Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.
Animals ; Animals, Wild ; Bias (Epidemiology) ; China* ; Codon, Initiator ; Codon, Terminator ; DNA, Intergenic ; Genes, rRNA ; Genetic Variation ; Genome ; Genome, Mitochondrial* ; Mammals ; Phylogeny ; RNA, Transfer ; Sequence Analysis* ; Tigers* ; Toxascaris*

To define the genes for production of catalytically active H. pylori urease, we camed out study to elucidate the structure of urease gene transcript, to delineate the genetic region which affected the extent of the expression and the activation of urease structural subunits. UreC and ureD were confirmed not to affect the expression of structural genes and active enzyme production, meaning that these genes are not components of the urease gene cluster of H. pylori. p-independent transcriptional stop signal was found in 12 bp down-stream of ureH stop codon. RNA extension test showed that the transcript starts with 267 bp upstream of ureA start codon. Although accessory genes did not affect the extent of the expression of the structural subunits, they were essential for assembling the active urease in E. coli. E. coli transformants of plasmid clones containing ureAB produced catalytically active urease when they are complemented with the plasmid clones of ureIEFGH or coexisted with ureIEFGH, meaning that accessory gene products could be trans-acting as well as cis-acting. The extent of production of urease structural subunits depended on the region of 241 to 57 bp upstream of ureA start codon. E. coli transformant of pBeloBACII clone containing the urease gene cluster, which is maintained with a single copy in host, did not express the urease. Proteins (60, 38, 30, 29, 27, and 24 kDa) that could hold nickel ions were identified in the cell extract of H. pylori. The results in this study will provide the basis to understand the control mechanism for urease gene expression and formation of the active urease.
Clone Cells ; Codon, Initiator ; Codon, Terminator ; Complement System Proteins ; Gene Expression ; Helicobacter pylori* ; Helicobacter* ; Ions ; Multigene Family ; Nickel ; Plasmids ; RNA ; Urea ; Urease*

Partially purified H. pylori ADH was used to determine the amino acid sequence of ADH N- terminus. The sequence of the ADH N-terminus was determined as MRVQSKGF. The genomic library of H. pylori that has been prepared by pTZ19U plasmid vector was screened with the deduced oligonucleotide probes to select the plasmid clone containing the entire ADH gene. The clone pTZ19U/ADH-6 was selected and its EcoRI-BamHI fragment (1.3 kb) was subcloned into pBluescript II K/S vector to determine nucleotide sequence. The length of H. pylori ADH gene was 1,044 bp. Ribosomal binding site was found in the upstream of start codon and rho- independent transcriptional stop signal was observed in the downstream of stop codon. The ADH gene encodes a protein of 348 amino acids, of which the predicted molecular size and pI value were 38.6 kDa and 7.1, respectively. ADH activity of E. coli transformant of pBluescript/ADH is 10-times greater compared to that of non-transformants. When H. pylori ADH gene was disrupted by pBluescript/ADH-KM whose internal region of 1.3 kb DNA fragment containing ADH gene was replaced by KM resistance sequence, the strain lost the ADH activity completely, despite the normal growth of the strain. This demonstrates that ADH gene is not essential for the viability of H. pylori.
Alcohol Dehydrogenase* ; Amino Acid Sequence ; Amino Acids ; Base Sequence* ; Binding Sites ; Clone Cells ; Cloning, Molecular* ; Codon, Initiator ; Codon, Terminator ; DNA ; Genes, vif* ; Genomic Library ; Helicobacter pylori* ; Helicobacter* ; Oligonucleotide Probes ; Plasmids

We have previously observed that a sequence in coat protein (CP) ORF of Turnip yellow mosaic virus (TYMV) is required for efficient replication of the virus. The sequence was predicted to take a stem-loop structure, thus termed SL2. While examining various SL2 mutants, we observed that all the modifications resulting in extension of translation beyond the CP ORF significantly suppressed subgenomic RNA accumulation. The genomic RNA level, in contrast, was not affected. Introduction of an in-frame stop codon in the CP ORF of these constructs restored the level of subgenomic RNA. Overall, the results suggest that the read-through makes the subgenomic RNA unstable.
Animals ; Brassica napus ; Codon, Terminator ; Ecthyma, Contagious ; RNA ; Tymovirus ; Viruses

The DNA sequence of a plasmid named pHP489 of Helicobacter pylori strain 489 was determined and analyzed to characterize its replication apparatus. The pHP489 plasmid consisted of 1,222 bp and had an overall G+C content of 33.1%. An ORF was predicted to encode the putative protein of 239 amino acid residues (28 kDa). A putative ribosomal binding site and a potential terminator sequence are located upstream and downstream of the ORF, respectively. However, the consensus sequence for a promoter in upstream of ORF was not found. A potential dna A box was found at 317 nt upstream of a start codon and followed by two-57 bp directed repeats and an inverted repeat. The DNA homology was found in the regions of less than 90 bp among pHPK255, pHPM180, and pHel1 of other H. pylori plasmids and Mycoplasma mycoides plasmids. pHP489K that was produced by pHP489 sequence and C. jejuni derived aph(3')-III, was transformed to various H. pylori isolates and were stably maintained in the H. pylori host without the addition of selective antibiotics for the 30-times subcultues. The plasmic vector, in which the ORF region of pHP489 DNA was deleted, could be transformed into H. pylori. However, the plasmid vector, whose the direct repeats region of pHP489 DNA was deleted, failed to be transformed. The direct repeats region of pHP489 DNA was confirmed to be bound with cytosolic factors of H. pylori. These results showed that the direct repeats region of pHP489 DNA is an essential apparatus by which the plasmid could be replicacted in H. pylori. And pHP489 plasmid was supposed to be replicated by host factors rather than plasmic-encoded factors.
Animals ; Anti-Bacterial Agents ; Base Composition ; Base Sequence ; Binding Sites ; Codon, Initiator ; Consensus Sequence ; Cytosol ; DNA ; Ecthyma, Contagious ; Helicobacter pylori* ; Helicobacter* ; Mycoplasma mycoides ; Plasmids* ; Repetitive Sequences, Nucleic Acid ; Sequence Analysis* ; Terminator Regions, Genetic

Nowadays, there are a lot of evidence that mutation of the p53 tumor suppressor gene is one of the most common genetic abnormalities in neoplastic progression. In this study, we analyzed 20 specimens of oral tumors (squamous cell carcinoma 14 cases, ameloblastoma 3 cases, adenoid cystic carcinoma 2 cases, malignant schwannoma 1 case) using polymerase chain reaction and direct sequencing which used an automated DNA sequencer and software for detection of mutations. Polymerase chain reactions were performed with 4 sets of primers encompassing exon 5, 6, 7, 8, and direct sequencing method was employed. The results were as followings. 1. We detected 10 piont mutations out of 20 specimens (50%). 2. The genetic alterations included 7 mis-sense mutations resulting in single amino acid subtitutions, 2 silent mutations, 1 non-sense mutations encoding a stop codon. 3. Mutations were mostly in exon 7(7 out of 10 mutations, 70%) and involved codons 225, 234, 235, 236, 238, 247. 4. Therse were 4 cases of T-->A transversion, 2 cases of C-->A transversion, A-->G transition, 1 case of C-->G, T-->G transversion respectively. 5. We could find out point mutations more conveniently using PCR-Automated Direct Sequencing method.
Ameloblastoma ; Carcinoma, Adenoid Cystic ; Codon ; Codon, Terminator ; DNA ; Exons ; Genes, p53 ; Genes, Tumor Suppressor* ; Neurilemmoma ; Point Mutation ; Polymerase Chain Reaction

The molecular defect of congenital lipoid adrenal hyperplasia has been discovered to be in the transport of cholesterol into mitochondria due to defective regulatory protein called "Steroidogenic Acute Regulatory Protein (StAR)", while the enzyme P450scc itself is normal. This study with EcoRII restriction enzyme aimed at elucidating more conveniently the molecular defect in the StAR gene. The genomic DNAs were extracted from their peripheral blood. We amplified the exon 7, hot spot, of the StAR gene with 1 set of primers by Polymerase Chain Reaction (PCR). Subsequently, a PCR product corresponding to target sequence (~437 bps) from the patient and her father have been sequenced by automatic sequence analyzer. The PCR-RFLP (Restriction Fragment Length Polymorphism) analysis after restriction digestion with EcoRII restriction enzyme was also performed on 12% polyacrylamide gel electrophoresis. The mutation was identified in the exon 7 of the StAR gene, substituting C for T at codon 258, consequently replacing glutamine by stop codon. This mutation alters EcoRII restriction site. In addition, we obtained the good result of PCR-RFLP (Restriction Fragment Length Polymorphism) analysis on 12% polyacrylamide gel electrophoresis. Therefore, the PCR-RFLP (Restriction Fragment Length Polymorphism) analysis with EcoRII restriction enzyme can be easily utilized to screen carrier, diagnose the patient prenatally or postnatally.
Cholesterol ; Codon ; Codon, Terminator ; Digestion ; DNA ; Electrophoresis, Polyacrylamide Gel ; Exons ; Fathers ; Glutamine ; Humans ; Hyperplasia ; Mitochondria ; Molecular Biology* ; Polymerase Chain Reaction

Pseudohypoparathyroidism type Ia (PHP Ia) is a disorder characterized by multiform hormonal resistance including parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO). It is caused by heterozygous inactivating mutations within the Gs alpha-encoding GNAS exons. A 9-year-old boy presented with clinical and laboratory abnormalities including hypocalcemia, hyperphosphatemia, PTH resistance, multihormone resistance and AHO (round face, short stature, obesity, brachydactyly and osteoma cutis) which were typical of PHP Ia. He had a history of repeated convulsive episodes that started from the age of 2 months. A cranial computed tomography scan showed bilateral calcifications in the basal ganglia and his intelligence quotient testing indicated mild mental retardation. Family history revealed that the patient's maternal relatives, including his grandmother and 2 of his mother's siblings, had features suggestive of AHO. Sequencing of the GNAS gene of the patient identified a heterozygous nonsense mutation within exon 11 (c.637 C>T). The C>T transversion results in an amino acid substitution from Gln to stop codon at codon 213 (p.Gln213*). To our knowledge, this is a novel mutation in GNAS.
Amino Acid Substitution ; Basal Ganglia ; Brachydactyly ; Child ; Codon ; Codon, Nonsense ; Codon, Terminator ; Exons ; Humans ; Hyperphosphatemia ; Hypocalcemia ; Intellectual Disability ; Intelligence ; Male ; Obesity ; Osteoma ; Parathyroid Hormone ; Pseudohypoparathyroidism* ; Siblings

BACKGROUND: Angiogenin, a 14.1-kDa protein isolated from the medium conditioned by HT-29 human colon carcinoma cells, induces the angiogenesis. In contrast to the human angiogenin, thought to have one homologue, the mouse angiogenin is known to have several angiogenin homologues. During we were investigating the target genes, overexpressed by the chimeric PAX3/FKHR transcription factor, new gene closely similar to the mouse angiogenin rather than angiogenin itself was obtained. We report this Ang-vl gene to understand the process of angiogenesis by comparing the mouse angiogenin family genes. METHODS: The representational difference analysis was used to investigate the target genes over expressed by the PAX3/FKHR chimeric transcription factor. The target genes were subcloned into the pBluescriptSK + and sequenced using the 73 and 77 vector itself primers. Analyses of the completed consensus nucleic acid and peptide sequences were performed using the intelligenetics and GCG software packages as well as BLAST algorithms. RESULTS: The Ang-vl gene, including the glutamine that becomes the N-terminal amino acid by the post-translational peptidase reaction and stop codon, was obtained. CONCLUSIONS: We cloned the one member of the mouse angiogenin family genes. From the point of protein chemistry, the mechanism of angiogenin or, for that matter, of any other blood vessel inducing proteins is not yet known. However, the homologues of the angiogenin might interact each other to regulate the angiogenesls. In this regard, the Ang-vl gene provides an opportunity to understand the mechanism of angiogenesis.
Animals ; Blood Vessels ; Chemistry ; Clone Cells ; Codon, Terminator ; Colon ; Consensus ; Glutamine ; Humans ; Mice* ; Transcription Factors*

IL-32 exists as seven mRNA transcripts that can translate into distinct individual IL-32 variants with specific protein domains. These translated protein domains of IL-32 variants code for specific functions that allow for interaction with different molecules intracellularly or extracellularly. The longest variant is IL-32γ possessing 234 amino acid residues with all 11 protein domains, while the shortest variant is IL-32α possessing 131 amino acid residues with three of the protein domains. The first domain exists in 6 variants except IL-32δ variant, which has a distinct translation initiation codon due to mRNA splicing. The last eleventh domain is common domain for all seven IL-32 variants. Numerous studies in different fields, such as inflammation, autoimmunity, pathogen infection, and cancer biology, have claimed the specific biological activity of individual IL-32 variant despite the absence of sufficient data. There are 4 additional IL-32 variants without proper transcripts. In this review, the structural characteristics of seven IL-32 transcripts are described based on the specific protein domains.
Autoimmunity ; Biology ; Codon, Initiator ; Inflammation ; Protein Structure, Tertiary ; RNA, Messenger