• Title/Summary/Keyword: DNA probe hybridization

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Construction of a Bacterial Artificial Chromosome Library Containing Large BamHI Genomic Fragments from Medicago truncatula and Identification of Clones Linked to Hypernodulating Genes

  • Park So-Yeon;Nam Young-Woo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.2
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    • pp.256-263
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    • 2006
  • In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

The Production of Sex Determined Cattle by Embryonic Sexing Using Fluorescence In Situ Hybridization Technique (FISH 기법을 이용한 소 수정란의 성감별과 산자 생산)

  • Sohn, S.H.;Park, H.
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2007.05a
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    • pp.39-50
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    • 2007
  • Sexing from bovine embryos fertilized in vitro implicates a possibility of the sex controlled cattle production. This study was carried out to produce the sex determined cattle through the embryonic sexing by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe constructed from the btDYZ-1 sequence. Using this probe, a male-specific signal was detected on 100% of Y-chromosome bearing metaphase specimens. The analyzable rate of embryonic sexing by FISH technique was about 93% (365/393) regardless of embryonic stages. As tested single blastomere by FISH and then karyotype with their biopsied embryos, the accuracy of sex determination with FISH was 97.6%. We tried the embryo transfer with sex determined embryos on 15 cattle. Among them, the 5 cattle delivered calf with expected sex last year.

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Alterations in Prolactin Messenger Ribonucleic Acid Level During the Rat Estrous Cycle: Effect of Naloxone (흰쥐 성주기간동안 Prolactin mRNA의 변화:Naloxone)

  • 안혜영;유선경;조병남;김경진;유경자;조완규
    • The Korean Journal of Zoology
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    • v.33 no.2
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    • pp.183-190
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    • 1990
  • The present study exarnines the physiological alteradons in prolactin (PRL) messenger ribonucleic acid (mRNA) and serum PRL levels during the rat estrous cycle and the effed of naloxone, an endogenous oploid peptide receptor antagonist, on PRL gene expression during the rat estrous cycle. Adult female rats exhibiting at least two consecutive 4-day estrous cycles were used in this study. A single injection of naloxone (2mg/kg b.w.) or saline was given sc 30 mm prior to decapitation. Animals were sacrificed at 10:00 h of each stage of the estrous cycle, and at 2-h intervals from 10:00 h to 20:00 h during the proestrus. PRL mRNA and serum PRL levels were determined by a RNA-blot hybridization with the rat PRL cDNA probe and by a PRL radjoimmunoassay, respectively. PRL mRNA and serum PRL levels were not dramatically altered in the morning of each stage of diestrus I, II and proestrus, and naloxone failed to modify the two parameters. During estrus naloxone clearly suppressed serum PRL levels, but it was unable to modify PRL mRNA levels. A more detailed examination of the proestrus stage revealed that PRL mRNA and serum PRL levels were fluctuated as a function of time: PRL mRNA levels reached a maximum level at 12:00 h and gradually decreased until 18:00 h. PRL mRNA levels then rose at 20:00 h. No difference of PRL mRNA levels between the control and naloxone-treated groups was observed. Changes in serum PRL levek during proestrus were conversely related to changes in PRL mRNA: serum PRL levels were low from 10:00 h to 14:00 h, then increased and reached a maximum level at 16:00-18:00 h. Following then, serum PRL levels were decreased. Naloxone was effective in suppressing the charaderistic afternoon surge of PRL from 16:00 h to 20:00 h. These data clearly showed that alterations in PRL mRNA levels were conversely correlated with changes mn serum PRL levels on proestrus, indicating a differential regulation of PRL gene expression and secretion.

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gyrA Mutations Found Among Ofloxacin-resistant Mycobacterium tuberculosis is Isolated from Korea

  • Kim Junho;Kim Yeun;Bae Kiho;Song Taek-Sun;Cho Sang-Nae;Lee Hyeyoung
    • Biomedical Science Letters
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    • v.11 no.4
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    • pp.465-471
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    • 2005
  • Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyrA corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyrA. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyrA gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes $(Asp{\to}Ala,\;Asp{\to}Gly,\;Asp{\to}His,\;Asp{\to}Tyr,\;and\;Asp{\to}Asn)$. Five isolates contained a mutation at codon 90 resulting $Ala{\to}Val$ change. Four isolates had mutations at codon 91 causing a $Ser{\to}Pro$ change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes $(Gly{\to}Cys,\;Gly{\to}Ala)$. On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyrA among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations atgyrA gene.

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Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Genetic Characteristics and Virulence Factors of Pandemic Vibrio parahaemolyticus Isolated in South Korea and Japan (한국과 일본에서 유행하는 장염비브리오의 병원성 인자와 유전자의 특성)

  • Hong, Seuk-Won;Moon, Ji-Young;Lee, Bok-Kwon;Kim, Yung-Bu
    • Journal of Life Science
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    • v.17 no.3 s.83
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    • pp.386-395
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    • 2007
  • A total of 27 strains of Vibrio parahaemolyticus (18 strains isolated from Korea and 9 strains from Japan) were serotyped and examined for biochemical characteristics, antimicrobial susceptibility patterns, cytotoxicity assay, thermostable direct hemolysin (TDH) production and molecular epidemiology. Using polymerase chain reaction (PCR) method and DNA probe hybridization method, the strains were tested for toxR, tdh, trh and ORF 8 genes. The V. parahaemolyticus isolated from patients were belonged to 8 different serotypes : O3:K6, O1:K38, O3:K57, O4:K9, O4:Kl2, O4:K68, O5:Kl5 and O6:K46. Urease-positive strain possessed the trh gene, and conversely, urease-negative strains lacked the gene, indicating that urease production by V. parahemolyticus strains strongly correlates with the possession of the trh gene. Most strains showed multiple resistant to more than three antibiotics and the antibiogram could be classified into 6 group (I to VI). All of the O3:K6 strains isolated in South Korea and Japan producted TDH at high levels. The TDH titers ranged between 256 and 2.048, and the average titer was 1009. To distinguish the new and increasingly common V. parahaemolyticus strains from clinical isolates, ORF 8 is a useful genetic marker. After Southern hybridization, the HindIII restriction fragment patterns of the tdh gene were grouped one type, respectively. One type showed two bands one of which was 4.3kb and the other was 11.5kb in size. Variation between the O3:K6 serotype are minor when compared to the differences seen with the non O3:K6 strains. The migration patterns of Not I -digested of the total DNA of the O3:K6 strains were similar, and only slight variations were observed between the serotypes. By contrast, the O3:K6 strains and non O3:K6 had markedly different profiles. In conclusion, Random amplified polymorphic DNA (RAPD) profile using appropriate primers was an effective epidemiological marker.

Telomeric Dynamics and Telomerase Activity in Early Bovine Embryos (소의 초기 배 발생단계별 Telomeric DNA 함량 및 Telomerase Activity 분석)

  • Jung, Yei-Hwa;Lee, Soo-Hee;Cho, Sang-Rae;Kong, Il-Keun;Cho, Jae-Dong;Sohn, Sea-Hwan
    • Clinical and Experimental Reproductive Medicine
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    • v.36 no.2
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    • pp.101-109
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    • 2009
  • Objective: This study was carried out to analyze the amount of telomeric DNA and telomerase activity in early bovine embryos. Methods: The amount of telomeric DNA in early bovine embryos at the 8 cell, morula and blastocyst stages was analyzed by Quantitative Fluorescence In Situ Hybridization (Q-FISH) technique using a bovine telomeric DNA probe. Telomerase activity was analyzed by Telomeric Repeat Amplification Protocol (TRAP assay). Results: The relative amount of telomeric DNA in early bovine embryos was gradually increased from 8 cell to blastocyst stage. It was not significantly associated with the grade of embryo quality. While telomerase activity was detected in the early bovine embryos at these stages, it significantly increased at morula stage and showed maximum activity at the blastocyst stage. Conclusion: The amount of telomeric DNA and the telomerase activity of bovine embryos increase during the progression of early embryogenesis, suggesting a positive correlation between telomeric DNA and telomerase activity. The telomerase activity seems to increase to maintain the levels of telomeric DNA through embryo development which are required for extensive cell division.

Cloning and Functional Expression in Escherichia coli of the Polyhydroxyalkanoate Synthase (phaC) Gene from Alcaligenes sp. SH-69

  • Lee, Il;Nam, Sun-Woo;Rhee, Young-Ha;Kim, Jeong-Yoon
    • Journal of Microbiology and Biotechnology
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    • v.6 no.5
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    • pp.309-314
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    • 1996
  • Alcaligenes sp. SH-69 can synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon source such as glucose. To clone the phaC gene from Alcaligenes sp. SH-69, a polymerase chain reaction was performed using the oligomers synthesized based on the conserved regions of the phaC genes from other bacteria. A PCR product (550 bp) was partially sequenced and the deduced amino acid sequence was found to be homologous to that of the phaC gene from Alcaligenes eutrophus. Using the PCR fragment Southern blotting of Alcaligenes sp. SH-69 genomic DNA digested with several restriction enzymes was carried out. To prepare a partial genomic library, about 5-Kb genomic DNA fragments digested with EcoRI, which showed a positive signal in the Southern blotting, were eluted from an agarose gel, ligated with pUC19 cleaved with EcoRI, and transformed into Escherichia coli. The partial library was screened using the PCR fragment as a probe and a plasmid, named pPHA11, showing a strong hybridization signal was selected. Restriction mapping of the insert DNA in pPHA11 was performed. Cotransformation into E. coli of the plasmid pPHA11 and the plasmid pPHA21 which has phaA and phaB from A. eutrophus resulted in turbid E. coli colonies which are indicative of PHA accumulation. This result tells us that the Alcaligenes sp. SH-69 phaC gene in the pPHA11 is functionally active in E. coli and can synthesize PHA in the presence of the A. eutrophus phaA and phaB genes.

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Telomeres Distribution and Telomerase Activity During Chick Embryonic and Developmental Stages

  • Cho, E.J.;Kang, M.Y.;Jung, G.S.;Sohn, S.H.
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.111-111
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    • 2003
  • Telomeres are the end of chromosomes and consist of a tandem repeat sequence of (TTAGGG)n and associated proteins. Telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. Even though telomeres and telomerase have been studied extensively, very little is known about telomere dynamics in embryonic cells. This study was carried out to analyze the telomeres distribution and telomerase activity of chicken cells during embryonic and developmental stages. The target cells for analysing were sperms, ovulated ova, early embryonic cells and the cells from brain, heart, liver, kidney and germinal tissue in fetus. Telomeres distribution on target cells was analyzed by Q-FISH (Quantitation-Fluorescence in situ Hybridization) techniques using a chicken telomere repeat probe. Telomerase activity was performed by TRAP assay (Telomeric repeat Amplification Protocol) with target DNA. In results, the telomeres of chicken were found at the ends of all chromosomes. In addition, chicken had interstitial telomeres on chromosomes 1, 2 and 3. Telomerase activity was highly detectable in early embryonic cells, germinal tissues and kidney cells. Whereas telomerase activity was gradually down-regulated when the organs, including brain, heart, and liver, were developed from embryos. In the distribution of telomeric DNA on the embryonic and developmental stages, most of the cells was gradually decreased in telomere quantity during ontogenesis.

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The Amount of Telomeres and Telomerase Activity on Chicken Embryonic Cells During Developmental Stages (닭의 발생 단계별 세포내 Telomere의 양적 분포양상과 Telomerase 활성도 분석)

  • Cho, E.J.;Choi, C.H.;Sohn, S.H.
    • Journal of Animal Science and Technology
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    • v.47 no.2
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    • pp.187-194
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    • 2005
  • Telomeres locate at the end of chromosomes and consist of a tandem repeat sequence of $(TIAGGG)^{n}$ and associated proteins. Telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. This study was carried out to analyze the amount of telomeres and telomerase activity of chicken cells during embryonic and developmental stages. The whole embryos and prenatal tissues such as brain, heart, liver, kidney and testis at different developmental stages were obtained from Korean Native Chicken. The amount of telomeres on embryonic cells was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) techniques using the chicken telomeric DNA probe. Telomerase activity was measured by telomeric repeat amplification protocol (TRAP) assay. Results indicated that the amounts of telomeric DNA on the most embryonic cells were gradually decreased during ontogenesis. Furthermore, the quantity of telomeres was quite different among embryonic tissues according to developmental origin. The relative amount of telomeres has more in regenerative cells such as embryonic disc and testicular cells than in non-regenerative cells such as liver, brain, heart and kidney cells. Telomerase activity was also highly detectable in most chicken cells at early embryonic stages. After 9 days of incubation, however, the telomerase activitie W