A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Phylo-geographic analysis of whitefly on the basis of mitochondrial cytochrome oxidase 1 gene

Abstract Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).

Resumo Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).

Phylo-geographic analysis of whitefly on the basis of mitochondrial cytochrome oxidase 1 gene / Análise filogeográfica da mosca-branca com base no gene citocromo oxidase 1 mitocondrial

Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).

Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).

A streamlined strategy for self-production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains.

Positive selection vectors carry a lethal gene encoding a toxic product that is harmful to most laboratory E. coli strains. Previously, we reported a strategy for in-house production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains. However, the strategy involves lengthy gel electrophoresis and extraction procedures to purify the linearized vector after digestion. Here, we streamlined the strategy to eliminate the gel-purification step. A uniquely designed short fragment called the Nawawi fragment was inserted into the coding sequence of the lethal gene of the pJET1.2 plasmid, resulting in the pJET1.2N plasmid that can be propagated in the E. coli strain DH5α. Digestion of the pJET1.2N plasmid with EcoRV released the Nawawi fragment, and the resulting blunt-ended pJET1.2/blunt cloning vector can be used directly for DNA cloning without prior purification. Cloning of a DNA fragment was not hindered by the Nawawi fragments carried over from the digestion step. After transformation, the pJET1.2N-derived pJET1.2/blunt cloning vector produced > 98% positive clones. The streamlined strategy accelerates the in-house production of the pJET1.2/blunt cloning vector and enables DNA cloning at a lower cost. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03647-3.

A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Key message: In-house production of a positive selection cloning vector could be simple, efficient and low cost. Abstract: DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03289-x.

Homogalacturonan and xylogalacturonan region specificity of self-cloning vector-expressed pectin methylesterases (AoPME1-3) in Aspergillus oryzae.

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

Variability in the immunogenic preS region of Pakistani hepatitis B virus isolates.

This paper reports the genetic variability of hepatitis B virus in Pakistan population. The worldwide prevalence of hepatitis B virus is estimated to be around 350 million that causes significant mortality especially in developing countries like Pakistan. In this study, genetic diversity of HBV was checked by using preS domain of HBV. About seventy-five samples were selected for study. Among these samples nine samples showed positive results after PCR and gel analysis. These nine samples were named SBS001-SBS008. After gel purification these samples were ligated in T/A cloning vector and transformed with E. coli DH5α. After successful cloning and positive restriction analysis these samples were subjected to DNA sequencing. Sequencing results showed that eight samples (SBS002-SBS008) have a deletion of 33 nucleotides at N-terminal that is characteristics of genotype D while SBS001 belongs to genotype C. Silent mutations and amino acid changes were also searched in this highly variable region of genome. Based upon this study it was concluded that genotype D is the most common genotype in Pakistan.

Stable Transformation of the Actinobacteria Frankia spp.

A stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genus Frankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into different Frankia strains by a filter mating with Escherichia coli strain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced into Frankia casuarinae strain CcI3 at a frequency of 4.0 × 10-3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced into F. casuarinae strain CcI3 and other Frankia strains at frequencies ranging from 10-2 to 10-4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerant Frankia sp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitive F. casuarinae strain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed in F. casuarinae strain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression in Frankia spp., overcoming a major hurdle in the field. This step in the development of genetic tools in Frankia spp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCE The absence of genetic tools for Frankia research has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids into Frankia spp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.

RecET Direct Cloning of Polysaccharide Gene Cluster from Gram-Negative Bacteria.

RecET direct cloning enables obtaining a large DNA region from genome. Here we describe its applications in cloning of polysaccharide gene cluster from gram-negative bacteria. Rapid and exact cloning of polysaccharide gene cluster can be achieved by this method.

Effect of sequential C-terminal tryptophans on green fluorescent protein fluorescence.

The effect of the addition of sequential C-terminal tryptophan residues on the fluorescence intensity of GFP was investigated. Tandem repeats of six tryptophan residues markedly decreased fluorescence intensity. This phenomenon is likely to occur because of the inhibition of GFP folding, resulting in insolubility. Exploiting this phenomenon, we constructed a cloning vector that facilitates the identification of recombinant colonies of Escherichia coli by the activation of GFP.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains.

Designing and Construction of a Cloning Vector Containing mpt64 Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis), remains as one of the leading causes of deaths worldwide, with nearly two million death cases annually. BCG (Bacille Calmette-Guerin) continues to be the most widely used vaccine in the world, but the protective immunity differs in different parts of the world. Accordingly, new strategies including DNA vaccines are essentially needed. This study was aimed to design and construct a cloning vector containing mpt64 gene of M. tuberculosis. MATERIALS AND METHODS: M. tuberculosis H37Rv was cultured on Lowenstein Jensen medium, and genomic DNA was extracted. The mpt64 gene was amplified by PCR using designed specific primers. After the digestion of mpt64 and pcDNA3.1 (+) by BamHI and EcoRI restriction enzymes, the mpt64 fragment was ligated into the digested vector using T4 DNA ligase enzyme. Then, the recombinant vector was transformed into competent Escherichia coli (E. coli) TOP10 strain. To confirm the colonies of transformed bacteria, antibiotic resistance, colony-PCR, restriction enzyme digestion and DNA sequencing were used. RESULTS: To confirm the clones, colony-PCR using mpt64 specific primers was performed and the fragment of 718 bp was observed by gel electrophoresis. Clones were also verified by restriction enzyme digestion using BamHI and EcoRI restriction enzymes and the 718 bp fragment was observed. Furthermore, results of DNA sequencing showed 100% homology with the mpt64 fragment of H37Rv in GenBank. CONCLUSION: In this study, the mpt64 fragment was successfully cloned in pcDNA3.1 (+) vector. This construct can be used in future studies as a DNA vaccine in animal models to induce immune system responses.

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site.

OBJECTIVE: Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant loxP sites present. RESULTS: An oligonucleotide containing a mutant loxP 2272 site and a XhoI restriction enzyme sequence was designed and inserted at the SfiI restriction site located approximately 200 basepairs upstream of the lacZ gene in pBeloBAC11. Clones containing the desired insert were identified by XhoI restriction digests since an additional band was generated. This transposon-mediated deletion technology allows researchers to identify the boundaries of cis-acting elements and genes.

pELMO, an optimised in-house cloning vector.

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vector's multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the SmaI restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500 bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.

Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria.

BACKGROUND: Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. RESULTS: To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. CONCLUSION: The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.

The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria.

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 10(7)), Lactobacillus plantarum (5.7 × 10(5)), Lactobacillus casei (2.3 × 10(5)), Leuconostoc citreum (2.7 × 10(5)), and Enterococcus faecalis (2.4 × 10(5)). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

Screening of recombinant Escherichia coli using activation of green fluorescent protein as an indicator.

A novel cloning vector that can be used to identify recombinant Escherichia coli colonies by activation of the green fluorescent protein gene (GFP) was constructed. Screening using the vector does not require special reagents. The recombinant plasmid activates GFP, and the rate of false-positive results is low.

Development of an oriC vector for use in Mycoplasma synoviae.

Mycoplasma synoviae, an important poultry pathogen, belonging to the class Mollicutes, causes airsacculitis, synovitis, decreased egg production and produces significant economic losses. Efforts to determine M. synoviae virulence factors and their role in pathogenicity require suitable tools for genetic manipulation of this pathogen. This study describes, for the first time, the identification and cloning of the origin of replication (oriC) of M. synoviae to develop a replicable oriC vector for this mycoplasma. Shuttle vectors containing different putative oriC regions along with tetracycline resistance gene tetM were constructed to transform M. synoviae. An oriC vector, pMAS-LoriC, harbouring the complete dnaA gene along with upstream and downstream DnaA boxes, successfully transformed M. synoviae at an average transformation frequency of 1.07×10(-8) transformants per colony-forming unit (CFU), and remained freely replicating as well as integrated at the chromosomal oriC. Plasmid copy number for pMAS-LoriC was estimated to be 62±29 (average±SD) per cell. This study also provided evidence of the occurrence of homologous recombination and the functionality of the heterologous tetM determinant in M. synoviae. The transformation technique and the oriC vector developed in this study have the potential to be used in targeted gene disruption, gene complementation and expression studies in this organism.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.