[
Worm Breeder's Gazette,
1990]
A number of improvements have been made in the gm automated DNA sequence analysis system: (1) The ratio of AT-containing to CG-containing dinucleotides has been added as a test for introns. This works better than AT frequency alone in C. elegans.(2) A branch site consensus sequence or an enhanced dinucleotide ratio can be required as an additional test on introns. (3) Predicted amino-acid sequence files are generated in a format appropriate for input to the Dana-Farber motif-identification program plsearch. (4) A graphic interface based on X-windows, version 11 is available as an option. (5) The complete analysis algorithm is significantly faster than the previous version. (6) A greedy model evaluation algorithm is available as an option. This algorithm generates the longest, non-overlapping models that cover a sequence and is much faster than the complete analysis algorithm. The program has been tested on Sun3, Sun4 and VAX machines running Unix (Ultrix on the VAX). Results for a series of tests run on a Sun 4/60 are shown in the table. [See Figure 1] gm can be run remotely on our machine, using the Internet. To do this, telnet to haywire.nmsu.edu, and logon as gm_guest with password gmuser. Read the README file for information on running gm. We are also distributing gm as C source code to nonprofit laboratories, either via anonymous ftp to haywire.nmsu.edu, or on tape. If you would like to receive gm on tape, send a 1/4' cartridge or 1/2' reel tape to: Chris Fields, Box 30001/3CRL, New Mexico State University, Las Cruces, New Mexico 88003-0001, USA; Telephone (505) 646-2848.
[
Worm Breeder's Gazette,
1997]
Mammalian Ras proteins regulate multiple effector molecules. They include Raf family (Raf-1, B-Raf, A-Raf), RalGDS family (RalGDS, RGL, RGF) and AF-6. C. elegans genetic studies contributed a lot to understanding of Ras function, especially regulation of Raf-MEK-MAPK pathway. However, studies have been limited to Raf and its downstream. Our long-term goal is to understand all pathways regulated by C. elegans Let-60. Here we report progress of our search for new Let-60 effectors and genetic study of their functions. We first screened for Let-60-binding proteins by using the yeast two-hybrid system. lACT-RB2, a random-primed mixed-stage C. elegans cDNA library was constructed in lACT that express cDNA clones fused to GAL4 activator domain. After conversion to the plasmid form (pACT-RB2), it was co-transformed into the test yeast strain CG-1945 with a plasmid pAS2-1-Let-60V12, expressing an activated Let-60 mutant as a fusion with GAL4 DNA-binding domain. After screening of approximately 106 transformants, 88 colonies were identified as both His+ and LacZ+. Plasmid clones were recovered and confirmed to confer both His+ and LacZ+ phenotypes when co-transformed again with pAS2-1-Let-60V12 but not when co-transformed with pAS2-1 vector. Inserts of confirmed clones were characterized by DNA sequencing. The sequencing data were used to identify cosmid clones containing overlapping genomic sequences by the BLASTN search (thanks to the C. elegans genome sequence project). The cDNA sequences together with the cosmid sequences were also used to identify EST clones containing overlapping sequences (thanks to Dr. Y. Kohara at NIG, Japan). When corresponding cosmid was not found, the cDNA sequences or the EST sequences were used to identify homologous proteins from the protein database by the BLASTX search. The clones comprised 6 classes. They included those encoding C. elegans Raf (Ce-Raf, 35 clones), C.elegans homologs of RalGDS (Ce-RalGDS, 29 clones), of AF-6 (Ce-AF-6, 6 clones), of Cdc25 (Ce-Cdc25, 10 clones) and of phospholipase Cb (Ce-PLCb, 2 clones), and those encoding other proteins (6 clones). Since lACT-RB2 represents 107 independent clones, this screen is not saturated yet. Predicted structures of newly found Let-60 effectors are shown in Fig. 1. We found Ce-RalGDS (F28B4.2) initially by simply performing a BLASTP search with mouse RalGDSA. Two-hybrid clones contained regions encoding the middle Cdc25 homology domain, the C-terminal Ras-interacting domain and a termination codon. pACT-RB2 plasmid library was used as a template to obtain sequence for N-terminal portion by PCR with a SL1 splice leader primer and a cDNA-specific primer. Clones for Ce-AF-6 contained the initiation codon and regions encoding the N-terminal Ras-interacting domain and the middle GLGF/DHR motif (no cosmid). Sequence for the C-terminal portion and the termination codon was found in a EST clone. Clones representing Ce-Cdc25 (T14G10.2/K04D7) encoded a Cdc25 homology domain most similar to that of Cdc25Mm/RasGRF. But regions around this domain were divergent from Cdc25Mm/RasGRF. It is possible that Ce-Cdc25 is a downstream effector of Ras, not a homolog of Cdc25Mm/RasGRF, an upstream regulator of Ras. Clones for Ce-PLCb (F31B12.1) encoded the X, Y and C2 domains found in the human counterpart. The Ras-binding domain was mapped to the C-terminal 300 amino acids by deletion analysis. F31B12.1 contains a very long N-terminal extension not found in human PLCb. But this prediction seems to be correct since PCR with a primer containing the predicted initiation codon and a downstream primer using the pACT-RB2 plasmid library gave a band with a predicted size. By computer analysis, Ponting and Benjamin recently proposed the existence of a family of Ras-associating domains (RA domain) including those of RalGDS and AF-6 [1]. Their list of proteins containing the RA consensus included Ce-RalGDS, Ce-Cdc25 and Ce-PLCb. However, Ce-AF-6 was not detected in their search, since it was not contained in any sequenced cosmids. Also, the list did not include Cdc25Mm/RasGRF, supporting the uniqueness of Ce-Cdc25. Search is in progress for mutant worms carrying Tc1-insertions within these genes. A PCR-based method was used to screen 108 plates, each started with ten MT3126 worms (thanks to the CGC). Two alleles each for Ce-RalGDS and Ce-PLCb were detected (we also found one allele for the previously reported Ach-1 gene[2]). After sib-selection, single worms were identified for all of them (thanks to Dr. Y. Andachi and the Japan C. elegans laboratory course at NIG). We are in the process of screening for worms carrying deletion of these genes by Tc1-excision.