Genome-walking PCR Research Articles

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Identification of a candidate gene responsible for male sterility conferred by CMS-T cytoplasm in onion (Allium cepa L.) and development of molecular markers for detection of CMS-T cytoplasm

Cytoplasmic male sterility (CMS) has been exclusively used as an emasculation tool in seed production of hybrids in onion (Allium cepa L.).Three types of onion CMS (CMS-S, CMS-R, and CMS-T) have been used in hybrid onion breeding. Male sterility conferred by both CMS-S and CMS-R is likely induced by open reading frame (orf) 725, a chimeric gene. However, this gene was not detected in onion accessions containing CMS-T cytoplasm. Instead, a region showing high homology with orfA501, which is located in the 3’ end of orf725, was detected in CMS-T. A novel chimeric gene, which we named orf219, was identified by genome walking PCR amplification based on the orfA501 homolog. The 684-bp open reading frame of orf219 consisted of an 128-bp of exon 1 of atp1 and a 556-bp sequence of an orfA501 homolog. The high-copy-number orf219 was detected only in the CMS-T cytoplasm (T cytotype). Analysis of RT-PCR products showed normal transcription of orf219 and eight RNA editing sites, one of which created a stop codon, resulting in a shorter amino acid sequence upon translation. Sequences of four hypervariable regions in the chloroplast genome and the organization of syntenic blocks in mitochondrial genome indicated that CMS-T was very closely related to the normal (N) male-fertile and CMS-R cytotypes. Based on these findings, new molecular markers were developed for the identification of cytotypes CMS-T, -R, -S, and -N. Cytotypes of 424 diverse onion accessions were identified using six molecular markers. The CMS-S and CMS-R cytotypes predominated among the analyzed accessions. In contrast, only five accessions possessed CMS-T, supporting earlier reports that CMS-T is rarely used in hybrid-onion breeding.

Development of signature-tagged mutagenesis in Riemerella anatipestifer to identify genes essential for survival and pathogenesis

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 1010 CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.

Recombinant Approaches for Microbial Xylanases: Recent Advances and Perspectives.

Xylanases are crucial enzymes to hydrolyse the xylan of plant hemicellulose in order to complete the carbon cycle. Xylanases have been used widely in a variety of industries ranging from food and feed industry to pulp and paper industry. Most of the industrial processes which using xylanase requires a thermostable and alkali stable enzyme. Therefore it is desired to produce high thermostable and alkali stable xylanase with high activity. In this review a number of molecular techniques are used in this genomic era have been utilized to enhance physiological properties of xylanases for greater commercial application in the industries. A brief outline of diverse molecular techniques such as genome-walking PCR, thermal asymmetric interlaced PCR (TAIL-PCR), staggered extension process (StEP) recombination method, site-directed mutagenesis together with metagenomic approaches have been discussed which are used to improve the charactestics of xylanases and its production. Metagenomic studies along with directed evolution by mutant creation have also been reported as an effective tool in improvement of xylanase activity and its properties. This review comprehensively describes the recent reports and different combinatorial approaches towards production of efficient xylanases.

The transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster (Pinctada fucata).

Matrix proteins play key roles in shell formation in the pearl oyster, but little is known about how these proteins are regulated. Here, two POU domain family members, Pf-POU2F1 and Pf-POU3F4, were cloned and characterized. Functional domain analysis revealed that both them have conserved POUS and POUH domains; these domains are important for transcription factor function. The tissue distributions of Pf-POU2F1 and Pf-POU3F4 mRNAs in pearl oyster revealed different expression patterns, and the expression of Pf-POU3F4 mRNA was relatively high in the mantle. The promoters of the matrix protein genes Aspein and Prismalin-14 were cloned using genome-walking PCR. Relatively high transcriptional activities of these promoters were detected in HEK-293T cells. In transient co-transfection assays, Pf-POU3F4 greatly up-regulated the promoter activities of the Aspein and Prismalin-14 genes in a dose-dependent manner. Structural integrity of Pf-POU3F4 was essential for its activation function. One region of the Aspein gene promoter, -181 to -77bp, and two binding sites in the Prismalin-14 gene promoter, -359 to -337 bp and -100 to -73bp, were required for activation of Pf-POU3F4. An electrophoresis mobility shift assay demonstrated that Pf-POU3F4 directly bound these sites. Pf-POU3F4 knockdown led to a decrease in Aspein and Prismalin-14 gene expression. Furthermore, expression levels for the Pf-POU3F4 gene were similar to those of the Aspein and Prismalin-14 genes during five development stages. Taken together, these results suggest that the transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster. The nucleotide sequence data of Pf-POU2F1 is available in the GenBank databases under the accession number KM588196. The nucleotide sequence data of Pf-POU3F4 is available in the GenBank databases under the accession number KM519606. The nucleotide sequence data of Aspein gene promoter is available in the GenBank databases under the accession number KM519607. The nucleotide sequence data of Prismalin-14 gene promoter is available in the GenBank databases under the accession number KM519601.

Clinicopathologic, Immunohistochemical, and Ultrastructural Findings of a Fatal Case of Middle East Respiratory Syndrome Coronavirus Infection in the United Arab Emirates, April 2014

Middle East respiratory syndrome coronavirus (MERS-CoV) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal MERS-CoV infection is unknown. We describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of MERS-CoV in the world, which was related to a hospital outbreak in the United Arab Emirates in April 2014. The main histopathologic finding in the lungs was diffuse alveolar damage. Evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. Double staining immunoassays that used anti–MERS-CoV antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of MERS-CoV antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. No evidence of extrapulmonary MERS-CoV antigens were detected, including the kidney. These results provide critical insights into the pathogenesis of MERS-CoV in humans.

Molecular cloning, gene expression profiling and in silico sequence analysis of vitamin E biosynthetic genes from the oil palm

Homogentisate geranylgeranyl transferase (HGGT) and homogentisate phytyltransferase (HPT) are the two key enzymes involved in condensation of homogentisic acid (HGA) with a prenyldiphosphate to produce tocotrienols and tocopherols in plants, respectively. The partial cDNAs encoding HGGT and HPT enzymes were successfully isolated from the two oil palm species, Elaeis guineensis and Elaeis oleifera by PCR amplification using degenerate primers. Subsequently, full length cDNA sequences were completed by rapid amplification of cDNA ends (RACE) and further annotated using various bioinformatics tools. The analysis revealed the presence of an UbiA prenyltransferase conserved domain in all four deduced amino acid sequences and suggested that oil palm HGGT and HPT are more evolutionarily related to their counterparts from other monocot plant species. Quantitative gene expression analysis was carried out to elucidate the transcript profiles of the oil palm HGGT and HPT in different oil palm tissues and at different developmental stages of the mesocarp. The HPT was constitutively expressed in all analyzed tissues except in 15w.a.a kernel whereas oil palm HGGT showed preferential expression in mesocarp and kernel tissues. However, HPT was highly expressed at the fruit ripening stage of 17w.a.a mesocarp when active oil deposition occurs. Genome-walking PCR successfully amplified the promoter regions of HGGT and HPT from E. guineensis. Computational analysis using PlantCare and PLACE databases revealed several cis-regulatory elements including phytohormone-responsive, light-responsive and abiotic factor-responsive elements which may be involved in coordinating expression of both genes. Taken together, this study provides useful information about important features of the cDNA and promoter sequences as well as an insight into the transcriptional regulation of these key vitamin E genes for future genetic improvement efforts.

Novel Protease-Resistant Exochitinase (Echi47) from Pig Fecal Environment DNA with Application Potentials in the Food and Feed Industries

A novel exochitinase gene (Echi47) was directly cloned from the pig fecal environment DNA using the genomic walking PCR technique and expressed in Escherichia coli BL21 (DE3). Echi47 has an open reading frame (ORF) of 1,161 bp encoding 386 amino acids. The amino acid sequence of Echi47 showed 36% identity with that of chitinase from Coprinellus congregatus. The recombinant exochitinase was purified with specific activity toward colloidal chitin of 6.84 U/mg. Echi47 was optimally active at pH 5.0 and 40 °C, respectively. When colloidal chitin was used as substrate, N-acetylchitobiose [(GlcNAc)2] was mostly produced at the initial stage, suggesting that it is an exochitinase. Echi47 exhibited excellent resistance to pepsin, trypsin, proteinase K, and flavor protease. Under simulated alimentary tract conditions, Echi47 was stable and active, releasing 21.1 mg of N-acetylchitooligosaccharides from 80 mg of colloidal chitin. These properties make Echi47 a potential additive in the food and feed industries.

Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

Maize (Zea mays L.) is one of the world's major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very difficult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog (GenBank accession no. EC864166) of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements (ECREs). Pro66 could drive expression of the β-glucuronidase (GUS)-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deficit, salt, copper, iron deficiency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile

As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF23 to ORF21) and PCRB (ORF21 to ORF20) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons.

A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum

Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Molecular cloning and heterologous expression of an acid stable xylanase gene from Alternaria sp. HB186

A new xylanase gene, named xyn186, was cloned by the genome-walking PCR method from the Alternaria sp. HB186. The sequence of xyn186 contains a 748 bp open reading frame separated by one intron with the size of 52 bp. The cDNA was obtained by DpnI-mediated intron deletion. The cDNA was cloned into pHBM905A and transformed into Pichia pastoris GS115 to screen xylanase-secreting transformants on RBB-xylan plates. The molecular mass of the enzyme was estimated to be 23 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 50°C, respectively. The K (m) and V (max) valued for birchwood xylan are 1.404 mg ml(-1) and 0.2748 mmol min(-1) mg(-1), respectively. The inhibitory effects of various metal ions were investigated, Cu(2+) and Hg(2+) ions inhibited most of the enzyme activity. The gene copy number of xyn186 in the genome of P. pastoris was estimated as two by the Real-time PCR. To date, xyn186 gene is the first xylanase gene cloned from the genus Alternaria.

Cloning and expression pattern of alkaline phosphatase during the development of Paralichthys olivaceus

Alkaline phosphatases are ubiquitous enzymes involved in many important biological processes. Mammalian tissue-nonspecific alkaline phosphatase has long been thought to feature in embryonic development and bone formation. In this study, an alkaline phosphatase (ALP) gene from Paralichthys olivaceus was identified by rapid amplification of cDNA ends and genome-walking PCR. The ALP gene extends 10,141bp and contains 11 exons and 10 introns. The open reading frame of the ALP transcript consists of 1,431bp, which encodes 476 amino acids products named as POALP. An analysis of its secondary and tertiary structure revealed that the POALP was conserved in different species, but one disulfide linkage made it possible to adapt to low-temperature environment. The ALP activity was found to be first detectable in the embryo before hatching. The POALP was distributed ubiquitously in the body of P. olivaceus and was particularly high in the digestive tract. These findings suggest the potential role of POALP in nutrient absorption and transportation. During the pre-metamorphosis (F stage), ALP gene expression is 2.5-folds of that in the pro-metamorphosis (E stage); but in the post-metamorphosis (I stage), it was 1.8-folds of that of pro-metamorphosis. Exogenetic thyroxine (T4) and thiourea (TU) influenced the ALP gene expression significantly during the metamorphosis. Bioinformatics analysis showed that Japanese flounder ALP promoter region contained promoter sequence and putative recognition site for several transcriptional factors, including SREBP-1, SYR, and CdxA. In vitro promoter assays employing EGFP reporter system demonstrated that the promoter of ALP was active.

Three cotton homeobox genes are preferentially expressed during early seedling development and in response to phytohormone signaling

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants. In this study, three cDNAs (designated as GhHB2, GhHB3 and GhHB4) encoding HD-Zip proteins were isolated from cotton cDNA library. GhHB2 gene encodes a protein of 300 amino acids, GhHB3 gene encodes a peptide with 254 amino acids, and GhHB4 gene encodes a protein of 281 amino acids. The deduced proteins, which contain the homeodomain and leucine-rich zipper motif, share relatively high similarities with the other plant HD-Zip proteins. Quantitative RT-PCR analysis indicated that GhHB3 and GhHB4 were preferentially expressed in hypocotyls and cotyledons, whereas GhHB2 gene was predominantly expressed in young stems, at relatively high levels in hypocotyls. Expressions of all the three genes were up-regulated in roots, hypocotyls and cotyledons after GA(3) treatments. Additionally, GhHB4 expression was enhanced by 6-BA treatment. A GhHB2 promoter fragment was isolated from cotton by Genome-Walking PCR. Expression of GUS gene controlled under GhHB2 promoter was examined in the transgenic Arabidopsis plants. Strong GUS staining was detected in cotyledon, veins of the emerging leaves and shoot apices of 5- to 15-day-old transgenic seedlings, but GUS activity became more and more weak as the seedlings further developed. In addition, the promoter activity was induced by exogenous GA, indicating that GhHB2 promoter is very active during early seedling development, and may be GA-inducible. The results suggested that the three HB genes may function in early seedling development of cotton and in response to gibberellin signaling.

PCR Amplification and Bioinformatics of a Novel Gene Coding Hydroxylamine Oxidoreductase*

A probable novel gene coding hydroxylamine oxidoreductase (HAO) was acquired by PCR and gene sequencing in Acinetobactor sp. YY-5. Four pairs of PCR primers were designed according to the conservative regions of reported HAO gene to amplify and sequence the partial sequence of the HAO gene; several primers for genome walking PCR were designed according to the partial sequence to acquire the flank region of the partial HAO sequence; and the acquired sequence was subjected to Blast analysis in NCBI and open read frame (ORF) prediction by the professional ORF -analyzing software, FGeneSB. A 462 bp DNA sequence was obtained by one of the 4 pairs of primers, and further 2 690 bp DNA sequence of the flank region of 462 bp sequence was acquired through two rounds of genome walking PCR. Four ORFs were found within the 3 152 bp DNA sequence, one of which was of 555 bp. The 555-bp ORF contained the original 462 bp sequence and corresponded to an amino acid sequence of Mr 20.2×103, and no obvious similar HAO gene was found in GenBank. Thus, a novel HAO gene was possibly acquired by PCR.

Expression and localization of GhH6L, a putative classical arabinogalactan protein in cotton (<italic>Gossypium hirsutum</italic>)

Arabinogalactan proteins (AGPs) are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X-Y-proline-proline-proline (where X is serine or alanine and Y is threonine or serine). Northern blotting analysis and quantitative RT-PCR results showed that it was preferentially expressed in 10 days post-anthesis (dpa) fibers and was also developmentally regulated. A promoter fragment was isolated from cotton (Gossypium hirsutum) by genome walking PCR. Expression of beta-glucuronidase (GUS) gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.

해충저항성 GM 배추에서 T-DNA와 식물체 게놈의 인접 부위 분석

아그로박테리움을 이용한 형질전환은 대다수의 쌍자엽과 몇몇 단자엽 식물의 게놈내로 외래유전자를 도입하는 성공적인 방법이다. 해충저항성 CryIAc 유전자가 도입된 배추형 질전환체를 아그로박테리움 형질전환법을 통해 얻은 후, 도입유전자의 수를 Southern 분석을 통하여 확인하였다. 배추형질전환체 46개 중에서 29개는 1 copy의 CryIAc 유전자가 도입된 것으로 확인되었다. 식물체의 게놈내로 T-DNA 결합에 관한 정보를 얻기 위해서 LB 인접서열을 genome walking PCR 방법을 통하여 분석하였다. 46개의 배추형질전환체중에서 37개는 운반체의 backbone 염기서열을 지니는 것으로 확인되었다. 이러한 결과는 도입 운반체의 LB 지점에서 제대로 종결이 이루어지지 않아서 운반체의 backbone 염기서열이 운반된 것으로 보여진다. 운반체의 backbone 염기서열이 도입되지 않은 9개체의 배추형질전환체를 LB 인접서열을 분석한 결과, 모든 LB 부위는 절단부위가 보존되지 않았고, 절단부위에서 36bp까지도 결실이 확인되었다. The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome, We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium-transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.

Environmental DNA as a source of a novel epoxide hydrolase reacting with aliphatic terminal epoxides

We describe a convenient method for amplification of novel epoxide hydrolase-encoding genes directly from the metagenome. In a first step, small specific regions of putative epoxide hydrolase genes were amplified by using PCR with degenerate consensus primers specific for prokaryotic epoxide hydrolases, and environmental DNA as template. In a second step, the sequence obtained from one randomly selected epoxide hydrolase gene fragment served as the starting point for genome-walking PCR. This technique enabled us to recover a complete novel epoxide hydrolase gene with a GC content of 64.7%. A database search revealed that this novel gene was 44% and 43% identical to two putative epoxide hydrolases from Ralstonia metallidurans and Ralstonia eutropha, respectively, at the amino acid level, the highest among all orthologs searched. The gene, which encodes a polypeptide with a molecular mass of 34kDa, was cloned and overexpressed in Escherichia coli. The recombinant enzyme showed hydrolyzing activity toward aliphatic terminal epoxides with chain lengths ranging from 6 to 10 carbon atoms. In all cases, the enantioselectivity of the enzyme was low. Determination of the regioselectivity coefficients αR and αS revealed that the oxirane ring was attacked almost exclusively at the non-substituted carbon of the R-epoxide. The preference for attack at the non-substituted ring carbon of the S-epoxide was dependent on the chain length of the substrate and ranged from 55% to 78%, resulting in a partially enantioconvergent reaction.

Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum

Here we present the identification and cloning of the NcBSR4 gene, the putative Neospora caninum orthologue to the Toxoplasma gondii TgBSR4 gene. To isolate NcBSR4, genome walking PCR was performed on N. caninum genomic DNA using the expressed sequence tag NcEST3c28h02.y1 sequence, which shares a 44% identity with the TgBSR4 gene, as a framework. Nucleotide sequencing of amplified DNA fragments revealed a single uninterrupted 1227bp open reading frame that encodes a protein of 408 amino acids with 66% similarity to the TgBSR4 antigen. A putative 39-residue signal peptide was found at the NH2-terminus, followed by a hydrophilic region. At the COOH-terminus, a potential site for a glycosylphosphatidylinositol anchor was identified at amino acid 379. A polyclonal serum against recombinant NcBSR4 protein was raised in rabbits, and immunolabelling demonstrated stage-specific expression of the NcBSR4 antigen in N. caninum bradyzoites produced in vitro and in vivo. Furthermore, RT-PCR analysis showed a slight increase of NcBSR4 transcripts in bradyzoites generated during in vitro tachyzoite-to-bradyzoite stage-conversion, suggesting that this gene is specifically expressed at the bradyzoite stage and that its transcription relies on the switch to this stage.