Cloning and characterization of a Dictyostelium gene encoding a small GTPase of the Rab11 family

Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism. J. Cell. Biochem. 70:29–37, 1998. © 1998 Wiley-Liss, inc.     

The amphioxus rab GDP-dissociation inhibitor (GDI) gene is neural-specific: implications for the evolution of chordate rab GDI genes.

The rab GDP-dissociation inhibitor (rab GDI) proteins are involved in the regulation of vesicle-mediated cellular transport. We isolated the amphioxus rab GDI gene, analyzed its expression during amphioxus development, and performed a phylogenetic analysis of the rab GDI family. In contrast to the two major rab GDI forms in mammals, the alpha and beta forms, there is only one rab GDI isoform in amphioxus. Our analysis indicates that the occurrence of the alpha and beta forms of rab GDI preceded the divergence of lineages leading to birds and mammals, and that the amphioxus rab GDI may have evolved directly from the common ancestor of both forms. While the mammalian rab GDI beta-genes are ubiquitously expressed, the rab GDI alpha genes are predominantly expressed in neural tissues. The expression analysis of the amphioxus rab GDI gene shows predominantly neural expression similar to that of the mammalian rab GDI alpha form, suggesting that the ancestral expression pattern of chordate rab GDI was neural. In addition, the chicken rab GDI beta-like gene also shows neural-specific expression, which indicates that the neural expression was retained in both early postduplication alpha and beta isoforms and that a novel function associated with ubiquitous expression may have evolved uniquely in mammals. These results reveal a likely scenario of functional divergence of the rab GDI genes after duplication of the ancestral gene. A similar pattern of evolution, in which one of the duplicated genes retained a role similar to that of the ancestral one while other genes were recruited into novel roles, was also observed in the analysis of chordate Otx and hedgehog genes. In the rab GDI, hedgehog, and Otx gene families, the gene retaining the ancestral role shows a lower rate of sequence evolution than its counterpart, which was recruited for a novel function.     

Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe.

The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.     

Rho GTPase signaling in Dictyostelium discoideum: insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.     

Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.     

Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.     

Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discoideum.

The cysteine proteinase 1 and 2 mRNA sequences of Dictyostelium discoideum encode proteins with a high degree of homology to plant and animal sulphydryl proteinases. The two mRNA sequences are co-ordinate in their regulation, both being first expressed late during cellular aggregation, prematurely induced in response to exogenous cAMP and several-fold enriched in prestalk over prespore cells. The two proteins are considerably diverged, with only 43% overall homology but all residues known to be important in catalysis are conserved and both contain a hydrophobic leader peptide which forms part of an N-terminal domain of just over 100 amino acids not found in the mature form of known cysteine proteinases. We have determined the sequence organization of both genes and find differences both in the number and position of introns. The close co-regulation of these two genes suggests that they may play a common role in Dictyostelium development, presumably in the autodigestion of cellular protein which occurs during differentiation. However, the low degree of sequence homology and major differences in gene organization indicate that they have undergone a considerable period of separate evolution and that they may differ in their precise function.     

The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.     

斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。