoptotic protease cascade by activation of procaspase-3. This paper studies the effect of Hsp90 on the formation of the active complex. Hsp90 is induced in response to many different apoptotic stimuli, and overexpression of Hsp90 has been shown to result in unregulated proliferation, suggesting a role for the protein in cell survival. Affinity chromatography showed that the Hsp90 protein associates with cytochrome c, and further immunodepletion studies showed that reduction of Hsp90 amounts in cell extracts inhibits the cytochrome c-mediated activation of caspase-3, by direct interaction between the Apaf-1 protein and Hsp90. To further study the role of Hsp90 in activation of caspase-3, L929 cells were transfected to express Hsp90. The transfected cells were then treated with staurosporine to induce apoptosis. The CaspACE™ Assay System, Colorimetric (Cat.# G7220), was used to analyze treated and untreated cell extracts for caspase-3 activity levels. These experiments show that Hsp90 inhibits stress-induced activation of caspase-3 and demonstrate a mechanism by which Hsp90 overexpression can protect cells from apoptosis.
Pandey, P.1, Saleh, A.2, Nakazawa, A.1, Kumar, S.1, Srinivasula S.M.2, Kumar, V.1, Weichselbaum, R.3, Nalin, C.4, Alnemri E.S.2, Kufe, D.1, and Kharbanda, S.1* (2000) EMBO J. 19, 4310-4322
1Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, 2Thomas Jefferson University, Kimmel Cancer Institute, Philadelphia, PA 19107, 3Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637 and 4Oncology Research, Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA
*To whom correspondence should be addressed. E-mail: surender_kharbanda@dfci.harvard.edu