Actinomycin Deffects on Termite Chromosomes: Induction of High Resolution Banding Patterns with silver Staining in Mitotic Stages

The treatment of termite male spermatogonia with actinomycin D induces highly elongated and finely banded late prophase and prometaphase chromosomes as evidenced by the silver staining method. Actinomycin D suppresses the silver staining of nucleolar organizing regions in prometaphase and reduces it in metaphase chromosomes.     

Differentiation of interphase nucleolus organizers in embryonic pig kidney cells (PK cell line)

The prometaphase karyotype of cell line PK contains two heteromorphous pairs of nucleolus organizers that belong to chromosomes 8a and 8, and to 10L and 10s. It was proposed that such heteromorphism may promote chromosome differentiating of interphase nucleolus organizers (INOs) with linear configuration. To test this assumption, we used two-dimensional (2D) preparations of methanol fixed PK cells surface stretched without hypotonic treatment. It was shown that in these preparations the large bulk of interphase PK cells contained 3-4 necklace-like linear structures arranged in nucleolar domains. The observed structures were positive in phase contrast and after DAPI-staining. Complimentary rDNA-FISH revealed that these structures were INOs, the largest iNO in individual cells containing prominent terminal rDNA FISH/DAPI signal. In accordance with the data on prometaphase analysis, the latter INOs belong to chromosomes 8a. As reported by Smetana and coworkers (1999), proteins of the nucleolar fibrillar center reacted preferentially with silver in methanol fixed unwashed smears of human peripheral lymphocytes. It was established that the same specific silver reaction is characteristic most probably of 2D preparations of methanol fixed PK cells. Both silver stained and rDNA-FISH linearized INOs had necklace-like or banded structure with different degrees of resolution. Banded INOs consisted of transverse argyrophilic structures: dense bands and loose interbands. High resolved banded INOs revealed a longitudinal splitting (binemic structure) of interband zone. Necklace-like INOs consisted of argyrophilic beads nearly two-fold more narrow than argyrophilic bands, and uninemic or silver-negative interbead zones. Our findings evidence that necklace-like INOs are typical for G1 and S phase cells, whereas banded INOs are characteristic of G2 cells. Among high resolved linear INOs, we found four reproducible patterns of silver staining, which could be combined it two homologous groups. Because each given pattern is unique for individual PK cells, we concluded that the patterns under study were chromosome specific. Using prometaphase analysis data, we determined chromosome affiliation for each of the four tested patterns of INO silver staining. High resolved INOs, belonging to different chromosomes, were further compared with regard to their average length and the mean of argyrophilic bead number per individual INO, in addition to the length and argyrophilic bead number ratios calculated for different INO pairs of individual cells. Surprisingly, we found that both the ratios, detected for most heteromorphous pair of homologous chromosomes 8a and 8, made only 1.26 +/- 0.02. In comparison, the similar length ratio for nucleolus organizers in chromosomes 8a and 8, calculated for individual prometaphase cells, reached 2.92 +/- 0.30.     

玉米rRNA基因的组织和表达模式

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号：荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。     

Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes

The present study provides specific cytogenetic information on prometaphase chromosomes of the alpaca (Lama pacos, fam. Camelidae, 2n = 74) that forms a basis for future work on karyotype standardization and gene mapping of the species, as well as for comparative studies and future genetic improvement programs within the family Camelidae. Based on the centromeric index (CI) measurements, alpaca chromosomes have been classified into four groups: group A, subtelocentrics, from pair 1 to 10; group B, telocentrics, from pair 11 to 20; group C, submetacentrics, from pair 21 to 29; group D, metacentrics, from pair 30 to 36 plus sex chromosomes. For each chromosome pair, the following data are provided: relative chromosome length, centromeric index, conventional Giemsa staining, sequential QFQ/C-banding, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization. The overall number of RBG-bands revealed was 391. Nucleolus organizer-bearing chromosomes were identified as pairs 6, 28, 31, 32, 33 and 34. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X and Y painting probes was also carried out to validate X-Y chromosome identification of alpaca and to confirm close homologies between the sex chromosomes of these two species.     

玉米rRNA基因的组织和表达模式

玉米(Zea mays)只有1对45S rDNA位点并在分裂期染色体形成次缢痕, 是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交(fluorescence in situ hybridization, FISH)、CPD(PI与DAPI组合)染色和银染技术, 分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号: 荧光强烈地位于核仁周边的纽, 而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出; 点的数目越多, 纽变得越小; 点的数目多少与细胞的活性呈正相关。研究结果表明, 纽代表了处于凝缩状态的非活性的rDNA染色质, 纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现; 不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示, 大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示, 同一间期细胞的2个同源rDNA位点的表达水平存在差异, 同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示, 早中期细胞的rDNA染色质相对解凝缩, 银染在所有早中期细胞和部分中期细胞显示了明显的核仁, 表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录, 其转录在晚中期才停止。     

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xy(p) type in D. nisus and D. semisquamosus and of the Xy (r) type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.     

Silver staining of human acrocentrics in metaphase does not reflect an induced inhibition in NOR activity

Cytological staining with silver nitrate was used in order to study the activity of the nucleolar organizer regions (NORs) in metaphase figures from human lymphocytes exposed to mercury chloride and actinomycin D. The cells were exposed to both compounds either during G1-early S phase, allowing recovery after the exposure, or from G1 until harvest; no recovery was thus allowed in the latter case. HgCl₂ as well as actinomycin D did not influence the silver staining of the acrocentric chromosomes on metaphases. As actinomycin D is known to be an inhibitor of rRNA, as for example confirmed by inhibition of silver staining on interphase cells, our results on metaphase chromosomes indicate that AgNO₃ precipitation, although being a good indicator for nucleolar activation, is not adequate in case of inactivation.     

Silver staining in clinical cytogenetics

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine whether genetic material has been gained or lost in chromosomal rearrangements, the origin of chromosomal aberrations, the origin of cells in tissue culture, the chromosomal location of single genes, clonal origin of tumors, the zygosity of twins, and paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.     

Silver Staining in Clinical Cytogenetics

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine 1) whether genetic material has been gained or lost in chromosomal rearrangements, 2) the origin of chromosomal aberrations, 3) the origin of cells in tissue culture, 4) the chromosomal location of single genes, 5) clonal origin of tumors, 6) the zygosity of twins, and 7) paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.     

Silver staining of Drosophila polytene chromosomes and the effect of hyaluronidase and lysozyme pretreatment

The Ag-AS technique was used for staining the polytene chromosomes of D. melanogaster and D. lummei. Bands were stained dark reddish-brown, interbands light yellow. A toromere was heavily stained on the sixth chromosome of D. lummei. The staining intensity of nucleoli was lower than that of chromosomes. During a prolonged staining ectopic threads and the nonhomogeneous structure of nucleoli were revealed. Pretreatment with RNase caused slight changes in the silver staining pattern of chromosomes; pretreatment with DNase did not result in any visible changes, while after preincubation with proteolytic enzymes chromosome morphology was destroyed. Hyaluronidase and lysozyme removed the silver-reducing components from chromosomes without destroying the general chromosome structure. Each of these two enzymes acts specifically: hyaluronidase affects the morphology of chromosomes, but not nucleoli and bands at heat shock puffs, whereas the action of lysozyme is probably evenly distributed between chromosomes and nucleoli.     

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.     

Nucleolar organizer activity in wheat,rye and derivatives analyzed by a silver-staining procedure

Nucleolar activity was analyzed in wheat (Triticum sp.), rye (Secale cereale) and several types of wheat-rye derivatives using a modified, highly reproducible, silver staining procedure (Lacadena et al. 1984). A comparative analysis of the nucleolar organizer regions (NORs) of somatic metaphase chromosomes was made by phase contrast, C-banding, and silver staining. The frequency distribution of the number of nucleoli visualized at interphase by silver staining was also used to infer the activity of NORs. The results agree quite well with data from in situ hybridization reported by other authors. The behavior of euploid, ditelosomic and nulli-tetrasomic plants of common wheat showed the relative nucleolar activity of the four organizer chromosomes to be: 6B > 1B > 5D > 1A. — Several types of wheat-rye derivatives were analyzed: interspecific hybrid, triticale, addition and substitution lines, and plants with the genome constitutions, AABBDR, ABDR + 5D, ABRR, and ABRRR. In all cases the nucleolar organizer chromosome 1R of rye was suppressed by the presence of wheat chromosomes.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

Use of the concanavalin-peroxidase-DAB system for identification of human chromosomes]

The effect of concanavalin A on the prometaphase chromosomes was investigated, using a staining reaction based on the peroxydase-diaminobenzidin-H2O2 system. After incubation with concanavalina A, the chromosomes telomeres as well as the centromeres and satellites of the acrocentric chromosomes were strongly stained. Sometimes the chromatids appeared to be coiled. In other respect, it must be noted that peroxydase alone can stain the chromatids, which probably means that this compounds is able to unite with the chromosomes, without the aid of concanavalin A.     

Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.     

Immunofluorescence and inhibitor studies on creatine kinase and mitosis

Immunofluorescence microscopy of mitotiv PtKl cells with antibody against M-line creatine kinase from chicken breast muscle revealed spindle staining largely identical with that of antibody against tubulin. Treatment with epoxycreatine, a creatine analog and covalent creatine kinase inhibitor, allowed the study of creatine kinase function in these non-muscle cells. Cells fixed at interphase following epoxycreatine treatment maintained actin stress fibers and creatine kinase localization on intermediate filaments. Treated prometaphase cells lacked spindles as observed by immunofluorescence microscopy, but displayed instead a central region of diffuse immunofluorescent staining surrounded by condensed chromosomes. Those observed in metaphase or anaphase appeared normal by phase-contrast microscopy, but lacked immunofluorescent staining. Living epoxycreatine-treated cells were observed by phase-contrast microscopy to form spindles and progress through mitosis following an extended prometaphase.     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

Differential silver staining of chromatin in metaphase chromosomes

The silver techniques used to demonstrate nucleolar organizer regions and cores in chromosomes can also differentially stain chromatin within chromosomes. Direct silver staining of mouse and human chromosomes resulted in preferential staining of centromeric regions and non-nucleolar secondary constrictions, both of which are composed of constitutive heterochromatin. After C-banding, these regions were no longer silver-stainable, suggesting that the biochemical constituents (presumably non-histone proteins) which contain the reaction sites for silver are extracted during the banding treatment. Light and electron microscopy of chromosomes G-banded with trypsin and then silver-stained revealed heavier deposits of silver over the condensed aggregates of chromatin within the band regions than over the more dispersed interband chromatin. At the ultrastructural level, chromatin fibres were covered with silver grains, indicating that there are many reaction sites for this metal along the fibres. These results suggest that the degree of silver staining in any region of the chromosome may be contingent upon the concentration of chromatin in that region. This finding may have important implications concerning the nature of the silver-stained core-like structure in chromosomes. If a preferential dispersion of chromatin fibres occurs at the periphery of the chromosome during slide preparation, leaving the central region of each chromatid relatively undispersed, this difference in the concentration of chromatin may account for the differential silver staining of these regions and the consequent appearance of a core-like structure.