Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.
Boraginaceae/genetics* ; Cloning, Molecular ; Coenzyme A ; Coenzyme A Ligases/genetics* ; Ligases ; Phylogeny

BRAP (BRCA1 associated protein) is one of BRCA1 (Breast cancer suppressor protein) associated cytoplasmic proteins. BRAP gene has been found to be associated with the risk of some cancers, and the associations between BRAP and cardiovascular diseases and metabolic syndrome is gradually attracting much attention. However, the explicit mechanisms involved remain to be fully elucidated. We reviewed the association between BRAP gene and cardiovascular diseases and metabolic syndromes and the biologic mechanisms in the regulation of metabolism, hoping to provide clues on our future researches.
Cardiovascular Diseases ; genetics ; Humans ; Ubiquitin-Protein Ligases ; genetics

OBJECTIVETo introduce a new technique for rapid construction of gene site-directed mutagenesis.

METHODSThree primers are synthesized. One is a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid are located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer, another PCR is performed using the previous PCR mutation segment as primer and the other flanking primer. The final PCR segment can be cloned into an appropriate plasmid by using the enzyme sites in the primers.

RESULTSTwo site-directed mutagenesis have been successfully constructed in the Parkin gene by this method.

CONCLUSIONThe method is effective and simple for construction of gene site-directed mutagenesis.

Base Sequence ; Cloning, Molecular ; methods ; DNA Primers ; genetics ; Ligases ; genetics ; Mutagenesis, Site-Directed ; Mutation ; Plasmids ; genetics ; Ubiquitin-Protein Ligases

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.
Carbon-Nitrogen Ligases ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Glutamates ; biosynthesis ; Ligases ; Phosphotransferases (Phosphate Group Acceptor) ; genetics

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).
Acyl Coenzyme A ; Butanones ; Chlamydomonas reinhardtii/genetics* ; Ligases ; Polyketide Synthases

The gene responsible for autosomal recessive parkinsonism, parkin, has recently been identified on chromosome 6q. It has been shown to be mutated in Japanese and European families, most of whom had early-onset parkinsonism. Here, we present a family with young-onset parkinsonism of an autosomal recessive inheritance. A homozygous exon 4 deletion in the parkin gene was found in 3 family members. To the best of the authors' knowledge, this is the first report in Korea of familial parkinsonism with the parkin gene mutation.
*Exons ; Female ; *Gene Deletion ; Genes, Recessive ; Human ; Ligases/*genetics ; Middle Aged ; Parkinsonian Disorders/*genetics ; *Ubiquitin-Protein Ligases

OBJECTIVETo construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene.

METHODSA pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing.

RESULTPCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles.

CONCLUSIONA lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.

Gene Targeting ; Genetic Vectors ; Lentivirus ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Ubiquitin-Protein Ligases ; genetics

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.
Exons/*genetics ; *Gene Deletion ; Parkinson Disease/*genetics ; Point Mutation ; Ubiquitin-Protein Ligases/*genetics

OBJECTIVE: To analyze the clinical and genetic characteristics of a boy featuring unexplained developmental delay, malnutrition and distinct facial appearance. METHODS: Physical examination was carried out for the child. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA and trio-whole exome sequencing. Candidate variants were verified by Sanger sequencing. RESULTS: The patient had facial dysmorphism including nasal alae aplasia, scalp defect and teeth deformities, in addition with recurrent diarrhea due to pancreatic exocrine insufficiency. DNA sequencing revealed that he has harbored compound heterozygous variants of the UBR1 gene, namely c.3167C>G (p.S1056X) and c.1911+14C>G, which were inherited from his father and mother, respectively. Database search has suggested the c.3167C>G to be a novel nonsense variant and c.1911+14C>G a known splicing variant. Based on the guidelines of the American College of Medical Genetics and Genomics, the two variants were predicted to be pathogenic and likely pathogenic, respectively. CONCLUSION The child was diagnosed with Johanson-Blizzard syndrome due to the compound heterozygous variants of the UBR1 gene. Above finding has enriched the mutational spectrum of the UBR1 gene and provided a basis for genetic counseling for this family.
Child ; Humans ; Male ; Ectodermal Dysplasia/genetics* ; Pancreatic Diseases/genetics* ; Ubiquitin-Protein Ligases/genetics*

Humans ; Mitochondria/genetics* ; Mutation/genetics* ; Parkinson Disease/genetics* ; Protein Kinases ; Ubiquitin-Protein Ligases