ABSTRACT
<p><b>OBJECTIVE</b>In Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.</p><p><b>METHODS</b>NAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.</p><p><b>RESULTS</b>The IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.</p><p><b>CONCLUSION</b>Feedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.</p>
Subject(s)
Animals , Arginine , Corynebacterium , Genetics , Metabolism , Escherichia coli , Feedback, Physiological , Gene Deletion , Mutagenesis, Site-Directed , Phosphotransferases (Carboxyl Group Acceptor) , Genetics , Proline , MetabolismABSTRACT
<p><b>OBJECTIVE</b>Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.</p><p><b>METHODS</b>Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.</p><p><b>RESULTS</b>Arginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.</p><p><b>CONCLUSION</b>The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.</p>
Subject(s)
Arginine , Bacterial Proteins , Chemistry , Genetics , Metabolism , Corynebacterium , Genetics , Metabolism , Gene Expression Regulation, Bacterial , Repressor Proteins , Chemistry , Genetics , MetabolismABSTRACT
Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')2 fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-Id) responses. Antigen-specific elution method was used for panning private anti-Id VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.
Subject(s)
Animals , Aflatoxin B1 , Allergy and Immunology , Amino Acid Sequence , Antibodies, Anti-Idiotypic , Chemistry , Camelids, New World , Allergy and Immunology , Immunoglobulin Heavy Chains , Chemistry , Molecular Sequence DataABSTRACT
A strip reader based lateral flow immunoassay (LFIA) was established for the rapid and quantitative detection of ractopamine (RAC) in swine urine. The ratio of the optical densities (ODs) of the test line (AT) to that of the control line (AC) was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration (IC50) of 0.59 ± 0.06 ng/mL. The limit of detection (LOD) of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.
Subject(s)
Animals , Immunoassay , Phenethylamines , Urine , Reagent Strips , Swine , UrineABSTRACT
OBJECTIVE@#To investigate the polymorphism of 11 canine STR loci.@*METHODS@#A fluorescent multiplex system with 11 STR loci (PEZ1, PEZ2, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, FH2010, FH2054, FH2132 and FH2611) was constructed independently and performed to amplify 105 samples from dogs. The character of these loci was analyzed with the PCR data.@*RESULTS@#The distributions of genotypes and allele frequencies of 11 STR loci were obtained. The total power of discrimination for the 11 loci in canine population was 0.9999999 and the cumulative probability of exclusion was 0.9330621. The observed heterozygosity and polymorphism information content (PIC) were 0.502 and 0.640, respectively.@*CONCLUSION@#Each of the eleven canine STR loci has a high genetic polymorphism and can be applied for the parentage testing and individual identification. The fluorescent multiplex system is a reliable method in forensic application.
Subject(s)
Animals , Alleles , Dogs/genetics , Forensic Genetics , Gene Frequency , Genotype , Microsatellite Repeats/genetics , Polymorphism, GeneticABSTRACT
<p><b>OBJECTIVE</b>To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus.</p><p><b>METHODS</b>Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smart trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'-RACE products.</p><p><b>RESULTS</b>This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced.</p><p><b>CONCLUSION</b>The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.</p>
Subject(s)
Amino Acid Sequence , Base Sequence , Blotting, Northern , Citrinin , Cloning, Molecular , DNA, Complementary , Chemistry , Fungal Proteins , Chemistry , Genetics , Gene Library , Molecular Sequence Data , Monascus , Genetics , Metabolism , Mycelium , Genetics , Metabolism , Pigments, Biological , RNA, Messenger , Metabolism , Sequence Alignment , Sequence Analysis, DNAABSTRACT
<p><b>OBJECTIVE</b>To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture.</p><p><b>METHODS</b>Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method.</p><p><b>RESULTS</b>Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaIII enzyme in inserts. There were seven repeated clones.</p><p><b>CONCLUSION</b>With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags > or = 10) and 143 unique tags to moderately expressed genes (repeat tags > or = 2).</p>
Subject(s)
Anti-Bacterial Agents , Citrinin , Expressed Sequence Tags , Gene Expression , Gene Expression Profiling , Gene Library , Monascus , Genetics , Metabolism , Polymerase Chain Reaction , RNA, Fungal , GeneticsABSTRACT
Serial analysis of gene expression (SAGE) is an effective method of determining gene expression profiles of tissues and organs under different conditions. In this paper, the detail protocol of SAGE was introduced and some modified procedure of SAGE was reviewed.