CSM News
Electronic Edition
Volume 5, number 2
July 8, 1995
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu.
Back issues of CSM-News, the CSM Reference database and other useful
information is available by anonymous ftp from worms.cmsbio.nwu.edu
[165.124.233.50], via Gopher at the same address, or by World Wide Web
at the URL "http://worms.cmsbio.nwu.edu/dicty.html"
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Announcement
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Dicty95
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Dicty95 in Dourdan France was an enormous success--a great meeting!
Our thanks go to the organizers Michel Sartre and Michel Veron. They
were ably assisted by Gerard Klein and members of their labs. For those
interested a few photos from the meeting have been posted on the web
page. Take a look!
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GLOBAL SEARCH SERVICE
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July 7, 1995
Discussions at the recent Dicty meeting in Dourdan resulted in a
plan to partially avoid the inefficiencies resulting from sequencing
developmental genes of Dictyostelium that are isolated in independent
laboratories. During the next few years the majority of such clones
will come from screens of REMI generated mutants and a detailed
proceedure has been agreed upon for these genes. However, genes
recovered by other means, such as PCR amplification from conserved
regions or two-hybrid screens, can be equally accommodated.
The basic plan is to have a central site where sequences as short
as 100 base pairs can be used to search all previously sequenced
fragments and genes so as to distinguish new genes from old
ones. These will include genes where the sequencing is in progress as
well as finished genes. There is sufficient information in 100 base
pairs, even with 5% error, that new sequences can be unequivocably
recognized as such. Any one, from any lab, can send in a sequence with
no further documentation and find out within days if anyone else has
encountered the same gene. The person submitting the sequence will be
informed whether it comes from a region previously submitted or is
novel. If the sequence has been previously submitted, both parties
will be informed of the fact. Novel sequences which have been
submitted for the search will be added to the file so that
subsequently submitted sequences can be checked against it. The file
will be kept confidential so as to encourage submission of sequences
rather than used to scan the sequences of others.
A Developmental Gene Program has been funded at UCSD in which Rick
Firtel, Doug Smith and I hope to help saturate the morphogenetic genes
that can be tagged by REMI. I have created a file containing the
sequences of all Dicty genes now in public data bases (GenBank etc)
together with all the sequences that we and others are in the process
of investigating. Sequences from regions flanking insertion sites
have been submitted by several laboratories including those of Gomer,
Kay, Kessin, Kuspa, Nellen, and Williams and in several cases we have
found that two labs have happened on the same gene. Further steps were
then worked out by consultations.
I am offering to serve as a GLOBAL SEARCH SERVICE for anyone who
wishes to know if their gene might have been encountered by someone
else. To keep the file meaningful, I request that sequences be
restricted to regions known from direct Southern data of wild-type and
mutant DNA to flank an insertion site of a REMI mutant. It will not be
necessary to describe the resulting phenotype (this protects the
privacy of the lab). Sequences from PCR or 2-hybrid screens can also
be submitted when accompanied by a brief description of the manner of
isolation. This total file will remain confidential.
As part of the Developmental Gene Program, a public access file,
DictyDB, will be made available on World Wide Web in a few months that
will contain all of the Dicty genes in public data bases as well as
the sequences of REMI tagged genes that have been finished and
confirmed. Newly isolated mutants will be made available to interested
parties.
It was also suggested at the meeting in Dourdan that another public
access file be generated in which sequences encountered in any manner
but of no great interest to the specific lab, such as "Space Clones"
found in PCR screens, cDNA clones that were isolated for no known
reason, regions flanking genes of interest but clearly the adjacent
gene etc. be deposited for the perusal of others who might find
something of interest. I would be willing to set up such an ATTIC of
left-over, throw-away sequences. There would be no restrictions or
responsibility for what is in the ATTIC, just the sequence and a note
of who submitted it. Having one centralized public access depository
would increase the usefulness of such partial sequences. ATTIC could
be scanned via the Web.
Submission of sequences either to GLOBAL SEARCH or ATTIC can be
made by e-mail to me, wloomis@UCSD.edu, or by disc. Since typing in
sequences is a bore, I cannot handle hard copy (typed) sequences. We
hope to stream-line the submission via the Web site this fall. Since
REMI can disrupt a given gene at sites up and down the ORF, it would
be useful to submit each kilobase of sequence to GLOBAL SEARCH as soon
as it is done so as to save others from sequencing a gene already
under analysis. In this way the file will grow as we go.
The whole scheme is open to suggestions and modifications since it
is aimed at making Dicty studies more efficient for all. I look
forward to receiving a lot of sequences from many different labs in
the near future.
Bill Loomis
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Abstracts
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Whole-mount in situ hybridization of Cell-type Specific mRNAs in
Dictyostelium
Ricardo Escalante and William F. Loomis
Department of Biology, University of California, San Diego, La Jolla
CA 92093
Devel. Biol. in press
ABSTRACT
We have been able to hybridize non-radioactive probes from
cell-type specific genes to fixed whole-mounts prepared at the mound,
slug and culminant stages of Dictyostelium development. The cellular
patterns of labelling with probes from the prespore gene, cotB, and
the prestalk genes, ecmA and ecmB, confirmed the patterns seen in
strains carrying reporter constructs in which the regulatory regions
of these genes drive b-galactosidase. This technique permits the
direct observation of protein synthetic capacity from characterized
genes without the need of generating transformed lines carrying
specific reporter constructs. Moreover, the pattern is not complicated
by previous developmental history of gene expression.
[Editor's Note: For those interested in this technique a copy of the
paper is available on the Web page]
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Dictyostelium vaults: disruption of the major proteins reveals growth and
morphological defects and uncovers a new associated protein.
Sanjay K. Vasu and Leonard H. Rome*
Department of Biological Chemistry and the Mental Retardation Research
Center, University of California School of Medicine, Los Angeles,
California 90024-1737.
(running title: disruption of mvpB. MvpB sequence data are available from
EMBL/GenBank/DDBJ under accession number Z37109. )
* to whom correspondence should be addressed
Tel: (310) 825-0709
FAX: (310) 206-5272
EMAIL: lrome@biochem.medsch.ucla.edu
J. Biol. Chem., in press
Abstract
Vaults are large cytoplasmic ribonucleoprotein particles which are
highly conserved in both morphology and protein composition. Protein
components of vaults isolated from Dictyostelium discoideum migrate on
SDS-PAGE gels as two bands, one at 94 kDa (MvpA) and the other at 92
kDa (MvpB). An MvpB cDNA clone was isolated from a Dictyostelium
expression library. MvpB shares 60% identity with MvpA at the amino
acid level. This cDNA has been used to disrupt the single mvpB gene in
both wild-type and mvpA- genetic backgrounds. Although the mvp- mutant
lines are viable, they show that loss of MvpA and/or MvpB interferes
with vault function sufficiently to impede growth under conditions of
nutritional stress. The resulting mutant cell lines reach stationary
phase in suspension culture at one third of the density of wild-type
cells. Ovoid structures, isolated from single mvp- mutant lines,
represent what remains of vaults in these cells. Similar ovoid
structures, isolated from the mvpA- mvpB- line, copurify with a newly
identified protein of MW 92 kDa (MvpC), which lacks cross-reactivity
with currently available anti-vault antibodies. Our results indicate
that the major vault proteins are necessary for optimal cell growth in
Dictyostelium, and reveal an unanticipated complexity in vault
composition.
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[End CSM-News, volume 5, number 1]