• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1823-1833
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• 2018

Fluorescence in-situ hybridization (FISH) is a common and popular method used to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes, CLZ and KCLZ, were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes was 30% (w/v). The specificity of the designed probes was high due to the use of pellets from pure reference strains. Feasibility was tested using samples of Chinese liquor from the famed Luzhou manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probes CLZ and KCLZ was 49.11% and 32.14%, respectively. Quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.369-375
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• 1997

Studies were conducted to determine the efficiency of decondensation protocols. Sperm obtained from seven normal donors was immediately washed after liquefaction and then decondensed using the method of West et al. (1989) and my original protocol. My optimized protocol entailed mixing 1 ml aliquots of semen with 4 ml phosphate buffered saline (PBS). Following centrifugation, pellets were resuspended in 1 ml PBS containing 6 mM EDTA. After centrifugation, pellets were resuspended in 1 ml PBS containing 2 mM dithiothreitol at $37^{\circ}C$ for 45 min. Following mixing with 2 ml PBS and centrifugation, pellets were resuspended by vortexing. While vortexing, 5 ml of fixative were gently added. Slide preparation was accomplished using the smear method and it was stored at $4^{\circ}C$. When comparing these protocols, the degree of sperm decondensation and head swelling was monitored by measuring nuclear length, area, perimeter, and degree of roundness using FISH analysis software. Apparent copy number for chromosome 1 and, separately, for the sex chromosomes was determined by FISH using satellite DNA probes for loci DIZ1, DXZ1 and DYZ3. Sperm treated by my decondensation protocol showed significant increases (p<0.05) in length, area, perimeter, and degree of roundness. There was a significant decrease (p<0.05) in the frequency of nuclei displaying no signal but no change in the frequency of nuclei with two signals in samples decondensed by my protocol. My data suggested that decondensation using my original protocol may lower the frequency of cells with spurious "nullisomy" due to hybridization failure without inducing spurious "disomy" resulting from increased distances between split signals.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Journal of the korean academy of Pediatric Dentistry
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• v.31 no.2
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• pp.304-313
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• 2004

Xylitol, a five-carbon natural sugar alcohol, is widely used non-cariogenic sugar substitute. In present study, the effects of xylitol on the expression of mRNA for glucosyltransferase which synthesizes glucan from sucrose were detected by Fluorescent in situ hybridization (FISH) and flow cytometry. FITC fluorescences for mRNA of gtfB, gtfC and gtfD were decreased further with increasing concentration of xylitol from 1% to 10% when detected by FISH. Flow cytometric analysis also showed that the expression of gtfB, gtfC and gtfD was increased by the addition of sucrose and decreased by the addition of xylitol to BHI broth containing 1% sucrose. In conclusion, the expression of gtfB, gtfC and gtfD mRNA was decreased by the addition of xylitol.

• The Korean Journal of Zoology
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• v.30 no.4
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• pp.417-431
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• 1987

Isozyme analysis, morphometric comparison, and artificial hybridization test wereperformed to elucidate the patterns of genic variation, morphological differentiation, genetic incompatibility, and a probable path in speciation between two MDH allelotypes (MM type and MS type) of the Dark Chub Zacco temmincki, a fresh water fish inhabiting in Korean waters. The degree of genic variation of MS type(HD=.023, HG=.021) was twofold higher than that of MM type (HD=.013, HG=.014) but both allelotypes were far less than the average genic variation of fresh water Bish in general. The average genetic similarities among 7 populations of MM type and 6 Populations of MS type were S=.947 and S=.966 respectively, whereas the value between two allelotypes was S=.853. Presumed divergent time of two allelotypes was estimated to be about 700 thousand years ago. Discriminant function analysis based on 18 morphometric characters of 302 specimens representing 12 populations revealed no morphological difference between two allelotypes. Artificial hybridization test indicates that there is an obvious genetic incogpatibility between two allelotypes and therefore it is assumed that isolating mechanism is completed.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.894-898
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• 2001

Genetics has played a pivotal role in increasing the world food production through revolutions in plant and animal sciences. Though the attention on fisheries has been inadequate but the growing importance of modern genetic manipulations and biotechnological innovations to aquaculture has been realized. Recent advances in fish genetics and molecular biology have provided a suite of useful techniques, which have several applications in aquaculture. This paper reviews the advancement in the applications of selection, hybridization, chromosome engineering, sex control, gene transfer and molecular technologies for enhanced aquaculture productivity.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4745-4749
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• 2012

The diagnosis of malignant mesothelioma (MM) remains a clinical challenge and the fluorescence in situ hybridization (FISH) assay has been reported to be one promising tool. The present meta-analysis aimed to establish the overall diagnostic accuracy of FISH for diagnosing MM. After a systematic review of English language studies, the sensitivity, specificity and other measures of accuracy of FISH in the diagnosis of MM were pooled using random-effects models. Summary receiver operating characteristic curves were applied to summarize overall test performance. Nine studies met our inclusion criteria, the pooled sensitivity and specificity for FISH for diagnosing MM being 0.72 (95% CI 0.67-0.76) and 1.00 (95% CI 0.98-1.00), respectively. The positive likelihood ratio was 34.5 (95% CI 14.5-82.10), the negative likelihood ratio was 0.24 (95% CI 0.16-0.36), and the diagnostic odds ratio was 204.9 (95% CI 76.8-546.6), the area under the curve being 0.99. Our data suggest that the FISH assay is likely to be a useful diagnostic tool for confirming MM. However, considering the limited studies and patients included, further large scale studies are needed to confirm these findings.

• Korean Journal of Ecology and Environment
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• v.37 no.4 s.109
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• pp.363-367
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• 2004

For comparative analysis of the eubacterial community structure at 8 sampling sites throughout the Nak-Dong River, FISH (fluorescence in situ hybridization) method was employed. The total ratio of each determined eubacterial group such as ${\alpha}\;{\cdot}\;{\beta}\;{\cdot}\;{\gamma}-subclasses$proteobacteria and Cytophaga-Flavobacterium(CF) group to total counts(DAPI) at each site varied 9.3-42.5% with the highest value at uppermost part. And each ratio of determined eubacterial groups reached mostly under 10% except that of CF group (23%) at uppermost part. Furthermore, compared to lower part, upper part represented unexpectedly higher proportions of ${\gamma}-subclass$ proteobacteria comprised almost fast growing bacteria on degradable organics. Also the variations of ammonia-oxidizing bacteria ranged from $2.7{\times}10^4$ to $18.0{\times}10^4$ cells $mL^{-1}$ with the lowest value in lower part and the highest value in mid part whereas those of nitrite-oxidizing bacteria varied 5.2-7.7{\times}10^4$ cells $mL^{-1}$ without noticeable differences throughout the sites. Additionally, the ratio of nitrifying bacteria to total counts ranged from 1.0% to 13.6% with no differences between ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. In conclusion, FISH method introduced in this study for monitoring, normally used for the quantitative analysis of bacteria, provided also good information on their environmental status in the Nak-Dong River.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Fisheries and Aquatic Sciences
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• v.8 no.3
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• pp.122-127
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• 2005

A DNA chip that rapidly and accurately detects the viral genes in rhabdovirus-infected fish was developed. The N, Ml, and G proteins of three rhabdovirus strains, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and flounder rhabdovirus (HIRRV), were selected for use as probes. The sequences of the corresponding genes were obtained, and probes were prepared by PCR using specific primer sets. The specificity of the probes was confirmed by cross PCR. The prepared probes were spotted on poly-L-lysine- or aminosilane-coated glass slides and hybridized with target DNA under several different conditions in order to determine the optimal hybridization temperature, glass-slide coating, and target cDNA concentration.