An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization （M-FISH） for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Dual fluorescence in situ hybridization in detection of HER-2 oncogene amplification in primary hepatocellular carcinoma

BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.

Application of fluorescence in situ hybridization in the detection of bladder transitional-cell carcinoma: A multi-center clinical study based on Chinese population

Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008.Receiver operating characteristic(ROC)curve analysis was performed and the area under curve(AUC)values were calculated for both the FISH and urine cytology tests.Results:A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population.A total of 4807 patients with hematuria were prospectively,randomly enrolled for the simultaneous analysis of urine cytology,FISH testing,and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen.Overall,the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%,while that of cytology was 33.4%(p<0.001).The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7%and 89.6%,respectively(p=0.004).The sensitivity values of FISH for low and high grade bladder cancer were 82.6%and 90.1%,respectively(p=0.002).Conclusion:FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages.Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.

Comparison of Fluorescence in situ Hybridization and Immunohistochemistry for Assessment of HER-2 Status in Breast Cancer Patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.

Niche Differentiation of Phenol-Degrading Microorganisms in UASB Granular Sludge as Revealed by Fluorescence in situ Hybridization

A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction.Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation.For accurate taxonomic assignment of Cryptanaerobacter clones,phylogenetic analysis using nearly full-length 16S ribosomal RNA(rRNA)gene sequences was necessary.Three oligonucleotide probes were designed to detect the following three taxonomic groups:Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus.FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea.However,a well-defined distribution of Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus was observed.Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other,while Syntrophorhabdaceae was located in the deeper part of the granules.Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate.Phenol degradation in the granular sludge was observed to be carried out in the following way.First,Cryptanaerobacter converts phenol to benzoate,which is then degraded by Syntrophus into acetate.This syntrophic degradation of phenol occurs near the surface of the granule,where the phenol concen-tration is high.In the deeper part of the granule,where the phenol concentration is lower,Syntrophorhabdaceae degrades phenol into acetate.We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms,which contradicts the theo-ries presented by previous studies.

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA （D1-D2） of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization （FISH） tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific （taxonomic） （Pmin0443 and Kmik0602） and the control probes （UniC0512 and UniR0499） were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence （p 〉0.05） was observed in the cell densities of the samples determined by FISH and light microscopy （LM）. All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

DETECTING EXPRESSION OF MRP-1/CD9 mRNA IN LUNG CANCERS USING TISSUE MICROARRAYS AND FLUORESCENCE IN SITU HYBRIDIZATION METHODS

Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.

Observation on In Situ Hybridization and Immunocytochemistry of Matrix Metalloproteinases in Rat Pancreas

In situ hybridization and immunocytochemical techniques were employed to examine the expression of matrix metalloproteinases 1 (MMP 1) and to identify the pattern of its distribution in rat pancreas. The results indicated that the signal of MMP 1 mRNA and MMP 1 positive immunoreaction were detected in some fiberoblasts around interlobular ducts and exocrine cell in margin acinus of some lobules, but the signal of MMP 1 mRNA and MMP 1 positive immunoreaction could not be detected in most of other acinus and islets of pancreas. It is concluded that the expression of MMP 1 in above cells of rat might play an important role in acinar proliferation and differentiation of rat pancreatic tissues.

FLUORESCENCE IN SITU HYBRIDIZATION COMBINED WITH IMMUNOFLUORESCENT STAINING FOR RAPID DETECTION OF Nmyc AMPLIFICATION IN NEUROBLASTOMA

Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.

Optimization and application of fluorescent in situ hybridization (FISH) process in EBPR fed with municipal wastewater

For sludge samples from EBPR reactor fed with municipal wastewater,fluorescent in situ hybridization (FISH) operation process including moisture chamber,pretreatment,treatment with lysozyme and Proteinase K and washing time was optimized and improved.Preserving box was chosen to be moisture chamber due to its bigger depth /radii ratio,good sealability and big volume contrast with Petri dish.3-5 mm diameter glass balls could disperse samples without destroying microorganism cells and community structure.Impurities and ECPs could be removed easily and sludge samples became thinner after dispersing which benefit the observation.Permeabilized cells with lysozyme and Proteinase K could enhance probe penetration before hybridization.Experiments of different treatment time with lysozyme and Proteinase K were carried out.Best results were observed when sludge samples treated with lysozyme 10 min/Proteinase K 20 min or lysozyme 20 min/Proteinase K 10 min.Slides were washed at 48 ℃ for 10,20,30,40 and 60 min in parallel.The best washing time was 20 min when washing temperature was 48 ℃.Fluorescent dye could residue when washing time was 10 min and washing out happened when washed for 30 min or more.

Literature Analysis on Fluorescence in situ Hybridization in China during 2002-2016

In order to explore researches about the chromosome karyotype analysis and fluorescence in situ hybridization(FISH) technology in China,using the bibliometric method,taking " fluorescence in situ hybridization(FISH) " and " chromosome" as key words,this paper made a statistical analysis on the literature published in China National Knowledge Infrastructure(CNKI) during 2002-2016.The results indicated that the number of papers published in 2002 was the smallest(37),while the number of papers published in 2012 was the largest(125).In terms of the distribution of organizations of authors,in 1201 papers,11 organizations published papers ≥15,accounting for 21.65%.In terms of distribution of papers published by different periodicals,11 periodicals published papers ≥10,accounting for 17.65%.In terms of the papers supported by foundation projects,in all papers searched,377 papers were supported by foundation projects,accounting for 31.39%.In terms of the distribution of doctoral and master's dissertations,259 papers were master's dissertations,accounting for 21.57%;92 papers were doctoral dissertations,accounting for 7.66%.

Mesenchymal-epithelial transition factor amplification correlates with adverse pathological features and poor clinical outcome in colorectal cancer

BACKGROUND Colorectal cancer(CRC)is the third most common cancer and the second most common cause of cancer-related mortality worldwide.Mesenchymal-epithelial transition factor(MET)gene participates in multiple tumor biology and shows clinical potential for pharmacological manipulation in tumor treatment.MET amplification has been reported in CRC,but data are very limited.Investigating pathological values of MET in CRC may provide new therapeutic and genetic screening options in future clinical practice.AIM To determine the pathological significance of MET amplification in CRC and to propose a feasible screening strategy.METHODS A number of 205 newly diagnosed CRC patients undergoing surgical resection without any preoperative therapy at Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences were recruited.All patients were without RAS/RAF mutation or microsatellite instability-high.MET amplification and c-MET protein expression were analyzed using fluorescence in situ hybridization(FISH)and immunohistochemistry(IHC),respectively.Correlations between MET aberration and pathological features were detected using the chi-squared test.Progression free survival(PFS)during the two-year follow-up was detected using the Kaplan-Meier method and log rank test.The results of MET FISH and IHC were com pared using one-way ANOVA.RESULTS Polysomy-induced MET amplification was observed in 14.4%of cases,and focal MET amplification was not detected.Polysomy-induced MET amplification was associated with a higher frequency of lymph node metastasis(LNM)(P<0.001)and higher tumor budding grade(P=0.02).In the survival analysis,significant difference was detected between patients with amplified-and non-amplified MET in a two-year follow-up after the first diagnosis(P=0.001).C-MET scores of 0,1+,2+,and 3+were observed in 1.4%,24.9%,54.7%,and 19.0%of tumors,respectively.C-MET overexpression correlated with higher frequency of LNM(P=0.002),but no significant difference of PFS was detected between patients with different protein levels.In terms of concordance between MET FISH and IHC results,MET copy number showed no difference in c-MET IHC 0/1+(3.35±0.18),2+(3.29±0.11)and 3+(3.58±0.22)cohorts,and the MET-to-CEP7 ratio showed no difference in three groups(1.09±0.02,1.10±0.01,and 1.09±0.03).CONCLUSION In CRC,focal MET amplification was a rare event.Polysomy-induced MET amplification correlated with adverse pathological characteristics and poor prognosis.IHC was a poor screening tool for MET amplification.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

Chromosomal mapping of 5S and 18S-5.8S-25S rRNA genes in Saccharina japonica(Phaeophyceae)as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH)technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23,whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.

DISTRIBUTION OF VEGF mRNA IN BREAST CANCER WITH NONRADIOACTIVE IN SITU HYBRIDIZATION AT ELECTRON MICROSCOPIC LEVELS

Object: To localize the mRNA coding for VEGF at Ultrastractural level in human breast cancer by using digoxigenin-labeled cDNA probes. Methods: Nonradioactive in situ hybridization at electron microscopic level was employed to detected VEGF mRNA in breast cancer. Result: Cancer cells and endothelial cell of angiogensis show dark color in experiment sections. No dark color can be found in control sections. Positive hybridization signals showed dark dot and were located in various compartments of the breast cancer cell and endothelial cell in experiment section. No labeling was observed in control sections. In experiment sections, the staining appeared concentrated in cytoplasm and nucleus of the breast cancer cell and endothelial cell. Conclusion: Nonradioactive in situ hybridization at electron microscopic level is efficient for direct observation of the target site mRNA of VEGF in the cytoplasm and nucleus.

The Cloning and Fluorescence In situ HybridizationAnalysis of Cotton Telomere Sequence

Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that keep chromosomes structure independency and completeness. The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown to have tandemly repeated sequence 5-TTTAGGG-3： The Arabidopsis-type telomere has been found in many plants, but several reports indicate that this sequence is absent in some plants. Up to now, no research has been conducted on the telomere of cotton. In this paper, the Arabidopsis-type telomere sequence was amplified and cloned using the primers designed based on the fragment containing telomere sequence in an Arabidopsis bacterial artificial chromosome （BAC）. Fluorescence in situ hybridization （FISH） with cotton metaphase chromosomes using the Arabidopsis-type telomere sequence as probes indicated that the signals were located at all chromosome ends of seven diploid and two tetraploid cotton species with different signal intensities among chromosome complements of different cotton species, even between long and short arms of the same chromosome. To identify the signals of FISH, the genome DNA of Xinhai 7, a cultivar of Gossypium barbadense, digested by BAL-31 nuclease was introduced in this study. The result of BAL-31 digestion indicated that the hybridization signals of FISH represent the outermost DNA sequence of each cotton chromosomes. So we first proved that the telomeric repeats of cotton cross-hybridize with that of Arabidopsis. The results of terminal restriction fragment （TRF） showed significant variation in telomere length among cotton species. The telomere length of cultivated cotton was close to 20 kb and was larger than those of wild cotton species whose telomere length rahged from 6 to 20 kb.