We have found that EEF1A2, the gene encoding protein elongation factor EEF1A2 (also known as eEF-1 alpha 2), is amplified in 25% of primary ovarian tumors and is highly expressed in approximately 30% of ovarian tumors and established cell lines. We have also demonstrated that EEF1A2 has oncogenic properties: it enhances focus formation, allows anchorage-independent growth and decreases the doubling time of rodent fibroblasts. In addition, EEF1A2 expression made NIH3T3 fibroblasts tumorigenic and increased the growth rate of ES-2 ovarian carcinoma cells xenografted in nude mice. Thus, EEF1A2 and the process of protein elongation are likely to be critical in the development of ovarian cancer.

Hydrogen-ion titrationswere conducted for hen-egg-white lysozyme in solutions of potassium chloride, over the range of pH 2.5-11.5 and for ionic strengths to 2. 0 M. The dependence oflysozyme's net proton charge, zP' on pH and ionic-strength in potassium-chloride solution is measured. From the ionic-strength dependence of zP' interactions of lysozynie with potassium and chloride ions are calculated using the molecular-thennodynamic theory of Fraaije and Lyklema 1. Lysozyme interacts preferentially with up to 12 chloride ions at pH 2.5. The observed dependence of ion-protein interactions on pH and ionic strength is explained in terms of electricdouble-layer theory. New experimental pKa data are reported for eleven ammo acids in potassium-chloride solutions of ionic strength to 3.0 M.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Polymerase eta (pol eta) is a low-fidelity DNA polymerase that is the product of the gene, POLH, associated with the human XP variant disorder in which there is an extremely high level of solar-induced skin carcinogenesis. The complete human genomic sequence spans about 40 kb containing 10 coding exons and a cDNA of 2.14 kb; exon I is untranslated and is 6 kb upstream from the first coding exon. Using bacterial artificial chromosomes (BACs), the gene was mapped to human chromosome band 6p21 and mouse band 17D. The gene is expressed in most tissues, except for very low or undetectable levels in peripheral lymphocytes, fetal spleen, and adult muscle; exon II, however, is frequently spliced out in normal cells and in almost half the transcripts in the testis and fetal liver. Expression of POLH in a multicopy episomal vector proved nonviable, suggesting that overexpression is toxic. Expression from chromosomally integrated linear copies using either an EF1-alpha or CMV promoter was functional, resulting in cell lines with low or high levels of pol eta protein, respectively. Point mutations in the center of the gene and in a C-terminal cysteine and deletion of exon II resulted in inactivation, but addition of a terminal 3 amino acid C-terminal tag, or an N- or C-terminal green fluorescent protein, had no effect on function. A low level of expression of pol eta eliminated hMre11 recombination and partially restored UV survival, but did not prevent UV-induced apoptosis, which required higher levels of expression. Polymerase eta is therefore involved in S-phase checkpoint and signal transduction pathways that lead to arrest in S, apoptosis, and recombination. In normal cells, the predominant mechanism of replication of UV damage involves pol eta-dependent bypass, and Mre11-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pol eta.