Well-spread Chromosomes Research Articles

Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.

Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.

Preparation of Male Meiotic Chromosomes for Fluorescence In Situ Hybridization and Immunodetection with Major Focus on Dogroses.

Meiosis is a very essential cell division resulting in the formation of four haploid gametes in plants. The preparation of meiotic chromosomes is a key step in plant meiotic research. Well-spread chromosomes, low background signal, and effective cell wall elimination give the best hybridization results. Dogroses (Rosa, section Caninae) are allopolyploids and frequently pentaploids (2n=5x=35) with asymmetrical meiosis. Their cytoplasm is enriched with organic compounds such as vitamins, tannins, phenols, essential oils, and many more. The cytoplasm is often a huge problem, avoiding successful cytogenetic experiments using fluorescence staining techniques. Here, we present a protocol with modifications for the preparation of male meiotic chromosomes suitable for fluorescence in situ hybridization (FISH) and immunolabeling with a major focus on dogroses.

Quantification of Chromosomal Aberrations in Mammalian Cells.

Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.

Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations.

Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest.

Chromosome Microdissection on Semi-Archived Material.

Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Optimization of the cytogenetic protocol for Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822).

To obtain well spread chromosomes, the cytogenetic protocol for Pangasianodon hypophthalmus and Clarias gariepinus were optimized. This includes, the colchicine concentration (0.01%, 0.025%, 0.05%)/exposure duration (1, 3, and 5 h), hypotonic solution (distilled water or 0.075M KCl solution)/exposure duration (30 min, 1, and 2 h), the time of cell suspension preparation (at hypotonic treatment or before slide preparation) and chromosome aging period (0, 3, and 7 days in Carnoy’s fixative). In addition, the type (i.e., fin, gill or kidney) and the amount of tissue (10, 50, 100 or 150 mg) were also investigated. Regardless of the species, the result obtained showed that well-spread chromosomes could be obtained using the following optimized protocol: Juveniles are injected with 0.05% colchicine (at one ml kg−1) and allowed to swim for 3 h. Then, 50 mg of gill tissue is made into cell suspension in 0.075M KCl for 1 h. The cell suspension is treated in Carnoy’s fixative (changed three times at 20 min interval) and then aged for 3 days. Finally, chromosome slides are made and stained with 10% Giemsa for 1 h.

Meiotic analysis and FISH with rDNA and rice BAC probes of the Thai KPS 01-01-25 sugarcane cultivar

The interspecific sugarcane hybrid “KPS 01-01-25” is one of Thailand’s most successful cultivars, but its genetics and genomic constitution are greatly complicated due to the highly polyploid nature of this crop. Here we analyzed the crop’s karyotype, studied chromosome pairing at meiosis I and performed fluorescent in situ hybridization (FISH) of rDNA and rice BACs as probes. Mitotic cell complements in early tapetal cells showed 110 well-spread chromosomes. Pollen mother cells at pachytene clearly demonstrated fully paired configurations of the long chromosomes, in which few pairing partner exchanges and loops could be discerned. In diakinesis cells we observed 74.1 % bivalents, 18.6 % univalents, 2.9 % trivalents and 4.4 % quadrivalents. During this stage secondary associations were abundant, but these threadlike structures disappear after metaphase I. FISH with 5S rDNA produced up to ten fluorescent spots, of which 2–5 are often very small, whereas the 45S rDNA probe showed foci on 8–9 chromosomes. Based on our results and that of related studies we estimate that this sugarcane is composed of 11–12 genomes. We also selected rice BACs containing chromosome 5 genes controlling economically important genes. Eight BACs with relatively strong dot-blot hybridization on genomic sugarcane DNA were selected for FISH on sugarcane chromosome slides. These cross-species chromosome BAC FISH revealed weak signals, and most of the foci were distributed over parts of the chromosomes. Based on these experiments we concluded that cross-species FISH in sugarcane can better be carried out with sorghum BACs as probes.

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.

Cytogenetic mechanism for reversion of triploids to heteroploid mosaics in Crassostrea gigas (Thunberg) and Crassostrea ariakensis

AbstractTriploid oyster induction has been very successful.However, during ¢eld culture, a certain proportionof triploids apparentlyloses one set of chromosomes,‘reverting’to diploid^triploid mosaics.The revertantswill lose theiraquaculture advantages due to the re-covery of reproductive ability. Mitotic chromosomeswere observed in mosaics of Crassostrea gigas andCrassostrea Ariakensis to elucidate the cytogeneticmechanism of reversion. Most individuals showed amodal number of 30 in triploid cells and varyingnumbers of chromosomes in other cells. From all15mosaics of C.gigas and16 mosaics ofC. ariakensis in-dividuals, high proportions of aneuploid cells withvarious numbers of chromosomes were observed.Most of the aneuploid cells were hypotriploid cellswith 26^29 chromosomes. Notably, in 10 out of 15C. gigas and 9 out of16 C. ariakensis ,somecellscon-sisted of 20^26 well-spread chromosomes and 3^10chromosome ‘clumps’. Individuals with more chro-mosome clumps tended to have higher percentagesof hypotriploid cells. The correlation between thesetwo categories of cells was signi¢cant (Po0.05) in C.gigasand highlysigni¢cant ( Po0.01) in C.ariakensis.Thus, we conclude that some chromosomes areeliminated during mitotic divisions because theyclump together and are thus unable to undergo nor-mal segregation.Keywords: triploid oyster, reversion, mechanism,chromosome eliminationIntroductionTriploid induction has become one of the most im-portant and successful genetic breeding techniquesin aquaculture (Thorgaard1983;Thorgaard & AllenJr1986).The use of triploidy is based onthe principlethattriploidsaresterilebecauseoftheoddnumberofchromosome sets (Thorgaard 1983; Allen Jr 1987).There are severaladvantages of sterility.The ¢rst ad-vantagederivesfromthefactthatsteriletriploidscanmaintain higher market quality and often highergrowth rates during the reproductive season, whennormal diploids consume much of their energy onproducing gametes, thereby reducing £esh qualityand the somatic growth rate.The second advantageis that undesirable population proliferation of cul-turedstockscanbecontrolled,reducingtheopportu-nityfor‘geneticpollution’.Thethirdadvantageisthatsterilityaiords the possibilityof biological control ofreproduction, i.e. genetically modi¢ed organisms ornon-native species can be introduced to foreign en-vironments without risktonative species.Triploidoysterhasbeenthemostsuccessfulexam-ple for the application of triploidy in aquaculture(AllenJr1988; AllenJr, Downing & Chew1989). Uptoa few years ago, the primary method for producingtriploid oysters was to inhibit the second polar body(PB2) extrusion of newly fertilized eggs with cyto-chalasin B (CB) (chemical triploids, Allen Jr et al.1989). This method does not produce100% triploidsbecause some of the eggs may escape treatment forvariousreasons.Recently,theproductionof100%tri-

Meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection in Populus spp.

In order to isolate meiotic chromosomes of Populus species meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection were studied. Pollen mother cells at diakinesis of Populus canadensis Moench were used as samples. Two methods were used to prepare meiotic chromosomes: in the first, cell suspensions were dropped on polyethylene-naphthalate or polyester membrane slides which had just been incubated at −20°C; in the second method, cell suspensions were also dropped on polyethylene-naphthalate or polyester membrane slides, but spread with the aid of high temperatures. The cells did not completely spread by the first method and chromosomes at diakinesis could not be individually distinguished. In contrast, well-spread diakinesis chromosomes were obtained by the second method, where chromosomes, connected with their nucleolus, were successfully isolated with the laser micro-dissection system. As well, we discuss the prospect of applications of laser micro-dissection in cytogenetics and molecular genetics in Populus species.

Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora

Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.

A comparative study of karyotypes in two species of Byblis (Byblidaceae)

Byblis filifolia and B. liniflora had the diploidal and the tetraploidal chromosome numbers of 2n=16 and 32, respectively. In condensation behavior from prophase to metaphase, most chromosomes of both species had early condensing segments at the proximal regions. At prometaphase and early-metaphase, all chromosomes except for some small sized chromosomes had decondense segments at the distal regions in both arms. At mid-metaphase, well-spread chromosomes were quite rare to obtain, because all chromosomes at mid-metaphase stage were quite sticky nature, but not earlier stages of metaphase and prophase. The metaphase chromosomes in both species showed a gradual decrease in size from the largest to the smallest. In B. filifolia, total chromosome length at early-metaphase was 40.5 μm, while total chromosome length at mid-metaphase was 23.3 μm. In contrast, B. liniflora showed total chromosome length of 106.1 μm at early-metaphase, and 54.0 μm at mid-metaphase. To compare to the same stage of metaphase, the total length in B. liniflora was nearly twice as long as that of B. filifolia. Two sat-chromosomes were observed in both species.

Chromosome numbers of four Pistacia (Anacardiaceae) species

SummaryThere is little cytological information for Pistacia species in the literature and they are old or require confirmation. This study aims to adapt a chromosome count procedure for Pistacia species and to determine chromosome numbers of four Pistacia species in root tip cells. Chromosome counts were performed for P. vera L., P. terebinthus L., P. atlantica Desf. and in a new species P. eurycarpa Yalt., which have been characterized at the molecular level. Dehulled Pistacia nuts were stratified and treated with 500 ppm GA3 and then germinated on plates. Growing root tips were used for counting at c-metaphase of mitosis cells. A good chromosome counting procedure was established for Pistacia species that provided well-spread and well-stained chromosome at metaphase of mitosis. Chromosome numbers of all four Pistacia species were found to be 2n = 30. Chromosome numbers of P. atlantica and P. eurycarpa (syn. P. atlantica subsp. kurdica) species are reported here for the first time as 2n = 30. This study provides important information on pistachio cultivars/rootstocks for breeders and cytogeneticists.

Somatic pairing of homologs in budding yeast: existence and modulation.

FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity. Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase. Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing. S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants. Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole). Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure. Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

A New Procedure to Prepare Slides of Metaphase Chromosomes of Roses

To our knowledge, there has been no published technique to produce consistently high-quality slides of somatic chromosomes of roses (Rosa sp.). Therefore, various pretreatments, fixatives, digestions, stains, and maceration and squashing methods were tested to identify a procedure to produce clear, well-spread chromosomes from shoot tips. The best results were obtained after pretreatment in a mixture of 0.1% colchicine and 0.001 m 8-hydroxyquinoline for 4 h, and fixation in 2 acetone: 1 acetic acid (v/v) with 2% (w/v) polyvinylpyrrolidone. The darkest-stained chromosomes were obtained with carbol-fuchsin staining of air-dried cell suspensions that had been spread in 3 ethanol: 1 acetic acid (v/v).

Use of acetylcholine (Ach) for spreading metaphase chromosomes and application to the cytogenetic analysis of human stomach cancers

Cytogenetic studies of biopsy specimens endoscopically obtained from gastric cancers were performed in our laboratory. The necessity for well-spread chromosomes for analysis has resulted in the development of a new technique, in which culture medium containing acetylcholine (Ach) is used; with this new technique, the number of metaphases of analyzable karyotypes was significantly increased (P < 0.01 compared with a previous method in which Ach was not used). The mean ratio of metaphase numbers of analyzable karyotypes in four cases in which Ach was used was 38.1 +/- 8.1%, a value more than four times the number in the seven cases in which Ach was not used. It is possible that Ach may decrease the viscosity of the cytoplasm in gastric cancer cells by disrupting microfilaments.

Tendrils as an Alternate Tissue Source for Chromosome Visualization

Tendrils were used as an alternate tissue source to determine the ploidy level of transgenic melon (Cucumis melo L.) plants. Young tendril tips were prepared for chromosome visualization using standard root tip preparation techniques. Well-spread chromosome preparations were obtained. Tendrils were found to be as amenable to chromosome visualization as root tips. The transformed melon plants, representing three transgenic melon lines, were tetraploid. The tendril technique also was applied successfully to several other tendril-producing species: cucumber (C. sativus L.), pea (Pisum sativum L.), and grape (Vitis vinifera L.). This technique can be very useful for chromosome visualization in tendril-producing species, and can be especially valuable when root tips or anthers cannot be obtained easily.

The Karyotype of the White-Winged Dove

Chiarelli and Capanna (1973) point out the significance of karyotypes as a species-specific aspect of the phenotype. They also discuss the potential of a species' karyotype to evolve largely independent of the genotype thereby providing an additional suite of characters for karyological analysis. This paper presents the first description of the karyotype of the White-winged Dove (Zenaida asiatica). Karyotypes were prepared and examined from three populations of White-winged Doves in southwestern Texas. These included locations in Apline (Brewster County), Pharr (Hidalgo County), and the Sierra Vieja Mountains (Presidio County). Standard mitotic karyotypes were prepared from bone marrow cells following the techniques of Patton (1967), Hsu and Patton (1969), and Lee (1969). Additional material was prepared from spleen tissue. Chromosomes were subsequently stained in 2% Giemsa solution. Elevation of the mitotic index was accomplished following the yeast technique described by Lee and Elder (1980). Differential staining of nucleolus organizer regions (NORs) of mitotic chromosomes was accomplished following the technique of Howell and Black (1980). Meiotic chromosome spreads, from testicular tissue of reproductively active males, were prepared in much the same manner as the mitotic except that a 1% sodium citrate solution was used for the hypotonic incubation phase following homogenization in several drops of 2.2% sodium citrate solution. Karyotypes of members of the genus Zenaida have a diploid chromosome number range of 2n = 76 (for Z. auriculata) (De Lucca and De Aguiar 1976) to 2n = 78 (for Z. macroura) (Benirschke and Hsu 1971). Analysis of well-spread metaphase chromosome sets from White-winged Doves establishes a diploid chromosome number that ranges from 2n = 76 to 2n = 80 (Fig. 1). This variation was noted within individuals as well as between individuals. The karyotype of the White-winged Dove contains five macrochromosomal autosome pairs that are biarmed with the remainder being acrocentric. These macrochromosome pairs (#1, #2, #4, #5, and the Z sex chromosome) are all classified as submetacentric (more metacentric). The #3 chromosome pair is classified as acrocentric. The remainder of the chromosome pairs and the W sex chromosome were small acrocentric microchromosomes. These designations were made on the basis of centromere indices and follow the terminology ofLevan et al. (1964). The fundamental number for the White-winged Dove ranged from 84 (when 2n = 76) to 88 (when 2n = 80). Haploid chromosome numbers ranged from n = 38 to n = 40. No occurrences of abnormal synapsis were observed. The mitotic chromosomes ranged in mean length from 7.7 tm to 4 tm. Group 2 consists of chromosome pairs #4, #5, and the Z sex chromosome, all of which have a mean

Development of an instrument for chromosome slide preparation.

For cytogenetic analysis of chromosome abnormalities, we have developed an instrument which can produce semi-automatically chromosome slides for microscopic observation. This instrument has automatic slide supplier and slide transfer system. Chromosome slides were obtained by the steam dry method; slides were sequentially exposed to different atmosphere controlled strictly in respect of temperature and humidity. Slides with well-spread chromosomes were obtained constantly in quality and rapidly in speed of 5 slides per minute. Precise controls of room temperature and humidity were attained by the use of 26 thermomodules. Accuracy of the controls was excellently high, showing a fluctuation of +/- 0.5 degrees C in temperature and +/- 3% in humidity for any setting in the use range. This instrument was proved to be very useful for rapid obtaining of a large number of chromosome slides with highly qualified metaphase spreads. This instrument will be utilized to assist the automation analysis system of chromosome abnormalities. Mechanism of chromosome spreading, problems in actual use, and future improvements were discussed.

Cytotype Distribution of Atriplex canescens (Chenopodiaceae) of Southern New Mexico and Adjacent Texas

Cytotype Distribution of Atriplex canescens (Chenopodiaceae) of Southern New Mexico and Adjacent Texas