A recombineering pipeline for functional genomics applied to Caenorhabditis elegans

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.     

A multi-step strategy for BAC recombineering of large DNA fragments

Recombineering techniques have been developed to modify bacterial artificial chromosomes (BACs) via bacterial homologous recombination systems, simplifying the molecular manipulations of large DNA constructs. However, precise modifications of a DNA fragment larger than 2-3 kb by recombineering remain a difficult task, due to technical limitations in PCR amplification and purification of large DNA fragments. Here, we describe a new recombineering strategy for the replacement of large DNA fragments using the commonly utilized phage/Red recombination host system. This approach involved the introduction of rare restriction enzyme sites and positive selection markers into the ends of a large DNA fragment, followed by its release from the donor BAC construct and integration into an acceptor BAC. We have successfully employed this method to precisely swap a number of large DNA fragments ranging from 6 to 40 kb between two BAC constructs. Our results demonstrated that this new strategy was highly effective in the manipulations of large genomic DNA fragments and therefore should advance the conventional BAC recombineering technology to the next level.     

A CRISPR/dCas9-assisted system to clone toxic genes in Escherichia coli

BackgroundThe cloning of toxic genes in E. coli requires strict regulation of the target genes' leaky expression. Many methods facilitating successful gene cloning of toxic genes are commonly exploited, but the applicability is severely limited.MethodsA CRISPR/dCas9-assisted system was used to clone toxic genes in E. coli. The plasmid-based and genome-integrated systems were designed in this study. And the green fluorescent protein characterization system was used to test the repression efficiency of the two systems.ResultsWe optimized the plasmid-based CRISPR/dCas9-assisted repression system via testing different sgRNAs targeting the Ptrc promoter and achieved inhibition efficiency up to 64.8%. The genome-integrated system represented 35.9% decreased GFP expression and was successfully employed to cloned four toxic genes from Corynebacterium glutamicum in E. coli.ConclusionsUsing this method, we successfully cloned four C. glutamicum-derived toxic genes that had been failed to clone in conventional ways. The CRISPR/dCas9-assisted gene cloning method was a promising tool to facilitate precise gene cloning of different origins in E. coli.General significanceThis system will be useful for cloning toxic genes from different origins in E. coli, and can accelerate the related research of gene characterization and heterologous expression in the metagenomic era.     

A new recombineering system for Photorhabdus and Xenorhabdus

Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.     

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.     

Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii

As we had found previously that thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-kappaB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-kappaB-alpha (IkappaB-alpha) was decreased and the nuclear translocation of NF-kappaB was increased. The thapsigargin-induced activation of NF-kappaB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-kappaB. Lipopolysaccharide (LPS)-induced activation of NF-kappaB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IkappaB-alpha. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-kappaB pathway.     

Use of conversion adaptors to clone antigen genes in lambda gt11

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.     

A new licence to clone

Many countries are still grappling with the issue of therapeutic cloning of human cells as the UN tackles this issue alongside reproductive cloning, but in the UK a new programme looking at the causes of motor neuron disease has recently been sanctioned. Michael Gross reports.     

Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.     

莱茵衣藻长链酰基辅酶A合成酶(LACS)同源基因的生物信息学分析

长链酰基辅酶A合成酶(LACS)能催化游离的脂肪酸形成酰基辅酶A硫脂,在油脂合成及降解途径中起着重要的作用.研究在莱茵衣藻(Chlamydomonas reinhardtii)中发现两个LACS基因,将其命名为CrLACS1和CrLACS2.生物信息学分析表明CrLACS1和CrLACS2在蛋白的理化性质及结构上都具有较高的相似性,而系统进化树分析显示CrLACS1和CrLACS2处于不同分枝,预测亚细胞定位也不相同.综合结果表明,CrLACS1和CrLACS2具有相似的结构,催化相同的反应,但具有不同的生物学功能.他们可能参与油脂代谢的不同途径:CrLACS1参与油脂的合成途径,而CrLACS2参与油脂的降解途径.     

Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts

The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.     

Nuclear ribosomal RNA genes and algal phylogeny--the Chlamydomonas example

The nuclear ribosomal RNA genes (rDNA) of Chlamydomonas reinhardtii, C. moewusii and C. eugametos were examined with restriction endonuclease fragment and direct rRNA sequencing analyses. These comparative molecular data confirm similarity between C. moewusii and C. eugametos, and dissimilarity between the strains and C. reinhardtii. For C. moewusii and C. eugametos, the fragment analysis of digests with 16 (six base pair recognition site) restriction endonucleases revealed either no or minor differences. These minor differences appear to be confined to length and site variation in the rapidly evolving intergenic spacer region of the algal rDNA repeat unit. In contrast, patterns of digests for C. reinhardtii were completely different from those of C. moewusii and C. eugametos for all enzymes tested. Over two regions of the 18S ribosomal RNA (spanning approx. 300 bases) in C. moewusii and C. eugametos, we observed three possible base substitutions and no insertion/deletion events. The same comparison between C. reinhardtii and C. moewusii (or C. eugametos) revealed 31 base substitutions and eight insertion/deletion events. Overall, the rDNA comparisons support the proposed conspecificity of C. moewusii and C. eugametos, as well as the hypothesis that intraspecific variation in the algal ribosomal RNA coding region is minimal and that comparisons of rDNA sequences at higher taxonomic levels can be useful indicators of algal phylogeny. The degree of difference in the sequences of the 18S coding region between C. reinhardtii and C. moewusii or C. eugametos is comparable to that between an angiosperm and Equisetum and may reflect an ancient divergence between two species in one algal genus.(ABSTRACT TRUNCATED AT 250 WORDS)