Direct labelling of BAC-DNA by rolling-circle amplification

Efficient amplification and labelling of probes are crucial for successful sequence detection by fluorescent in situ hybridization (FISH). In particular, chromosome painting to visualize chromosome segments or entire chromosomes by FISH requires large amounts of probes derived from extended templates. There are a number of techniques for probe labelling. The most widespread is nick translation, based on the replicational incorporation of modified nucleotides. Here we demonstrate successful rolling-circle amplification (RCA) of very low amounts of long circular template sequences (single bacterial artificial chromosomes (BACs) or pools of BACs). The amplicons were suitable for labelling by nick translation and subsequent FISH. A novel achievement is the use of RCA for simultaneous amplification and labelling of single BACs or BAC pools in a labour- and cost-effective manner.     

The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidiumparvum oocysts

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0·998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum -specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.     

Oligonucleotides, part 5+: synthesis and fluorescence studies of DNA oligomers d(AT)5 containing adenines covalently linked at C-8 with dansyl fluorophore.

The synthesis of oligodeoxynucleotides d(AT)5 in which specific adenines are linked at C-8 position with dansyl fluorophores via a variable polymethylene spacer chain are reported. This was achieved by a strategy involving prelabelling at the monomeric stage followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled oligonucleotides. Several mono and polydansyl d(AT)5 derivatives in which the fluorophore is linked via ethylene, tetramethylene and hexamethylene spacer arms were synthesised for a systematic study of their fluorescence characteristics. It was observed that (i) enhancements in fluorescence intensity and emission quantum yields are seen due to multiple labelling, (ii) the magnitude of enhancements are related to labelling configuration and (iii) quenching efficiency is minimal with shorter and rigid spacer arms. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.     

Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

Karyotype,chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n = 20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

Number and sites of rDNA loci of Guizotia abyssinica (L. f.) Cass. as determined by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on somatic chromosome preparations of Guizotia abyssinica using a DNA probe of the 18S-5.8S-26S rRNA genes including the transcribed and non-transcribed spacer sequences. A maximum of six major and two minor signal sites of rDNA was observed. However, the minor signals were not persistently detected. The positions of the FISH signals coincided with the sites of the nucleolar organizer regions and their adjacent C-banded heterochromatin when present.     

Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana

A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.     

Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a sensitive technique for molecular diagnosis of chromosomes on single cells and can be applied to sex determination of embryos. The objective has been to develop an accurate and reliable bovine Y chromosome-specific DNA probe in order to sex biopsed blastomeres derived from IVF bovine embryos by FISH. Bovine Y chromosome-specific PCR product derived from BtY2 sequences was labeled with biotin-16-dUTP (BtY2-L1 probe), and FISH was performed on karyoplasts of biopsed blastomeres and matched demi-embryos. Our FISH signal was clearly detected in nuclei of blastomeres of male embryos. FISH analysis of bovine embryos gave high reliability (96%) between biopsied blastomeres and matched demi-embryos. These results indicated that the BtY2-L1 bovine Y chromosome-specific FISH probe was an effective probe for bovine embryo sexing, and the FISH technique of probe detection could improve the efficiency and reliability.     

Localization of DNA probes for human ribosomal genes on barley chromosomes]

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

Phenology of cryptomonads and the CRY1 lineage in a coastal brackish lagoon (Vistula Lagoon,Baltic Sea)

Cryptomonadales have acquired their plastids by secondary endosymbiosis. A novel clade—CRY1—has been discovered at the base of the Cryptomonadales tree, but it remains unknown whether it contains plastids. Cryptomonadales are also an important component of phytoplankton assemblages. However, they cannot be readily identified in fixed samples, and knowledge on dynamics and distribution of specific taxa is scarce. We investigated the phenology of the CRY1 lineage, three cryptomonadales clades and a species Proteomonas sulcata in a brackish lagoon of the Baltic Sea (salinity 0.3–3.9) using fluorescence in situ hybridization. A newly design probe revealed that specimens of the CRY1 lineage were aplastidic. This adds evidence against the chromalveolate hypothesis, and suggests that the evolution of cryptomonadales’ plastids might have been shorter than is currently assumed. The CRY1 lineage was the most abundant cryptomonad clade in the lagoon. All of the studied cryptomonads peaked in spring at the most freshwater station, except for P. sulcata that peaked in summer and autumn. Salinity and concentration of dissolved inorganic nitrogen most significantly affected their distribution and dynamics. Our findings contribute to the ecology and evolution of cryptomonads, and may advance understanding of evolutionary relationships within the eukaryotic tree of life.     

A combined PRINS-FISH technique for simultaneous localisation of DNA sequences on plant chromosomes

A novel approach for simultaneous localization of two DNA sequences on plant chromosomes is described. The approach is based on a combined use of primed in situ DNA labelling (PRINS) with fluorescent in situ hybridization (FISH). Traditionally, this has been done using FISH with two probes labelled by two different marker molecules. Compared to this method, the combined PRINS-FISH procedure is faster. Furthermore, because one of the DNA sequences is localized by PRINS with specific primers, only one labelled probe is needed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of a chromosomal rearrangement responsible for producing "apparent" XY-female fall-run Chinook salmon in California

Fluorescence in situ hybridization (FISH) was used to identify the X and Y chromosomes of offspring produced by normal and "apparent" XY-female fall-run Chinook salmon (Oncorhynchus tshawytscha) from California. FISH experiments were performed using probes to 2 sex-linked loci, growth hormone pseudogene (GH-Psi), and OtY1, as well as a probe to a sex-linked microsatellite (Omy7INRA). Comparison of FISH staining patterns between the offspring produced by normal and apparent XY-females revealed that the apparent XY-female examined transmitted a "Y-like" chromosome with an attenuated OtY1 and GH-Psi signal to half of its offspring. Segregation analysis of microsatellites derived from rainbow trout (Oncorhynchus mykiss) with respect to phenotypic sex was carried out for 2 normal and 2 apparent XY-female Chinook salmon families. Inheritance patterns of Omy7INRA were consistent with this locus being closely linked to GH-Psi in males and in apparent XY-females carrying the Y-like chromosome. Another microsatellite locus (Omm1077) was closely linked to the primary sex-determining locus (SEX) in males but not to GH-Psi/OtY1 in apparent XY-females. The FISH analyses suggest that apparent XY-female fall-run Chinook salmon in California are not the product of a Y chromosome to autosome translocation. Despite the combined FISH and inheritance analyses, we were unable to differentiate between 2 alternative explanations for apparent XY-females, namely, recombination of markers between the sex chromosomes, or a Y chromosome with a dysfunctional or missing sex-determining region.     

Development of a PNA probe for fluorescence in situ hybridization detection of Prorocentrum donghaiense

Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1-D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.     

Localization of Human Ribosomal Gene DNA Probes on Barley Chromosomes

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S–28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barleyHordeum vulgareL. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 70% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.     

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.     

丙烯酰胺非整倍体诱发效应的荧光原位杂交和CREST染色的研究

本文以小鼠着丝粒次要卫星DNA探针FISH和抗着丝粒CREST染色,研究了可疑的非整倍体毒剂丙烯酰胺(AA)诱导的小鼠NIH3T3细胞微核(MN)的着丝粒组成情况和小鼠骨髓染色体畸变(CA)情况。结果发现AA在100—400μg/ml诱导的MN约52.7％—71.6％为FISH阳性,60.5％—68.2％的MN为CR-EST阳性,两种结果均显示AA具有较强的非整倍体诱发效应。小鼠骨髓CA的FISH表明,AA既能诱导染色体结构畸变,又能诱导非整倍体形成,而以非整倍体诱发效应更为明显。     

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.