Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas.

Acceleration of lymphomagenesis in oncogene-bearing transgenic mice by slow-transforming retroviruses has proven a valuable tool in identifying cooperating oncogenes. We have modified this protocol to search for genes that can collaborate effectively with the transgene in later stages of tumor development. Propagation of tumors induced by Moloney murine leukemia virus (M-MuLV) in E mu-Pim1 or H2-K-myc transgenic mice by transplantation to syngeneic hosts permitted proviral tagging of 'progression' genes. Molecular cloning of common proviral insertion sites that were detected preferentially in transplanted tumors led to the identification of a novel gene, designated Frat1. The initial selection for integrations near Frat1 occurs in primary tumor cells that have already acquired proviruses in other common insertion sites, yielding primary lymphomas that contain only a minor fraction of tumor cells with an activated Frat1 allele. Transplantation of such primary lymphomas allows for a further expansion of tumor cell clones carrying a proviral insertion near Frat1, resulting in detectable Frat1 rearrangements in 17% of the transplanted E mu-Pim1 tumors and 30% of the transplanted H2-K-myc tumors, respectively. We have cloned and sequenced both the mouse Frat1 gene and its human counterpart. The proteins encoded by Frat1 and FRAT1 are highly homologous and their functions are thus far unknown. Tumor cell lines with high expression of Myc and Pim1 acquired an additional selective advantage in vivo upon infection with a Frat1-IRES-lacZ retrovirus, thus underscoring the role of Frat1 in tumor progression, and the ability of Frat1 to collaborate with Pim1 and Myc in lymphomagenesis.     

Separation of a New Deoxyribonucleic Acid Polymerase from Vaccinia-infected HeLa Cells

HeLa cell deoxyribonucleic acid (DNA) polymerase was purified about 100-fold by sequential column chromatography on phosphocellulose, hydroxylapatite, and Bio Rex 70. A new form of DNA polymerase found in vaccinia virus-infected cells was separated from HeLa DNA polymerase by chromatography on diethylamino-ethyl cellulose. The new form was also purified approximately 100-fold in the same manner as the HeLa DNA polymerase. In addition to chromatographic differences, the two enzymes differed with regard to primer response, relative activity at high pH, inactivation by heat and p-chloromercuribenzoate, and inhibition by vaccinia antiserum.     

Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G₂-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin''s intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Molecularly cloned c-mos(rat) is biologically active.

A unique rat cellular gene, c-mos(rat), homologous to the transforming sequences, v-mos, of Moloney murine sarcoma virus (M-MSV) was detected by hybridization to a v-mos specific probe. The c-mos(rat) gene was cloned together with its flanking sequences in an 11-kbp EcoRI DNA fragment inserted in vector Charon 4A. Two probes were used to investigate the position and orientation of c-mos(rat) in the clone examined ( D3e ), namely pMSV -31 which contains the sequences specific for the transforming sequences of M-MSV and pCS-1 which harbors 0.5 kbp of 5'-terminal sequences of c-mos(mouse) as well as 0.7 kbp of its flanking sequences. After ligation of a restriction fragment of clone D3e containing c-mos(rat) to a fragment containing the long terminal repeat of M-MSV and transfection of the DNA onto rat cells, we detected foci of transformed cells, thus showing that c-mos(rat) is biologically active. Using DNA framents derived from clone D3e , we studied the conservation of c-mos and of its flanking sequences in several species. c-mos(rat) as well as some of its flanking sequences appeared to be highly conserved in the species studied.     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.     

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.