Cloned DNA Fragments Research Articles

A geminivirus crosses the monocot-dicot boundary and acts as a viral vector for gene silencing and genome editing

IntroductionMembers of the family Geminiviridae have been reported to infect either a monocot plant or a dicot plant, but not both. This study reports a geminivirus, Wheat Dwarf India Virus (WDIV), first identified in wheat, that is capable of infecting both monocot and dicot plants and acting as a viral vector. ObjectivesThis study was aimed at developing a broad host range viral vector system for reverse genetics and genome editing. MethodsHere we used a wheat isolate of WDIV and Ageratum yellow leaf curl betasatellite (AYLCB) for infectivity assays and vector development. We performed Agrobacterium-mediated inoculation of WDIV and AYLCB in wheat, oat, barley, corn, soybean, and tobacco. To examine the potential of WDIV to act as a viral vector, we modified the WDIV genome and cloned DNA fragments of the phytoene desaturase (PDS) genes from wheat and tobacco, separately. For gene editing experiments, tobacco lines expressing Cas9 were infiltrated with a WDIV-based vector carrying gRNA targeting the PDS gene. ResultsAbout 80 to 90% of plants inoculated with infectious clones of WDIV alone or WDIV together with AYLCB showed mild symptoms, whereas some plants showed more prominent symptoms. WDIV and AYLCB were detected in the systemically infected leaves of all the plant species. Furthermore, the inoculation of the WDIV vector carrying PDS fragments induced silencing of the PDS gene in both wheat and tobacco plants. We also observed high-efficiency genome editing in the Cas9-expressing tobacco plants that were inoculated with WDIV vector-carrying gRNA. ConclusionDetection of WDIV in naturally infected wheat, barley, and sugarcane in the field and its ability to systemically infect wheat, oat, barley, corn, soybean, and tobacco under laboratory conditions, provides compelling evidence that WDIV is the first geminivirus identified with the capability of infecting both monocot and dicot plant species. The wide host range of WDIV can be exploited for developing a single vector system for high-throughput genome editing in many plant species.

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors.

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.

Transcriptional Regulation of Liver-Type OATP1B3 (Lt-OATP1B3) and Cancer-Type OATP1B3 (Ct-OATP1B3) Studied in Hepatocyte-Derived and Colon Cancer-Derived Cell Lines

Due to alternative splicing, the SLCO1B3 gene encodes two protein variants; the hepatic uptake transporter liver-type OATP1B3 (Lt-OATP1B3) and the cancer-type OATP1B3 (Ct-OATP1B3) expressed in several cancerous tissues. There is limited information about the cell type-specific transcriptional regulation of both variants and about transcription factors regulating this differential expression. Therefore, we cloned DNA fragments from the promoter regions of the Lt-SLCO1B3 and the Ct-SLCO1B3 gene and investigated their luciferase activity in hepatocellular and colorectal cancer cell lines. Both promoters showed differences in their luciferase activity depending on the used cell lines. We identified the first 100 bp upstream of the transcriptional start site as the core promoter region of the Ct-SLCO1B3 gene. In silico predicted binding sites for the transcription factors ZKSCAN3, SOX9 and HNF1α localized within these fragments were further analyzed. The mutagenesis of the ZKSCAN3 binding site reduced the luciferase activity of the Ct-SLCO1B3 reporter gene construct in the colorectal cancer cell lines DLD1 and T84 to 29.9% and 14.3%, respectively. In contrast, using the liver-derived Hep3B cells, 71.6% residual activity could be measured. This indicates that the transcription factors ZKSCAN3 and SOX9 are important for the cell type-specific transcriptional regulation of the Ct-SLCO1B3 gene.

First Report of Cotton Leafroll Dwarf Virus Infecting Hibiscus syriacus in South Korea.

Three cotton leafroll dwarf virus (CLRDV; genus Polerovirus, family Solemoviridae) genotypes have recently been identified (Tabassum et al., 2021; Ramos-Sobrinho et al., 2021). This virus is widespread in the United States (Thiessen et al., 2020; Aboughanem-Sabanadzovic et al., 2019; Tabassum et al., 2020) and has also been reported to infect chickpea (Cicer arietinum) in Uzbekistan (Kumari et al., 2020). As well, CLRDV was detected from 23 weed species (16 families), including Hibiscus sabdariffa (Sedhain et al., 2021, Hagan et al., 2019). From June to September 2019, virus-like symptoms, including mild leaf stunting, crinkling, and deformation, were observed in multiple plants (n=14) in several provinces of South Korea (e-Xtra Table. 1). To characterize the associated viruses, pooled leaf tissues from all 14 samples were used for total RNA isolation, followed by paired-end high-throughput sequencing (HTS) on the Illumina NovaSeq 6000 platform (Macrogen, South Korea). A total of 614,424,952 trimmed and high-quality reads were assembled into 506,024 contigs using Trinity de novo transcriptome assembly. The resulting contigs were compared with viral sequences in the GenBank database using BLASTx analysis. Several viral contigs were identified, including cucumber mosaic virus, apple stem pitting virus, apple stem grooving virus, cherry virus A, and CLRDV. The CLRDV contig of 5,800 nucleotides (nt) with an average coverage of 307x shared 92.1% identity (query coverage: 99%) with the CLRDV isolate CN-S5 (KX588248). To confirm CLRDV infection and to obtain its complete genome sequence, total RNA was extracted from each of the 14 samples and used for reverse transcription (RT)-PCR with six overlapping sets of primers designed from the HTS contig (e-Xtra Table. 2). The expected product sizes were obtained only for the Hibiscus syriacus L. (family: Malvaceae) sample showing foliar mild vein clearing symptoms on the leaves (e-Xtra Fig.1). All RT-PCR products were cloned using the RBC TA Cloning vector (Taipei, Taiwan) and at least five positive clones per cloned DNA fragment were sequenced. The 5 and 3 termini sequences were determined as described previously (Zhao et al. 2016). The complete genome of CLRDV isolate SK (OK073299) was determined to be 5,862 nt and it shared 89-91% complete genome identity with 12 other CLRDV isolates based on pairwise comparisons (e-Xtra Table. 3). Maximum likelihood phylogenetic analysis based on the complete genome and P3-CP aa sequences showed that CLRDV-SK is more closely related CN-S5 (e-Xtra Fig. 2). In the fall of 2021, additional H. syriacus samples (n=18) with mild chlorosis, blistering and crinkling symptoms were collected from 2 provinces of South Korea and tested by RT-PCR using the primers: CLRDV-SK-101-120 For & CLRDV-SK-1021-1040 Rev targeting a region of the ORF0. Two of 18 samples (11.1%) tested positive for CLRDV. The 16 negative samples only showed symptoms of mild yellowing. The RT-PCR products were cloned and sequenced. In pairwise comparisons, the obtained sequences (OM339522-23) were 95.85% and 96.06% identical to the corresponding sequences of CLRDV isolate SK. This is the first report of CLRDV occurrence in H. syriacus in South Korea to the best of our knowledge. Our findings will assist further studies on the epidemiology and sustainable management of diseases caused by CLRDV. Acknowledgments This work was supported by IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries; Project No. AGC1762111), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. References Tabassum, A., et al., 2021. PloS One. 16: e0252523 Ramos-Sobrinho, R., et al., 2021. Viruses. 13:2230 Thiessen, L.D., et al. 2020. Plant Dis. 104:3275 Aboughanem-Sabanadzovic, N., et al. 2019. Plant Dis. 103:1798 Tabassum, A., et al. 2020. Microbiol. Res. Announce. 9:e00812-20 Kumari, S.G., et al. 2021. Plant Dis. 104:2532 Sedhain, N.P., et al. 2021. Crop protection 144:105604 Hagan, A., er al. 2019. Alabama Cooperative Extension System. ANR:2539 Zhao, F., et al. 2016. Arch. Virol. 161:2047 Conflict of interest The authors declare that they have no conflict of interest.

ОКИСЛИТЕЛЬНАЯ МОДИФИКАЦИЯ ДНК ПЛАЗМИД СПОСОБСТВУЕТ ИХ ПРОНИКНОВЕНИЮ В ЦИТОПЛАЗМУ МЕЗЕНХИМНЫХ СТВОЛОВЫХ КЛЕТОК ЧЕЛОВЕКА

It is well known that the action of ionizing radiation causes oxidative stress in the cells of the body and leads to the synthesis of reactive oxygen species, which lead to multiple damage to cellular DNA, including the formation of oxidized bases. In this paper, we consider the effect of radiation-induced oxidative modification of plasmid DNA on their penetration into human mesenchymal stem cells. To study the role of oxidative DNA modification in cell penetration, a genetic construct was created based on the pEGFP-C1 (pC1) vector, GenBankAccession: U55763, containing as an insert an artificially synthesized DNA fragment containing a poly-G region (p12G) serving as target for efficient oxidation of the cloned DNA fragment. As a "marker", the selected vector contains the EGFP fluorescent protein (GFP) gene. Flow cytometry and fluorescence microscopy showed that recombinant constructs based on the pEGFP vector containing duplicated poly-G regions and Gn repeats penetrate cells much more efficiently than the original pEGFP vector. Exposure to radiation at a dose of 50 cGy, which causes an increase in the level of 8-oxodG in plasmids after irradiation, leads to a more intense penetration of oxidized plasmids compared to a similar experiment without irradiation.

High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development

The concept of DNA transfer between bacteria was put forth by Griffith in 1928. During the dawn of molecular cloning of DNA in the 1980s, Hanahan described how the transformation of DNA plasmids into bacteria would allow for cloning of DNA fragments. Through this foundational work, it is widely taught that a typical transformation produces clonal bacterial colonies. Using low concentrations of several plasmids that encode different fluorescent proteins, under the same selective antibiotic, we show that E. coli bacteria readily accept multiple plasmids, resulting in widespread aclonality and reveal a complex pattern of colony development. Cotransformation of plasmids occurs by either CaCl2 or by electroporation methods. A bacterium rod transformed with three plasmids—each expressing a high level of a unique fluorescent protein—and replated on agar, appears to reassign a random number of the three fluorescent plasmids to its daughter cell during cell division. The potential to simultaneously follow multiple lineages of clonally related bacteria in a bacteria colony would allow for mosaic analysis of gene function. We show that clonally related bacterium rods self-organize in a fractal growth pattern and can remain linked during colony development revealing a potential target against microbiota growth.

Efficient Generation of Genome-wide Libraries for Protein-ligand Screens Using Gibson Assembly.

Genome-wide screens using yeast or phage displays are powerful tools for identifying protein-ligand interactions, including drug or vaccine targets, ligand receptors, or protein-protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 10 5 -10 7 DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system.

Emergence of transferable ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae due to a novel CMY AmpC β-lactamase in China

ObjectivesTo evaluate the molecular mechanisms of ceftazidime/avibactam (CAZ/AVI) resistance in six Klebsiella pneumoniae strains that co-produce K. pneumoniae carbapenemase (KPC)-2 and a novel variant of CMY cephalosporinase in a Chinese hospital. MethodsAntimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing (WGS) was performed to investigate potential resistance determinants. Plasmid conjugation, electroporation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) hybridization and cloning experiment were carried out to investigate the resistance plasmids and genes. ResultsA high level of CAZ/AVI resistance was observed in six KPC-Kp strains (MIC 128 mg/L). Five strains were isolated in 2015 and one in 2016, before the approval of CAZ/AVI in China. Sequence analysis indicated that all the strains belonged to sequence type (ST) 11 and uniformly carried a novel CMY AmpC β-lactamase gene, designated blaCMY-172. When compared with CMY-2, CMY-172 has a deletion of three consecutive amino acids (K290, V291 and A292) in the R2-loop region and a non-synonymous amino acid substitution at position 346 (N346I). The blaCMY-172-bearing plasmid, pKPCZA02_4, was 93.3 Kb, IncI1-I type, and conjugative; blaCMY-172 was located in an IS1294-mediated transposon. Plasmid conjugation and DNA fragment cloning proved that blaCMY-172 was responsible for CAZ/AVI resistance. ConclusionsOur study identified conjugative plasmid-mediated blaCMY-172 as a new mechanism for CAZ/AVI resistance in clinical KPC-Kp strains. Careful monitoring of CAZ/AVI susceptibility is imperative for preventing the spread of the resistance gene.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Molecular phylogeny based on its sequences of nrDNA of some species belonging to dodder (Cuscuta L.) genus from various ecological sites of Turkey

Nuclear ribosomal DNA (nrDNA) sequence data of the Cuscuta genus, which have been considered as one of the most popular sequences for phylogenetic inference in plants, have been studied from a phylogenetic perspective in agricultural and non-agricultural lands of Turkey. The samples of Cuscuta spp. were collected from different geographical regions of Turkey between the years of 2013-2015. Some other species, not available locally, were taken from the herbarium samples of some research units. In order to study the phylogenetic relations of collected species, DNA isolations were made from body tissue samples. Conserved regions on ribosomal DNA (rDNA) were amplified by universal primers via PCR method and cloned into a proper cloning vector. The cloned DNA fragments were sequenced and analysed by web-based and computer programs. DNA sequences of certain species were recorded to the National Center for Biotechnology Information (NCBI) database. Based on the morphological examination and molecular analyses of fresh and the herbarium specimen, 8 species were identified. The identified species were C. hyalina (Gene bank accession no. KY020420), C. monogyna (KY020421), C. europaea (KY020422), C. palaestina (KY020423), C. approximata (KY020424), C. kurdica (KY020427), C. kotschyana (KY020430) and C. babylonica (KY020431). The ITS (Internal Transcribed Spacer) region contains several indels in identified Cuscuta species with the length varying from 668 to 730 bp. Sequence divergence ranges from 1.00% to 8.00% within Cuscuta spp. Based on our findings, the ITS sequences provided phylogenetically informative results in combination with the secondary structures.

Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine

The gene coding for antimicrobial peptides produced by probiotic Lactobacillus reuteri KUB-AC5 located on the cloned DNA fragment I-C46 containing 2 open reading frames I-C46-F2.1 and I-C46-F2.2 were designed for strain-specific primers P1 and P2, respectively, and assessed by real-time quantitative polymerase chain reaction. According to the obtained results, primer P1 has limited strain specificity. Primer P2 exhibited high efficacy and specificity at annealing temperature of 70°C while P1 annealed at 57°C causing nonspecific bands. Hence, P2 was selected for quantitative polymerase chain reaction assay by isothermal annealing and extension reaction at high temperature of 70°C resulting in linearity for its DNA sequences ranging from 102 to 107 target copy numbers per assay, and displaying a detection limit of 6.17 log cfu/g of cecal digesta. Using spike testing, this system was able to detect 7.88 ± 0.06 to 11.78 ± 0.06 log copy number/g of digesta, higher than cultivation assay at about 1 log cfu/g, with good correlation of 0.99. These results suggested possible detection of strain KUB-AC5 in the gastrointestinal tract of chicken to evaluate the efficacy and persistence of a probiotic strain which requires correct inclusion rates in the feed.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

Design and fabrication of a two-phase diamond nanoparticle aided fast PCR device

Polymerase Chain Reaction (PCR) is an important and prevalent technique in biotechnology because of its crucial role in cloning DNA fragments and diagnostic applications. In the present study, a high-throughput two-phase PCR device is designed and fabricated which utilizes a serpentine microchannel together with a spiral structure. The former is for the droplet-generation and mixing and the latter is for the thermal cycling process. Moreover, the effect of diamond nanoparticles (diamondNP) on the performance of PCR is also investigated while using commercial PCR devices and the fabricated PCR device designed in this study. Using numerical simulation, it is shown that within the simple and easy-to-make microchannel structures designed in this study, there are different transient secondary flows which can help mixing the contents of the droplets including PCR solution and diamondNP. Moreover, a new fabrication process is used for the design of the droplet-generation chip which is applicable for the fabrication of all high-pressure microfluidic devices. It is found that the effect of diamondNPs on the PCR performance depends on the nanoparticle concentration; diamaondNPs can improve the PCR performance more than 5 times. On the other hand, if a high concentration of diamondNPs is used, PCR performance decreases significantly. Moreover, it is shown that the fabricated device is able to do the same PCR procedure faster than the commercial PCR devices.

Improvement of photo-fermentative hydrogen production performance by expressing tetracycline resistance genes tetAR in Rhodobacter sphaeroides HY01

A typical expression plasmid, pRK415, was widely used to introduce cloned DNA fragment into a broad range of Gram-negative bacteria, including Rhodobacter sphaeroides. Tetracycline (Tc) is required for stable inheritance of the plasmid, but it is subject to photooxidation, inhibitory to the growth of the host strain. However, in this study, the introduction of pRK415 into R. sphaeroides HY01 under tetracycline stress showed clear enhancement on H2 production performance of the recombinant strain pRK415/HY01. The maximum H2 production rate of pRK415/HY01 was enhanced by 10%–70% compared with the control group in the absent of Tc. Supplementing with Tc, the wild type strain HY01 showed repressed cell growth and reduced H2 production performance. The tetAR genes knockout test demonstrated that the expression of tetAR genes on pRK415 promoted the H2 production performance. And within tolerance concentration (Tc < 2.5 mg/L), higher Tc concentration led to higher H2 production performance of pRK415/HY01. Expression of tetAR genes in the genome of R.sphaeroides HY01 by substituting partial of hupSL genes enhanced the H2 production performance as well. The mechanism of the H2 production performance enhancement was discussed.

Structural and Functional Analysis of a Bidirectional Promoter from Gossypium hirsutum in Arabidopsis.

Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.

Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits

Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging’s multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). We examined if foraging’s complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, we cloned DNA fragments upstream of each of foraging’s transcription start sites and generated four separate forpr-Gal4s. Supporting our hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. In the CNS, forpr1-Gal4 and forpr4-Gal4 were expressed in neurons while forpr2-Gal4 and forpr3-Gal4 were expressed in glia cells. In the gastric system, forpr1-Gal4 and forpr3-Gal4 were expressed in enteroendocrine cells of the midgut while forpr2-Gal4 was expressed in the stem cells of the midgut. forpr3-Gal4 was expressed in the midgut enterocytes, and midgut and hindgut visceral muscle. forpr4-Gal4’s gastric system expression was restricted to the hindgut. We also found promoter specific expression in the larval fat body, salivary glands, and body muscle. The modularity of foraging’s molecular structure was also apparent in the phenotypic rescues. We rescued larval path length, triglyceride levels (bordered on significance), and food intake of for0 null larvae using different forpr-Gal4s to drive UAS-forcDNA. In a foraging null genetic background, forpr1-Gal4 was the only promoter driven Gal4 to rescue larval path length, forpr3-Gal4 altered triglyceride levels, and forpr4-Gal4 rescued food intake. Our results refine the spatial expression responsible for foraging’s associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging’s pleiotropy arises at least in part from the individual contributions of its four promoters.

The plasmid vectors, pBS2ndd and pBS3ndd, for versatile cloning with low background in Escherichia coli.

For decades, diverse plasmid vectors have been continuously developed for molecular cloning of DNA fragment in the bacterial host cell Escherichia coli. Even with deliberate performances in vector preparation, the cloning approaches still face inevitable background colonies, or false positive clones, that may be arisen from intact or self-ligated plasmid molecules. To assist in such problem, two plasmids, pBS2ndd and pBS3ndd, which resistant to ampicillin and kanamycin respectively, were developed in this study as more advantageous cloning vector. The plasmids carry ndd, a lethal gene from bacteriophage T4 coding for nucleoid disruption protein that binds to the host chromosome and progressively kill the cell. The deadly toxicity of Ndd inhibits host cells that obtain intact or ndd-religated vector from growing, which results in low background and dramatically reduces the effort for selection of recombinants. Moreover, their identical multiple cloning site was designed to support various cloning strategies. Digestion of plasmids with XcmI allows for in vitro T/A ligation, while with EcoRV permits blunt-end ligation, with capability of blue-white colony screening. In vivo homologous recombination cloning is also utilizable by amplification of insert fragments using primers containing homology arms and transformation into capable E. coli strains. To demonstrate their advantages, the plasmids were used to clone PCR product samples for DNA sequencing with low-background and versatile cloning strategies. Such rapid and cost-effective cloning procedures are also proposed here. Finally, the cloning for protein expression with blue-white selection was also possible using egfp as a model regulated by lac and T7 promoters on the plasmid or other build-in promoters with the insert.

Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps.

In recent years, with the advent of polymerase chain reaction and the development of highly efficient methods of cloning and DNA sequencing, the need to prepare large quantities of plasmid vectors and recombinants has greatly diminished. In consequence, this protocol, at one time in common use, has been largely replaced by faster and easier column-based purification methods. In this protocol, plasmid DNA is purified from the cleared bacterial lysate by centrifugation to equilibrium in CsCl gradients containing ethidium bromide. These gradients have an aesthetic quality that justifies the continued presentation of a protocol that in many other respects is an antique. The typical yield of high-copy-number plasmid vectors or of amplified low-copy-number vectors prepared by this method is ∼3-5 µg of DNA/mL of original bacterial culture. The yield of recombinant plasmids containing inserts of foreign DNA is usually slightly lower, depending on the size and nature of the cloned DNA fragment.

Biophysical characterization of the association of histones with single-stranded DNA

BackgroundDespite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. MethodsNon-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. ResultsHistones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (−) strand. ConclusionsAt physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. General significanceIn the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.