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

OBJECTIVETo detect the relationship between point mutations on exon 2 of parkin gene and sporadic early-onset Parkinson's disease.

METHODSThe point mutations on exon 2 of parkin gene were detected using polymerase chain reaction(PCR), agarose electrophoresis, single strand conformation polymorphism(SSCP), DNA sequencing and analysis of restrict enzyme in DNA of 60 Parkinson's disease patients with an onset age under 50 and 120 normal controls.

RESULTSOne homozygous mutation (G(237)-->C) on exon 2 was found by sequencing and verified by analysis of restrict enzyme, whereas no mutation was found in normal controls.

CONCLUSIONPoint mutations on exon 2 of parkin gene are likely to be related to sporadic early-onset Parkinson's disease.

Adult ; Aged ; Aged, 80 and over ; Exons ; Female ; Humans ; Ligases ; genetics ; Male ; Middle Aged ; Parkinson Disease ; genetics ; Point Mutation ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; Ubiquitin-Protein Ligases

To investigate the distribution of possible novel mutations from parkin gene in variant subset of patients with Parkinson's disease (PD) in China and explore whether parkin gene plays an important role in the pathogenesis of PD, 70 patients were divided into early-onset group and late-onset group; 70 healthy subjects were included as controls. Genomic DNA from 70 normal controls and from those of PD patients were extracted from peripheral blood leukocytes by using standard procedures. Mutations of parkin gene (exon 1-12) in all the subjects were screened by PCR-single strand conformation polymorphism (SSCP), and further sequencing was performed in the samples with abnormal SSCP results, in order to confirm the mutation and its location. A new missense mutation Gly284Arg in a patient and 3 abnormal bands in SSCP electrophoresis from samples of another 3 patients were found. All the DNA variants were sourced from the samples of the patients with early-onset PD. It was concluded that Parkin point mutation also partially contributes to the development of early-onset Parkinson's disease in Chinese.
DNA Mutational Analysis ; Exons ; Genotype ; Parkinson Disease/*genetics ; *Point Mutation ; Polymorphism, Single-Stranded Conformational ; Ubiquitin-Protein Ligases/biosynthesis ; Ubiquitin-Protein Ligases/*genetics

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/β-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/β-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.
DNA Repair ; Down-Regulation ; Embryonic Stem Cells ; Humans ; Ligases ; Proteolysis ; Public Sector ; Stem Cells ; Ubiquitin ; Ubiquitin-Protein Ligases ; Ubiquitin-Specific Proteases ; Ubiquitination

OBJECTIVETo investigate the distribution of possible novel mutation of parkin gene in variant subset patients with Parkinson's disease (PD) in China and to explore the role of parkin gene in the pathogenesis of PD.

METHODSSeventy patients were divided into early onset and late-onset groups, and 70 healthy subjects were included as controls. Genomic DNA from 70 normal controls and from those of PD patients were extracted from peripheral blood leukocytes with standard procedures. Mutations of parkin gene (exons 1-12) in all subjects mentioned above were screened by PCR-SSCP. And in the samples with abnormal SSCP result, further sequencing was performed to confirm the mutation and its location.

RESULTSA new missense mutation (Gly284Arg) on exon 7 was found in a sample, while 4 samples from all subjects exhibited abnormal mobility shift on SSCP electrophoresis. All mentioned DNA variants were sourced from the samples of the patients with early-onset PD.

CONCLUSIONPoint mutation in parkin gene also contributes partly to the development of early-onset Parkinson's disease in Chinese population.

Adult ; Age of Onset ; Aged ; Base Sequence ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Ligases ; genetics ; Male ; Middle Aged ; Mutation, Missense ; Parkinson Disease ; genetics ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Ubiquitin-Protein Ligases

4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis.
Amino Acid Sequence ; Angelica sinensis ; genetics ; Base Sequence ; Cloning, Molecular ; Coenzyme A Ligases ; genetics ; DNA, Complementary ; chemistry ; Molecular Sequence Data

Two oligonucleotide probes are permitted to anneal to the nucleic acid target of interest so that the ends of two probes immediately become adjacent to each other. The ligase can then efficiently join the two juxtaposed oligonucleotide probes by the formation of a phosphodiester bond if and only if perfectly matched base-pairs at the nick are present. During past 20 years, many ligase-mediated techniques have been developed for analyzing various bio-molecules, such as known/unknown point mutations, small-scale insertions and deletions, CpG islands methylation, large sets of single nucleotide polymorphisms (SNPs), specific proteins and DNA regions with which some other proteins can interact. Since the ligation reaction can be easily integrated into other techniques, certain advances have been already achieved. These novel approaches retain high accuracy through multiple hybridization and enzymatic processing events, and provide inherent quality control checking. In this article, we provide a comprehensive review of the ligase-mediated techniques for bio-molecular analysis.
CpG Islands ; genetics ; DNA Methylation ; Ligases ; metabolism ; Molecular Probe Techniques ; Mutation ; Oligonucleotide Probes ; biosynthesis ; chemical synthesis ; genetics ; Polymorphism, Single Nucleotide

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.
Animals ; Cullin Proteins/genetics* ; DNA-Binding Proteins/genetics* ; Factor VIII ; Male ; Mice ; Mice, Knockout ; Spermatogenesis/genetics* ; Ubiquitin-Protein Ligases

OBJECTIVE: To provide genetic testing for two brothers with mental retardation and epilepsy. METHODS: Array comparative genomic hybridization (aCGH) was used to detect copy number variations in the two patients, their parents and maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was utilized to delineate the deleted region in the pedigree. RESULTS: A 138 kb deletion in 15q11.2 region was detected by aCGH in both patients, which encompassed part of the UBE3A gene. MS-MLPA has narrowed down the region to exons 8 to 14 of the UBE3A gene. The same deletion was also found in their mother and grandfather. CONCLUSION The pathogenesis of this rare form of recurrent Angelman syndrome may be attributed to the partial deletion of maternal UBE3A gene.
Angelman Syndrome ; Comparative Genomic Hybridization ; DNA Copy Number Variations ; Female ; Gene Deletion ; Humans ; Male ; Sequence Deletion ; Ubiquitin-Protein Ligases

OBJECTIVE: To explore the genetic basis for a Chinese pedigree where three siblings were affected with Parkinson's disease. METHODS: Multiple ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS) were employed to detect the causative mutation. Sanger sequencing of cDNA was also used for verify the effect of mutation on the transcription of RNA. RESULTS: Heterozygous deletion of exon 3 of the PARK2 gene was detected by MLPA, while a heterozygous splice site variant c.619-3G>C was detected by NGS. Both mutations were shown to result in aberrant transcripts of the PARK2 gene (loss of exons 3 and 6, respectively) by Sanger sequencing of cDNA. Both mutations have co-segregated with the disease in the pedigree. CONCLUSION Compound heterozygous mutations of the PARK2 gene probably underlie the disease in this pedigree. Identification of the splice site variant c.619-3G>C has expanded the mutation spectrum of the PARK2 gene.
Asian Continental Ancestry Group ; China ; DNA Mutational Analysis ; Exons ; Heterozygote ; Humans ; Mutation ; Parkinson Disease ; genetics ; Pedigree ; Ubiquitin-Protein Ligases ; genetics