BACKGROUND: Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance. METHODS: 28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced. RESULTS: All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological suscaptibility test, 20 strains were resistant, and one was susceptible, and the other failed to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position - 11 upstream of the start codon. CONCLUSION: The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M.tuberculosis, and for demonstrating the epidemiological relatedness of the PZA-resistant M.tubersulosis strains.
Adenine ; Codon, Initiator ; Codon, Nonsense ; Enzyme Assays ; Frameshift Mutation ; Guanine ; Mycobacterium tuberculosis* ; Mycobacterium* ; Open Reading Frames ; Promoter Regions, Genetic ; Pyrazinamide*

The primary clue for locating protein-coding regions is the open reading frame and the determination of ORFs (Open Reading Frames) is the first step toward the gene prediction, especially for prokaryotes. In this respect, we have developed a web-based ORF search tool called ORF Miner. The ORF Miner is a graphical analysis utility which determines all possible open reading frames of a selectable minimum size in an input sequence. This tool identifies all open reading frames using alternative genetic codes as well as the standard one and reports a list of ORFs with corresponding deduced amino acid sequences. The ORF Miner can be employed for sequence annotation and give a crucial clue to determination of actual protein-coding regions.
Amino Acid Sequence ; Animals ; Ecthyma, Contagious ; Genetic Code ; Open Reading Frames ; Resin Cements

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Molecular genomic studies of HCMV have been attempted in order to understand the pathogenesis and evolution of HCMV. However, studies on HCMV strains of Asian origin are limited. In this study, it was attempted to understand the genomics of HCMV isolated from Korea. Clinical strain LCW isolated from Korean patient was passaged in vitro cell culture, and subjected to next-generation sequencing. Complete genome sequence was obtained and compared with other HCMV strains. The LCW genome was found to contain 170 open reading frames (ORFs) and two ORF (RL5A and RL13) of the strain LCW were found to be truncated due to early stop codon. Phylogenetic analysis suggested that the strain LCW was closely related with Asian strains such as HCMV strains JHC and HAN. Common nucleotide sequences among the 3 Asian strains distinguishable from other strains were detected at 197 sites including 104 sites in ORFs.
Animals ; Asian Continental Ancestry Group ; Base Sequence ; Cell Culture Techniques ; Codon, Terminator ; Cytomegalovirus* ; DNA ; Ecthyma, Contagious ; Genome* ; Genomics ; Humans* ; In Vitro Techniques ; Korea ; Open Reading Frames

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*

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

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly characterized by distinctive facial features, upper limb malformations, growth and cognitive retardation. The diagnosis of the syndrome is based on the distinctive clinical features. The etiology is still not clear. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A (also called SMC1L1) and SMC3 have been suggested as probable cause of this syndrome. We experienced a case of newborn with CdLS showing bushy eyebrows and synophrys, long curly eyelashes, long philtrum, downturned angles of the mouth and thin upper lips, cleft palate, micrognathia, excessive body hair, micromelia of both hands, flexion contracture of elbows and hypertonicity. We detected a NIPBL gene mutation in a present neonate with CdLS, the first report in Korea.
Codon, Nonsense ; Codon, Terminator ; De Lange Syndrome/diagnosis/*genetics/ultrasonography ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Proteins/*genetics ; Sequence Analysis, DNA ; Tomography, X-Ray Computed

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

Mutations in the p53 tumor suppressor gene have been shown to be one of the most common genetic abnormalities in human cancers. Loss of p53 tumor suppressor gene function can occur through gene mutation or interaction with early proteins of oncogenic viruses such as E6 protein of human papillomaviruses(HPV). We studied 24 squamous cell carcinomas of the larynx(20) and hypopharynx(4) for p53 gene mutations as well as for the presence of oncogenic HPV DNA. Exon 5 through 8 of the p53 gene were examined using polymerase chain reaction-direct sequencing methods. HPV detection was done using polymerase chain reaction amplification with HPV L1 consensus primer. HPV type was determined by the same method using HPV-16 and -18 type-specific E6 primers. The results were as follows: 1) Eight of 24 tumors(33%) had p53 mutations, one of which had 2 mutational sites. All cases of which had p53 mutations or HPV DNA detection were larynx cancer. 2) p53 genetic alteration in larynx cancer included seven missense mutations resulting in single amino acid substitutions, one nonsense mutation encoding a stop codon and one silent mutation. Six of 9(66.7%) mutations occurred in two distinct regions of the genes, codon 245-248(3 mutations) and codon 283-294(3 mutations). 3) The p53 mutational spectrum observed was characterized by G to T transversion(4 of 9), T to A transversion(2 of 9), C to A transversion(1 of 9), G to A transition(1 of 9) and C to T transition(1 of 9). 4) HPV DNA was detected in 3 of 24(12.5%) tumors. According to the type of HPV DNA, HPV-16 was detected in 1 case and HPV non-16, -18 was detected in 2 cases, one of which had p53 mutation. 5) There was no relationship between p53 mutations or HPV DNA detection and clinicopathologic parameters. These results suggest that p53 mutations may play an important role in carcinogenesis of laryngeal cancer. Further study is necessary to clarify the role of p53 mutation and oncogenic HPV infection on clinical outcome of laryngeal cancer.
Amino Acid Substitution ; Carcinogenesis ; Carcinoma, Squamous Cell ; Codon ; Codon, Nonsense ; Codon, Terminator ; Consensus ; DNA* ; Exons ; Genes, p53* ; Genes, Tumor Suppressor ; Human papillomavirus 16 ; Humans* ; Laryngeal Neoplasms ; Larynx* ; Mutation, Missense ; Oncogenic Viruses ; Pharyngeal Neoplasms* ; Pharynx* ; Polymerase Chain Reaction

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