Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase

Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases

bHLH Transcription factors regulate organ morphogenesis via activation of an ADAMTS protease in C. elegans

AbstractThe ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases plays important roles in animal development and pathogenesis. However, transcriptional regulation of ADAMTS proteins during development remains largely unexplored. Here we show that basic helix–loop–helix (bHLH) transcription factors regulate the expression of an ADAMTS protease that is required for gonad development in Caenorhabditis elegans. Mutations in the gene mig-24 cause shortened and swollen gonad arms due to a defect in gonadal leader cell migration, although leader cell specification appears to occur normally. The MIG-24 protein is a bHLH transcription factor of the Achaete–Scute family and is specifically expressed in gonadal leader cells. MIG-24 can physically interact with HLH-2, an E/Daughterless family bHLH transcription factor and bind the promoter region of gon-1, which encodes an ADAMTS protease required for gonadal leader cell migration. Mutations or RNA interference of mig-24 and hlh-2 severely impaired gon-1 expression and forced expression of GON-1 in leader cells in mig-24 mutants partially rescued the gonadal elongation defect. We propose that, unlike most previously characterized Achaete–Scute transcription factors that are involved in cell fate specification, MIG-24 acts with HLH-2 in specified cells to control cell migration by activating the expression of the GON-1 ADAMTS protease

Regulation of checkpoint kinases through dynamic interaction with Crb2

ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3–Chk1 and Rad3–Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases