Rapid expression screening of Caenorhabditis elegans homeobox open reading frames using a two-step polymerase chain reaction promoter-gfp reporter construction technique.

In this paper a description is given of the expression pattern of the Caenorhabditis elegans homeobox gene ceh-38 using GFP reporter constructs, which were generated using a two-step polymerase chain reaction (PCR) procedure. This method allows fast analysis of genes of interest by looking at their expression in vivo using their putative promoter region to control the expression of a reporter gene. In this case the method was applied to screen C. elegans homeobox-containing genes to identify those that are expressed in the head and nervous system. The C. elegans genome project has made rapid progress, and more than 79 megabases of genomic data with several thousand open reading frames are available. This information can be used to design primers from putative promoter regions, which are amplified using long-range PCR. The long-range PCR product is then directly joined to the vector in a long-range Fill-in PCR. Since many genome projects are advancing rapidly, this approach should also be applicable for other model systems, and the method lends itself to automation, since no gel-purification steps are necessary. ceh-38 is a member of the ONECUT class of homeobox genes. Expression of ceh-38 starts during embryogenesis. In larvae and adults, expression was seen in many different types of tissues, such as the pharynx, gut, hypodermis and many nerve cells.

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation.

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing.