Cloning and heterologous expression of a hydrophobin gene Ltr. hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.

First Molecular Diagnosis of a Patient with Unverricht-Lundborg Disease in Korea

Unverricht-Lundborg disease (ULD) is a form of progressive myoclonus epilepsy characterized by stimulation-induced myoclonus and seizures. This disease is an autosomal recessive disorder, and the gene CSTB, which encodes cystatin B, a cysteine protease inhibitor, is the only gene known to be associated with ULD. Although the prevalence of ULD is higher in the Baltic region of Europe and the Mediterranean, sporadic cases have occasionally been diagnosed worldwide. The patient described in the current report showed only abnormally enlarged restriction fragments of 62 dodecamer repeats, confirming ULD, that were transmitted from both her father and mother who carried the abnormally enlarged restriction fragment as heterozygotes with normal-sized fragments. We report the first case of a genetically confirmed patient with ULD in Korea.

A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.

Distribution and molecular epidemiologic characterics of insertion sequence IS1301 in Neisseria meningitidis isolated from China / 中华流行病学杂志

Objective To research the distribution and molecular epidemiology of insertion sequence IS1301 in Neisseria (N.) meningitidis strains in China, so as to provide scientific and available evidence for a new method of genotyping in N.meningitidis strains with IS1301. Methods Examined the IS1301 by PCR in 219 N.meningitidis strains from 16 provinces and 3 cities during 2007 and 2008 in China, productions of amplification were sent for sequencing. The positive N.meningitidis strains were analyzed by pulse field gel electrophoresis (PFGE) and nucleic acid blotting hybridization(Southern blot) by electrophoresis. Results The positive rates with IS1301 were 15.53%, 11.11%, 20.75%, 6.17% and 28.57% for four serotypes (A, B, C, N) respectively. The sequence comparability between the amplification productions and No.Z49092.1 N.meningitidis which registered in GenBank was 94%-100%. There were two types of clusters devided by cladogram analysis. There appeared large IS1301 sequence difference between the serotype C and others. The number of IS1301 replica ranged from 6-17 per strain at least. The number of IS1301 replica changed in the same type of PFGE N.meningitidis respectively. Conclusion Typing by IS1301 combined with PFGE could comprehend the homology and genetic polymorphism of N.meningitidis epidemic strains at the molecular level.

Genotype and Drug Resistance of ?-Lactamases in Escherichia coli / 中华医院感染学杂志

OBJECTIVE To study the antibiotic sensitivities and characterization of ?-lacamase activity and the antibiotic resistance mechanisms.METHODS The extended-spectrum ?-lactamases were detected.Agar dilution method was used to determine the minimum inhibitory concentration(MIC)of severel antibiotics.The plasmids of clinical isolates were cloned into competent cells(K-12).RESULTS Isoelectric focusing(IEF):The last 5 clinical isolates have all got an activity at a high pH 9 which could be presumptively identified as AmpC.The last two clinical isolates had a weak activity with pH of about 8.0,which belonged to SHV type ?-lactamases.All the isolates had a group of activities at the lower pH range of the gel(pH5).These could be presumptively identified as TEM ?-lactamases.CONCLUSIONS The data suggest that each isolate clearly originate from the same population but possess mutations that leads to antibiotic resistance.The combinations of mutations in same isolates suggest that an earlier strain have infected other compartments and become the founder for later antibiotic resistant variants.

Clustering distribution of the specific fragment R049 of uropathogeulc Escherichia coil / 中华微生物学和免疫学杂志

Objective To study the distribution of the specific fragment R049 of uropathogenic E. coli(UPEC) 132 in UPEC and fecal E. coli strains. Methods The specific fragment R049 was amplified by PCR from 20 UPEC strains and 40 fecal E. coli strains, and 5 genes encoding virulence factors (papC, fimH, hly, aer, cnf1) were detected from fragment R049 positive strains. Pulse field gel electronphoresis (PFGE) was applied for isolating the Xba Ⅰ restriction fragments of the genomes of fragment R049 positive strains, and then Southern blot was applied for analyzing the distribution features of the positive hybridization bands by digoxin-labeled R049 ORF probe. Results The specific fragment R049 was amplified from 8 of 20 UPEC strains (40%) and 3 of 40 fecal E. coli strains (7.5%), and statistics analysis showed significant difference (P<0.01). The specific fragment 11049 was closely related with 5 virulence factors of UPEC in the fragment R049 positive strains. Southern blot showed the sizes of positive bands were 150 kb, 15 kb and 240 kb in 3 fecal E. coli strains, 350 kb in 6 of 8 UPEC strains, and 280 kb and 25 kb in the rest two UPEC strains. Conclusion The specific fragment R049 of UPEC132 possessed the feature of clustering distribu-tion in domestic isolated UPEC strains.

Molecular and Clinical Characteristics of Myotonic Dystrophy Type 1 in Koreans / 대한진단검사의학회지

BACKGROUND: Myotonic dystrophy type 1 (DM1) is an autosomal-dominant muscular dystrophy caused by expansion of cytosine-thymine-guanine (CTG) trinucleotide repeats in the myotonic dystrophy protein kinase (DMPK) gene. The clinical features of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations. The aim of this study was to characterize the molecular and clinical spectra of DM1 in Koreans. METHODS: The CTG repeats of 283 Korean individuals were tested by PCR fragment analysis and Southern blot. The following characteristics were assessed retrospectively: spectrum of CTG expansions, clinical findings, genotype-phenotype correlation, anticipation, and genetic instability. RESULTS: One-hundred twenty-four patients were confirmed as DM1 by molecular tests, and the CTG expansions ranged from 50 to 2,770 repeats (median 480 repeats). The most frequent clinical features were myotonia, muscular weakness, and family history. Patients with muscular weakness or dysfunction of the central nervous system harbored larger CTG expansions than those without each symptom (P<0.05). The age of onset was inversely correlated with the size of the CTG expansion (gamma=-0.422, P<0.001). The instability of CTG expansion representing as the maximum difference between sibships was observed from 50 to 700 repeats in nine families. Clinical anticipation and the increase in CTG repeat were significantly higher in maternally transmitted alleles (P=0.002). CONCLUSIONS: Molecular genetic tests are not only essential for diagnosis, but also helpful for suggesting the spectrum and relationship between genotype and phenotype in Korean DM1 patients.

Chemiluminescent Southern blot System for Detection of HBV Replication in vitro and Its Application for Analysis the Drugs Inhibition to HBV / 中国生物工程杂志

Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109,?-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53?mol/L,0.41?mol/L,0.01?mol/L.Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese-midicine.

Cloning and Characterization of Swamp Buffalo SRY Gene / 中国生物工程杂志

The SRY gene from buffalo (Bubalus bubalis) genome was amplified by the polymerase chain reaction ( PCR) with primers based on the sequence of Hostein SRY gene. The amplified fragment was 2005 bp include 5UTR ( 1 - 504bp) and 3'UTR(1196 - 2005bp). And the amplified fragment was cloned and sequenced. Sequence analysis showed that the coding region of SRY gene (505 - 1195bp) from buffalo was highly homologous with those of other bovine counterpart genes (96% homology) , especially in the HMG box region (99%homology). It was found that there were only signal on male buffalo genome on Southern blot,which indicate SRY gene are highly conservative on evolves.

Changes of type Ⅰ fibroblast growth factor receptor gene during development / 免疫学杂志

Objective To find the changes of human fibroblast growth factor receptor 1 genone during development.Method Southern blot analysis of genomic DNA isolated from adult and fetal tissues. Result Adult FGFR1 gene structure is different from its embryonic counterpart. Conclusion The disserence might lead to changes of FGFR1 expression as wel as functions of the cells.

Incidence of Fragile X Syndrome in Korean Patients with Mental Retardation / 대한산부인과학회잡지

Fragile X syndrome is the most common cause of inherited mental retardation. It accounts for 0.2% - 2.7% of patients with mental retardation, based upon the molecular genetic diagnosis. However, the exact prevalence of fragile X syndrome in Korean patients with mental retardation is unknown. We have performed cytogenetic and molecular analysis for fragile X syndrome in 212 Korean patients with mental retardation. Among them, six patients (2.8%) was identified as carrying fragile X syndrome by both cytogenetic and molecular analysis. The results by cytogenetic analysis was identical to those by molecular analysis. Cytogenetic analysis of 6 carriers (mothers of patients with proven fragile X syndrome) showed a fragile X chromosome in one patients (16.7%) while molecular analysis revealed premutation in all patients. PCR method using Klentaq1 Pfu polymerase showed the same results as those by PCR method using Exo(-) Pfu polymerase, but the former method is recommended because of its simplicity in technical aspect. These data suggest that the prevalence of fragile X syndrome in Korean patients with mental retardation is 2.8%, not significantly different from those in Caucasians.

Amplication of C-myc and rearrangement of erb B2 in bladder cancer / 대한해부학회지

In order to eluciate the involvement of oncogene and tumor suprrssor gene in the tumorigenesis of human bladder tumor, I examined eleven bladder tumor tissues with Southern blot hybridization. For Southern blot c-myc, erb B2, c-fos, N-ras, K-ras, v-mos, and p53 were used as probes. In DNA analysis amplication of c-myc and rearrangement of erb B2 were detected each one case respectively. No significant alternations were obserbed in the other probes. Even though a small number of samples were examined, these finging may expect that the amplication of c-myc and the rearrangement of erb B2 play their roles in the development of bladder tumors. Futher more samples and oncogene or tumor supressor gene probes should be used to understand the tumorigenesis of the bladder cancer.

Diagnosis of Fragile X Syndrome and DMD by using DIG System / 대한산부인과학회잡지

The aim of this study was to develop a rapid and safe non-radioactive DIG DNA labeling and detection for Southern blot analysis for fragile X syndrome and Duchenne muscular dystrophy (DMD). Southern blot analysis is accurate test showing expression of the (CGG)n repeat and abnormal methylation pattern of CpG island in hagile X syndrome, and good confirmative secondary test in case of deletion in DMD. But in terms of test rapidity, these conventional radioactive Southern analysis may not be feasible for rapid screening of prenatal samples and at-risk populations to determine their status and to provide genetic counseling to their families. As an alternative radioactive Southern blotting, DIG DNA labeling and detection system does not require handling of radioactive material nor require learning any new technology. The complete procedure of labeling the DNA and hybridization to detection of the first visible signal can be compbsbed witbin 7 days. In addition, hybridization solutions containing labeled DNA can be reused several times after renewed denaturation.

Homozygous Deletion of CDK4I in Acute Leukemia / 대한혈액학회지

BACKGROUND: The CDK4I (cyclin dependent kinase 4 inhibitor) gene is on the chromosome 9p21. It encodes p16, which binds to CDK4, and inhibits phosphorylation of retinoblastoma protein (pRb) by D-type cyclin-CDK4 complex. D-type cyclin-CDK4 complex phosphorylates pRb which regulates transition of G1 to S phase of the cell cycle. So inactivation of the CDK4I gene leads to the dysregulation of the cell cycle and may contribute to the malignant progression of cells. Homozygous deletion of CDK4I gene was frequently found in human hematologic malignancies. The purpose of this study is to find the relationship between the CDK4I gene homozygous deletion and diverse acute leukemia. METHODS: We analyzed homozygous deletions of the CDK4I gene from 21 cases of acute leukemia by means of Southern blot analysis (10 B-cell acute lymphocytic leukemias, 6 T-cell acute lymphocytic leukemias, 5 acute myelogenous leukemias). Hybridization of EcoRI digested DNA using CDK4I probes (exon 1 and exon 2) obtained by PCR of human control DNA was performed. RESULTS: 1) We observed 4 cases (19%) of homozygous deletions in 21 cases of acute leukemia. 2) ALL 4 cases of homozygous CDK4I deletion were T-cell ALL which accounted for 66% (4/6 cases) of T-cell ALLs. 3) Homozygous CDK4I deletions were not found in any cases of B-ALLs (0/10 cases) and AML (0/5 cases). 4) In 4 cases of homozygous deletion, consistent results were obtained by 2 different CDK4I probes (pv 336-bp probe, pv 142-bp probe). CONCLUSION: The high frequency of CDK4I homozygous deletions in T-ALLs supports that the inactivation of these genes plays an important role in the pathogenesis of T- ALL.

The Analysis of Mitochondrial DNA in the Failing and Normal Hearts / 대한내과학회지

OBJECTIVE: The aim of this study is to analyze the mitochondrial DNA in failing and normal hearts. METHODS: Genomic DNA was extracted from 18 failing and 4 normal hearts. The DNA was digested with each 50 units of BamH I, Pvu II, Pst I, and hybridized using DNA fragments encoding CO II (cytochrome oxidase II) and CO IU. They were detected using 'Fluorescein Gene Images' system. RESULTS: The light microscopic feature of failing myocardium was compatible with that of primary cardiomyopathy. In southern blot analysis, there was no significant difference in mitochondrial DNA amounts between normal and failing hearts. The amount of mitochondrial DNA in hearts, whether normal or failing, was greater than that in lymphocytes. There were no abnormal bands except 16.6kb-normal band using the enzyme BamH I, Pvu II from failing and normal hearts. After digesting with Pst I, 2.1kb band was found using probe CO II and 14.5kb band using probe CO III. CONCLUSION: The amount of mitochondrial DNA in hearts, whether normal or failing, was greater than that in lymphocytes, which suggests that the heart is an active organ in the energy metabolism. Abnormal band was not found in southern blot analysis of the mitochondrial DNA from failing and normal hearts. The more sensitive method such as PCR is required to detect the presence of sma11 amount of mutated DNA.

Amplification of Cyclin D1 in Head and Neck Squamous Cell Carcinomas / 대한이비인후과학회지

BACKGROUND: Amplification of the chromosome 11q13 region has been observed in a variety of human cancers, including head and neck squamous cell carcinoma and carcinomas of breast, esophagus, lung, bladder, and liver. The chromosome 11q13 region has various putative oncogenes, of which cyclin D1 is most consistently amplfied and overexpressed. OBJECTIVES: To estabilsh the frequency and clinicopathologic correlations of cyclin D1 amplification in head and neck squamous cell carcinomas. MATERIALS AND METHODS: Fresh tissue samples were obtained from 26 patients with head and neck cancers undergoing surgery or biopsy. Amplification of cyclin D1 was evaluated in 26 head and neck squamous cell carcinomas by Southern blotting. The presence or absence of amplification was correlated with anatomic site, tumor stage, and differentiation pattern. RESULTS: Five tumors of 26(19%) showed a twofold to 4-fold amplification of cyclin D1 compared with beta-actin control prove. Amplified and nonamplified groups revealed no differences in anatomic primary site, stage, N stage, and pathologic differentiation. CONCLUSION: We showed a significant incidence of cyclin D1 amplification in head and neck squamous cell carcinomas, but cannot demonstrate an association with clinical presentation and pathologic findings.

Neuroepithelial Tumor Relevant Genes

Cancer may be a disease of genes, arising from genetic damage of diverse sorts-recessive and dominant mutations, large rearrangement of DNA and gene translocation on chromosomes, all leading to distorisions of either the expression or biochemical function of genes. The search for these genetic damage in neoplastic cells now is the most important in cancer research. It has been found that the cancer relevant genes were located on the specific regions of chromosomes. To determine whether epidermal growth factor receptor(EGFR), P53 and bcr genes located in chromosomes 7, 17 and 22 are altered, we examined 12 neuroepithelial tumor with Southern blot analysis(five low grade astrocytoma, two high grade astrocytoma, two medulloblastoma, on oligodendroglioma, one ependymoma, one choroid plexus papilloma). The loss of heterozygosity(LOH) of EGFR gene was detected in two cases of medulloblastoma. The rearrangement of EGFR gene was detected in a case of ependymoma. The LOH of P53 gene was found in a case of choroid plexus papilloma and low grade astrocytoma. The rearrangement of P53 gene was founs id a case of oligodendroglioma. The LOH of bcr gene was observed in two cases of medulloblastoma and low grade astrocytoma. The rearrangement of bcr gene was observed in two cases of high grade astrocytoma. These results suggested that tumorigenesis and tumor development in the neuroepithelial tumor may invlove specific gene changes in chromosomes 7, 17 and 22.

Enhanced expression of the c-myc protooncogene in human intracranial meningiomas

We examined the alteration and expression of c-myc protooncogene in 11 human intracranial meningiomas using Southern blot, Northern blot and immunohistochemical techniques. Southern blot showed neither amplification nor rearrangement but Northern blot and immunohistochemical study revealed enhanced expression of the c-myc gene. Immunohistochemically, c-myc product was found in all of the 11 cases and seven of these cases showed an above moderate degree of immunoreaction in semiquantitative analysis. Loss of heterozygosity at IGLC2 locus on chromosome 22 was detected in four of the 8 informative cases. But extent and intensity of immunoreactivity did not correlated with loss of heterozygosity on chromosome 22. These genetic changes may play important roles in the pathogenesis of human intracranial meningioma.

Diagnosis of x-linked ichthyosis and detection of its carriers with southern blot hybidization / 대한피부과학회지

BACKGROUND: The skin changes of X-linked recessive ichthyosis are cnused by the deficiency of the enzyme steroid sulfatase, which usually results from deletions of this gene in Caucasian populations. OBJECTIVE AND MEHTODS: To disgnose X-linked recessive ichthyosis and detect its carrier, we have investigated distinctive gene deletion and measured gene dosage of steroid sulfatase gene by southern blot hybridization in Korean patients with X-linked recessive ichthyosis. RESULTS: Patients from 8 of 9 unrelated families exhibited deletions, if the steroid sulfatase gene. Of 6 families showing a family history compatible with X-linked recessive inheritance, One family exhibited a normal pattern of hybridization. All but one family showed deletion of steroid sulfatase gene. All three patients lacking a fami1y history of the disease exhibited gene deletions. The ratio of the steroid sulfatsse specific band density to the Factor VIII specific band density was measured in 8 obligate carriers using a laser densitometer. The average ratio exhibited by the car riers was less than half that of normal women. Conclusian: These results suggest that the X-linked recessive ichth osis patient and its carrier can also be diagnosed and detected by Southern blot hybridization of steroid sulfatase gene in Korea.

Loss of Heterozygosity of Chromosome 17p in Brain Tumors

With the methods of restriction fragment length polymorphisms(RFLPs) and southern blot analysis, gene deletion of chromosome 17p in 16 cases of brain tumors, was investigated. There were 4 cases of glioblastoma multiforme, 1 case of anaplastic astrocytoma, 4 cases of low grade astrocytoma, 3 cases of oligodendroglioma, and 4 cases of meningioma. Among restriction fragment length polymorphism(RFLP) DNA located in chromosome 17p, p144D 6 and p SNZ 22 were imployed as the probes. In eight of 16 cases(50%) constitutional heterozygosity was observe dfor p144 D6 probe on the short arm of chromosome 17, and in nine of 16 cases(56%) for PYNZ 22.1 probe. With both probes constitutional heterozygosity was observed in thirteen of 16 cases(81%). And the loss of constitutional heterozygosity was detected in two of 14 informative cases. Although, with the malignant gliomas, including 4 cases of glioblastoma multiforme and 1 case of anaplastic astrocytoma, two of 4 informative cases showed loss of constitutional heterozygosty, None of 9 informative cases showed loss of heterozygosity with the other brain tumors(low grade astrocytoma, oligodendroglioma, and meningioma).