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1.
Article in Chinese | WPRIM | ID: wpr-879513

ABSTRACT

OBJECTIVE@#To analyze the molecular characteristics of a recombinant allele of the ABO blood group.@*METHODS@#The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing.@*RESULTS@#The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived.@*CONCLUSION@#An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.


Subject(s)
ABO Blood-Group System/genetics , Alleles , Blood Grouping and Crossmatching , Female , Fucosyltransferases/genetics , Genotype , Humans , Phenotype , Recombination, Genetic
2.
Asian Journal of Andrology ; (6): 562-571, 2021.
Article in English | WPRIM | ID: wpr-922359

ABSTRACT

Repairing DNA double-strand breaks (DSBs) with homologous chromosomes as templates is the hallmark of meiosis. The critical outcome of meiotic homologous recombination is crossovers, which ensure faithful chromosome segregation and promote genetic diversity of progenies. Crossover patterns are tightly controlled and exhibit three characteristics: obligatory crossover, crossover interference, and crossover homeostasis. Aberrant crossover patterns are the leading cause of infertility, miscarriage, and congenital disease. Crossover recombination occurs in the context of meiotic chromosomes, and it is tightly integrated with and regulated by meiotic chromosome structure both locally and globally. Meiotic chromosomes are organized in a loop-axis architecture. Diverse evidence shows that chromosome axis length determines crossover frequency. Interestingly, short chromosomes show different crossover patterns compared to long chromosomes. A high frequency of human embryos are aneuploid, primarily derived from female meiosis errors. Dramatically increased aneuploidy in older women is the well-known "maternal age effect." However, a high frequency of aneuploidy also occurs in young women, derived from crossover maturation inefficiency in human females. In addition, frequency of human aneuploidy also shows other age-dependent alterations. Here, current advances in the understanding of these issues are reviewed, regulation of crossover patterns by meiotic chromosomes are discussed, and issues that remain to be investigated are suggested.


Subject(s)
Cell Division/physiology , Chromosome Segregation/physiology , Humans , Meiosis/genetics , Recombination, Genetic
4.
Article in Korean | WPRIM | ID: wpr-816642

ABSTRACT

The 2019 novel coronavirus disease (COVID-19) outbreaks that emerged in Wuhan city, Hubei province, have led to a formidable number of confirmed cases that resulted in >5,700 deaths globally, including 143 countries in all 6 continents. The World Health Organization declared a Public Health Emergency of International Concern with a very high level of global risk assessment. Severe acute respiratory syndrome (SARS)-coronavirus-2 (SARS-CoV-2), the agent of COVID-19, has >79% nucleotide sequence homology to SARS-CoV; therefore, both belong to the genus betacoronavirus and subgenus sarbecovirus. The S1 domains of the two appeared to share the cellular receptor ACE2, but revealed a much higher S1-ACE2 binding affinity. As seen in many other human coronaviruses, SARS-CoV-2 also shows respiratory infection, but the basic reproductive number (R₀) in transmission and the clinical latency are quite dissimilar from those of SARS- or MERS-CoVs. Many scientists infer that the time point of cross-barrier transfer from bats to mediate animals or to humans should be a rather recent event based on the full-length genome analyses obtained from the very first patients. Copy-choice polymerization, which often leads to a significant genome recombination rate in most coronaviruses, predicts the continued emergence of novel coronaviruses.


Subject(s)
Animals , Base Sequence , Chiroptera , Coronavirus , Disease Outbreaks , Emergencies , Genome , Humans , Middle East Respiratory Syndrome Coronavirus , Molecular Biology , Polymerization , Polymers , Public Health , Recombination, Genetic , Risk Assessment , SARS Virus , Severe Acute Respiratory Syndrome , World Health Organization
5.
Chinese Journal of Biotechnology ; (12): 1761-1770, 2019.
Article in Chinese | WPRIM | ID: wpr-771755

ABSTRACT

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.


Subject(s)
Escherichia coli , Escherichia coli Proteins , Genetic Engineering , Plasmids , Recombination, Genetic
6.
Chinese Journal of Biotechnology ; (12): 827-836, 2019.
Article in Chinese | WPRIM | ID: wpr-771327

ABSTRACT

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10⁵ U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10⁶ cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10⁴ recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.


Subject(s)
Cloning, Molecular , Escherichia coli , Genetics , Exonucleases , Genetics , Recombinant Proteins , Metabolism , Recombination, Genetic
7.
Article in Korean | WPRIM | ID: wpr-785901

ABSTRACT

Antibacterial drugs are one of the most important therapeutic agents of bacterial infections but multidrug resistant Escherichia coli (MDREC) is an increasing problem worldwide. Major resistance mechanism of MDREC is horizontal gene transfer of R plasmids harboring integrons, which the integron integrase (IntI) catalyzes gene cassette insertion and excision through site specific recombination. In this study, resistance profiles of integron harboring E. coli isolated in Korea and the genetic environments of integron gene cassettes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of integron harboring E. coli. Resistance rates of integron harboring E. coli, including β-lactams, aminoglycosides, and fluoroquinolones and MDR frequencies were significantly higher than that of E. coli without integron (p < 0.01). Majority (80%) of integron harboring E. coli showed resistance transfer by conjugation. Most (80%) of E. coli had dfrA17-aadA5 cassette array and PcH1 hybrid promoter; 16.7% of E. coli had dfrA12-orfF-aadA2 cassette array and PcW promoter. The higher prevalence of weak Pc variants among most (96.7%) of integron harboring MDREC suggests that a flexible cassette array is more important than enhanced expression. All the integrons had LexA binding motif suggests that SOS responses control the expression of these integrons. In conclusion, the genetic bases of integrons were diverse, and the spread and the expression of prevalent gene cassette arrays may be deeply related with strengths of Pc promoters in integrons. These informations will provide important knowledge to control the increase of integron harboring MDREC.


Subject(s)
Aminoglycosides , Bacterial Infections , Escherichia coli , Escherichia , Fluoroquinolones , Gene Transfer, Horizontal , Integrases , Integrons , Korea , Polymerase Chain Reaction , Prevalence , R Factors , Recombination, Genetic , SOS Response, Genetics
8.
Article in English | WPRIM | ID: wpr-760363

ABSTRACT

Two infectious bronchitis virus (IBV) K046-12 and K047-12 strains were isolated and the nearly complete genomes of them were sequenced. Sequence comparisons showed that the K046-12 genome was most similar to Korean IBV strains, and the K047-12 genome was most similar to QX-like IBV strains. Phylogenetic analysis showed that nearly all K046-12 and most K046-12 genes were placed in the same cluster as Korean IBV isolates, but the S1 region was placed in the same cluster as Mass-type IBVs. For K047-12, nearly all K047-12 and most K047-12 genes were located in the same cluster as QX-like IBVs, but the M region was located in the same cluster as Korean IBV isolates with K047-12. Recombination analysis confirmed that K046-12 is a recombinant strain with the primary parental sequence derived from Korean IBVs and minor parental sequence derived from Mass-type IBV, and K047-12 is a recombinant strain with the major parental sequence derived from QX-IBV and minor parental sequence derived from Korean IBVs. This study showed that new IBV recombinants are constantly generated among various IBVs, including those used for vaccination. Therefore, genetic analysis of new virus isolates should be performed for effective infectious bronchitis control and appropriate vaccine development.


Subject(s)
Bronchitis , Genome , Humans , Infectious bronchitis virus , Korea , Parents , Recombination, Genetic , Vaccination
9.
Article in English | WPRIM | ID: wpr-760350

ABSTRACT

Enterovirus species G (EV-G) is highly diverse, and is ubiquitous in pig populations, usually without diarrhea. The present study aimed to investigate the presence of novel EV-G recombinants with the torovirus papain-like cysteine protease (PLCP) in Jeju pig herds. EV-G1-PLCP mono-infections were most prevalent in diarrheic weaned piglets. The PLCP genes of the Jeju isolates varied in size and junction sequences, and were greatly heterogeneous, with 77.0–90.7% homology amongst all recombinants. Our results suggest that the exogenous PLCP gene has undergone continuous rapid mutation in the individual EV-G genomes following cross-order recombination, thereby causing clinical disease in swine.


Subject(s)
Cysteine Proteases , Diarrhea , Enterovirus , Genome , Korea , Prevalence , Recombination, Genetic , Swine , Torovirus
10.
Immune Network ; : e10-2018.
Article in English | WPRIM | ID: wpr-740202

ABSTRACT

Interaction between pathogen-associated molecular patterns and pattern recognition receptors triggers innate and adaptive immune responses. Several studies have reported that toll-like receptors (TLRs) are involved in B cell proliferation, differentiation, and Ig class switch recombination (CSR). However, roles of TLRs in B cell activation and differentiation are not completely understood. In this study, we investigated the direct effect of stimulation of TLR1/2 agonist Pam3CSK4 on mouse B cell viability, proliferation, activation, Ig production, and Ig CSR in vitro. Treatment with 0.5 µg/ml of Pam3CSK4 only barely induced IgG1 production although it enhanced B cell viability. In addition, high-dosage Pam3CSK4 diminished IgG1 production in a dose-dependent manner, whereas the production of other Igs, cell viability, and proliferation increased. Pam3CSK4 additively increased TLR4 agonist lipopolysaccharide (LPS)-induced mouse B cell growth and activation. However, interestingly, Pam3CSK4 abrogated LPS-induced IgG1 production but enhanced LPS-induced IgG2a production. Further, Pam3CSK4 decreased LPS-induced germline γ1 transcripts (GLTγ1)/GLTε expression but increased GLTγ2a expression. On the other hand, Pam3CSK4 had no effect on LPS-induced plasma cell differentiation. Taken together, these results suggest that TLR1/2 agonist Pam3CSK4 acts as a potent mouse B cell mitogen in combination with TLR4 agonist LPS, but these 2 different TLR agonists play diverse roles in regulating the Ig CSR of each isotype, particularly IgG1/IgE and IgG2a.


Subject(s)
Animals , B-Lymphocytes , Cell Proliferation , Cell Survival , Hand , Immunoglobulin Class Switching , Immunoglobulin E , Immunoglobulin G , In Vitro Techniques , Mice , Pathogen-Associated Molecular Pattern Molecules , Plasma Cells , Receptors, Pattern Recognition , Recombination, Genetic , Toll-Like Receptors
11.
Article in English | WPRIM | ID: wpr-758815

ABSTRACT

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.


Subject(s)
Alleles , Animals , Endogenous Retroviruses , Genes, MHC Class I , Genes, MHC Class II , Genome , Heterografts , Leukocytes , Mass Screening , Recombination, Genetic , Retroviridae , RNA , Swine , Transgenes , Transplantation, Heterologous , Viremia
12.
Article in English | WPRIM | ID: wpr-758810

ABSTRACT

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER(T2)/LCMV-EGFP(LoxP) and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreER(T2)) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER(T2)-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER(T2)/EGFP(LoxP) recombination system via SCNT.


Subject(s)
Animals, Genetically Modified , Astrocytes , Central Nervous System , Cerebellum , Cerebrum , DNA , Embryo Transfer , Embryonic Development , Estrogens , Female , Fibroblasts , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein , Humans , Nuclear Transfer Techniques , Ovulation , Polymerase Chain Reaction , Pregnancy , Real-Time Polymerase Chain Reaction , Recombination, Genetic , Regenerative Medicine , RNA, Messenger , Skin , Swine , Tissue Donors , Toxicity Tests , Transgenes
13.
Article in Korean | WPRIM | ID: wpr-715377

ABSTRACT

Cell permeable peptide (CPP) is able to transport itself or conjugated molecules such as nucleotides, peptides, and proteins into cells. Since short peptide of human immunodeficiency virus-1 Tat has been discovered as CPP, it has been continuously studied for their ability to transport heterologous cargoes into cells. In this study, we have focused on the fusion protein of respiratory syncytial virus (RSV), which has six basic amino acids in multi basic furin-dependent cleavage site (MBFCS) required to be cationic CPP. To develop more efficient CPP, the sequence, which linked two MBFCS, was synthesized (called RS-CPP). To assess cell permeable efficiency of RS-CPP or MBFCS, the peptides was conjugated with fluorescein isothiocyanate, and cell permeable efficiency was measured by fluorescence-activated cell sorting. Cell permeability of RS-CPP or MBFCS was increased in a dose-dependent manner, but RS-CPP showed more efficient cell permeability than MBFCS in MDCK, HeLa, Vero E6, and A549 cells. To evaluate whether RS-CPP can transport its conjugated functional peptide (VIVIT) in CD8+ T cell, it was confirmed that IL-2 and β-galactosidase expression were significantly inhibited through selective block of nuclear factor activated T-cell. To investigate endocytic pathways, Cre-mediated DNA recombination (loxP-STOP-loxP-LacZ reporter system) was investigated with divergent endocytosis inhibitors in TE671 cells, and RS-CPP endocytosis is occurred via binding cell surface glycosaminoglycan and clathrin-mediated endocytosis, or macropinocytosis. These results indicated that RS-CPP could be a novel cationic CPP, and it would help understanding for delivery of biologically functional molecules based on viral basic amino acids.


Subject(s)
Amino Acids, Basic , DNA , Endocytosis , Flow Cytometry , Fluorescein , Humans , Interleukin-2 , Nucleotides , Peptides , Permeability , Recombination, Genetic , Respiratory Syncytial Viruses , T-Lymphocytes
14.
Immune Network ; : e25-2018.
Article in English | WPRIM | ID: wpr-716242

ABSTRACT

γδ T cells are abundant in the gut mucosa and play an important role in adaptive immunity as well as innate immunity. Although γδ T cells are supposed to be associated with the enhancement of Ab production, the status of γδ T cells, particularly in the synthesis of IgA isotype, remains unclear. We compared Ig expression in T cell receptor delta chain deficient (TCRδ⁻/⁻) mice with wild-type mice. The amount of IgA in fecal pellets was substantially elevated in TCRδ⁻/⁻ mice. This was paralleled by an increase in surface IgA expression and total IgA production by Peyer's patches (PPs) and mesenteric lymph node (MLN) cells. Likewise, the TCRδ⁻/⁻ mice produced much higher levels of serum IgA isotype. Here, surface IgA expression and number of IgA secreting cells were also elevated in the culture of spleen and bone marrow (BM) B cells. Germ-line α transcript, an indicator of IgA class switch recombination, higher in PP and MLN B cells from TCRδ⁻/⁻ mice, while it was not seen in inactivated B cells. Nevertheless, the frequency of IgA+ B cells was much higher in the spleen from TCRδ⁻/⁻ mice. These results suggest that γδ T cells control the early phase of B cells, in order to prevent unnecessary IgA isotype switching. Furthermore, this regulatory role of γδ T cells had lasting effects on the long-lived IgA-producing plasma cells in the BM.


Subject(s)
Adaptive Immunity , Animals , B-Lymphocytes , Bone Marrow , Immunity, Innate , Immunoglobulin A , Immunoglobulin Class Switching , Lymph Nodes , Mice , Mucous Membrane , Peyer's Patches , Plasma Cells , Receptors, Antigen, T-Cell, gamma-delta , Recombination, Genetic , Spleen , T-Lymphocytes
15.
Laboratory Animal Research ; : 166-175, 2018.
Article in English | WPRIM | ID: wpr-719077

ABSTRACT

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.


Subject(s)
Animals , Atrophy , B-Lymphocytes , Bone Marrow , Genome , Homologous Recombination , Humans , Immunoglobulins , Killer Cells, Natural , Lymph Nodes , Lymphocytes , Lymphopenia , Megakaryocytes , Methods , Mice , Mice, Knockout , Negotiating , Phenotype , Receptors, Antigen, T-Cell , Recombination, Genetic , Sensitivity and Specificity , Severe Combined Immunodeficiency , Spleen , T-Lymphocytes , Thymus Gland
16.
Electron. j. biotechnol ; 30: 64-70, nov. 2017. ilus, graf, tab
Article in English | LILACS | ID: biblio-1021461

ABSTRACT

Background: The development of a potential single culture that can co-produce hydrogen and ethanol is beneficial for industrial application. Strain improvement via molecular approach was proposed on hydrogen and ethanol co-producing bacterium, Escherichia coli SS1. Thus, the effect of additional copy of native hydrogenase gene hybC on hydrogen and ethanol co-production by E. coli SS1 was investigated. Results: Both E. coli SS1 and the recombinant hybC were subjected to fermentation using 10 g/L of glycerol at initial pH 7.5. Recombinant hybC had about 2-fold higher cell growth, 5.2-fold higher glycerol consumption rate and 3-fold higher ethanol productivity in comparison to wild-type SS1. Nevertheless, wild-type SS1 reported hydrogen yield of 0.57 mol/mol glycerol and ethanol yield of 0.88 mol/mol glycerol, which were 4- and 1.4-fold higher in comparison to recombinant hybC. Glucose fermentation was also conducted for comparison study. The performance of wild-type SS1 and recombinant hybC showed relatively similar results during glucose fermentation. Additional copy of hybC gene could manipulate the glycerol metabolic pathway of E. coli SS1 under slightly alkaline condition. Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.


Subject(s)
Ethanol/metabolism , Escherichia coli/metabolism , Hydrogen/metabolism , Hydrogenase/metabolism , Recombination, Genetic , Biodegradation, Environmental , Culture Media , Escherichia coli/enzymology , Alkalinization , Fermentation , Glucose/metabolism , Glycerol/metabolism , Hydrogenase/genetics
17.
Electron. j. biotechnol ; 30: 110-117, nov. 2017. graf, tab, ilus
Article in English | LILACS | ID: biblio-1021571

ABSTRACT

Background: Catalase (CAT) is an important enzyme that degrades H2O2 into H2O and O2. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized. Results: After optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20­70°C and pH 5.0­11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC­MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml. Conclusions: To our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.


Subject(s)
Serratia marcescens/enzymology , Catalase/metabolism , Recombination, Genetic , Serratia marcescens/genetics , RNA, Ribosomal, 16S , Kinetics , Catalase/isolation & purification , Catalase/genetics , Chromatography, Liquid , Sequence Analysis, DNA , Electrophoresis , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Hydrogen Peroxide/metabolism
18.
Electron. j. biotechnol ; 30: 118-124, nov. 2017. tab, ilus, graf
Article in English | LILACS | ID: biblio-1021652

ABSTRACT

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.


Subject(s)
Zymomonas/genetics , Homologous Recombination , Plasmids , Recombination, Genetic , Alcohol Dehydrogenase/metabolism , Zymomonas/enzymology , Electroporation , Ethanol/metabolism , Gene Knockout Techniques , Mutation
19.
Electron. j. biotechnol ; 25: 64-69, ene. 2017. tab, graf, ilus
Article in English | LILACS | ID: biblio-1008601

ABSTRACT

Background: MicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b. Results: We found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-ß1, and ß-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α. Conclusion: miR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.


Subject(s)
Animals , Hair Follicle/growth & development , MicroRNAs/metabolism , Recombination, Genetic , Goats , Adenoviridae , Tumor Necrosis Factor-alpha/metabolism , Hair Follicle/cytology , Hair Follicle/metabolism , MicroRNAs/genetics , Fibroblast Growth Factor 5/metabolism , Enzyme Assays , Luciferases
20.
Article in English | WPRIM | ID: wpr-217324

ABSTRACT

OBJECTIVE: Interleukin (IL)-17 is a pro-inflammatory cytokine that has pleiotropic effects on multiple target cells and thereby contributes to the development of immune-mediated inflammatory disorders. However, the role of IL-17 in the humoral immune response has not been clearly elucidated. METHODS: Mice deficient in IL-17A (IL-17A knockout [KO] mice) and wild type (WT) C57BL/6 mice were compared. Distinct B cell (mature/precursor and marginal zone/follicular) and plasma cell populations were compared using fluorescence-activated cell sorting (FACS) and confocal immunostaining. Immunoglobulin production was assessed by enzyme-linked immunosorbent assay. RESULTS: There was no difference in B cell and plasma cell populations between IL-17A KO and WT mice. However, after T cell-dependent antigen challenge, IL-17A KO mice produced lower levels of immunoglobulin (Ig)G1 than wild-type animals. IL-17A KO mice also showed reduced germinal center (GC) formation and lower expression of activation-induced cytidine deaminase, the essential enzyme for class switch recombination (CSR). IL-17 had no effect on the proliferation or survival of naïve B cells in in vitro functional studies. However, IL-17 treatment promoted naïve B cell differentiation into plasma cells in synergy with IL-4, although IL-17 alone had no effect. CONCLUSION: Our findings suggest that IL-17 contributes to the humoral immune response by enhancing GC formation, CSR to IgG1, and plasma cell differentiation in synergy with IL-4.


Subject(s)
Animals , B-Lymphocytes , Cell Differentiation , Cytidine Deaminase , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Germinal Center , Immunity, Humoral , Immunoglobulin G , Immunoglobulins , In Vitro Techniques , Interleukin-17 , Interleukin-4 , Interleukins , Mice , Plasma Cells , Recombination, Genetic
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