Showing papers on "Cloning published in 2012"
TL;DR: A novel cloning method that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction is described and SLiCE overcomes the sequence limitations of traditional cloning methods.
Abstract: We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.
440 citations
TL;DR: Direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET is described.
Abstract: Functional genomics requires facile methods to recover sequences of interest. Fu et al. show that the phage proteins RecE and RecT mediate recombination between linear DNA fragments and can facilitate natural product discovery. Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.
368 citations
TL;DR: One-step sequence- and ligation-independent cloning (SLIC) is developed as a simple, cost-effective, time-saving, and versatile cloning method that achieves highly efficient and directional cloning.
Abstract: We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.
334 citations
TL;DR: Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding and provides numerous opportunities for agriculture and biomedicine.
Abstract: Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No 258/97 Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved
92 citations
TL;DR: It is shown that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb, and enables simultaneous cloning of multiple inserts.
Abstract: We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.
78 citations
TL;DR: The results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.
Abstract: In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.
74 citations
TL;DR: Cl cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.
Abstract: The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ~200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.
66 citations
TL;DR: This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker and an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants.
Abstract: Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
46 citations
TL;DR: Cloning of whole genomes of the genus Mycoplasma in yeast was extended to bacteria that use the universal genetic code and it was discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast.
Abstract: Cloning of whole genomes of the genus Mycoplasma in yeast has been an essential step for the creation of the first synthetic cell. The genome of the synthetic cell is based on Mycoplasma mycoides, which deviates from the universal genetic code by encoding tryptophan rather than the UGA stop codon. The feature was thought to be important because bacterial genes might be toxic to the host yeast cell if driven by a cryptic promoter active in yeast. As we move to expand the range of bacterial genomes cloned in yeast, we extended this technology to bacteria that use the universal genetic code. Here we report cloning of the Acholeplasma laidlawii PG-8A genome, which uses the universal genetic code. We discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast. This gene was inactivated in order to clone and stably maintain the A. laidlawii genome as a centromeric plasmid in the yeast cell.
43 citations
TL;DR: The discovery of an efficient mechanism of homologous recombination between two linear DNA substrates provides a new method for direct cloning.
Abstract: The discovery of an efficient mechanism of homologous recombination between two linear DNA substrates enables direct cloning of large genomic sequences
43 citations
TL;DR: A non-methylating E. coli strain, JTU007, is constructed by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B and transferred using high-throughput conjugation to the methyl-restriction S. coelicolor.
Abstract: Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm− Dam− strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.
TL;DR: A one-step cloning method for the insertion of two arms into a KO vector using exonuclease III is developed, which has great potential for high-throughput construction of KO vectors.
Abstract: Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116) and HDAC2 KO cells from the colorectal cancer cell line (DLD1). Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.
TL;DR: The use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats.
TL;DR: A competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background.
TL;DR: Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.
Abstract: Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phage mid-vectors. Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome. Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings. Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.
07 Mar 2012
TL;DR: The function of genes isolated using map-based cloning of mutant alleles has been confirmed by functional complementation using genetic transformation, enabling the introduction of foreign genes into crop plants, expeditiously creating new genetically modified organisms.
Abstract: Genome sequencing of several organisms has resulted in the rapid progress of genomic studies. Genetic transformation is a powerful tool and an important technique for the study of plant functional genomics, i.e., gene discovery, new insights into gene function, and investigation of genetically controlled characteristics. In addition, the function of genes isolated using map-based cloning of mutant alleles has been confirmed by functional complementation using genetic transformation. Furthermore, genetic transformation enables the introduction of foreign genes into crop plants, expeditiously creating new genetically modified organisms. Gene transformation and genetic engineering contribute to an overall increase in crop productivity (Sinclair et al., 2004).
TL;DR: A combination of bioinformatic analysis and the Gateway cloning system was shown to be a useful tool for large-scale screening of nuclear targeting proteins with NLSs in H. pylori, which can be used to better understand the H.pylori-directed host cell pathology.
TL;DR: It is demonstrated that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embriesos.
Abstract: Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.
TL;DR: This study generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus, and assessed the cloning efficiency associated with adult or fetal fibroblasts as a nuclear source.
Abstract: Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.
TL;DR: The isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli are described.
Abstract: Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.
01 Jan 2012
TL;DR: This study aimed to design a system for protein expression and detection using a new super-folder derivative of GFP (sfGFP) as a fusion partner, which will provide an efficient tool for the expression, purification and detection of many proteins, having problems in solubility and stability.
Abstract: Green fluorescent protein (GFP) is widely used as an excellent expression tag for fusion proteins, which can improve their expression while preserving their function and native-like structure. Fusion protein method allows the purification and the detection of a protein of interest even when no specific antibody is available. This study aimed to design a system for protein expression and detection using a new super-folder derivative of GFP (sfGFP) as a fusion partner. This included the construction of the protein expression plasmid pRSET-sfGFP by cloning sfGFP gene downstream of the N-terminal 6×His tag in the T7 promoter-plasmid pRSET. The soluble expressed sfGFP protein (27 kDa) from this plasmid in the cytoplasm of E. coli was purified using metal affinity chromatography, as shown after SDS-PAGE separation and blue gel staining. In order to detect sfGFP, as single or in fusion proteins, in ELISA and immunoblotting analysis, sfGFP-specific polyclonal antibodies were produced in rabbit after immunization with three injections with the pure sfGFP prepared in Freund’s adjuvant. Two-step antibody purification, using protein A-Sepharose and sfGFP-coupled Sepharose affinity chromatography columns, was performed to obtain highly reactive and pure sfGFP-specific IgG. This system will provide an efficient tool for the expression, purification and detection of many proteins, having problems in solubility and stability, as fusion partners with sfGFP.
TL;DR: Two novel HLA-A alleles differing from their closest related alleles by nucleotide exchanges in introns, A*29-02-02:02 and A*68-01-01:02, were identified.
Abstract: Two novel HLA-A alleles differing from their closest related alleles by nucleotide exchanges in introns, A*29:02:01:02 and A*68:01:01:02, were identified.
TL;DR: In this article, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region and a promoter for constitutive expression of cloned genes.
Patent•
19 Dec 2012TL;DR: In this paper, the isolation of nucleic acid segments encoding antibody variable domains and insertion of the isolated nucleic acids segments in eukaryotic expression plasmids is performed without the intermediate isolation and analysis of clonal intermediate plasmid.
Abstract: In the method as reported herein the isolation of nucleic acid segments encoding antibody variable domains and the insertion of the isolated nucleic acid segments in eukaryotic expression plasmids is performed without the intermediate isolation and analysis of clonal intermediate plasmids. Thus, in the method as reported herein the intermediate cloning, isolation and analysis of intermediate plasmids is not required, e.g. by analysis of isolated transformed E.coli cells.
TL;DR: Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions and further studies are needed, however, to clarify the substrate specificity and role of the CYP 26B1 splice variant in health and disease.
Abstract: Background: All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development ...
TL;DR: A major quantitative trait locus (QTL) was mapped between two simple sequence repeats encompassing a 459-kb region on chromosome 3 and should result in the eventual cloning of paternal sorting of mitochondria and provide insights about nuclear control of organellar-DNA sorting.
Abstract: Mitochondria are organelles that have their own DNA; serve as the powerhouses of eukaryotic cells; play important roles in stress responses, programmed cell death, and ageing; and in the vast majority of eukaryotes, are maternally transmitted. Strict maternal transmission of mitochondria makes it difficult to select for better-performing mitochondria, or against deleterious mutations in the mitochondrial DNA. Cucumber is a useful plant for organellar genetics because its mitochondria are paternally transmitted and it possesses one of the largest mitochondrial genomes among all eukaryotes. Recombination among repetitive motifs in the cucumber mitochondrial DNA produces rearrangements associated with strongly mosaic (MSC) phenotypes. We previously reported nuclear control of sorting among paternally transmitted mitochondrial DNAs. The goal of this project was to map paternal sorting of mitochondria as a step towards its eventual cloning. We crossed single plants from plant introduction (PI) 401734 and Cucumis sativus var. hardwickii and produced an F2 family. A total of 425 F2 plants were genotyped for molecular markers and testcrossed as the female with MSC16. Testcross families were scored for frequencies of wild-type versus MSC progenies. Discrete segregations for percent wild-type progenies were not observed and paternal sorting of mitochondria was therefore analyzed as a quantitative trait. A major quantitative trait locus (QTL; LOD >23) was mapped between two simple sequence repeats encompassing a 459-kb region on chromosome 3. Nuclear genes previously shown to affect the prevalence of mitochondrial DNAs (MSH1, OSB1, and RECA homologs) were not located near this major QTL on chromosome 3. Sequencing of this region from PI 401734, together with improved annotation of the cucumber genome, should result in the eventual cloning of paternal sorting of mitochondria and provide insights about nuclear control of organellar-DNA sorting.
TL;DR: ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design.
Abstract: Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired. This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP. ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.
TL;DR: The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H2O2 treatment, SA, antimycin A and KCN, suggesting a role of Hs aOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain.
Abstract: A novel alternative oxidase (AOX1) gene, designated HsAOX1 (GenBank accession number JF440341) was cloned by RT-PCR from wild barley (Hordeum spontaneum). The full length of HsAOX1 is 1115 bp with an open reading frame of 987 bp, encoding a protein of 328 amino acids with molecular weight of 36.89 kDa and a theoretical isoelectric point of 6.81. As found in other plant AOX1 proteins, sequence alignment showed that HsAOX1 had conserved metal binding and hydrophobic α-helix regions and had high homology to other AOX1 in plants. The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H2O2 treatment, SA, antimycin A and KCN. These results showed that HsAOX1 functions not only during inhibition of cytochrome electron transport but also during oxidative stresses, thus suggesting a role of HsAOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain. The cloning and characterization of the HsAOX1 gene will be useful for further studies of biological roles of HsAOX1 in plants.