Genomic structure of the Dictyostelium rac family. 73.1 kb genomic DNA has been assembled in 15 contigs. Contigs are aligned at the start codon of rac genes. Bars indicate coding sequences and are extended beyond the start and stop codon to indicate untranslated but transcribed sequences, when supported by EST clones. Genes other than rac genes have been named according to their position relative to the rac gene of the corresponding contig (see Table S1 in Supplementary Material for a description). Lines indicate intergenic and intron sequences. When more than one termination site was identified, the most distant has been depicted. A frameshift caused by a 5 nt deletion in ΨracK is indicated by #. Two introns of racB are not covered by genomic clones (indicated by slashes). Less than half of the protein ORFs identified correspond to genes whose function is known in Dictyostelium or in other species. Accession numbers for the contigs: rac1a, AF309947; rac1b, AF310884; rac1c, AF310885; racA, AF310886; racB, AF310887; racC, AF310888; racD, AF310889; racE, AF310890; racF1, AF037042; racF2, AF310892; racG, AF310893; racH, AF310894; racI, AF310895; racJ, AF310896; racL, AF310897. The contig for racE extends sequence U41222. The contig for racH overlaps with sequence AF104350, which lacks the racH gene. The contig for racF2 overlaps with sequence AF019108.

Patterns of mRNA expression of rac genes during the developmental cycle. Dictyostelium cells were allowed to develop on nitrocellulose filters and total RNA was extracted at the indicated time points. Northern blots containing 20 µg total RNA for each time point were hybridised with the corresponding ³²P-radiolabelled probe. Two different blots were used; for racA, racE, racI, racJ and racL the same blot was used; for racG and racH a separate blot was used. The lane corresponding to 4 h was slightly overloaded. The blots were stripped and tested for absence of radioactive signals prior to hybridisation with a new probe. The arrows mark the size of the transcripts in kilobases. The developmental stages corresponding to the time points are depicted below. The transcripts of some of the rac genes analysed here, most notably racJ and racL, are present after the aggregation stage. Very long exposure times were required in order to detect racA, racI, racJ and racL.

Alternative splicing of racC. Comparison of genomic and EST sequences allowed the identification of extensive alternative splicing in the racC gene. At least three promoters and two terminators account for the presence of diverse EST species (only informative EST clones are presented).

Multiple alignment of Dictyostelium Rho-related proteins. Sequences were aligned with ClustalX and default parameters, and the output file was subsequently edited manually. RacA has been truncated at residue 202 to include only the GTPase domain. The deduced amino acid sequence of ΨRacK is also included for comparison (## denotes the deletion of two residues). Dashes indicate gaps introduced for optimal alignment. Residues identical or similar in at least 60% of the sequences are boxed in black or grey, respectively. Secondary structure elements as determined for human RhoA are indicated at the top of the aligned sequences: A1–5, α-helices; B1–6, β-strands; H1–3, 3₁₀-helices. Functional regions are indicated above the structural elements and consensus sequences for some of them are indicated below the aligned sequences. Symbols denote residues involved in phosphate binding (triangles), magnesium ion binding (squares) and nucleotide binding or stabilisation (circles). The Rho insert is boxed.

(Opposite and above) Multiple alignment and sequence comparison of RhoBTB proteins. (A) Multiple alignment and domain structure of RhoBTB proteins. A search of GenBank databases with the 400 residues long C-terminal part of DdRacA led to the identification of the complete sequence of one orthologue in Drosophila and three in human. Proteins of this novel subfamily are composed of a GTPase domain, a proline-rich region, two BTB domains (the first one divided into two subdomains by intervening sequences; dashed line) and a C-terminal domain of unknown function. Note that only HsRhoBTB3 ends with a prenylation signal. Sequences were aligned with ClustalX. Dashes indicate gaps introduced for optimal alignment. Residues identical or similar in at least three sequences appear in a black or grey background, respectively. Accession numbers are as follows: DmRhoBTB, AF217287; HsRhoBTB1, KIAA0740; HsRhoBTB3, KIAA0717; HsRhoBTB3, KIAA0878. (B) Comparison of DdRacA with other RhoBTB proteins. The numbers in each corresponding-colour region represent the percent similarity of each major domain of RacA to any of the other RhoBTB proteins. For the first BTB domain both subdomains have been calculated separately.

Phylogenetic tree of the Rho family. Representative species for which fairly complete sets of Rho proteins are available have been selected. Besides the 15 D.discoideum (Dd) Rho proteins, we also retrieved for this study sequences of six Rho proteins from E.histolytica (Eh), six from S.cerevisiae (Sc), 13 from A.thaliana (At), five from C.elegans (Ce), seven from D.melanogaster (Dm), six from G.gallus (Gg) and 19 from H.sapiens (Hs). Amino acid sequences of the GTPase core were aligned and a bootstrapped unrooted phylogenetic tree was constructed using the neighbour joining method as described in Materials and Methods. A consensus tree obtained with the parsimony method yielded comparable results. The innermost node of each branch supported by >50% bootstraps has been marked with a circle. The scale bar indicates 10% divergence. Dictyostelium Rho-related proteins are highlighted in black. Well-defined subfamilies have been marked on the right, using dashed lines to include closely related members not clearly supported by bootstrapping. Except for DdRac1a/1b/1c, RacF1/F2 and, to a lesser extent, RacB and RacA, which can be grouped in the Rac subfamily, neither of the additional Dictyostelium Rho-related proteins fall into any of the other subfamilies. Note that the DdRacA GTPase domain is more closely related to Rac proteins than to RhoBTB proteins, where RacA actually belongs. The closest relatives of DdRacC and DdRacE are EhRacC and AtU88402, respectively.

Analysis of intron positions in Dictyostelium rac genes. (A) Locations of introns relative to regions encoding structural and functional elements in the rac genes. Only introns placed in coding regions are shown. There are no introns beyond helix H3. Positions of introns are depicted as triangles. Introns residing in the same codon but at different locations are discriminated by their shading. Positions shared by two or more genes have been labelled with circled letters. (B) Evolutionary relationships among some Dictyostelium rac genes. The tree is based on the analysis of shared intron positions combined with analyses of protein sequences. Nodes represent duplication events or, for racA, fusion with the BTB domain-containing C-terminal moiety. Letters at the branches denote acquired (+) or lost (–) introns. The nomenclature for the intron positions is as in (A). Branch lengths are not proportional to evolutionary distance. The Dictyostelium rac family probably arose by a complex process of duplications and acquisition and loss of introns.