Leukaemia disease genes: large-scale cloning and pathway predictions.

Retroviral insertional mutagenesis in BXH2 and AKXD recombinant inbred mice induces a high incidence of myeloid or B- and T-cell leukaemia and the proviral integration sites in the leukaemias provide powerful genetic tags for disease gene identification. Some of the disease genes identified by proviral tagging are also associated with human disease, validating this approach for human disease gene identification. Although many leukaemia disease genes have been identified over the years, many more remain to be cloned. Here we describe an inverse PCR (IPCR) method for proviral tagging that makes use of automated DNA sequencing and the genetic tools provided by the Mouse Genome Project, which increases the throughput for disease gene identification. We also use this IPCR method to clone and analyse more than 400 proviral integration sites from AKXD and BXH2 leukaemias and, in the process, identify more than 90 candidate disease genes. Some of these genes function in pathways already implicated in leukaemia, whereas others are likely to define new disease pathways. Our studies underscore the power of the mouse as a tool for gene discovery and functional genomics.

The yeast SEN1 gene is required for the processing of diverse RNA classes.

A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.

The essential yeast Tcp1 protein affects actin and microtubules.

Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and act1-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcp1p partially suppressed growth defects conferred by act1-1 and act1-4. Furthermore, Tcp1p was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcp1p is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components.