两栖动物线粒体基因组结构特征分析

对2007年6月13日以前公布于GenBank上的78种两栖动物的线粒体基因组全序列进行了总结、比较和分析。78种基因组中基因的数量从35~41个不等;根据基因的数量、种类及其排列顺序的差别将其分为22种基因组类型,其进而聚为3组,其中类型4为两栖纲与其它脊椎动物的常见类型,类型8为两栖纲中现生3个目的公有类型。与类型4比较,其余21种线粒体基因组类型涉及基因变动的基因共有18个,其中变动比较多的是tRNA基因,移位、增多和缺失的发生频率都较大,而蛋白编码基因比较稳定,主要是移位。78种两栖动物中,蚓螈目的线粒体基因组均小于18000bps,多数在15000~16000bps;有尾目和无尾目均大于16000bps,其中有尾目多数在16000~17000bps,无尾目的多数在17000~18000bps。     

A thyroid peroxidase (TPO) mutation in dogs reveals a canid-specific gene structure

Congenital hypothyroidism with goiter (CHG) occurring as an autosomal recessive disorder is typically due to a defect of thyroid hormone synthesis (aka dyshormonogenesis). Thyroid peroxidase (TPO) is a multifunctional, heme-containing enzyme whose activity is required, and several inactivating TPO mutations causing CHG in humans and dogs have been described. Recently, two half-sib Spanish water dog (SWD) pups were diagnosed with CHG based on clinical signs, endocrine testing, and thyroid histology. TPO enzyme activity was absent, and immuno-cross-reactive TPO was undetectable in affected-dog thyroid tissue. A single guanosine insertion was observed in the first exon of the affected-dog TPO cDNA at a site not previously thought to be within the coding sequence. The insertion allele segregated with the deduced disease allele in the SWD breed and was not observed in unrelated dogs of various breeds. Comparison of the insertion site (an 8-nt poly-G tract) with the orthologous sequences of other mammalian reference genomes revealed that the octa-G tract obliterated the intron 1 splice acceptor site and the exon 2 translation initiation codon found at that position in other species. An in-frame ATG in strong Kozak consensus context was observed in the normal dog sequence 12 codons 5′ of the usual mammalian start site, suggesting that dogs have lost the noncoding exon 1 demonstrated in human and mouse. A survey of TPO sequences in other carnivore species indicates that the poly-G tract necessitating an alternative translation initiation site is a canid-specific feature.     

ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data

Background

Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.

Results

ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.

Conclusions

ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.     

I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3′ overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.     

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

Cleavage site determinants in the mammalian polyadenylation signal.

Using a series of position and nucleotide variants of the SV40 late polyadenylation signal we have demonstrated that three sequence elements determine the precise site of 3-end cleavage in mammalian pre-mRNAs: an upstream AAUAAA element, a down-stream U-rich element consisting of five nucleotides, at least four of which are uridine, and a nucleotide preference at the site of cleavage in the order A > U > C >> G. Cleavage occurs no closer than 11 bases, but no further than 23 bases from the AAUAAA element. The downstream U-rich element is usually located 10-30 bases from the cleavage site. The relative position of the AAUAAA and the U-rich elements define the approximate region within a 13 base domain in which cleavage will occur. The exact position of cleavage is then determined by the local nucleotide sequence in the order of preference noted above. This model accounts for nearly three quarters of polyadenylation signals surveyed and is consistent with previous experimental observations.     

Genetic diversity in cultured and wild marine cyanomyoviruses reveals phosphorus stress as a strong selective agent

Viruses that infect marine cyanobacteria–cyanophages–often carry genes with orthologs in their cyanobacterial hosts, and the frequency of these genes can vary with habitat. To explore habitat-influenced genomic diversity more deeply, we used the genomes of 28 cultured cyanomyoviruses as references to identify phage genes in three ocean habitats. Only about 6–11% of genes were consistently observed in the wild, revealing high gene-content variability in these populations. Numerous shared phage/host genes differed in relative frequency between environments, including genes related to phosphorous acquisition, photorespiration, photosynthesis and the pentose phosphate pathway, possibly reflecting environmental selection for these genes in cyanomyovirus genomes. The strongest emergent signal was related to phosphorous availability; a higher fraction of genomes from relatively low-phosphorus environments–the Sargasso and Mediterranean Sea–contained host-like phosphorus assimilation genes compared with those from the N. Pacific Gyre. These genes are known to be upregulated when the host is phosphorous starved, a response mediated by pho box motifs in phage genomes that bind a host regulatory protein. Eleven cyanomyoviruses have predicted pho boxes upstream of the phosphate-acquisition genes pstS and phoA; eight of these have a conserved cyanophage-specific gene (PhCOG173) between the pho box and pstS. PhCOG173 is also found upstream of other shared phage/host genes, suggesting a unique regulatory role. Pho boxes are found upstream of high light-inducible (hli) genes in cyanomyoviruses, suggesting that this motif may have a broader role than regulating phosphorous-stress responses in infected hosts or that these hlis are involved in the phosphorous-stress response.     

Novel Protein-Protein Contacts Facilitate mRNA 3′-Processing Signal Recognition by Rna15 and Hrp1

Precise 3′-end processing of mRNA is essential for correct gene expression, yet in yeast, 3′-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3′-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3′-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.     

Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity

Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes.     

Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster

Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes.