A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

Studies over the past decades have uncovered fusion genes, a class of oncogenes that provide immense diagnostic and therapeutic advantages because of their tumor-specific expression. Originally associated with hemotologic cancers, fusion genes have recently been discovered in a wide array of solid tumors, including sarcomas, carcinomas, and tumors of the central nervous system. Fusion genes are attractive as both therapeutic targets and diagnostic tools due to their inherent expression in tumor tissue alone. Therefore, the discovery and elucidation of fusion genes in various cancer types may provide more effective therapies in the future for cancer patients.
Chromosomal Instability ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Humans ; Neoplasms ; diagnosis ; genetics ; therapy ; Oncogene Proteins, Fusion ; genetics ; Oncogenes ; Recombination, Genetic ; genetics ; Translocation, Genetic ; genetics

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; Homologous Recombination

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

X-linked agammaglobulinemia (XLA) is characterized by early onset of recurrent bacterial infection, markedly reduced levels of all major classes of immunoglobulins in the serum and few mature B cells in the blood. XLA is known to be associated with mutations in Bruton's tyrosin kinase (Btk). The Btk protein consists of 5 functional domains; the pleckstrin homology (PH) domain, the Tec homology (TH) domain, the Src homology 3 (SH3) domain, the SH2 domain, and the kinase (SH1) domain. Mutations in all domains of the Btk gene have been shown to cause XLA. The large number of Alu elements within the human genome provides abundant opportunities for unequal homologous recombination events between Alu repeats, resulting in human disease. We present a case of XLA with deletion of introns 15-18 of Btk gene which were mediated by an Alu-Alu recombination event.
Agammaglobulinemia* ; Alu Elements ; B-Lymphocytes ; Bacterial Infections ; Genome, Human ; Homologous Recombination ; Humans ; Immunoglobulins ; Introns* ; Phosphotransferases ; Recombination, Genetic* ; src Homology Domains

X-linked agammaglobulinemia (XLA) is characterized by early onset of recurrent bacterial infection, markedly reduced levels of all major classes of immunoglobulins in the serum and few mature B cells in the blood. XLA is known to be associated with mutations in Bruton's tyrosin kinase (Btk). The Btk protein consists of 5 functional domains; the pleckstrin homology (PH) domain, the Tec homology (TH) domain, the Src homology 3 (SH3) domain, the SH2 domain, and the kinase (SH1) domain. Mutations in all domains of the Btk gene have been shown to cause XLA. The large number of Alu elements within the human genome provides abundant opportunities for unequal homologous recombination events between Alu repeats, resulting in human disease. We present a case of XLA with deletion of introns 15-18 of Btk gene which were mediated by an Alu-Alu recombination event.
Agammaglobulinemia* ; Alu Elements ; B-Lymphocytes ; Bacterial Infections ; Genome, Human ; Homologous Recombination ; Humans ; Immunoglobulins ; Introns* ; Phosphotransferases ; Recombination, Genetic* ; src Homology Domains

In order to prevent phage contamination in amino acid fermentation, we introduced the restriction and modification system cglI gene complex into Corynebacterium crenatum and studied their phage-resistance. The cglI gene complex was amplified from Corynebacterium glutamicum by PCR and constructed into pJL23 vector. The recombinant strains were obtained by transformation of the recombinant plasmid pJL23-cglI into C. crenatum. Results showed that the recombinant strains possessed strong phage-resistance activity and broad phage-resistance spectrum, demonstrating the feasibility of using cglI gene complex for construction of phage-resistance recombinant C. crenatum strains and presenting a powerful way to solve the problem of phage contamination in amino acid fermentation industry.
Amino Acids ; biosynthesis ; Bacterial Proteins ; genetics ; Bacteriophages ; growth & development ; Corynebacterium ; genetics ; virology ; Corynebacterium glutamicum ; genetics ; metabolism ; DNA Restriction-Modification Enzymes ; genetics ; Fermentation ; Galectins ; genetics ; Recombination, Genetic ; Transformation, Bacterial

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180degrees and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.
DNA End-Joining Repair ; DNA Transposable Elements ; DNA* ; Enhancer Elements, Genetic ; Hand ; Homologous Recombination ; Retroelements

We developed a method to construct a gene mutant pool in Pichia pastoris based on in vivo homologous recombination. It was an absolute PCR-dependent method (PDM) and could avoid the disadvantages of traditional mutant pool construction process such as long-experimental period, low pool capacity and inadequate abundance. The method consisted of four steps: (1) construction of recombinant expression plasmid of target gene; (2) design of long primers that have 40-70 bp of homology to expression vector fragments at both ends and amplification of target gene by error-prone PCR, DNA Shuffling or other methods; (3) PCR amplification of expression vectors fragments; (4) mixture of gene and vectors by appropriate mole ratio, electroporation, formation of expression cassette in vivo, homologous recombination with host genome and achievement of mutant pool. Screening from this library, we obtained mutants with improved enzyme activity, protein expression level and thermostability. In conclusion, PDM was very efficient and convenient with advantages of shortened pool construction cycle from 2 weeks to 3 days, enlarged pool capacity from the original 10(3)-10(4) to more than 10(5), with a positive rate of more than 95%.
Gene Library ; Genetic Vectors ; genetics ; Homologous Recombination ; genetics ; Mutation ; Pichia ; genetics ; Polymerase Chain Reaction ; methods

BACKGROUNDTo investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.

METHODSThe recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT.

RESULTSThe adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5 x 10(11) pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P > 0.05), but indices in Group A were significantly lower than those in the latter three groups (P < 0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P < 0.01).

CONCLUSIONSThe transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.

Adenoviridae ; Animals ; Bone Marrow Transplantation ; Fas Ligand Protein ; Female ; Genetic Vectors ; Leukemia, Experimental ; Leukocytes, Mononuclear ; Male ; Membrane Glycoproteins ; genetics ; Mice ; Mice, Inbred C57BL ; Recombination, Genetic ; Transduction, Genetic ; Transfection ; Tumor Escape ; physiology