Insertion Of Fragment Research Articles

Insertion of Metal-Substituted Silylene into Naphthalene's Aromatic Ring and Subsequent Rearrangement for Silaspiro-Benzocycloheptenyl and Cyclobutenosilaindan Derivatives.

Synthesis of silacycle compounds are of fundamental and application importance. Herein we report the first example of insertion of metal-substituted silylene fragment into naphthalene's aromatic ring. More significantly, this insertion is followed by interesting rearrangements to yield silaspiro-benzocycloheptenyl and cyclobutenosilaindan derivatives. The formation of cyclobutenosilaindan derivative includes the C-C bond cleavage and 4π electrocyclization steps; the formation of silaspiro-benzocycloheptenyl derivative is more complicated, including the C-C bond cleavage, reversible 4π electrocyclization, C-H bond activation and C-Si bond cleavage. DFT investigations were carried out to shed light on the mechanistic aspects of these two rearrangements. The formed cyclobutenosilaindan potassium can readily react with PhOH, MeOTf, EtOTf, PhCH2 Cl or PhCOCl at room temperature to afford the hydrogen, alkyl, benzyl or benzoyl substituted cyclobutenosilaindans in high yields.

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

SummaryThe vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vriΔ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vriΔ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

PSX-39 Late-Breaking Abstract: Characterization of epigenetic and transcriptional landscape in heat stressed rats using ATAC-seq and RNA-seq

Abstract Understanding animal physiology and identifying reliable biomarkers may help to establish effective management strategies for the prevention of heat stress (HS). However, little is known about the molecular mechanism of mammal tolerance to high temperatures. In a previous study with Sprague-Dawley rats, we performed RNA-seq assays on the liver of rats in control (CT; 22 ℃, n = 5) and heat stress (HS120; 42 ℃ for 120 min, n = 5) groups. A total of 3,909 differential expression genes (DEGs, Q < 0.05) were observed. This study was conducted to further examine the epigenetic landscape in the liver of rats under HS and identify transcription factors (TFs), as well as their regulated genes. Three liver tissues were selected from the RNA-seq samples and performed an Assay for Transpose Accessible Chromatin (ATAC-seq). Peaks meeting criteria of P< 0.05 and |Fold Change| >1.5 were considered as differential peaks. All ATAC-seq libraries generated an expected distribution of the insert fragment lengths, with the majority of fragments being small, which characterize inter-nucleosomal open chromatin, and progressively fewer fragments of larger size, which are spanning nucleosomes. The accessibility of transcriptional start sites (TSS) was significantly enriched. After merging data, 2,356 differential peaks showed CT having more accessible TSS than H120 and only 230 differential peaks showed H120 group having more accessible TSS than CT. Thirty-six and 22 TF motifs were predicted by up- and down-regulated differential peaks in H120 vs. CT. Together with the previous DEG results, we proposed candidate TFs annotated to Cebpa, Foxa4, and Sp3 DEGs, which are involved in the regulation of oxidative stress. In summary, we showed that nuclear chromatin in the liver of heat stressed rats was less open than that of control rats. We suggest that the TFs (Cebpa, Foxa4, and Sp3) may be involved in the physiological regulation of HS.

Rice Protein Tagging Project: A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

Rice Protein Tagging Project: A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

Legal and practical challenges to authorization of gene edited plants in the EU

According to a predominant interpretation of the C-528/16 judgment of the Court of Justice of the European Union, mutants resulting from gene editing, even those featuring only single nucleotide variants, should be subject to the authorization procedures designed for organisms developed through genetic modification (i.e. insertion of large DNA fragments). In this article, we illustrate practical problems with the authorization of products of gene editing in the EU. On the basis of these problems, we analyze the influence of the current interpretation of EU legislation and judgment on the practical ability to authorize and detect such products on the EU market. We show that the predominant interpretation of the judgment leads to legally unacceptable consequences, in particular to the violation of the principle of proportionality with regard to individuals who wish to develop and market products of gene editing. As a result of our considerations, we show that the C-528/16 judgment did not need to be interpreted in the dominant way.

Isolation and molecular typing of Mycobacterium avium subsp. paratuberculosis from faeces of dairy cows.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis mainly in domestic and wild ruminants; paratuberculosis is also known as Johne's disease. This disease is endemic all over the world generating significant economic losses, especially in dairy herds, although, MAP is the cause of infection in many other species including primates. Currently, MAP mycobacteria are recognized as pathogens transmitted by food. They are a potential threat to animal and human health. Infected animals excreting mycobacteria with faeces are the main source of MAP. The development of control strategies and disease control are based on determination of the genetic diversity of the MAP strains causing Johne's disease. This study describes 43 strains isolated from a herd of dairy cows located in northern Poland. The types of MAP were determinted based on the polymorphism analysis of two insertion fragments: IS900 and IS1311. The polymorphism of IS900 was analyzed with the use of a PCR multiplex according to Collins' method and the IS1311 polymorphism with the use of the PCR-REA method. Based on the differences observed, the strains isolated were classified into two MAP types, cattle (C-type) and sheep (S-type), with the predominance of the cattle type.

Yellow-fruited phenotype is caused by 573 bp insertion at 5' UTR of YFT1 allele in yft1 mutant tomato

The yft1 tomato mutant has a yellow-fruited phenotype controlled by a recessive gene of YFT1 allele, which has been shown by map-based cloning to be a homolog of ETHYLENE INSENSITIVE 2 (EIN2). Genetic lesion of YFT1 allele in yft1 is attributed to a 573 bp DNA fragment (IF573) insertion at 1,200 bp downstream of the transcription start site. Transcriptomic analysis revealed that YFT1 lesion resulted in 5,053 differentially expressed genes (DEGs) in yft1 pericarp compared with the M82 wild type cultivar. These were annotated as being involved in ethylene synthesis, chromoplast development, and carotenoid synthesis. The YFT1 lesion caused a reduction in its own transcript levels in yft1 and impaired ethylene emission and signal transduction, delayed chromoplast development and decreased carotenoid accumulation. The molecular mechanism underlying the downregulated YFT1 allele in yft1 was examined at both RNA and DNA levels. The IF573 event appeared to introduce two negative regulatory sequences located at -272 to -173 bp and -172 to -73 bp in the YFT1 allele promoter, causing alterative splicing due to introduction of aberrant splicing sites, and breaking upstream open reading frames (uORF) structure in the 5'-UTR. Those results a new provided insight into molecular regulation of color formation in tomato fruit.

Statistical testing of hypotheses about the form of the factor law of influence by the Kolmogorov criterion

When affected by the impact of emergency sources on the same type of elementary objects, the depth of the introduction of shrapnel into the barrier changes randomly. It is necessary to determine the law of distribution of random depth of inserting fragments into the barrier. In the course of the work, hypotheses about three distribution laws were tested. Based on the results of experimental studies, it is proposed to assess the suitability of the law of distribution, the statistics of which, according to the Kolmogorov criterion, is intended to check the alignment of empirical distribution with this continuous theoretical distribution. With a small number of tests on the criterion Kolmogorov confirmed the consent of even clearly incompatible samples. However, with a large number of observations (more than 50) the Kolmogorov Criterion is able to detect any deviation from the hypothesis.

Enhancing fragment-based protein structure prediction by customising fragment cardinality according to local secondary structure

BackgroundWhenever suitable template structures are not available, usage of fragment-based protein structure prediction becomes the only practical alternative as pure ab initio techniques require massive computational resources even for very small proteins. However, inaccuracy of their energy functions and their stochastic nature imposes generation of a large number of decoys to explore adequately the solution space, limiting their usage to small proteins. Taking advantage of the uneven complexity of the sequence-structure relationship of short fragments, we adjusted the fragment insertion process by customising the number of available fragment templates according to the expected complexity of the predicted local secondary structure. Whereas the number of fragments is kept to its default value for coil regions, important and dramatic reductions are proposed for beta sheet and alpha helical regions, respectively.ResultsThe evaluation of our fragment selection approach was conducted using an enhanced version of the popular Rosetta fragment-based protein structure prediction tool. It was modified so that the number of fragment candidates used in Rosetta could be adjusted based on the local secondary structure. Compared to Rosetta’s standard predictions, our strategy delivered improved first models, + 24% and + 6% in terms of GDT, when using 2000 and 20,000 decoys, respectively, while reducing significantly the number of fragment candidates. Furthermore, our enhanced version of Rosetta is able to deliver with 2000 decoys a performance equivalent to that produced by standard Rosetta while using 20,000 decoys. We hypothesise that, as the fragment insertion process focuses on the most challenging regions, such as coils, fewer decoys are needed to explore satisfactorily conformation spaces.ConclusionsTaking advantage of the high accuracy of sequence-based secondary structure predictions, we showed the value of that information to customise the number of candidates used during the fragment insertion process of fragment-based protein structure prediction. Experimentations conducted using standard Rosetta showed that, when using the recommended number of decoys, i.e. 20,000, our strategy produces better results. Alternatively, similar results can be achieved using only 2000 decoys. Consequently, we recommend the adoption of this strategy to either improve significantly model quality or reduce processing times by a factor 10.

Enhancement of homology-directed repair with chromatin donor templates in cells.

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.

Identification of genomic insertion and flanking sequences of the transgenic drought-tolerant maize line "SbSNAC1-382" using the single-molecule real-time (SMRT) sequencing method.

Safety assessment of genetically modified (GM) crops is crucial at the product-development phase before GM crops are placed on the market. Determining characteristics of sequences flanking exogenous insertion sequences is essential for the safety assessment and marketing of transgenic crops. In this study, we used genome walking and whole-genome sequencing (WGS) to identify the flanking sequence characteristics of the SbSNAC1 transgenic drought-tolerant maize line “SbSNAC1-382”, but both of the two methods failed. Then, we constructed a genomic fosmid library of the transgenic maize line, which contained 4.18×105 clones with an average insertion fragment of 35 kb, covering 5.85 times the maize genome. Subsequently, three positive clones were screened by pairs of specific primers, and one of the three positive clones was sequenced by using single-molecule real-time (SMRT) sequencing technology. More than 1.95 Gb sequence data (~105× coverage) for the sequenced clone were generated. The junction reads mapped to the boundaries of T-DNA, and the flanking sequences in the transgenic line were identified by comparing all sequencing reads with the maize reference genome and the sequence of the transgenic vector. Furthermore, the putative insertion loci and flanking sequences were confirmed by PCR amplification and Sanger sequencing. The results indicated that two copies of the exogenous T-DNA fragments were inserted at the same genomic site, and the exogenous T-DNA fragments were integrated at the position of Chromosome 5 from 177155650 to 177155696 in the transgenic line 382. In this study, we demonstrated the successful application of the SMRT technology for the characterization of genomic insertion and flanking sequences.

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method.

Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.

An intergenic "safe haven" region in Aspergillus fumigatus.

Aspergillus fumigatus is the most common opportunistic human fungal pathogen responsible for invasive aspergillosis. Gene manipulation is critical for the investigation of A. fumigatus biology and pathogenesis at the molecular level, and it often requires integration of the introduced DNA into the fungal genome. Here we have searched and identified two potential "safe haven" regions, SH1 and SH2, based on A. fumigatus genome annotation and transcriptome data. When a DNA fragment carrying a fluorescent protein gene mNeonGreen (mNG) and a drug selection marker was inserted into SH1 or SH2, the expression of mNeonGreen was easily detected, indicating that SH1 and SH2 are not surpressive genetic regions. We found that insertion of this DNA fragment into SH1 did not cause any significant changes in the expression of neighboring genes. Insertion of this DNA into either SH1 or SH2 did not significantly alter any of the phenotypes that we analyzed comparing to the wild type control. By comparison, transformants with random ectopic integration of the same DNA fragment showed a wider range of variation in mNeonGreen expression and in virulence in an insect infection model. Having identified predetermined "safe-haven" regions in A. fumigatus could therefore help reduce experimental variations and increase reproducibility, as it has been for the C. neoformans field.

Association of a new 99-bp indel of the CEL gene promoter region with phenotypic traits in chickens

Carboxyl ester lipase (CEL) encodes a cholesterol ester hydrolase that is secreted into the duodenum as a component of pancreatic juice. The objective of this study was to characterize the CEL gene, investigate the association between the CEL promoter variants and chicken phenotypic traits, and explore the CEL gene regulatory mechanism. An insertion/deletion (indel) caused by a 99-bp insertion fragment was shown for the first time in the chicken CEL promoter, and large differences in allelic frequency were found among commercial breeds, indigenous and feral birds. Association analysis demonstrated that this indel site had significant effects on shank length, shank girth, chest breadth at 8 weeks (p < 0.01), evisceration weight, sebum weight, breast muscle weight, and leg weight (p < 0.05). Tissue expression profiles showed extremely high levels of the CEL gene in pancreatic tissue. Moreover, the expression levels of the genes APOB, MTTP, APOV1 and SREBF1, which are involved in lipid transport, were significantly reduced by adding a 4% oxidized soybean oil diet treatment at the individual level and transfecting the embryonic primary hepatocytes with a CEL-overexpression vector. Interestingly, the results showed that the expression level of the II homozygous genotype was significantly higher than that of the ID and DD genotypes, while individuals with DD genotypes had higher phenotypic values. Therefore, these data suggested that the CEL gene might affect body growth by participating in hepatic lipoprotein metabolism and that the 99-bp indel polymorphism could be a potentially useful genetic marker for improving the economically important traits of chickens.

Mechanisms of fosfomycin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

ObjectivesOlder antimicrobials such as fosfomycin are being considered as alternative agents in the treatment of drug-resistant organisms. However, there are limited data on the usefulness of fosfomycin against carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the prevalence of fosfomycin resistance and associated mechanisms in CRKP. MethodsA total of 99 clinical CRKP isolates were collected in the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2017 and June 2018. Fosfomycin susceptibility testing was performed by the agar dilution method. Carbapenemase and fosfomycin resistance genes were detected by PCR. Analysis of the murA, glpT, uhpT, uhpA, ptsI and cyaA genes was performed by PCR and sequencing of four fosfomycin-resistant fosA3-negative CRKP strains. Conjugation experiments were employed to determine the mobility of the fosA3 gene. ResultsOf the 99 CRKP isolates, fosfomycin-non-susceptibility was detected in 48 (48.5%) isolates, among which the fosA3 gene was detected in 44. Among the four fosfomycin-resistant fosA3-negative CRKP isolates, one isolate possessed a single nucleotide insertion and deletion mutations as well as 219 nucleotide substitution mutations in murA, two isolates possessed deletion or mutation of large DNA fragments in glpT, and one isolate possessed a fragment insertion sequence in glpT. Transfection into Escherichia coli J53 via plasmid conjugation was successful for 19 (43.2%) of the 44 fosA3-positive CRKP isolates. ConclusionThe fosA3 gene is the primary mechanism of fosfomycin resistance in CRKP and can be transmitted widely by plasmid in hospitals. Mutations in murA and glpT were found in fosfomycin-resistant fosA3-negative CRKP.

Insertions and Duplications in the Polyproline Region of the Hepatitis E Virus.

Recombinant strains of hepatitis E virus (HEV) with insertions of human genomic fragments or HEV sequence duplications in the sequence encoding the polyproline region (PPR) were previously described in chronically infected patients. Such genomic rearrangements confer a replicative advantage in vitro but little is known about their frequency, location, or origin. As the sequences of only a few virus genomes are available, we analyzed the complete genomes of 114 HEV genotype 3 strains from immunocompromised (n = 85) and immunocompetent (n = 29) patients using the single molecular real-time sequencing method to determine the frequency, location, and origin of inserted genomic fragments, plus the proportions of variants with genomic rearrangements in each virus quasispecies. We also examined the amino acid compositions and post-translational modifications conferred by these rearrangements by comparing them to sequences without human gene insertions or HEV gene duplications. We found genomic rearrangements in 7/114 (6.1%) complete genome sequences (4 HEV-3f, 1 HEV-3e, 1 HEV-3 h, and 1 HEV-3chi-new), all from immunocompromised patients, and 3/7 were found at the acute phase of infection. Six of the seven patients harbored virus-host recombinant variants, including one patient with two different recombinant variants. We also detected three recombinant variants with genome duplications of the PPR or PPR + X domains in a single patient. All the genomic rearrangements (seven human fragment insertions of varying origins and three HEV genome duplications) occurred in the PPR. The sequences with genomic rearrangements had specific characteristics: increased net load (p < 0.001) and more ubiquitination (p < 0.001), phosphorylation (p < 0.001), and acetylation (p < 0.001) sites. The human fragment insertions and HEV genome duplications had slightly different characteristics. We believe this is the first description of HEV strains with genomic rearrangements in patients at the acute phase of infection; perhaps these strains are directly transmitted. Clearly, genomic rearrangements produce a greater net load with duplications and insertions having different features. Further studies are needed to clarify the mechanisms by which such modifications influence HEV replication.

Characteristics of gene mutations in Chinese single-center pulmonary adenocarcinoma patients and comparison with European and American pulmonary adenocarcinoma populations

Objective To explore the characteristics and significances of gene mutations in pulmonary adenocarcinoma, and to provide evidence for targeted medication. Methods High throughput sequencing based target-capture sequencing was performed in 104 patients with pulmonary adenocarcinoma to detect the mutational status of 56 cancer-related genes. All patients were diagnosed in the First People's Hospital of Kunshan from May 2017 to August 2018. The mutational characteristics of pulmonary adenocarcinoma was analyzed and compared with European and American pulmonary adenocarcinoma populations. The correlations between mutational characteristics and clinical features were analyzed, and the mutation sites for targeted medication were screened. Results Among 104 patients with pulmonary adenocarcinoma, totally 34 mutational genes were detected in 84 patients (81%, 84/104). Highly frequent mutations included epidermal growth factor receptor (EGFR) (49%, 51/104), TP53 (21%, 22/104), KRAS (13%, 14/104), and BRAF (6%, 6/104). Among all the 187 variants, 76% (142/187) were non-synonymous missense mutations, 13% (24/187) were small fragment deletions, 6% (12/187) were copy number variants, 3% (5/187) were small fragment insertions, and 2% (4/187) were nonsense site mutations. Among 104 patients with pulmonary adenocarcinoma, 34 targeted drug-associated mutations of 13 genes were detected in 68 patients (65%), and 19 (18%) patients harbored ≥ 2 targeted drug-associated mutations. EGFR mutations were more common in female patients than in male patients [62% (34/55)vs. 35% (17/49), χ2= 7.629, P= 0.006], while KRAS mutations were more frequent in male patients than in female patients [22% (11/49) vs. 5% (3/55), χ2= 6.424, P= 0.011]. The mutation frequencies of gene EGFR, TP53, KRAS, and CDKN2A in Chinese single-center (the First People's Hospital of Kunshan) and European and American adenocarcinoma populations were significantly different (all P < 0.05). Conclusions The molecular mutational characteristics of pulmonary adenocarcinoma are complex, and vary greatly among different populations. High throughput sequencing-based multiple-gene detection can reveal its mutational features comprehensively, and that has important roles in personal targeted medication guidance, drug-resistance monitoring and prognosis evaluation. Key words: Lung neoplasms; Adenocarcinoma; High-throughput nucleotide sequencing; Mutation; Molecular targeted therapy

Screening and Spontaneous Mutation of Pickle-Derived Lactobacillus plantarum with Overproduction of Riboflavin, Related Mechanism, and Food Application.

Riboflavin, also known as vitamin B2, plays an important role in human cell metabolism and participates in various redox reactions and in energy utilization. In this study, 90 riboflavin-producing lactic acid bacteria (LAB) were screened out from pickle juices. The yields of riboflavin in these LAB were about 0.096–0.700 mg/L, and one strain, Lactobacillus plantarum RYG-YYG-9049, was found to produce the highest riboflavin content. Next, roseoflavin was used to induce the spontaneous mutation of RYG-YYG-9049, and selected roseoflavin-resistant colonies generally produced higher riboflavin contents, ranging from 1.013 to 2.332 mg/L. The No. 10 mutant, L. plantarum RYG-YYG-9049-M10, had the highest riboflavin content. Next, the molecular mechanism of enhancing riboflavin production in RYG-YYG-9049-M10 was explored, leading to the finding that roseoflavin treatment did not change the rib operons including the ribA, ribB, ribC, ribH, and ribG genes. Unexpectedly, however, this mechanism did induce an insertion of a 1059-bp DNA fragment in the upstream regulatory region of the rib operon, as compared to the wild-type RYG-YYG-9049. To the best of our knowledge, this is the first report that roseoflavin could induce an insertion of DNA fragment in LAB to increase riboflavin content, representing a new mutation type that is induced by roseoflavin. Finally, in order to fortify riboflavin content in soymilk, RYG-YYG-9049 and RYG-YYG-9049-M10 were used to ferment soymilk, and several fermentation parameters were optimized to obtain the fermented soymilk with riboflavin contents of up to 2.920 mg/L. In general, roseoflavin induction is an economical and feasible biotechnological strategy to induce riboflavin-overproducing LAB, and this strategy can be used to develop LAB-fermented functional foods that are rich in riboflavin.

Molecular characteristics of the re-emerged avian leukosis virus in China, 2018-2019.

Since early 2018, avian leukosis virus (ALV) has re-emerged throughout six provinces in Northeast and East of China and caused huge economic losses. In different farms, there are significant differences in clinical symptoms, including morbidity, mortality and location of tumours, on affected animals, which implies that the present strains may have different origins and molecular characteristics. In this study, a systematic epidemiological investigation was conducted in 21 farms in six provinces. Results showed that the virus strains present in this outbreak are highly consistent but carry different mutations. All the strains shared 97.0%-99.0% identity with each other and were highly similar to the GD14J2 strain isolated previously, while different insertion fragments can be found in the env gene of different strains, suggesting that the strains of ALV in this outbreak may have the same ancestors but have gone through different evolutionary trajectories. This study demonstrated that these viruses may point to multiple sources of infection, and all should be identified and taken seriously in the formulation of control plans.

Boundaries in metagenomic screenings using lacZα-based vectors.

Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZα -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZα-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZα-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies.