Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

The location of repeated DNA sequences in the chromosomes of Chironomus tentans

Polytene chromosomes of Chironomus tentans were hybridized in situ with in vivo labelled nuclear and chromosomal RNA. Nuclear RNA formed hybrids preferentially in five distinct regions considered to contain clustered, repeated DNA sequences. These are the two nucleolar organizer regions, Balbiani ring 1 and 2, and the 5 S RNA genes in region 2A of chromosome II, which together comprised almost 70% of the total number of grains over the complement. The remaining grains were diffusely distributed over the chromosomes. There was a significant difference in the distribution of grains when RNA from different chromosomes was used for hybridization. Chromosome I RNA hybridized preferentially with chromosome I, and chromosome II+III RNA preferentially with chromosome II+III. Some regions within the chromosomes hybridized significantly more chromosomal RNA than other regions. A considerable cross-hybridization of RNA from one particular type of chromosome with the other chromosomes was also found. It is concluded that repeated DNA sequences which hybridize with heterogeneous chromosomal RNA in C. tentans are widely dispersed in the genome. Some of these sequences have a delimited localization, others are dispersed, and some sequences which are transcribed in one particular chromosome are present also in the other chromosomes.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

The number and location of genes for 5S ribonucleic acid within the genome of Drosophila melanogaster

DNA was prepared from wild-type and two mutant stocks of Drosophila melanogaster that differed in their dosage of the nucleolar organizer region. The relative amounts of DNA from the nucleolar organizer region in these preparations of DNA were determined by hybridization with (3)H-labelled 28S rRNA. As expected, the amount of (3)H-labelled 28S rRNA that hybridized was directly related to the dosage of nucleolar organizer region. No positive correlation was observed between the amount of (3)H-labelled 5S RNA that hybridized and the dosage of nucleolar organizer region. Thus genes for 5S RNA are located primarily, if not exclusively, outside the nucleolar organizer region. The haploid genome of the wild-type D. melanogaster used in this work has 106 genes for 28S rRNA and 96-105 genes for 5S RNA.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Placement of genes for ribosomal RNA within the nucleolar organizing body of Zea mays

A maize strain that carried a reciprocal translocation between chromosome 6 and a B chromosome (TB6a) was used in this study. The break in chromosome 6 transected the nucleolar organizing body at approximately the cytological midpoint, and the break in the B was one third the distance from its distal end. As a result both chromosomes 6-B and B-6 contained a portion of the nucleolar organizing body. Because of nondisjunction of chromosome B-6 at the second microspore division after meiosis, crosses between plants carrying six to eight of these chromosomes and homozygous for chromosome 6-B, produced progeny that had between one and about nine chromosomes B-6. Thus a quantitative series of nucleolar organizing body fragments was produced. Molecular hybridization experiments with ribosomal-RNA and DNAs extracted from these plants revealed 1) that genes coding for rRNA were located in the nucleolar organizer fragments on either side of the original translocation breakpoint and 2) that with each additional nucleolar organizer fragment provided by the chromosomes B-6, there is a proportional increase in ribosomal-DNA content. The most important conclusion to be derived from these studies is that the vast majority, if not all, of the ribosomal-RNA genes are unambiguously located within the nucleolar organizing body [with possibly a small percentage of them in the adjacent achromatic gap (Givens and Phillips, 1973, abstract)]. This placement is consistent with that of Givens and Phillips who used a quite different cytogenetic approach. The preciseness of previous determinations in Drosophila, and Xenopus allowed their placement only to the region of the nucleolar organizer. This study showed no evidence for a disproportionate replication of rDNA as a function of different amounts of nucleolar organizing material.Abbreviations rDNA ribosomal-DNA - rRNA ribosomal-RNA - NO nucleolar organizer     

Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations

Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function.     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Objectives: Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo‐PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. Materials and methods: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere‐specific PNA probe conjugated with Cy5 fluorochrome, Cy5‐OO‐(CCCTAA)₃. After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Results: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo‐PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. Conclusion: According to our experience, oligo‐PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.     

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Karyotypic characterization and constitutive heterochromatin in the grasshopper Stiphra robusta (Orthoptera proscopiidae)

Conventional analysis, C-banding, silver nitrate and base specific fluorochrome staining with chromomycin A3 (CMA3) were used to analyse the meiotic chromosomes of the grasshopper Stiphra robusta. Diploid numbers of 2n = 19 in the males and 2n = 20 in the females were observed. The chromosome complement comprised a graded series of uniarmed chromosomes, and the X chromosome was medium sized. The nucleolar organizer regions, restricted to the bivalent chromosomes 6, 7 and 8, were CMA3 positive.     

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.     

Improved in situ hybridization and G-banding by pretreatment with Denhardt''s solution and gelatin-chrome alum

Summary Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity duringin situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline—citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 x SSC for 2 min at 70° C. A³H-labelled, cloned DNA fragment of the highly, repetitive AGM component DNA was hybridized to the chromosomes by incubation at 45° C for 16 h. Treatment with gelatinchrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 x Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin afterin situ hybridization.     

Continuous occurrence of intra-individual chromosome rearrangements in the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Analysis of the holocentric mitotic chromosomes of the peach-potato aphid, Myzus persicae (Sulzer), from clones labelled 50, 51 and 70 revealed different chromosome numbers, ranging from 12 to 14, even within each embryo, in contrast to the standard karyotype of this species (2n?=?12). Chromosome length measurements, combined with fluorescent in situ hybridization experiments, showed that the observed chromosomal mosaicisms are due to recurrent fragmentations of chromosomes X, 1 and 3. Contrary to what has generally been reported in the literature, X chromosomes were frequently involved in recurrent fragmentations, in particular at their telomeric ends opposite to the nucleolar organizer region. Supernumerary B chromosomes have been also observed in clones 50 and 51. The three aphid clones showed recurrent fissions of the same chromosomes in the same regions, thereby suggesting that the M. persicae genome has fragile sites that are at the basis of the observed changes in chromosome number. Experiments to induce males also revealed that M. persicae clones 50, 51 and 70 are obligately parthenogenetic, arguing that the reproduction by apomictic parthenogenesis favoured the stabilization and inheritance of the observed chromosomal fragments.