Cloning Of Fragments Research Articles

Amplification and Cloning of Large cDNA Fragments of the Citrus tristeza virus Genome.

Citrus tristeza virus (CTV) is probably the most destructive viral pathogen of citrus. It causes chronic losses to commercial citrus production in all citrus-growing areas. The complete sequences of at least 42 genomes of different CTV strains have been obtained using different technologies including sequencing of multiple overlapped RT-PCR-amplified fragments with sizes of less than 4kb, or from small viral RNA (svRNA), through next-generation high-throughput sequencing (NGS) technologies. The large size of CTV genome (>19.2kb) makes it impractical to obtain and amplify full-length cDNA in a single step. The strategy of ligation of multiple cDNA fragments to assemble a full-length cDNA clone involves several serial cloning steps and sometimes subcloning phases using enzymatic digestion with restriction nucleases and ligation reactions. In this protocol, we describe a strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products. These 5'- and 3'-genomic halves, which were designed to be overlapped in 15nt in their 3'- and 5'-ends, respectively, were used as templates for further overlapped PCR to amplify the entire ~20kb CTV genome. The resultant full cDNA PCR product was then inserted into pCAMBIA-binary vector.

Cosmid Library Construction and Functional Cloning.

Cosmid libraries can represent an entire genome in a library of circular DNA molecules, allowing for the faithful amplification, cloning and isolation of large genomic DNA fragments. Moreover, using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.

Development of a high efficient promoter finding method based on transient transfection.

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.

Bacteria associated with planktonic diatoms from Lake Baikal

Algal-bacterial associations were studied in unialgal xenic cultures of Synedra acus subsp. radians, Asterionella formosa and Fragillaria crotonensis planktonic diatoms from Lake Baikal, using epifluorescent and scanning electron microscopy. It was found that rod- and ovoid-shaped bacteria colonized cell walls of diatoms. Cloning and sequencing of fragments of 16S rRNA gene in diatom cultures revealed members of Gammaproteobacteria (Pseudomonas sp.), Betaproteobacteria (Janthinobacterium sp., Hydrogenophaga sp., Methylophilus sp.), Bacteroidetes (Flavobacterium sp., Pedobacter sp.), and Acinobacteria (Nocardioides sp.).

Transposable Elements in the Organization and Diversification of the Genome of Aegilops speltoides Tausch (Poaceae, Triticeae).

Repetitive DNA—specifically, transposable elements (TEs)—is a prevailing genomic fraction in cereals that underlies extensive genome reshuffling and intraspecific diversification in the wild. Although large amounts of data have been accumulated, the effect of TEs on the genome architecture and functioning is not fully understood. Here, plant genome organization was addressed by means of cloning and sequencing TE fragments of different types, which compose the largest portion of the Aegilops speltoides genome. Individual genotypes were analyzed cytogenetically using the cloned TE fragments as the DNA probes for fluorescence in situ hybridization (FISH). The obtained TE sequences of the Ty1-copia, Ty3-gypsy, LINE, and CACTA superfamilies showed the relatedness of the Ae. speltoides genome to the Triticeae tribe and similarities to evolutionarily distant species. A significant number of clones consisted of intercalated fragments of TEs of various types, in which Fatima (Ty3-gypsy) sequences predominated. At the chromosomal level, different TE clones demonstrated sequence-specific patterning, emphasizing the effect of the TE fraction on the Ae. speltoides genome architecture and intraspecific diversification. Altogether, the obtained data highlight the current species-specific organization and patterning of the mobile element fraction and point to ancient evolutionary events in the genome of Ae. speltoides.

Методические аспекты изучения биоресурсных коллекций редких и хозяйственно ценных растений

A full range of classical and modern methods of evaluation of biological peculiarities and economic characteristics of samples including in the bioresource collection is presented. The main directions of their study: seasonal development, ontomorphogenesis, reproductive biology and genetic certi fi cation. It is shown that for trees, shrubs and perennials forming shoots of different types during the vegetative period, it is preferable to construct separate phonological spectra of vegetative and generative development. The adaptive potential is estimated on the basis of studying the structure of shoot systems and preservation of them in the unfavorable periods of weather; the formation of life form in ontogenesis; and upon reaching the generative state, it is done, taking into account the peculiarities of intrabud and extrabud organogeneses. The applied aspect of these studies is substantiation of time and methods of rejuvenation of objects for preservation of them in the bioresource collections. When studying reproductive biology, the potential importance of samples for breeding as paternal and maternal parental forms is determined. The fertility and viability of pollen are studied in detail in paternal forms, the terms and methods of pollen storage are elaborated. Fruit setting with different methods of pollination is evaluated in maternal forms. The fi nal stage is to estimate the potential and actual seed productivity, determine the type of dormancy of seeds and develop ways to overcome it. Based on the example of model objects, representatives of generic complexes of Rhaponticum and Trollius, the advantages of molecular-genetic method of ISSR-marking, based on the analysis of polymorphic DNA sites between microsatellites are shown. Its use allows to detect the greater number of polymorphic loci, and does not require prior cloning and sequencing of fragments for the selection of primers. The chemical composition of three species for cellulose, lignin, pentosans and ash content was studied in the model Miscanthus object, which is a promising bioenergetic culture. It was shown that samples with high cellulose content but low lignin content are of interest for technological processes.

Molecular studies on African swine fever virus from Brazilian isolates

ABSTRACT: African swine fever (ASF) is a devastating viral infirmity that affects domestic and wild swine caused by the ASF virus (ASFV) that belongs to the family Asfaviridae in the Asfavirus genus. Studies for genotypic and antigenic determination of ASFV including samples from Brazilian outbreaks were carried out outside Brazil. Here, we have reviewed studies on the molecular aspects of Brazilian isolates from 1978 and 1979. Results obtained from restriction fragment analysis, cloning and gene sequencing display the genotypic variation of viral samples. Viral genotyping based on sequences of the 3’ region of the p72 gene included in genotype I Brazilian samples, reinforcing the suggestion of the European origin for the virus that infected Brazilian herds and having low virulence potential. Corroborating those findings, at the American station PIADC, the infection of healthy pigs with the Brazilian strain induced ASF sub acute disease with low mortality and a low-virulence. Those results were similar with epidemiological vigilance forms of Brazilian swineherd in good health conditions having at least one ASFV isolation, and the ASF pioneer’s studies on the low mortality in the Brazilian herds affected by ASF. The ASFV spreading in Eastern Europe and Russia triggered a greater concern with intensifying the risk of viral dissemination from country to country. The low virulence ASF strains can increase the problem because of hidden viral reservoirs - which further reinforces the need for safety and preventive measures in virus-free countries. Finally, the problem is further compounded by the lack of vaccines and other immunological resources.

Impacts of long-term nitrogen addition, watering and mowing on ammonia oxidizers, denitrifiers and plant communities in a temperate steppe

Global changes in nitrogen (N) deposition, precipitation patterns and land use could have an impact on the biogeochemical N cycle mediated by microorganisms. Microbial responses to global changes have been generally evaluated in short-term studies, while the long-term effects are not well understood. In this study, the interactive effects of multiple global change factors on N-cycling microorganisms and their interactions with plants were investigated in a 9-year field experiment with N addition, watering and mowing in a temperate steppe in northern China. Quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP) and clone library were used to analyze the abundance and community structure of ammonia oxidizers and denitrifiers, respectively. Bacterial 16S rRNA gene abundance significantly increased under mowing, but the functional gene abundances including amoA, nirS, nirK and nosZ genes were not affected. Watering significantly affected the ammonia-oxidizing archaea (AOA) community and N addition showed a strong effect on soil ammonia-oxidizing bacteria (AOB) and nosZ-containing denitrifier community structure. No significant interactive effects of mowing, N addition and watering on communities of soil ammonia oxidizers and denitrifiers were observed. Structure equation models suggested that plant biomass and community were significantly correlated with microbial communities and activities. These findings suggest that there are complex interactions between plants and soil microorganisms in grassland ecosystems.

A novel LRAT mutation affecting splicing in a family with early onset retinitis pigmentosa

Background and purposeRetinitis pigmentosa is an important cause of severe visual dysfunction. This study reports a novel splicing mutation in the lecithin retinol acyltransferase (LRAT) gene associated with early onset retinitis pigmentosa and characterizes the effects of this mutation on mRNA splicing and structure.MethodsGenome-wide linkage analysis followed by dideoxy sequencing of the linked candidate gene LRAT was performed in a consanguineous Pakistani family with autosomal recessive retinitis pigmentosa. In silico prediction and minigene assays were used to investigate the effects of the presumptive splicing mutation.ResultsARRP in this family was linked to chromosome 4q31.21-q32.1 with a maximum LOD score of 5.40. A novel homozygous intronic mutation (NM_004744.4: c.541-15T>G) was detected in LRAT. In silico tools predicted that the AG-creating mutation would activate an intronic cryptic acceptor site, but cloning fragments of wild-type and mutant sequences of LRAT into Exontrap Cloning Vector pET01 and Expression Cloning Vector pCMV-(DYKD4K)-C showed that the primary effect of the sequence change was to weaken the nearby authentic acceptor site and cause exon skipping, with only a small fraction of transcripts utilizing the acceptor site producing the reference transcript.ConclusionsThe c.541-15T>G mutation in LRAT results in aberrant splicing and is therefore predicted to be causal for the early onset retinitis pigmentosa in this family. In addition, this work suggests that minigenes adapted to the specific gene and exon may need to be designed for variants in the first and last exon and intron to mimic the authentic splicing mechanism in vivo.

Bug-proneness and late propagation tendency of code clones: A Comparative study on different clone types

Code clones are defined to be the exactly or nearly similar code fragments in a software system’s code-base. The existing clone related studies reveal that code clones are likely to introduce bugs and inconsistencies in the code-base. However, although there are different types of clones, it is still unknown which types of clones have a higher likeliness of introducing bugs to the software systems and so, should be considered more important for managing with techniques such as refactoring or tracking. With this focus, we performed an empirical study that compared the bug-proneness of the major clone-types: Type 1, Type 2, and Type 3.According to our experimental results on thousands of revisions of nine diverse subject systems, Type 3 clones exhibit the highest bug-proneness among the three clone-types. The bug-proneness of Type 1 clones is the lowest. Also, Type 3 clones have the highest likeliness of being co-changed consistently while experiencing bug-fixing changes. Moreover, the Type 3 clones that experience bug-fixes have a higher possibility of evolving following a Similarity Preserving Change Pattern (SPCP) compared to the bug-fix clones of the other two clone-types. From the experimental results it is clear that Type 3 clones should be given a higher priority than the other two clone-types when making clone management decisions. Our investigation on the relatedness between bug-proneness and late propagation in code clones implies that bug-proneness of code clones is not primarily related with late propagation. The possibility that a bug-fix experienced by a clone fragment will be related with late propagation is only 1.4%. Moreover, for only 10.76% of the cases, a late propagation experienced by clone fragments can be related with a bug. Thus, late propagation contributes to a very little proportion of the bugs in code clones. We believe that our study provides useful implications for ranking clones for management such as refactoring and tracking.

Diversity and Dynamics of Microbial Community Structure in Different Mangrove, Marine and Freshwater Sediments During Anaerobic Debromination of PBDEs.

Little is known about the diversity and succession of indigenous microbial community during debromination of polybrominated diphenyl ethers (PBDEs). This study examined the diversity and dynamics of microbial community structure in eight saline (mangrove and marine) and freshwater sediment microcosms exhibiting different debrominating capabilities for hexa-BDE 153, a common congener in sediments, using terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses. The results showed that microbial community structure greatly differed between the saline and freshwater microcosms, likely leading to distinct variations in their debrominating capabilities and pathways. Higher relative abundances of Chloroflexi and Deltaproteobacteria succeed by Alphaproteobacteria and Betaproteobacteria were detected in the two mangrove microcosms with the fastest debrominating capabilities mainly via para pathway, respectively; the dominance of Alphaproteobacteria resulted in less accumulation of tetra-BDEs and more complete debromination of lower brominated congeners (from di- to tetra-BDEs). Meanwhile, the shifts in both microbial community structure and PBDE profiles were relatively small in the less efficient freshwater microcosms, with relatively more ortho and meta brominated products of BDE-153 resulted. Coincidently, one of the freshwater microcosms showed sudden increases of Chloroflexi and Deltaproteobacteria by the end of incubation, which synchronized with the increase in the removal rate of BDE-153. The significant relationship between microbial community structure and PBDEs was confirmed by redundancy analysis (18.7% of total variance explained, P = 0.002). However, the relative abundance of the well-known dechlorinator Dehalococcoides showed no clear correlation with the debrominating capability across different microcosms. These findings shed light in the significance of microbial community network in different saline environments on enhancement of PBDE intrinsic debromination.

Limitations of the Echinococcus granulosus genome sequence assemblies for analysis of the gene family encoding the EG95 vaccine antigen.

Echinococcus granulosus is an important zoonotic parasite that is distributed worldwide. The EG95 vaccine was developed to assist with control of E. granulosus transmission through the parasite's livestock intermediate hosts. The vaccine is based on a recombinant antigen encoded by a gene which is a member of a multi-gene family. With the recent availability of two E. granulosus draft genomes, we sought to map the eg95 gene family to the genomes. We were unable to map unequivocally any of the eg95 gene family members which had previously been characterized by cloning and sequencing both strands of genomic DNA fragments. Our inability to map EG95-related genes to the genomes has revealed limitations in the assembled sequence data when utilized for gene family analyses. This study contrasts with the expectations expressed in often high-profile publications describing draft genomes of parasitic organisms, highlighting deficiencies in currently available genomic resources for E. granulosus and provides a cautionary note for research which seeks to utilize these genome datasets.

Responses of N2O reductase gene (nosZ)-denitrifier communities to long-term fertilization follow a depth pattern in calcareous purplish paddy soil

The effect of long-term fertilization on soil denitrifying communities was analysed by measuring the abundance and diversity of the nitrous oxide (N2O) reductase gene, nosZ. Soil samples were collected from plots of a long-term fertilization experiment established in 1982 in Suining City, China. The fertilizer treatments were no fertilizer (CK), three chemical fertilizer (CF) treatments (N, NP, NPK), manure (M) alone, and manure with chemical fertilizers (NM, NPM, NPKM). The abundance and diversity of the denitrifying bacteria were assessed by real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nosZ genes. The diversity and abundance of nosZ-denitrifiers was higher in soil amended with manure and chemical fertilizers (CFM) than in soil amended with CF alone, and the highest in topsoil (0–20 cm). The nosZ-denitrifier community composition was more complex in CFM soil than in CF soil. Specific species were detected only in the CFM soil. The abundance of nosZ-denitrifier in the NPKM treatment was approximately two times higher than that in the CK, N, and NPK treatments. Most of the cloned nosZ sequences were closely related to nosZ sequences from Bradyrhizobiaceae and Rhodospirillaceae in Alphaproteobacteria. Of the measured abiotic factors, soil organic matter correlated significantly with the abundance (P<0.01); available phosphorus correlated significantly with the topsoil community composition (P<0.01), whereas soil organic matter correlated significantly with the subsoil (20–90 cm) community composition (P<0.01). This study demonstrated that long-term CFM fertilization affected both the abundance and composition of the nosZ-denitrifier community.

Machine‐Learning Aided Analysis of Clone Evolution

Code clones are similar code fragments appearing in software. As software evolves, code clones may be subjected to changes as well; we term this clone evolution. There have not been many investigations into clone evolution characteristics. Therefore, we tackle this by exploring useful information associated with changes of clones during evolution. We focus on three perspectives of clone evolution, ranging from individual clone changes to characterization of clone genealogies. With the help Xmeans clustering, we establish associations between clone changes and life of clones. Our experimental results on two softwares show that clones are mostly stable throughout software evolution. For the relatively smaller group of “unstable” clones, changes usually happen after several versions, and consistent changes appear more frequently than inconsistent ones. We suggest that developers should pay more attention to relatively longer genealogies, and should consider applying changes consistently to clone group when a constituent clone fragment has undergone change.

Comparing Software Bugs in Clone and Non-clone Code: An Empirical Study

Code cloning is a recurrent operation in everyday software development. Whether it is a good or bad practice is an ongoing debate among researchers and developers for the last few decades. In this paper, we conduct a comparative study on bug-proneness in clone code and non-clone code by analyzing commit logs. According to our inspection of thousands of revisions of seven diverse subject systems, the percentage of changed files due to bug-fix commits is significantly higher in clone code compared with non-clone code. We perform a Mann–Whitney–Wilcoxon (MWW) test to show the statistical significance of our findings. In addition, the possibility of occurrence of severe bugs is higher in clone code than in non-clone code. Bug-fixing changes affecting clone code should be considered more carefully. Finally, our manual investigation shows that clone code containing if-condition and if–else blocks has a high risk of having severing bugs. Changes to such types of clone fragments should be done carefully during software maintenance. According to our findings, clone code appears to be more bug-prone than non-clone code.

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus cereus VCRC B540.

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518bp PCR product, and analysis of the sequence revealed the presence of 1482bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398bp. Theoretical molecular weight (51.7kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.

Interstitial telomeric repeats-associated DNA breaks

ABSTRACTDuring a cell's lifespan, DNA break formation is a common event, associated with many processes, from replication to apoptosis. Most of DNA breaks are readily repaired, but some are meant to persist in time, such as the chromosome ends, protected by telomeres. Besides them, eukaryotic genomes comprise shorter stretches of interstitial telomeric repeats. We assumed that the latter may also be associated with the formation of DNA breaks meant to persist in time. In zebrafish and mouse embryos, cells containing numerous breakage foci were identified. These breaks were not associated with apoptosis or replication, nor did they seem to activate DNA damage response machinery. Unlike short-living, accidental sparse breaks, the ones we found seem to be closely associated, forming discrete break foci. A PCR-based method was developed, allowing specific amplification of DNA regions located between inverted telomeric repeats associated with breaks. The cloning and sequencing of such DNA fragments were found to denote some specificity in their distribution for different tissue types and development stages.

Evaluation of pEASY-Uni Seamless Cloning and Assembly Kit to clone multiple fragments of Elaeis guineensis DNA

Several seamless DNA assembly kits based on in vitro homologous recombination activity are commercially available in recent years for efficient and rapid construction of expression vectors, subsequent transformation and investigation of gene functionality. This study was performed to estimate the efficiency of uni seamless cloning system, through cloning of multiple DNA fragments derived from Elaeis guineensis stearoyl-ACP desaturase (SAD) and metallothionein-like protein genes (MET1 and MET2) into a plasmid cloning vector. PCR fragments were assembled based on homologous overlapping regions of 25–30bp into a pUC19 linearized vector. The successful cloning of the three DNA fragments was validated by blue white screening, colony cracking and colony PCR. The observed overall cloning efficiency for the three assembled DNA fragments of about 2.7kb in size was above 65%. This system produces seamless junctions, is directional and does not rely on restriction enzyme digestion and any specific site within the DNA fragments.

Mercury methylation in the soils and sediments of Three Gorges Reservoir Region

Previous studies demonstrated that microorganisms had an important role in the mercury (Hg) methylation process in the water and sediments of Three Gorges Reservoir (TGR). The purpose of this research was to analyze the microbial methylation of Hg in the soils and sediments of the water-level-fluctuating zone (WLFZ) of TGR. Different types of soils, sediment (≤ 155 m), semi-inundated soil (≥ 155 m), and non-inundated soil (≥ 175 m) of the WLFZ of Shibao (S), Zhenxi (Z), and Tujing (T) were investigated. Real-time PCR, terminal restriction fragment length polymorphism (T-RFLP), cloning, sequencing, and phylogenetic analysis were used to analyze the abundance and diversity of dsrB, hgcA, and mcrA genes, and their relationship with the levels of total Hg (THg), MeHg, and several biogeochemical factors that probably affected microbial methylation reaction. THg concentrations in different soil types of the WLFZ of TGR did not show significant differences (p > 0.05) in site S, while there were significant differences (p < 0.05) of MeHg levels in different soil types of the three sites. Phylogenetic analyses found that the dominant groups of microorganisms with dsrB in the sediment and non-inundated soil in site S differed remarkably. Microorganisms that probably related with Hg methylation mainly distributed in the sediment, with δ-proteobacteria as the dominant class. Real-time PCR found that soil MeHg levels correlated positively with the resident quantities of microorganisms with dsrB. The abundance of dsrB was much higher than that of hgcA which may indicate that only a small part of sulfate-reducing bacteria (SRB) related with Hg methylation. Soil MeHg levels correlated positively with the resident quantities of microorganisms with the dsrB gene. The phylogenetic analysis indicated that SRB that probably related with Hg methylation distributed mainly in the sediment, rather than in the non-inundated soil.

Fragments of Functional Clones

In [1] we came up with some approach and problems associated with subsets of functional clones on a fixed set that consist of functions occurring in a clone with a fixed restriction on their arity. This approach receives further development.