Genome Walking Method Research Articles

Characterisation of large F9 deletions in seven unrelated patients with severe haemophilia B.

Large deletions in the F9 gene are detected in approximately 5% of patients with severe haemophilia B, but only a few deletion breakpoints have been characterised precisely until now. In this study we identified a total of seven large F9 deletions in the index patients and nine female carriers by the AccuCopy technique. We also successfully characterised the exact breakpoints for each large deletion including four deletions encompassing the entire F9 gene by the genome walking method combined with primer walking strategy. The extents of deletion regions ranged from 11.1 to 884 kb. Microhomologies ranged from 2 to 6 bp were identified in the breakpoint junctions of six deletions. The other deletion occurred between two highly homologous sequences of the same long interspersed nuclear element 1 (LINE/L1). Non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) may be the main causative mechanisms for the six large deletions with microhomologies. Non-allelic homologous recombination (NAHR) may mediate the deletion occurred between the two tandem LINEs in the other large deletion. Repetitive elements and non-B DNA forming motifs identified in the junction regions may contribute to DNA breakage leading to large deletions.

Randomly broken fragment PCR with 5′ end-directed adaptor for genome walking

Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5′ end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5′ end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5′ end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.

Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.

Efficient retrieval of recombinant sequences of GM plants by Cauliflower Mosaic Virus 35S promoter-based bidirectional LT-RADE

Identification of genetically modified (GM) plants remains a difficult task, especially when few information on the GM content in a sample is available. The CaMV P-35S is a commonly applied genetic element in GM plants and represents as such a suitable starting point for the tracing of GM plants. Here, the application of a CaMV P-35S LT-RADE genome walking method on five different GM crops is documented: MON810 maize, LLRICE62 rice, T45 rapeseed, A2704-12 soybean and LLCOTTON25 cotton. Two sets of oligonucleotide primers are presented as a potential forward and reverse genome walking strategy with genomic DNA as template. The method’s applicability for assessing the recombinant nature of P-35S positive signals is discussed in detail. This study demonstrates the general use of LT-RADE in molecular characterization of GMOs retrieving several previously unknown transgenic insert nucleotide sequences. Current limitations and future developments are discussed.

A tailing genome walking method suitable for genomes with high local GC content

The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5′ polycytosine and 3′ polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content.

RT-qPCR analysis of dexB and galE gene expression of Streptococcus alactolyticus in Astragalus membranaceus fermentation

Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A. membranaceus by S. alactolyticus had some similar properties to those from A. membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A. membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) during A. membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S. alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A. membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S. alactolyticus strain FGM were involved in the regulation of A. membranaceus fermentation and they might play some roles in the increase of polysaccharides.

Conversion of Microsatellite Markers to Single Nucleotide Polymorphism (SNP) Markers for Genetic Fingerprinting of Theobroma cacao L.

The majority of the world's cacao for chocolate manufacture is produced in West Africa. Cocoa breeding programs in West Africa need genetic markers to reduce the time needed for improving cocoa by screening seedlings for the presence of the markers rather than mature plants for the phenotypic traits (i.e., marker-assisted selection [MAS]). For MAS to be successful, the breeder must have both access to markers linked to desired traits and a convenient marker-assay system that can be performed locally. In this study, microsatellite markers that flanked disease resistance quantitative trait loci (QTL) but could not be assayed conveniently in West Africa were converted using a genome walking method into single nucleotide polymorphism (SNP) markers that could be assayed locally. The SNP and microsatellite markers were equally effective in identifying off-types in two different mapping populations of cacao. Also, SNPs cast doubt on whether all microsatellite markers are identical by descent.

Molecular cloning, characterization and promoter analysis of a novel MDR-type ABC transporter gene (GbMDR1) from Ginkgo biloba

A novel MDR-type ABC transporter cDNA (designated as Gbmdr1) was cloned and characterized for the first time from the gymnosperm plant species, Ginkgo biloba, using RACE technique. The full-length cDNA of Gbmdr1 was 4275 bp, and it contained a 3840 bp open reading frame (ORF) encoding a polypeptide of 1279 amino acid with a predicted isoelectric point of 8.22 and molecular mass of 139.6 kDa. The deduced GbMDR1 protein consisted of four domains in- cluding two TMDs (74-351, 716-993) and two NBDs (428-607, 1067-1251). The promoter of the Gbmdr1 was also cloned by the Genome Walking method. Sequence analysis demonstrated that there were many regulatory elements in the Gb- mdr1 promoter and the TATA box was located at -52~-56 bp upstream of the transcriptional start site. Sequence alignment and molecular evolution analysis revealed that GbMDR1 was a plant MDR-like ABC transporter protein, and that it has a further relationship with most other MDRs of plant species. Southern blot analysis indicated that Gbmdr1 belonged to a small multi-gene family. Tissue expression analysis indicated that Gbmdr1 expression was high in stems and leaves but low in roots. These results show that GbMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease

Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind.

Genome walking by next generation sequencing approaches.

Genome Walking (GW) comprises a number of PCR-based methods for the identification of nucleotide sequences flanking known regions. The different methods have been used for several purposes: from de novo sequencing, useful for the identification of unknown regions, to the characterization of insertion sites for viruses and transposons. In the latter cases Genome Walking methods have been recently boosted by coupling to Next Generation Sequencing technologies. This review will focus on the development of several protocols for the application of Next Generation Sequencing (NGS) technologies to GW, which have been developed in the course of analysis of insertional libraries. These analyses find broad application in protocols for functional genomics and gene therapy. Thanks to the application of NGS technologies, the original vision of GW as a procedure for walking along an unknown genome is now changing into the possibility of observing the parallel marching of hundreds of thousands of primers across the borders of inserted DNA molecules in host genomes.

LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named “Rapid Amplification of genomic DNA Ends” (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.

Isolation of the promoter region of <I>HAK</I> gene from <I>Aeluropus littoralis</I> and functional analysis in rice

The AlHAK1 gene encoding a high-affinity K+ transporter was isolated from Aeluropus littoralis (Gouan) Parl, a graminaceous halophyte, and plays a crucial role in nutrition and ion homeostasis in plant cell. To investigate the regulation role of AlHAK1 on the transcriptional level, an about 1.3 kb 5'-flanking region of the AlHAK1 gene containing a putative promoter was cloned by genome walking method. Cis-regulatory elements analysis showed AlHAK1-promoter region contained typical TATA and CAAT boxes, and some growth and development relative motifs, as well as environmental re-sponsive elements. To reveal the function and regulating role, the AlHAK1 promoter was fused to the β-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector and introduced into rice via Agrobacterium-mediated transformation. Histo-chemical staining indicated that the GUS expression directed by AlHAK1 promoter was observed in leaves, stems, roots, anther, lemma, and palea. GUS quantitative fluorometric analysis indicated that GUS activity directed by AlHAK1 promoter was lower than CaMV35S and Ubiquitin constitutive promoters; however, in the roots and stems the GUS activity was rela-tively high and displayed a tissue-specific expression pattern. Under ABA, high temperature or drought stress, the GUS activity directed by AlHAK1 promoter was inducible in the roots and stems, suggesting the elements of HSE (-682 bp) and MybBS (-1 268 bp) might play a role in the inducible regulation.

Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP

A 1272 bp upstream sequence of MsZFN gene was cloned from alfalfa, which was designed as MsZPP (Genbank accession number: FJ 161979.2) using an adaptor-mediated genome walking method. A sole transcription start site was located 69 bp upstream of the translation start site. Its pattern of expression included roots, stem vascular tissues, floral reproductive organs, and leaves, but the promoter did not express in seeds, petals or sepals. Transcription levels can be stimulated by dark, MeJA, and IAA. However, GUS fusion activities had no change by treatments of GA, ABA, drought and high salt for 3 days. Deletion analysis revealed that all sections of the promoter can drive gus gene expression in the root, stem, leaves and floral reproductive organs; however, only fragments longer than the -460 bp promoter can stimulate strong gus gene expression in these organs. In addition, the -460 bp promoter fragment can drive gus expression not only in the vascular tissue, but also in leaf guard cells. The results suggest that the promoter MsZPP plays roles in the regulation of transgene expression, particularly due to its darkness, MeJA, and IAA responsiveness.

Genome walking in eukaryotes

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T-DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

Cloning and Recombinant Expression of Chitosanase Gene from Bacillus Sp. S-1

In this work, we report the characterization of a chitosanase-producing bacterium isolated from soil. This strain was grouped under the genus Bacillus by virtue of its morphological, physiological properties and 16S rDNA gene sequence and named it Bacillus sp. S-1. According to the information of chitosanase full-length sequences deposited in NCBI, a pair of degenerated primes was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction (PCR) using Bacillus sp. S-1 genome DNA as the template. A genome walking library was constructed followed as the protocol provided by CLONTECH Company. The flanking sequences of the 5’ and 3’ terminal was obtained by genome walking method and two-step PCR technique. After overlapped and confirmed, the full-length sequence of chitosanase from Bacillus sp. S-1 was achieved and it contained 1362 bp coding 453 amino acids (accession number is EU924147). The predicted amino acid sequence was 96% similar to that of Bacillus cereus ATCC 14579 (accession number is NC_004722). The fusion protein containing BSCHITO was produced in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified rBSCHITO degraded the chitosan (the degree of deacetylation of 99%) to produce mixture of chitooligosaccharides. The BSCHITO is thus an endo-chitosanase.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia

Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

An affinity-based genome walking method to find transgene integration loci in transgenic genome

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.

Genes Associated with 2-Methylisoborneol Biosynthesis in Cyanobacteria: Isolation, Characterization, and Expression in Response to Light

The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater.

Characterization of Copper/Zinc-Superoxide Dismutase (Cu/Zn-SOD) Gene from an Endangered Freshwater Fish Species Hemibarbus mylodon (Teleostei; Cypriniformes)

Gene structure of copper/zinc-superoxide dismutase (Cu/Zn-SOD; sod1) was characterized in Hemibarbus mylodon (Teleostei; Cypriniformes), an endangered freshwater fish species in Korean peninsula. Full-length cDNA of H. mylodon SOD1 consisted of a 796-bp open reading frame sequence encoding 154 amino acids, and the deduced polypeptide sequence shared high sequence homology with other orthologs, particularly with regard to metal-coordinating ligands. Genomic structure of the H. mylodon sod1 gene (hmsod1; 1,911 bp from the ATG start codon to the stop codon) was typical quinquepartite (i.e., five exons interrupted by four introns); the lengths of the exons were similar among species belonging to various taxonomic positions. The molecular phylogeny inferred from sod1 genes in the teleost lineage was in accordance with the conventional taxonomic assumptions. 5'-flanking upstream region of hmsod1, obtained using the genome walking method, contained typical TATA and CAAT boxes. It also showed various transcription factor binding motifs that may be potentially involved in stress/immune response (e.g., sites for activating proteins or nuclear factor kappa B) or metabolism of xenobiotic compounds (e.g., xenobiotic response element; XRE). The hmsod1 transcripts were ubiquitously detected among tissues, with the liver and spleen showing the highest and lowest expression, respectively. An experimental challenge with Edwardsiella tarda revealed significant upregulation of the hmsod1 in kidney (4.3-fold) and spleen (3.1-fold), based on a real-time RT-PCR assay. Information on the molecular characteristics of this key antioxidant enzyme gene could be a useful basis for a biomarker-based assay to understand cellular stresses in this endangered fish species.

Isolating promoters of multigene family members from the polyploid sugarcane genome by PCR-based walking in BAC DNA

The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.