CSM News Electronic Edition Volume 8, number 1 January 4, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Mutational analysis of the role of Rap1 in regulating cytoskeletal function in Dictyostelium. P. J. Rebstein1, J. Cardelli3, G. Weeks1,2 and G.B. Spiegelman1,2*, Departments of Microbiology and Immunology1 and Medical Genetics2,University of British Columbia, Vancouver, V6T 1Z3, Canada and Department of Microbiology and Immunology3, Louisiana State University Medical Center, Shreveport, Louisiana, 71130, U.S.A. Exp Cell Res in press It was shown previously that increased expression of the ras-related rap1 gene in D. discoideum altered cell morphology (Rebstein et al., Dev. Genetics, 1993. 14: 347-355). Vegetative cells were more flattened and spread than normal cells and had increased F-actin near the cell periphery. In addition, transformants were inhibited in the rapid cell contraction that occurs upon refeeding starving cells with nutrient media. In this communication, we show that expression of Rap markedly reduces the azide contraction response and have used this assay, cell morphology and the refeeding response to analyze mutants of Rap1 generated by site directed mutagenesis. The substitution G12V, predicted to increase the proportion of protein binding GTP, did not alter the effect of Rap on cell morphology or on its ability to inhibit the contraction response to azide, but modestly enhanced the ability of Rap1 to inhibit cell rounding in response to nutrient media. The substitution S17N, predicted to restrict the protein to the GDP-bound state, did not produce the flattened cell morphology and abolished the inhibitory effect of Rap on cell contraction. These results are consistent with a requirement of GTP binding for the Rap induced effects. Transformants carrying the in Rap-S17N protein had a more polar morphology than the parental Ax-2 cells, suggesting the possibility that Rap-S17N interferes with the ability of endogenous Rap to regulate the cytoskeleton. Substitutions at amino acid 38, within the presumptive effector domain, reduced but did not abolish the effects of Rap1 on cell contraction while the substitution T61Q had no effect on Rap1 activity. Taken together the results suggest a regulatory role for Rap in Dictyostelium cytoskeletal function. -------------------------------------------------------------------- Dictyostelium discoideum myosin II: Characterisation of functional myosin motor fragments S. E. Kurzawa, D. J. Manstein=A7, and M. A. Geeves* Max-Planck-Institut fur Molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany; Max-Planck-Institut fur Medizinische Forschung, Jahnstr. 29, 69120 Heidelberg, Germany, and National Institute for Medical Research, the Ridgeway, Mill Hill, London NW7 1AA, U.K. Biochem. in press. The transient kinetic properties of the recombinant myosin head fragments M761 and M781, that both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781 and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behaviour of the myosin head. In contrast loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A:, Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two actinin repeats has very little effect on the behaviour of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts makes it an ideal construct for systematic studies of the structure, kinetics and function of the myosin motor. ---------------------------------------------------------------------- [Ed. Note: The following contribution, submitted by Bill Loomis, is for the benefit of the Dictyostelium community and will be published only in the CSM News.] Partial Analysis of a cDNA Library Prepared from Post-Aggregative Stage Dictyostelium discoideum Danny Fuller, Negin Iranfar, Diane Foster, Douglas Smith and William F. Loomis Developmental Gene Program, Center for Molecular Genetics, University of California San Diego, La Jolla, CA 92093 [no plans for publication] As part of an effort to characterize the genetic information of Dictyostelium discoideum, we have sequenced and analyzed 42 inserts carried in plasmids isolated from a lambda ZAP cDNA library. cDNAs were generated from polyA+ RNA isolated from an equal number of tipped aggregates (12 hours of development) and migrating slugs (48 hours of development). Forty-two plasmids were excised from randomly chosen plaques and digested with EcoRI such that the size of the inserts could be determined by gel electrophoresis. The average insert was about 1 kb but individual inserts ranged from 300 bp to 2.1 kb. A total of 45 kb of finished sequence was derived from sequencing on an ABI 377 machine using generic primers (T3 and T7) and synthesized internal primers. Ten of the inserts had no significant open reading frame (ORF). Many of these were very A/T rich and one (FC30) was derived from the large ribosomal RNA of mitochondria. Five of these inserts terminated in polyA. Of the remaining 32 cDNA clones, 14 were found to be derived from Dictyostelium genes that had been previously characterized and deposited in GenBank. The 19 new Dictyostelium genes were conceptually translated and compared to genes in the public data bases using the BLAST and FASTA programs. Twelve of the newly sequenced Dictyostelium slug cDNAs showed significant similarity to previously characterized genes of other organisms. In many cases the similarity is sufficiently high over an extended region that they are likely to have a common function. The individual clones and their homologs are given in Table 1. Where appropriate the sequences have been deposited in GenBank. The cDNA clones are available upon request. All but 2 of the cDNAs with ORFs appeared to extend to the 3' end since we could recognize the stop codon followed by a very high A/T stretch. At least a dozen cDNAs covered the complete open reading frame as judged by comparison to previously deposited sequences. In the cases where we encountered previously sequenced Dictyostelium genes we could accurately judge the amount of the gene that was carried in our cDNAs. The amount missing depended on the size of the gene. For instance, 12 kb was missing from the 14 kb dynein mRNA, while only 30 bp was missing from the 460 bp cofilin mRNA. Four of our clones appear to have resulted from the ligation of independent cDNAs prior to insertion such that two separate genes are represented in the same plasmid. For example, FC8 carries copies from the unlinked genes nagA and rapA. These were recognized as unrelated genes by the results from our BLAST analysis. Since the cDNA clones were chosen at random from the library, many are likely to come from the most highly abundant class of mRNAs. We encountered pspD (PL3) twice in our sample of 32 cDNAs (one was in a composite clone of two genes). However, no other gene gave multiple isolates. Nor did we encounter cDNAs from genes that are highly expressed in growing cells such as actin. It appears that by 12 hours of development, most abundant vegetative mRNAs have turned over. It is of interest that 14 of the 32 cDNAs were derived from previously characterized Dictyostelium genes. This frequency suggests that almost half of the abundant mRNAs present in the post-aggregative slug stages may have already been recognized. It is also interesting that only 7 of the new genes failed to show significant similarity to one or more genes in the public data bases. It appears that many of the abundant mRNAs have been well conserved during evolution. One of them, cdcD [the homolog of CDC48] has hardly changed in a billion years! While many of the results from our partial analysis of cDNAs present in post-aggregative cells are only applicable to the specific library from which they were derived, it is likely that other cDNAs libraries prepared from the slug stage of Dictyostelium will carry the same sorts of genes in about the same proportions. Only when several thousand cDNAs have been isolated and sequenced will it be possible to make definitive statements concerning the mRNA complexity at various stages. While the sequences of newly encountered mRNAs may give hints as to their function, detailed genetic and biochemical studies will be necessary to fully document the role of each gene. The sequences with no ORFs are of interest. They make up about 20% of all the clones and may result from imperfect transcriptional control. As long as they are not translated, such RNAs would not be detrimental. The energy wasted in polymerizing such useless RNA would be a trivial component of the economy of a cell where most of the ATP is used in bulk movement. A cell is only as effcient as it needs to be and there may have been little selection for tighter control of transcription. This work was supported by the Developmental Gene Program [NICHD 30892]. Table 1 : Characterization of Random Slug cDNAs cDNA - locus Homolog Genbank/EMBL Accession FC1 - no ORF ---- na FC2 - no ORF ---- na FC3 - vilA villin U78754 FC4 - pspD Dicty pspD (PL3) U25144 _____________________________________________________________________ FC5 - rsc5 SEC14 U82515 FC6 - rsc6 none U82514 FC7 - no ORF ---- na FC8 - nagA/rapA Dicty nagA + rapA J04065 + X54291 FC9 - no ORF ---- na _____________________________________________________________________ FC10 - limA 3 "lim" domains U83086 FC11 - rsc11 YqkD (yeast ORF) U78758 FC12 - rsc12 none U83087 FC13 - pinA NIPP/dodo U78757 FC14 - profilin Dicty profilin X61581 ____________________________________________________________________ FC15 - rpgG ribosomal S3 U78756 FC16 - no ORF ---- na FC17 - no ORF ---- na FC18 - cofA Dicty cofilin D37981 FC19 - rsc19 none U82511 ____________________________________________________________________ FC20 - myoI Dicty myosin VII U83089 FC21 - rsc21 none U82512 FC22 - new none U82516 FC23 - mvpB Dicty mvpB Z37109 FC24 - csbA Dicty csbA M27588 ____________________________________________________________________ FC24 - prpD prpD (propionyl metab) U83088 FC25 - rsc25 none U82513 FC26 - dynein Dicty dynein P34036 FC27 - tubA Dicty tubA L13999 FC28 - no ORF ---- na ____________________________________________________________________ FC29 - no ORF ---- na FC30 - no ORF Dicty 23S mito rDNA na FC31 - no ORF ---- na FC32 - grfB RAS-GRF2 (CDC25) U82517 FC33 - secG SEC7 U78755 ____________________________________________________________________ FC34 - gtpA GTP binding protein U83248 FC35 - GAPDH Dicty GAPDH U55243 FC36 - dutA Dicty dutA D16417 FC37 - fpaA Dicty fpaA U18063 FC38 - ndkA Dicty ndkA P22887 _____________________________________________________________ FC39 - csbB Dicty csbB P16643 FC40 - symA sugar symport U78760 FC41 - cdcD cdc48 U83085 FC42 - ifdA eIF4A U78759 --------------------------------------------------------------------- [End CSM News, volume 8, number 1]