Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

A phenotype-based screen for embryonic lethal mutations in the mouse

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.

Predicted highly expressed and putative alien genes of Deinococcus radiodurans and implications for resistance to ionizing radiation damage

Predicted highly expressed (PHX) and putative alien genes determined by codon usages are characterized in the genome of Deinococcus radiodurans (strain R1). Deinococcus radiodurans (DEIRA) can survive very high doses of ionizing radiation that are lethal to virtually all other organisms. It has been argued that DEIRA is endowed with enhanced repair systems that provide protection and stability. However, predicted expression levels of DNA repair proteins with the exception of RecA tend to be low and do not distinguish DEIRA from other prokaryotes. In this paper, the capability of DEIRA to resist extreme doses of ionizing and UV radiation is attributed to an unusually high number of PHX chaperone/degradation, protease, and detoxification genes. Explicitly, compared with all current complete prokaryotic genomes, DEIRA contains the greatest number of PHX detoxification and protease proteins. Other sources of environmental protection against severe conditions of UV radiation, desiccation, and thermal effects for DEIRA are the several S-layer (surface structure) PHX proteins. The top PHX gene of DEIRA is the multifunctional tricarboxylic acid (TCA) gene aconitase, which, apart from its role in respiration, also alerts the cell to oxidative damage.

An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro.

We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways.

Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.