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" ============== Announcement ============== Dicty95 ------- 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! ----------------------------------------------------------------------- GLOBAL SEARCH SERVICE ---------------------- 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 ------------------------------------------------------------------- =========== Abstracts =========== 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] -------------------------------------------------------------------- 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. --------------------------------------------------------------------- [End CSM-News, volume 5, number 1]