The complete genomic sequence analysis of human norovirus NVgz01 strain in Guangzhou

The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims in the GenBank, and the nomvirus genome was amplified by RT-PCR. The PCR- products were cloned into T vector and sequenced, and the genomic nucleotide sequences were analyzed with the programs CLUSTAL W/X, DNASTAR and RAT （Recombination Analysis Tool）. The NVgz01 strain genome is 7558 bp in length and encodes three open reading frames （GenBank accession No. is DQ369797）. The genomic sequences of NVgz01 were compared with those of nomvirus in GenBank, which revealed that the homology with genogroup Ⅱ ranges between 76%-90%, and genogroup Ⅰ between 43%-44%. The ORF1 region shared 94% and 88% identity with Mc37 and Famiington strains, respectively; the capsid region （ORF2） shared 65% and 94% identity with Mc37 and Farmington strains, respectively. Phylogenetic trees were reconstructed by the neighbor-joining method. Comparative complete sequence analysis of the NVgz01 with reported human norovirus genomic sequences revealed that this isolate belongs to genogroup Ⅱ . The ORF1 and ORF2 regions shared different identity with Mc37 and Fannington strains, suggesting NVgz01 could be a recombinant virus.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

DNA End Resection:Facts and Mechanisms

DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways in eukaryotic cells.A vital step in HR repair is DNA end resection,which generates a long 30single-stranded DNA（ss DNA） tail that can invade the homologous DNA strand.The generation of 30 ss DNA is not only essential for HR repair,but also promotes activation of the ataxia telangiectasia and Rad3-related protein（ATR）.Multiple factors,including the MRN/X complex,C-terminal-binding protein interacting protein（Ct IP）/Sae2,exonuclease 1（EXO1）,Bloom syndrome protein（BLM）/Sgs1,DNA2 nuclease/helicase,and several chromatin remodelers,cooperate to complete the process of end resection.Here we review the basic machinery involved in DNA end resection in eukaryotic cells.