Homogenous Recombination Research Articles

Genetic Spectrum and Distinct Evolution Patterns of SARS-CoV-2.

Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.

Research on the roles of genes coding ATP-binding cassette transporters in Porphyromonas gingivalis pathogenicity.

Porphyromonas gingivalis, as a major pathogen of periodontitis, could rapidly adhere to and invade host gingival epithelial cells (GECs) for the induction of infection. One ATP-binding cassette (ABC) transporter gene was found to be upregulated during this infection process, however, the molecular mechanisms remain unclear. In this study, we systemically investigated the messenger RNA level changes of all ABC transporter family genes in P. gingivalis while being internalized within GECs by real-time polymerase chain reaction. We identified that two ABC transporter genes, PG_RS04465 (PG1010) and PG_RS07320 (PG1665), were significantly increased in P. gingivalis after coculturing with GECs. Mutant strains with knockout (KO) of these two genes were generated by homogenous recombination. PG_RS04465 and PG_RS07320 KO mutants showed no change in the growth of bacteria per se. Knockdown of PG_RS07320, but not PG_RS04465, caused decreased endotoxin level in the bacteria. In contrast, both mutant strains showed decreased Arg- and Lys-gingipains activities, with significantly reduced adhesion and invasion capabilities. Secreted interleukin-1β (IL-1β) and IL-6 levels in GECs cocultured with PG_RS04465 or PG_RS07320 KO mutants were also decreased, whereas, only the cells cocultured with PG_RS07320 KO mutants showed significant decrease. In addition, virulence study using mouse revealed that both KO mutant strains infection caused less mouse death than wild-type strains, showing reduced virulence of two KO strains. These results indicated that ABC transporter genes PG_RS04465 and PG_RS07320 are positive regulators of the virulence of P. gingivalis.

Immunocytological analysis of meiotic recombination in two anole lizards (Squamata, Dactyloidae).

Although the evolutionary importance of meiotic recombination is not disputed, the significance of interspecies differences in the recombination rates and recombination landscapes remains under-appreciated. Recombination rates and distribution of chiasmata have been examined cytologically in many mammalian species, whereas data on other vertebrates are scarce. Immunolocalization of the protein of the synaptonemal complex (SYCP3), centromere proteins and the mismatch-repair protein MLH1 was used, which is associated with the most common type of recombination nodules, to analyze the pattern of meiotic recombination in the male of two species of iguanian lizards, Anolis carolinensis Voigt, 1832 and Deiroptyx coelestinus (Cope, 1862). These species are separated by a relatively long evolutionary history although they retain the ancestral iguanian karyotype. In both species similar and extremely uneven distributions of MLH1 foci along the macrochromosome bivalents were detected: approximately 90% of crossovers were located at the distal 20% of the chromosome arm length. Almost total suppression of recombination in the intermediate and proximal regions of the chromosome arms contradicts the hypothesis that “homogenous recombination” is responsible for the low variation in GC content across the anole genome. It also leads to strong linkage disequilibrium between the genes located in these regions, which may benefit conservation of co-adaptive gene arrays responsible for the ecological adaptations of the anoles.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Construction of mutant strains of methicillin resistant Staphylococcus epidermidis with psm-mec gene deletion

Objective To construct mutant strains of methicillin resistant Staphylococcus epidermidis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene. Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloramphenicol sensitive clinical strains of MRSE, whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A. The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid. After being identified, the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electroporation, and then transformed into the selected clinical isolates of MRSE. The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination. The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships between the psm-mec gene and biofilm formation in MRSE strains. Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis. The mutants constructed via homogenous recombination were screened out and identified. Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation, demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE. Conclusion The Δpsm-mec mutant strains were successfully constructed. The psm-mec gene played an important role in the biofilm formation of Staphylococcus epidermdis. Key words: Staphylococcus epidermidis; psm-mec; Homologous recombination; Mutant; Biofilm formation

Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans.

The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae.

High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease PacI and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with PacI and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000. Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture. After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Possibilities and limits of axial lifetime control by radiation induced centers in fast recovery diodes

Device simulation, based on an extended recombination model, is used as a design tool for lifetime-controlled power diodes with different lifetime profiles. Homogenous and local recombination center profiles are considered. The sensitivity of important device properties, such as the trade-off between stationary and dynamical characteristics, to the recombination center peak position is investigated. The occurrence of dynamic impatt oscillations is analyzed.

Construction of mutant k-ras gene recombinant adenovirus

To construct the recombinant adenovirus of mutant k-ras by using the method of homogenous recombination in bacteria. Mutant k-ras gene was liberated from the vector of pcDNA3-k-ras 12(Val) via KpnI+XhoI digestion, and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack CMV/k-ras 12(Val). Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with PacI and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad-k-ras 12(Val) was measured with the aid of GFP expression. There were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack-CMV/k-ras 12 (Val) and pAdEasy-1 by method of CaCl₂. PCR test indicated each the recombinant adenovirus contained the insert of k-ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10¹² pfu/ml. The method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad-k-ras 12(Val) paves a sound foundation for further study.

Logistic regression approach to modelling the variability of recombination rate

The objective of the paper is to quantify the relationship between recombination rate and factors such as ‘sex of sperm’, paternal halfsib family, and individual, using logistic regression modelling. The analysed data set consists of 2214 single bovine sperm cell samples. Haplotypes at each single sperm were determined at eleven marker loci forming eight intervals located on chromosomes 6, 23 and X/Y. The experimental design comprises six paternal halfsib families. Six logistic regression models are fitted to the data from each interval. Departure from commonly assumed homogenous recombination is detected for one marker interval on chromosome 23 – influence of individual and ‘sex of sperm’ by family interaction, and for both intervals mapped to sex chromosomes – influence of ‘sex of sperm’ and paternal halfsib family.