Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes

Herpesviruses are common but important pathogens in humans and animals. These viruses have large complex genomes encoding genes with diverse functions in different phases of their life cycle and associated diseases. In the last decade, genomes of herpesviruses cloned as infectious bacterial artificial chromosomes (BACs) have become powerful tools for delineating the functions of viral genes and understanding the pathogenesis of their associated diseases. Here we review the history of herpesviral genetics and recent advances in methods for cloning herpesviral genomes as infectious BACs.     

A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes

Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.     

Analysis of herpesvirus host specificity determinants using herpesvirus genomes as bacterial artificial chromosomes

Almost all mammalian alphaherpesviruses can grow in cells derived from several types of animals in vitro . However, FHV-1 can only infect feline cell lines. For this reason, FHV-1 should be a good model to investigate species barriers to herpesviruses in vivo . To apply bacterial mutagenesis of FHV-1, we cloned the FHV-1 genome as a BAC. Using λ and flp recombinations, we introduced a monomeric red fluorescence protein into the C-terminus of glycoprotein D. Although GFP in the constructed recombinant FHV-1, a transfectant of the bacmid of FHV-1 that possessed the GFP, acted in non-feline cell lines, the virus could not enter non-feline cell lines, demonstrating that the host specificity of FHV-1 was restricted in an early step of infection. The host range of canine herpesvirus is limited to dogs in vitro and in vivo ; it cannot enter non-canine cell lines as a result of infection but the GFP is active by transfection, revealing the same result that the restriction step is at an early stage of infection. These results suggest the possibility of breaking species barriers of FHV-1 and CHV by modifying the gene(s) that act at the early stage of infection.     

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

Recent advances in bacterial cellulose production

Bacterial cellulose (BC), which is produced by some bacteria, has unique structural, functional, physical and chemical properties. Thus, the mass production of BC for industrial application has recently attracted considerable attention. To enhance BC production, two aspects have been considered, namely, the engineering and genetic viewpoints. The former includes the reactor design, nutrient selection, process control and optimization; and the latter the cloning of the BC synthesis gene, and the genetic modification of the speculated genes for higher BC production. In this review, recent advances in BC production from the two viewpoints mentioned above are described, mainly using the bacteriumGluconacetobacter xylinus.     

Oncogenicity of virulent Marek's disease virus cloned as bacterial artificial chromosomes

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U(S)2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.     

Cloning of infectious adeno-associated virus genomes in bacterial plasmids

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.     

Modified bacterial artificial chromosomes for zebrafish transgenesis

Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.     

Recent structural advances in bacterial chemotaxis signalling

Bacterial chemosensory arrays have served as a model system for in-situ structure determination, clearly cataloguing the improvement of cryo-electron tomography (cryoET) over the past decade. In recent years, this has culminated in an accurately fitted atomistic model for the full-length core signalling unit (CSU) and numerous insights into the function of the transmembrane receptors responsible for signal transduction. Here, we review the achievements of the latest structural advances in bacterial chemosensory arrays and the developments which have made such advances possible.     

海洋细菌生态学的若干前沿课题及其研究新进展

海洋细菌在海洋生态系统中的重要作用随着微食物环的提出被深入认识和充分肯定。本文概述了海洋细菌在微食物环中的重要生态作用及微食物环的研究进展,海洋细菌在碳的生物地球化学循环中的重要性,海洋细菌的活性及其群落结构与功能,分析了藻际环境特性和藻际微生物在赤潮多发海域的生态作用,提出了我国海洋细菌生态学研究的若干新思考与新任务,强调了基于"以菌治藻"的新理念,开展针对于赤潮灾害防除的"微食物环-赤潮-关键微生物菌群"耦合互作这一重要科学问题研究的必要性及紧迫性。     

Recent advances in bacterial heme protein biochemistry

Recent progress in genetics, fed by the burst in genome sequence data, has led to the identification of a host of novel bacterial heme proteins that are now being characterized in structural and mechanistic terms. The following short review highlights very recent work with bacterial heme proteins involved in the uptake, biosynthesis, degradation, and use of heme in respiration and sensing.     

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.     

植物抗病基因克隆研究进展

随着分子生物学及其相关技术的飞速发展,人们对植物与病原微生物相互作用的分子机制了解得越来越透彻。本文对植物过敏性反应和系统获得抗性作了简要概述,并着重讨论了植物抗病基因克隆的进展,涉及到转座子标签技术、定位克隆技术、染色体步行、染色体登陆等方法和策略,归纳了克隆到的植物抗病基因及其产物结构,概述了这些基因产物所共有的特点,并简要介绍了植物抗病基因工程的进展。     

Rapid modification of bacterial artificial chromosomes by ET-recombination

We present a method to modify bacterial artificial chromosomes (BACs) resident in their host strain. The method is based on homologous recombination by ET-cloning. We have successfully modified BACs at two distinct loci by recombination with a PCR product containing homology arms of 50 nt. The procedure we describe here is rapid, was found to work with high efficiency and should be applicable to any BAC modification desired.     

Rapid engineering of bacterial artificial chromosomes using oligonucleotides

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90-260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple.     

Smallest angiosperm genomes found in lentibulariaceae, with chromosomes of bacterial size

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.     

Recent advances with recombinant bacterial vaccine vectors

Bacille Calmette-Guerin (BCG), Listeria monocytogenes, Salmonellae and Shigellae have shown promise as vaccine vectors in experimental animal models. Although disappointing results in humans and non-human primates stalled the development of this vaccination strategy, interest in this approach was reinvigorated recently by the development of bacterial DNA-vaccine-vectors. The purpose of this review is to highlight the strengths and weaknesses of bacterial vaccine vectors, and to discuss the future prospects of these vaccine delivery systems.     

High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes

Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.     

Point mutation of bacterial artificial chromosomes by ET recombination

Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.