The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Aberration of X chromosome in liver neoplasm detected by fluorescence in situ hybridization

BACKGROUND: A diverse range of cytogenetic alterations of autosomal chromosomes has been reported to date. However, few studies have addressed the abnormalities of X chromosome in hepatocellular carcinoma (HCC) except sporadic reports on the deletion of band F1 in X chromo- some , and the clonal analysis of methylation pattern of the X chromosome-linked human androgen receptor gene. Identification of specific X chromosome alterations during the course of neoplastic development would be essential to defining the genetic basis of HCC. Therefore, we studied the regularity of aberration of X chromosome in liver canc- er. METHODS: Hepatocarcinoma cellular lines and tumor tis- sues were detected respectively through DNA probes of X chromosome after fluorescence in situ hybridization ( FI- SH). RESULTS: Increased copies of X chromosome were ob- served in all samples, and four signals of hybridization were of the major type. CONCLUSIONS: Increased copy number of X chromo- some frequently occur in liver cancer. The relationship be- tween copy number of X chromosome and liver cancer genesis needs further investigation. This study is the first of its kind determining the copy number of X chromosome in liver cancer by using FISH.

Chromosome 11 aneusomy in esophageal cancers and precancerous lesions-an early event in neoplastic transformation:An interphase fluorescence in situ hybridization study from south India

AIM: To detect aneusomic changes with respect to chromosome 11 copy number in esophageal precancers and cancers wherein the generation of cancer-specific phenotypes is believed to be associated with specific chromosomal aneuploidies. METHODS: We performed fluorescence in situ hybridization (FISH) on esophageal tissue paraffin sections to analyze changes in chromosome 11 copy number using apotome-generated images by optical sectioning microscopy. Sections were prepared from esophageal tumor tissue, tissues showing preneoplastic changes and histologically normal tissues (control) obtained from patients referred to the clinic for endoscopic evaluation. RESULTS: Our results demonstrated that aneusomy was seen in all the cancers and preneoplastic tissues, while none of the controls showed aneusomic cells. There was no increase in aneusomy from precancers to cancers. CONCLUSION: Our results suggest that evaluation of chromosome 11 aneusomy in esophageal tissue using FISH with an appropriate signal capture-analysis system, can be used as an ancillary molecular marker predictive of early neoplastic changes. Future studies can be directed towards the genes on chromosome 11,which may play a role in the neoplastic transformation of esophageal precancerous lesions to cancers.

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%.

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.

In situ aneuploidy assessment in human sperm:the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

Both the primed in situ(PRINS)and the peptide nucleic acid-fluorescence in situ hybridization(PNA-FISH) techniques constitute alternatives to the conventional(fluorescence in situ hybridization,FISH)procedure for chro- mosomal investigations.The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction.Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones.The two procedures present several advantages(specificity,rapidity and discriminating ability)that make them very attractive for cytogenetic purposes.Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.(Asian J Androl 2006 Jul;8:387-392)

Fiber-fluorescence in situ hybridization analyses as a diagnostic application for orientation of microduplications

Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization(FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.

Association of chromosome 7 aneuploidy measured by fluorescence in situ hybridization assay with muscular invasion in bladder cancer

Background:The preoperative prediction of muscular invasion status is important for adequately treating bladder cancer(BC)but nevertheless,there are some existing dilemmas in the current preoperative diagnostic accuracy of BC with muscular invasion.Here,we investigated the potential association between the fluorescence in situ hybridization(FISH)assay and muscular invasion among patients with BC.A cytogenetic-clinical nomogram for the individualized preoperative differentiation of muscle-invasive BC(MIBC)from non-muscle-invasive BC(NMIBC)is also proposed.Methods:All eligible BC patients were preoperatively tested using a FISH assay,which included 4 sites(chromosome-specific centromeric probe[CSP]3,7,and 17,and gene locus-specific probe[GLP]-p16 locus).The correlation between the FISH assay and BC muscular invasion was evaluated using the Chi-square tests.In the training set,univariate and multivariate logistic regression analyses were used to develop a cytogenetic-clinical nomogram for preoperative muscular invasion prediction.Then,we assessed the performance of the nomogram in the training set with respect to its discriminatory accuracy and calibration for predicting muscular invasion,and clinica usefulness,which were then validated in the validation set.Moreover,model comparison was set to evaluate the discrimination and clinical usefulness between the nomogram and the individual variables incorporated in the nomogram.Results:Muscular invasion was more prevalent in BC patients with positive CSP3,CSP7 and CSP17 status(OR[95%CI],2.724[1.555 to 4.774],P<0.001;3.406[1.912 to 6.068],P<0.001 and 2.483[1.436 to 4.292],P=0.001,respectively).Radiologydetermined tumor size,radiology-determined clinical tumor stage and CSP7 status were identified as independent risk factors of BC muscular invasion by the multivariate regression analysis in the training set.Then,a cytogenetic-clinical nomogram incorporating these three independent risk factors was constructed and was observed to have satisfactory discrimination in the training(AUC 0.784;95%CI:0.715 to 0.853)and validation(AUC 0.743;95%CI:0.635 to 0.850)set.The decision curve analysis(DCA)indicated the clinical usefulness of our nomogram.In models comparison,using the receiver operator characteristic(ROC)analyses,the nomogram showed higher discriminatory accuracy than any variables incorporated in the nomogram alone and the DCAs also identified the nomogram as possessing the highest net benefits at wide range of threshold probabilities.Conclusion:CSP7 status was identified as an independent factor for predicting muscular invasion in BC patients and was successfully incorporated in a clinical nomogram combining the results of the FISH assay with clinical risk factors.

Chromosome G-banding in SituHybridization of RFLP Marker umc58 Linked with the Gene hm1 Dictating Helminthosporium carbonum Susceptibility1 in Maize

The technique of simultaneous G banding and in situ hybridization has been developed in plants for the first time.Using this technique.RFLP marker umc58 closely linked with the hm1 gene dictating Helminthosporium carbonum susceptibility1 was localized onto 1L3(chromosome 1,long arm,the third band from the centromere to the end of the arm),5L5 and 9L5.Theresults demonstrated that umc58 was a tripli cated sequence.It was deduced that umc58 probably was in a duplicated region that includes a part of Helminthosporium carbonum susceptibility genes(hm1 and hm2),as the hybridization sites of umc58 in chro mosomes 1 and 9 were those at which the genes localize.The techniques of simultaneous G banding and ISH in plants are discussed.

Chromosomal cryptic insertion of the terminal region and its formative mechanism determined by fluorescence in situ hybridization

OBJECTIVE: To determine the karyotype of a cryptic structural abnormality and explore the mechanism of apparent chromosomal terminal deletion. METHODS: Fluorescence in situ hybridization(FISH) with a whole chromosome 7 painting probe and a 7q subterminal probe (7q36-->qter), prepared by chromosome microdissection technique, was used to analyze a case with a history of spontaneous abortion and 7q terminal deletion detected by conventional G-banding technique. RESULTS: The case was a maternal cryptic insertional translocation between chromosome region 1p32 and 7q32-->q35. The region of chromosome 7q36-->qter was not inserted in to chromosome 1, and the abnormal chromosome 7 was not a terminal deletion but an interstitial deletion. CONCLUSIONS: Chromosome insertion of the terminal region retains its telomere, which is consistent with the concept of a three-break rearrangement. Interstitial deletion may be regarded as another mechanism for terminal deletion in the chromosome banding level. Combined with chromosome microdissection, FISH technique could be a powerful diagnostic tool for detecting chromosome structural abnormalities.

Detection of Embryo Sex Chromosome by Dual Color Fluorescent In-Situ Hybridization

In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual color fluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomal mosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. In conclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient , and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t-1 DNA was extracted from its genomic DNA, labeled with biotin-1 1-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization （FISH） signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both Cot-1 DNA FISH banding patterns and chromosome morphology.

Establishment of a Multi-color Genomic in situ Hybridization Technique to Simultaneously Discriminate the Three Interspecific Hybrid Genomes in Gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.

Identification and transferring of a new Fusarium head blight resistance gene FhbRc2 from Roegneria ciliaris 3ScL chromosome arm into common wheat

Fusarium head blight(FHB)threatens wheat production worldwide.Utilization of FHB resistant varieties is the most effective solution for disease control.Owing to the limited sources of FHB resistance,mining of novel resistance genes is crucial.Here,we report an FHB resistance gene from a wild wheat relative species,Roegneria ciliaris and developed FHB resistant germplasm containing this gene.Wheat-R.ciliaris disomic addition line DA3S^(c) showed enhanced type II FHB resistance compared to its sister line 3S^(c)-Null without chromosome 3S^(c),indicating that the resistance was contributed by the addition of 3S^(c).The resistance gene on 3S^(c) was validated using F2 and F2:3 populations derived from the cross between DA3S^(c) and susceptible Aikang 58(a susceptible cultivar),demonstrating that the lines with 3S^(c) had significantly enhanced FHB resistance compared to the individuals without 3S^(c).This was the second resistance gene identified in R.ciliaris,designated FhbRc2.To transfer FhbRc2 to common wheat,we produced a doublemonosomic chromosome population by crossing DA3S^(c) with the Chinese Spring nulli-tetrasomic line N3DT3B.Eight alien chromosome lines containing 3S^(c) were identified using genomic/fluorescence in situ hybridization and 3S^(c)-specific marker analysis.Only the lines carrying the long arm of 3S^(c) conferred FHB resistance,further locating FhbRc2 on 3S^(c)L.A compensating wheat-R.ciliaris Robertsonian translocation line T3DS·3S^(c)L harboring FhbRc2 is developed and provides a potential genetic resource in wheat breeding for enhanced FHB resistance.

Chromosomal changes detected by fluorescence in situ hybridization in patients with acute lymphoblastic leukemia

Objectives To investigate patients with acute lymphoblastic leukemia (ALL) for TEL/AML1 fusion, BCR/ABL fusion, MLL gene rearrangements, and numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ hybridization (FISH) and to determine the relationship and the significance of those findings.Methods Fifty-one American patients (34 men and 17 women) were included in this study. Of them there were 41 patients with pro-B cell type ALL, 9 with B cell type ALL and 1 with T cell type ALL. Chromosome metaphases of each sample were prepared according to standard protocols. Fluorescence in situ hybridization was performed using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, specific chromosome centromere probes and dual color/multiple color translocation fusion probes. The digital image analysis was carried out using Cytovision and Quips FISH programs.Results An overall incidence of chromosomal anomalies, including t (9; 22), MLL gene rearrangements, t (12;21), and numerical chromosomal anomalies of chromosomes 4, 10, 17 and 21 was found in 33 patients (65%). Thirty-one of them were pediatric patients and two adults. The t (12;21) was the commonest chromosomal anomaly detected in this population; 14 out of the 45 pediatric patients (31%) were positive for TEL/AML1 fusion, among which three had an additional derivative 21 [t (12;21) ], four had a deletion of 12p and two had an extra copy of chromosome 21. All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping. The percentage of cells with fusion signals ranged from 20% to 80%. All fourteen patients positive for TEL/AML1 gene fusion were mosaic. Three out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. All pediatric patients with pre-T or T cell lineage and the six adults were negative for TEL/AML1 fusion. One patient had double Philadelphia chromosomes, three had a rearrangement or a deletion of the MLL gene, one had t (4;11) and two had a deletion of the MLL. One of the patients with an MLL deletion also had a large ring of chromosome 21, and r (21) was caused by AML1 gene tandemly duplicated at least five times. The second case with the MLL deletion was also unique, the patient had at (12;21) as well. A total of 20 patients had numerical changes (gain or loss) of chromosomes 4,10,17 and 21. Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ ABL or the MLL gene rearrangement; one did have the TEL/AML1 gene fusion. Eleven patients with pro-B cell or 8 cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21), among them, 10 patients had no structural alteration of chromosome 21, and one was combined by t (12; 21). Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1. Conclusions FISH plays an important role in detecting chromosome changes, especially in some cryptic chromosome translocations and patients with culture failures. This study found a trend towards a division between patients who had structural changes such as t (12;21) or a ring chromosome 21 and those who had numerical changes of chromosome 21 as well as the patients with TEL/AML1 fusion and patients with the coexistence of numerical chromosomal changes of chromosomes 4, 10 and 17. In our opinion there are two separate mechanisms which lead to the development or progression of leukemia.

Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5±1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.

Chromosomal in situ hybridization of Cyprinus carpio genomic DNA repetitive sequence CR1

Common carp Cyprinus carpio genomic DNA repetitive sequence CR1 has been DIG-labeled and hybridized in situ against chromosomes of red common carp ( Cyprinus carpio L. Xingguo red var.). It is found that the repetitive sequence CR1 is mainly localized at the cen-tromeric regions of chromosomes of the red common carp. The application of the

Isolation and Chromosomal Mapping of a Corn B Chromosome Specific RAPDs

B染色体存在于多种动植物中 ,具有很多独特的性状。B染色体与正常染色体在DNA组成方面十分相似 ,寻找B染色体特异序列一直是B染色体研究的难点和热点。通过对含有和不含有B染色体的两种玉米 (ZeamaysL .)基因组进行了RAPD分析 ,筛选到一个B染色体特异性分子标记B480。该标记与玉米的自主复制起始序列ARS1和ARS2同源 ,特别是该序列中的 2 5bp出现在多种模式生物基因组中。FISH的结果显示 。

Meiotic Behavior of 1BL/1RS Translocation Chromosome and Alien Chromosome in Two Tri-genera Hybrids

The behavior of wheat-rye translocation chromosome and alien chromosome including Thinopyrum and Haynaldia chromosome at meiosis was investigated in two hybrids by fluorescence in situ hybridization (FISH). Misdivision of translocation chromosome at anaphase I and rye chromatin micronucleus at tetrad stage were observed, A plant with one normal 1BL/1RS translocation chromosome and one 1BL/1RS translocation chromosome deleted about 1/3 of rye chromosome arm in length was identified. One plant with wheat-Thinopyrum non-Robertson translocation chromosome was also detected in the F-2 population of Yi4212 x Yi4095. That could be the results of unequal misdivision of wheat-rye 1BL/1RS translocation chromosome and Thinopyrum chromosome during meiosis. No interaction between translocation chromosome and alien chromosome at meiosis was supported by the data of the distribution frequencies of translocation chromosome and Thinopyrum or Haynaldia chromosome in the progeny of two hybrids. The results may be useful to cultivate new germplasms with different length of rye 1R short arm and wheat-alien non-Robertson translocation tines under wheat background.