基于5 993个核基因的被子植物系统发育关系研究

系统发育关系的构建对被子植物分类及进化研究非常重要。长期以来,被子植物系统发育的研究,大多使用质体基因、线粒体基因或少数保守的单拷贝核基因。该研究从已注释基因组或转录组中搜集88种被子植物(包含58目)的核基因集;通过对其进行同源基因聚类及去旁系同源基因,获得了5 993个一对一的直系同源基因家族(即对于每个基因家族,每种植物最多一条序列,最少包含50个物种);使用截取各种不同数目基因集的DNA或氨基酸序列,采用串联法(concatenation)和溯祖法(coalescence),共构建了20棵进化树。比较这些进化树,虽然大部分结果支持APG IV中描述的被子植物主要支系之间的关系[(真双子叶植物,单子叶植物),木兰类植物],但真双子叶植物内部各目分支的演化关系与APG IV有一个很大的不同,即认为檀香目和石竹目是蔷薇类植物的姊妹群。基于这些进化树,估算了被子植物各目分支的分化时间,结果表明被子植物的起源时间为237.78百万年前(95%置信区间为202.6～278.08),与主流观点认为的225百万年至240百万年前一致。以上结果为构建进化树提供了一种可行性策略,这种方法允许使用基因数目更多而计算速度更快。     

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.     

Mammalian mitochondrial nitric oxide synthase: characterization of a novel candidate

Recently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development.     

Cloning of a FLOWERING LOCUS T Ortholog in Wasabia japonica (Matsum)

A FLOWERING LOCUS T ortholog (WjFT) was identified in Wasabia japonica. Heterologous expression of WjFT remarkably promoted the flowering of Arabidopsis. The expression of WjFT was examined in field-grown wasabi in October and November of 2009, and February of 2010 because the differentiation of flower buds occurs in autumn in field-grown wasabi. No expression of WjFT was detected in October, it was slightly increased in November, and highly increased in February. WjFT might be useful for examining the flowering response of wasabi.     

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse

During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein--Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis.     

In vivo evidence for a regulatory role of the kinase activity of the <Emphasis Type="Italic">linotte</Emphasis>/<Emphasis Type="Italic">derailed</Emphasis> receptor tyrosine kinase,a <Emphasis Type="Italic">Drosophila</Emphasis> Ryk ortholog

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The linotte/derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, we have investigated the role of the cytoplasmic domain and the kinase activity of the linotte/derailed receptor tyrosine kinase in adult brain development. Our results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member.Edited by C DesplanEmmanuel Taillebourg and Caroline Moreau-Fauvarque contributed equally to this work     

Subfunctionalisation of paralogous genes and evolution of differential codon usage preferences: The showcase of polypyrimidine tract binding proteins

Gene paralogs are copies of an ancestral gene that appear after gene or full genome duplication. When two sister gene copies are maintained in the genome, redundancy may release certain evolutionary pressures, allowing one of them to access novel functions. Here, we focused our study on gene paralogs on the evolutionary history of the three polypyrimidine tract binding protein genes (PTBP) and their concurrent evolution of differential codon usage preferences (CUPrefs) in vertebrate species. PTBP1-3 show high identity at the amino acid level (up to 80%) but display strongly different nucleotide composition, divergent CUPrefs and, in humans and in many other vertebrates, distinct tissue-specific expression levels. Our phylogenetic inference results show that the duplication events leading to the three extant PTBP1-3 lineages predate the basal diversification within vertebrates, and genomic context analysis illustrates that local synteny has been well preserved over time for the three paralogs. We identify a distinct evolutionary pattern towards GC3-enriching substitutions in PTBP1, concurrent with enrichment in frequently used codons and with a tissue-wide expression. In contrast, PTBP2s are enriched in AT-ending, rare codons, and display tissue-restricted expression. As a result of this substitution trend, CUPrefs sharply differ between mammalian PTBP1s and the rest of PTBPs. Genomic context analysis suggests that GC3-rich nucleotide composition in PTBP1s is driven by local substitution processes, while the evidence in this direction is thinner for PTBP2-3. An actual lack of co-variation between the observed GC composition of PTBP2-3 and that of the surrounding non-coding genomic environment would raise an interrogation on the origin of CUPrefs, warranting further research on a putative tissue-specific translational selection. Finally, we communicate an intriguing trend for the use of the UUG-Leu codon, which matches the trends of AT-ending codons. Our results are compatible with a scenario in which a combination of directional mutation–selection processes would have differentially shaped CUPrefs of PTBPs in vertebrates: the observed GC-enrichment of PTBP1 in placental mammals may be linked to genomic location and to the strong and broad tissue-expression, while AT-enrichment of PTBP2 and PTBP3 would be associated with rare CUPrefs and thus, possibly to specialized spatio-temporal expression. Our interpretation is coherent with a gene subfunctionalisation process by differential expression regulation associated with the evolution of specific CUPrefs.