Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions

There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

Small membrane proteins found by comparative genomics and ribosome binding site models

The correct annotation of genes encoding the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short open reading frames have been confirmed to correspond to synthesized proteins. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16–50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated genes by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16–50 amino acids are synthesized. We summarize the properties of these small proteins; remarkably more than half of the proteins are predicted to be single‐transmembrane proteins, nine of which we show co‐fractionate with cell membranes.     

Isolation and sequencing of cDNA clones encoding the Dictyostelium discoideum 30,000-dalton actin-bundling protein

The Dictyostelium 30,000-dalton protein is a calcium-regulated actin filament-bundling protein which has been suggested to contribute to the structure and reorganization of filopodia and pseudopodia accompanying cell movements. cDNAs encoding this protein were isolated using antibody and oligonucleotide probes to screen cDNA libraries in phage lambda. The sequence of the cDNA predicts a protein of 295 amino acids with a molecular weight of 33,355. The sequence reveals two EF-hand calcium-binding regions that provide a structural explanation for calcium regulation of the activity of this protein. The putative calcium-binding region of the 30,000-dalton protein has similarity to sequences of other calcium-regulated actin-binding proteins such as alpha-actin and fimbrin. One region of the sequence with similarity to both Dictyostelium gelation factor (ABP 120) and fructose bisphosphate aldolase is a potential actin-binding sequence. A highly charged region of the protein is similar to a sequence in human cytovillin that is repeated eight times in chicken gizzard caldesmon. No strong homology to previously identified actin-binding sequences of other actin-binding proteins is apparent. Results from Southern blot experiments indicate that the 30,000-dalton protein is encoded by a single gene in the Dictyostelium genome.     

A developmentally regulated cysteine proteinase in Dictyostelium discoideum.

We have determined the sequence of a Dictyostelium mRNA encoding a protein with a high degree of homology to plant and animal cysteine proteinases. The degree of homology is highest in the region of the cysteine residue which is transiently acylated during peptide hydrolysis but all other residues known to be important in catalysis are also conserved. We have named this protein cysteine proteinase 1. There is a hydrophobic signal peptide of 18 amino acids and an additional 99 amino acids at the N terminus, which are not present in other cysteine proteases and which may be cleaved off during processing of the enzyme. There is a single copy of the gene in the Dictyostelium genome. The cysteine proteinase 1 mRNA is absent from growing cells and from cells isolated during the first 6 h of development but it constitutes approximately 1% of cellular mRNA by 10-12 h of development. During the development of Dictyostelium a major fraction of cellular protein is degraded to provide amino acids and a source of energy. Cysteine proteinase 1 may play a role in this auto-digestion.     

Manifestations of multicellularity: Dictyostelium reports in

The recent release of the Dictyostelium genome sequence is important because Dictyostelium has become a much-favoured model system for cell and developmental biologists. The sequence has revealed a remarkably high total number of approximately 12 500 genes, only a thousand fewer than are encoded by Drosophila. Previous protein-sequence comparisons suggested that Dictyostelium is evolutionarily closer to animals and fungi than to plants, and the global protein sequence comparison, now made possible by the genome sequence, confirms this. This review focuses on several classes of proteins that are shared by Dictyostelium and animals: a highly sophisticated array of microfilament components, a large family of G-protein-coupled receptors and a diverse set of SH2 domain-containing proteins. The presence of these proteins strengthens the case for a relatively close relationship with animals and extends the range of problems that can be addressed using Dictyostelium as a model organism.     

Widespread protein sequence similarities: origins of Escherichia coli genes.

To learn more about the evolutionary origins of Escherichia coli genes, we surveyed systematically for extended sequence similarities among the 1,264 amino acid sequences encoded by chromosomal genes of E. coli K-12 in SwissProt release 26 by using the FASTA program and imposing the following criteria: (i) alignment of segments at least 100 amino acids long and (ii) at least 20% amino acid identity. Altogether, 624 extended alignments meeting the two criteria were identified, corresponding to 577 protein sequences (45.6% of the 1,264 E. coli protein sequences) that had an extended alignment with at least one other E. coli protein sequence. To exclude alignments of questionable biological significance, we imposed a high threshold on the number of gaps allowed in each of the 624 extended alignments, giving us a subset of 464 proteins. The population of 464 alignments has the following characteristics expressed as median values of the group: 254 amino acids in the alignment, representing 86% of the length of the protein, 33% of the amino acids in the alignment being identical, and 1.1 gaps introduced per 100 amino acids of alignment. Where functions are known, nearly all pairs consist of functionally related proteins. This implies that the sequence similarity we detected has biological meaning and did not arise by chance. That a major fraction of E. coli proteins form extended alignments strongly suggests the predominance of duplication and divergence of ancestral genes in the evolution of E. coli genes. The range of degrees of similarity shows that some genes originated more recently than others. There is no evidence of genome doubling in the past, since map distances between genes of sequence-related proteins show no coherent pattern of favored separations.     

Evolutionary analyses of ABC transporters of Dictyostelium discoideum

The ABC superfamily of genes is one of the largest in the genomes of both bacteria and eukaryotes. The proteins encoded by these genes all carry a characteristic 200- to 250-amino-acid ATP-binding cassette that gives them their family name. In bacteria they are mostly involved in nutrient import, while in eukaryotes many are involved in export. Seven different families have been defined in eukaryotes based on sequence homology, domain topology, and function. While only 6 ABC genes in Dictyostelium discoideum have been studied in detail previously, sequences from the well-advanced Dictyostelium genome project have allowed us to recognize 68 members of this superfamily. They have been classified and compared to animal, plant, and fungal orthologs in order to gain some insight into the evolution of this superfamily. It appears that many of the genes inferred to have been present in the ancestor of the crown organisms duplicated extensively in some but not all phyla, while others were lost in one lineage or the other.     

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.     

MicroRNA enrichment among short 'ultraconserved' sequences in insects

MicroRNAs are short (approximately 22 nt) regulatory RNA molecules that play key roles in metazoan development and have been implicated in human disease. First discovered in Caenorhabditis elegans, over 2500 microRNAs have been isolated in metazoans and plants; it has been estimated that there may be more than a thousand microRNA genes in the human genome alone. Motivated by the experimental observation of strong conservation of the microRNA let-7 among nearly all metazoans, we developed a novel methodology to characterize the class of such strongly conserved sequences: we identified a non-redundant set of all sequences 20 to 29 bases in length that are shared among three insects: fly, bee and mosquito. Among the few hundred sequences greater than 20 bases in length are close to 40% of the 78 confirmed fly microRNAs, along with other non-coding RNAs and coding sequence.     

Dof基因家族调节植物生长发育功能的研究进展

Dof（DNA binding with one finger）蛋白是一类植物特异性转录因子,通常含有200～400个氨基酸和2个主要结构域。该家族成员的N 末端为高度保守的单锌指Dof结构域,具有与DNA和蛋白质相互作用的双重功能,其C末端的氨基酸序列则较为多变,是Dof蛋白重要的特异转录调控结构域。研究表明,Dof蛋白作为转录激活物或阻遏物参与了多方面的植物生长发育过程。随着基因组测序技术的发展,已有大量的Dof基因从植物基因组数据库中鉴定出来。该文对近年来国内外有关Dof基因家族的结构特点、全基因组鉴定、蛋白互作以及生物学功能等方面的研究进展进行综述,以期为Dof转录因子的深入研究提供参考。     

cDNA and gene sequence of Manduca sexta arylphorin, an aromatic amino acid-rich larval serum protein. Homology to arthropod hemocyanins

The serum (storage) proteins produced by insect larvae at the end of the feeding cycle are hexameric blood proteins with one or more type of subunits. The cDNA and gene structure of the aromatic amino acid-rich larval serum protein arylphorin from the tobacco hornworm, Manduca sexta, has been determined. In M. sexta arylphorin there are two subunits alpha and beta, which have 686 and 687 amino acids, respectively, and whose amino acid sequences are 68% identical. The two genes, separated by 7.1 kilobases of chromosomal DNA, are transcribed in the same direction. Based on the alignment of the amino acid sequence, the rate of nucleotide substitution between the two coding regions predicts that the two genes diverged about 100 million years ago. Both genes contain 5 exons and the upstream region contains a sequence, TGATAAA, which is similar to a sequence found in all other storage protein genes for which information is available. When the National Biomedical Research Foundation protein sequence data base was searched, it was found that the arylphorin subunits showed significant similarity to the arthropod hemocyanins, which are hexameric oxygen-carrying proteins. Based on the alignment of the sequence of M. sexta arylphorin and the hemocyanin from the spiny lobster (Panulirus interruptus), for which a 3.2 A structure has been determined, it was observed that the highest concentration of conserved residues were found in those regions of the sequence which are involved in subunit interactions in the hexameric protein. It is suggested that the insect storage proteins and the arthropod hemocyanins have evolved from a common ancestor.     

Tick-borne encephalitis virus: primary structure of DNA copies of the genes for strain 205 structural proteins

The nucleotide sequences of the cDNAs of the genes for the structural proteins C, preM, M and E of the tick-borne encephalitis viral strain 205 have been determined. The complete nucleotide sequence of the 5'-end nonstructural region of the viral genome has been studied for the first time. The difference in the amino acids sequences of the structural proteins from different strains (205, Sofiin and Neidorf) of the virus is discussed.