Worm Breeder's Gazette 14(5): 34 (February 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | Department of Physiology II, Kobe University School of Medicine, Kobe 650, Japan |
| 2 | Department of Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK73104 |
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.
Fig.1. Predicted structures of newly found Let-60 effectors. Open and
filled triangles represent inserted Tc1 at the forward and reversed
orientations, respectively. All insertions were actually found in
introns.
References:
[1] Ponting, C.P. and Benjamin, D.R., A novel family of Ras-binding
domains, TIBS 21, 422-425, 1996.
[2] Kariya, K., Yamawaki-Kataoka, Y., Shibatohge, M., Okada, T., Shima,
F., Watari, Y., Goshima, M., Akasaka, K., Suzuki, N. and Kataoka, T.,
WBG 14, 78-79, 1995.