Chromosomes of Sequoia sempervirens; 8-Hydroxy-Quinoline-Castor Oil Pretreatment for Improving Preparation

Living root tips of coast redwood were pretreated for 6 hr at 10-14 C in a homogenized mixture of 0.003 M aqueous solution of about 5 ml of 8-hydroxyquinoline and a drop of castor oil. They were rinsed 2-3 times in Carnoy's alcohol-chloroform-acetic acid (3:2:1), and fixation allowed to continue in this fluid for 1 hr at 60 C. They were then hydrolysed in 1 N HC1 at 60 C for 45 min; washed with distilled water, and squashed in a drop of aceto-carmine or aceto-orcein to make a temporary slide, subsequently made permanent by a quick-freezing method. Our work to date seems to confirm 2n = 66 for S. sempervirens.     

Staining Air Dried Protoplasts for Study of Plant Chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poorly stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

流式细胞术分析和分拣植物染色体

流式细胞术是当染色体、细胞核和细胞等颗粒随着流动的液体（水或缓冲液）通过一个测量点时,被探测器探测到,这样根据颗粒的物理和化学特征而将不同的颗粒分开并计数分拣的技术。流式细胞分析在人类基因组计划中发挥了重要作用,流式细胞技术的应用也适用于植物,目前这个技术应用范围包括流式核型分析,分拣纯化染色体,定位基因,构建文库等。文章综述了流式细胞术在植物基因组分析方面的研究进展。     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

A Technique for C-Banding Plant Chromosomes with Wright Stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Studies on the Silver-Staining of Plant Chromosomes

In the rye, barley and wheat, besides the Ag-NORs, some other Ag-stained regions including the telomeres, the centromeres and the middle of arms have been found. The phenomenon that the rye and barley have their own Ag-staining pattern is first described in this paper.     

Studies on G-bands and Macrocoils in Plant Chromosomes

Four different methods including trypsin urea, SDS and NaOH are presented for the in situ induction of G-bands and macrocoils on the chromosomes of Secale cereale, Hordeum vulgare and Vicia faba. The bands obtained were numerous and along the whole chromosome, the number of the G-bands was much interrelated with the condensation of chromosomes. The bands of homologous chromosomes in some cells were matchable. The G-banded chromosomes in late prophase have nearly reached high resolution level. When incubation periods were beyond critical time for G-banding, macrocoils were often revealed. Gyre number changed with chromosome condensation and the direction of coils has showed different patterns. Transformation of G-bands into macrocoils was first reported in plant chromosomes. Some chromosomes showing G-bands under light microscope appeared spiral patterns under scanning electron microscope. In this paper the relationship between G-bands and macrocoils in plant chromosomes is also discussed.     

An Enzymic Method of Preparing Plant Chromosomes for In Situ Hybridization

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily G-banded.     

Interaction of 8-Hydroxyquinoline with Soil Environment Mediates Its Ecological Function

Background

Allelopathic functions of plant-released chemicals are often studied through growth bioassays assuming that these chemicals will directly impact plant growth. This overlooks the role of soil factors in mediating allelopathic activities of chemicals, particularly non-volatiles. Here we examined the allelopathic potential of 8-hydroxyquinoline (HQ), a chemical reported to be exuded from the roots of Centaurea diffusa.

Methodology/Principal Findings

Growth bioassays and HQ recovery experiments were performed in HQ-treated soils (non-sterile, sterile, organic matter-enriched and glucose-amended) and untreated control soil. Root growth of either Brassica campestris or Phalaris minor was not affected in HQ-treated non-sterile soil. Soil modifications (organic matter and glucose amendments) could not enhance the recovery of HQ in soil, which further supports the observation that HQ is not likely to be an allelopathic compound. Hydroxyquinoline-treated soil had lower values for the CO₂ release compared to untreated non-sterile soil. Soil sterilization significantly influenced the organic matter content, PO₄-P and total organic nitrogen levels.

Conclusion/Significance

Here, we concluded that evaluation of the effect of a chemical on plant growth is not enough in evaluating the ecological role of a chemical in plant-plant interactions. Interaction of the chemical with soil factors largely determines the impact of HQ on plant growth.     

Staining Plant and Animal Chromosomes by the Feulgen-Acetocarmine Sequence

A technic, some fundamentals of which were first worked out on brome grass, has been considerably extended and adapted to the somatic chromosomes of salmon. Fresh salmon eggs were quickly pierced in 45% acetic acid and fixed therein for 4 minutes. The eggs were then placed in N HCl at 60°C. for 8 minutes and thereafter transferred to Feulgen stain for 30 to 45 minutes. Subsequently, each stained embryo was dissected out and divided in two, each half being placed on a slide in a drop of acetocarmine stain. The pieces were well macerated and, after covering with a cover slip, maceration was completed by tapping. Heavy pressure was gradually applied to the cover slip in order to flatten the chromosome complements. A square screw-type laboratory hose clamp was then used to maintain this pressure while a liquid gelatin seal was applied around the edges. The slide, with the clamp on, was placed in the refrigerator overnight. Before the slide was scanned, the clamp was removed permanently. After each scanning period the slide was returned to the refrigerator. Photomicrographs of well-spread chromosomes in one optical plane were enlarged and tracings made from them. These tracings together with the photomicrographs were used for chromosome analysis.     

Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

原发性肝癌染色体8p、16q遗传变异的研究

目的：分析人肝癌（HCC）组织中染色体8p、16q部分基因及染色体片段的遗传变异及与临床病理关系,初步筛选HCC相关的抑癌基因。方法：应用聚合酶链反应-变性聚丙烯酰胺凝胶-银染法分析45例HCC组织标本中染色体8p和16q的杂合性丢失（LOH）及微卫星不稳定性（MSI）。结果：发生LOH的总频率为68.89%（31/45）,其中D16S511位点的发生LOH率最高为53.33%（24/45）,其次是D8S261（39.02%,16/41）和D8S499（34.88%,15/43）。MSI出现的总频率为11.11%（5/45）,出现在三个微卫星位点（D8S261、D8S499及D16S511）上。结论：染色体16q23、8p22-21.3及8p12区域的LOH发生频率高,其可能存在与HCC发生发展相关的新的抑癌基因,特定位点的遗传变异可能与HBV感染、临床病理恶性程度等预后因素相关。     

原发性肝癌染色体8p、16q 遗传变异的研究

目的:分析人肝癌(HCC)组织中染色体8p、16q部分基因及染色体片段的遗传变异及与临床病理关系,初步筛选HCC相关的抑癌基因。方法:应用聚合酶链反应-变性聚丙烯酰胺凝胶-银染法分析45例HCC组织标本中染色体8p和16q的杂合性丢失(LOH)及微卫星不稳定性(MSI)。结果:发生LOH的总频率为68.89%(31/45),其中D16S511位点的发生LOH率最高为53.33%(24/45),其次是D8S261(39.02%,16/41)和D8S499(34.88%,15/43)。MSI出现的总频率为11.11%(5/45),出现在三个微卫星位点(D8S261、D8S499及D16S511)上。结论:染色体16q23、8p22-21.3及8p12区域的LOH发生频率高,其可能存在与HCC发生发展相关的新的抑癌基因,特定位点的遗传变异可能与HBV感染、临床病理恶性程度等预后因素相关。     

An Improved Method for the Study of Chromosomes

It has been felt strongly that the current methods for the fixation of chromosomes are inadequate, inasmuch as the reagents are unduly long in reaching the important elements of the cells, particularly the nucleus. A realization of this situation has been at the bottom of the development of the so-called smear method. This method can, however, be advantageously employed only where loose cells are available, as in the reproductive elements of plants and animals. The present author has found that cutting up the material in very thin slices of almost microscopical tenuity, and passing them immediately into the best fixing solutions, namely those containing osmic acid, gives excellent results. The material is then assembled on cards in accordance with the present author's mass method (for which see the original article), and after embedding in nitrocellulose is sectioned 5μ or even thinner. Practically all methods of staining may be used, but the most satisfactory results have been achieved with Heidenhain's hematoxylin bleached almost to disappearance and followed by prolonged treatment with an aqueous safranin. This differentiates the chromosomal structures extremely well. It is clear by this method that chromosomes in all cases consist of a ground substance in which are situated two chromatids. In the somatic tissues these chromatids are apparently generally spiral and run in opposite directions. The crossing points of these spirals are responsible for the optical illusion which has been designated the gene.