Identification of a novel adrenomedullin gene family in teleost fish

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.     

Knockdown of human MCM10 exhibits delayed and incomplete chromosome replication

In model organisms, MCM10 is required for forming the pre-initiation complex for initiation of chromosome replication and is involved in the elongation step. To investigate the role of MCM10 in human chromosome replication, we used small interfering RNA (siRNA) in MCM10-knockdown experiments and found that knockdown accumulated S and G2 phase cells. The chromosome replication of MCM10-knockdown cells was slowed during early and mid S phases, although Cdc45, Polα, and PCNA proteins were loaded onto the chromatin, and was aberrant during late S phase. Our results indicate that MCM10 is essential for the efficient elongation step of chromosome replication.     

Computational identification of a new SelD-like family that may participate in sulfur metabolism in hyperthermophilic sulfur-reducing archaea

Background

Selenium (Se) and sulfur (S) are closely related elements that exhibit similar chemical properties. Some genes related to S metabolism are also involved in Se utilization in many organisms. However, the evolutionary relationship between the two utilization traits is unclear.

Results

In this study, we conducted a comparative analysis of the selenophosphate synthetase (SelD) family, a key protein for all known Se utilization traits, in all sequenced archaea. Our search showed a very limited distribution of SelD and Se utilization in this kingdom. Interestingly, a SelD-like protein was detected in two orders of Crenarchaeota: Sulfolobales and Thermoproteales. Sequence and phylogenetic analyses revealed that SelD-like protein contains the same domain and conserved functional residues as those of SelD, and might be involved in S metabolism in these S-reducing organisms. Further genome-wide analysis of patterns of gene occurrence in different thermoproteales suggested that several genes, including SirA-like, Prx-like and adenylylsulfate reductase, were strongly related to SelD-like gene. Based on these findings, we proposed a simple model wherein SelD-like may play an important role in the biosynthesis of certain thiophosphate compound.

Conclusions

Our data suggest novel genes involved in S metabolism in hyperthermophilic S-reducing archaea, and may provide a new window for understanding the complex relationship between Se and S metabolism in archaea.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-908) contains supplementary material, which is available to authorized users.     

Identification and characterization of the novel centrosomal protein centlein

Centrosomes function as the major microtubule (MT)-organizing center. They are composed of a pair of centrioles which are surrounded by the pericentriolar material. Here, we describe the molecular characterization of a novel protein, named centlein (centrosomal protein). Centlein was a protein of 721 amino acids with a calculated molecular weight of 82,717 and possessed coiled-coil domains. Western blot analysis indicated that centlein was ubiquitously expressed. Endogenous centlein as well as enhanced green fluorescent protein-fused centlein was localized at centrosomes in interphase and mitosis. When centrosomes were isolated from cells treated with nocodazole, an MT-disrupting agent, centlein and the centrosomal protein, γ-tubulin, were enriched in the same fractions. These data indicate that centlein is a widespread centrosomal protein and that its association with centrosomes is independent of MTs. Centlein appeared to be enriched in the mother centriole in G1 phase, suggesting possible involvement of centlein in mother-centriole-related functions, such as duplication of centrioles and generation of primary cilia.     

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: “What have we learned from this vast amount of new genomic data?” Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity—even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.     

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.     

Conservation of novel <Emphasis Type="Italic">Mahya</Emphasis> genes shows the existence of neural functions common between Hymenoptera and Deuterostome

Honeybees have been shown to exhibit cognitive performances that were thought to be specific to some vertebrates. However, the molecular and cellular mechanisms of such cognitive abilities of the bees have not been understood. We have identified a novel gene, Mahya, expressed in the brain of the honeybee, Apis mellifera, and other Hymenoptera. Mahya orthologues are present in Deuterostomes but are absent or highly diverged in nematodes and, intriguingly, in two dipteran insects (fruit fly and mosquito) and Lepidoptera (silk moth). Mahya genes encode novel secretory proteins with a follistatin-like domain (Kazal-type serine/threonine protease inhibitor domain and EF-hand calcium-binding domain), two immunoglobulin domains, and a C-terminal novel domain. Honeybee Mahya is expressed in the mushroom bodies and antennal lobes of the brain. Zebra fish Mahya orthologues are expressed in the olfactory bulb, telencephalon, habenula, optic tectum, and cerebellum of the brain. Mouse Mahya orthologues are expressed in the olfactory bulb, hippocampus, and cerebellum of the brain. These results suggest that Mahya may be involved in learning and memory and in processing of sensory information in Hymenoptera and vertebrates. Furthermore, the limited existence of Mahya in the genomes of Hymenoptera and Deuterostomes supports the hypothesis that the genes typically represented by Mahya were lost or highly diverged during the evolution of the central nervous system of specific Bilaterian branches under the specific selection and subsequent adaptation associated with different ecologies and life histories.     

Pairwise interactions of the six human MCM protein subunits

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p. Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs. The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed. However, the architecture of the human MCM heterohexameric complex is still unknown. We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells. Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex. We also propose models for the structures of subcomplexes. Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.     

The genome of the diploid anuran <Emphasis Type="Italic">Xenopus tropicalis</Emphasis> contains a novel array of sarcoplasmic myosin heavy chain genes expressed in larval muscle and larynx

The sarcomeric myosin heavy chain (MyHC) proteins are a family of molecular motors responsible for the transduction of chemical energy into mechanical work in striated muscle. The vertebrate genome contains multiple copies of the MyHC gene, and expression of different isoforms correlates with differences in the physiological properties of muscle fibers. Most MyHC isoforms are found in two arrays, one containing the "fast-twitch" skeletal muscle isoforms and the other the "slow-twitch" or cardiac isoforms. To extend our understanding of MyHC evolution, we have examined the genome of the anuran Xenopus tropicalis. The X. tropicalis genome includes15 full-length MyHC genes organized in seven genomic locations. One unique array of MyHC genes is similar to the mammalian fast-skeletal array, but is not found in amniotes. The isoforms in this array are expressed during larval stages and in muscles of the adult larynx. Duplication of the fast-skeletal MyHC array appears to have led to expression divergence of muscle proteins in the larval and adult stages of the anuran life cycle. A striking similarity of gene order between regions flanking X. tropicalis MyHC arrays and human arrays was evident; genomic organization of MyHC isoforms may thus be highly conserved across tetrapods.     

Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

A novel centrosome-related protein CrpF46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein CrpF46 has an apparent molecular mass of ~60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-CrpF46 monoclonal antibody revealed that CrpF46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using CrpF46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense CrpF46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that CrpF46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism.     

Identification of a novel EGF-sensitive cell cycle checkpoint

The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.     

Quantitative determination of gene strand bias in prokaryotic genomes

Comparative genometrics of microorganisms is a relatively new area, in which genome properties are translated into numerical indexes. Such indexes can be used for a comprehensive and comparative analysis of microbial genomes, contributing to the understanding of their evolution. This work presents a new method for quantitative determination of gene strand bias in prokaryotic chromosomes, in which data transformation of gene position skew leads to a numerical index that can be applied to quantitative comparisons of genome organization. It was applied in the comparative analysis of 49 completely sequenced Firmicutes genomes, allowing the distinction of groups defined according to their patterns of gene strand preference. The resulting groups revealed that, regarding gene strand bias, reduced genomes are, in general, the more disordered among Firmicutes, while genomes of extremophile organisms comprehend those with the highest degree of genome organization in this phylum.     

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.     

Position-Associated GC Asymmetry of Gene Duplicates

It is well known that repositioning of a gene often exerts a strong impact on its own expression and whole development. Here we report the results of genome-wide analyses suggesting that repositioning may also radically change the evolutionary fate of gene duplicates. As an indicator of these changes, we used the GC content of gene pairs which originated by duplication. This indicator turned out to be duplicate-asymmetric, which means that genes in a pair differ significantly in GC content despite their apparent origin from a common ancestor. Such an asymmetry necessarily implies that after duplication two originally identical genes mutated in opposite directions—toward GC-rich and GC-poor content, respectively. In mammalian genomes, this trend is definitely associated with presumably methylated hypermutable CpG sites, and in a typical GC-asymmetric gene pair, its two member genes are embedded in GC-contrasting isochores. However, we unexpectedly found similar significant GC asymmetry in fish, fly, worm, and yeast. This means that neither methylation alone nor methylation in combination with isochores can be counted as a primary cause of the GC asymmetry; rather they represent specific realizations of some universal principle of genome evolution. Remarkably, genes from pairs with the greatest GC asymmetry tend to be on different chromosomes, suggesting that the mutational difference between gene duplicates is associated with translocation of a new gene to a different place in the genome, whereas GC symmetric pairs demonstrate the opposite tendency. A recently emerged extra gene copy is usually on the same chromosome as is its parent but quickly, by 0.05 substitution per synonymous site, either has perished or occupies a different chromosome. During this earliest posttranslocation period, the ratio of nonsynonymous/synonymous base substitutions is unusually high, suggesting a rapid adaptive evolution of novel functions. In a general context of evolution by gene duplication, our interpretation of this position-dependent GC asymmetry between duplicated genes is that evolution of redundant genes toward a new function has often been associated with their very early, postduplication repositioning in the genome, with a concomitant abrupt change in epigenetic control of tissue/stage-specific expression and an increase in the mutation rate. Of eight eukaryotic genomes studied, the most distinguished in this respect is the human genome.Reviewing Editor: Dr. Manyuan Long