Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

Repetitious DNA in some Anemone Species

The DNA from several Anemone species, which contain different amounts of heterochromatin as revealed by Giemsa staining, was analysed by ultra-centrifugation and renaturation. No satellite band was observed in any of the samples centrifuged in cesium chloride gradients. Renaturation studies showed the presence of repetitive sequences. The proportion of repetitive DNA per genome varied from 53% to 67% and did not correlate with either the DNA content per cell or the relative amount of heterochromatin.     

Supernumerary heterochromatic segments associated with the nucleolar chromosomes of Pyrgomorpha conica (Orthoptera) contain methylated rDNA sequences

The two nucleolus organizing chromosome pairs of the grasshopper Pyrgomorpha conica can carry a proximal supernumerary heterochromatic segment. We employed different cytological techniques to characterize and analyze the possible origin of this segment. The supernumerary segment and the nucleolus organizing regions (NORs) show similar responses after C-banding plus either Giemsa or acridine orange, and chromomycin A₃/distamycin A staining to detect GC-rich chromosome regions. Fluorescence in situ hybridization with a biotinylated rDNA probe demonstrated that the segment originated by amplification of the rDNA genes. However, as the silver staining indicates, the ribosomal genes present in the segment are not active since no nucleolus is formed. The use of in situ digestion with the isoschizomeric MspI and HpaII restriction endonucleases and subsequent Giemsa, ethidium bromide or chromomycin A₃/distamycin A staining, suggests that the segment has been inactivated by DNA methylation.     

The histologic detection of Helicobacter pylori in seropositive subjects is affected by pathology and secretory ability of the stomach

Background

Helicobacter pylori is unevenly distributed in hypochlorhydric environments. The study aim was to elucidate the risk factors for a negative Giemsa staining finding in seropositive subjects by measuring the secretory ability of the stomach.

Methods

Subjects aged over 18 years were included consecutively after endoscopic biopsy at gastric lesions with color or structural changes. Blood was sampled for the serum pepsinogen (PG) assay and H. pylori serology test. After excluding the subjects with past H. pylori eradication, the risk factors for a negative Giemsa staining finding in seropositive subjects were analyzed.

Results

Among 872 included subjects, a discrepancy between the serum anti‐H. pylori IgG and Giemsa staining findings was found in 158 (18.1%) subjects, including 145 Giemsa‐negative, seropositive subjects. Gastric adenocarcinoma/adenoma (OR = 11.090, 95% CI = 3.490‐35.236) and low serum PG II level (OR = 0.931, 95% CI = 0.899‐0.963) were the independent risk factors for a negative Giemsa staining finding in seropositive subjects. The cutoff value of serum PG II level was 7.45 ng/mL (area under curve [AUC] = 0.904, 95% CI = 0.881‐0.927). Follow‐up studies of Giemsa staining at different sites of the stomach revealed that 75% of the Giemsa‐negative seropositive subjects with adenocarcinoma are positive, whereas none of those with low serum PG II level of <7.45 ng/mL revealed positive findings.

Conclusions

The risk of a negative Giemsa staining finding in seropositive subjects is increased in gastric adenocarcinoma/adenoma specimens and in subjects with a diminished gastric secretory ability with low serum PG II level of <7.45 ng/mL. A false‐negative Giemsa staining finding is common in subjects with adenocarcinoma, and therefore, additional biopsies at different sites should be performed in these subjects.     

Reverse sister chromatid differential staining by Coomassie Brilliant Blue R-250

A sister chromatid differential staining pattern is observed if chromosomes replicate for two cycles in the presence of 5-bromodeoxyuridine (BUdR) and are subsequently stained in Hoechst 33258, irradiated with black light, and then stained in Coomassie Brilliant Blue R-250. In this pattern the chromatids containing DNA that is bifilarly substituted with BrdUrd are darkly stained and the chromatids with DNA that is unifilarly substituted are lightly stained. This staining pattern is the reverse of that found when slides are stained in Hoechst plus Giemsa. Slides stained with either Giemsa or Coomassie Blue can be destained and restained repeatedly with the other stain to alternate the pattern observed.     

Giemsa C-banding within the genus Glossina (Diptera,Glossinidae)

The Giemsa C-banding staining pattern of members of both the palpalis and morsitans groups of tsetse flies are described. Similarities in the staining reaction of the heterochromatic Y and supernumerary chromosomes within species have been observed which suggests a common origin. The presence of useful markers in all species/sub-species should assist in the recognition of species/sub-species hybrids.     

The role of chromosomal proteins in the induction of a differential staining of sister chromatids by light

Summary Fixed chromosomes of human lymphocytes, cultured in the presence of bromodeoxyuridine (BrdU) during two cell cycles, were exposed to near-ultraviolet irradiation, stained with Giemsa, and after destaining, were subjected to either Coomassie Blue or Feulgen-Schiff staining. A differential reaction of sister chromatids was first revealed by Coomassie Blue staining. Differential staining with Giemsa required a longer irradiation time. This appeared to be reduced after the addition of dithiodipyridine to the cells during their last few hours of culture. The differential pattern obtained after Coomassie Blue staining was the inverse of that obtained after Giemsa staining. From these findings we concluded that the induction of sister chromatid differentiation by light in BrdU-substituted DNA containing chromosomes occurs primarily via chromosomal proteins, presumably by differential breakage of their disulphide bonds. The results of the Feulgen-Schiff staining indicated that differential depurination of BrdU-containing DNA could occur, although only after very prolonged irradiation. A faint though distinctly differential Feulgen-Schiff pattern of sister chromated staining, resulting from differential removal of DNA, was observed after photosensitization by specific DNA-binding dyes. Thus, DNA seems to be affected only under more extreme conditions.     

Differentielle Darstellung der Metaphasechromosomen von Cypripedium debile mit Chinacrin- und Giemsa-Färbung

The somatic metaphase chromosomes of C. debile stain differentially with quinacrine and Giemsa respectively. After quinacrine staining the heterochromatic regions show fainter fluorescence than euchromatic regions. With Giemsa the heterochromatic regions are more deeply stained than the euchromatic regions. This is true also for slides which were pre-treated with trypsin or trichloroacetic acid. From these findings it may be supposed, that the heterochromatic regions are G-C rich. Furthermore, differences in protein composition are expected between hetero- and euchromatic regions. The results of differential staining of metaphase chromosomes of C. debile, including those which were published in previous papers, are summarized here.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

Reciprocal Giemsa staining of late DNA replicating regions produced by low and high pH sodium phosphate

A procedure is described whereby late replicating, BUdR-substituted chromosome regions stain intensely with Giemsa, thus producing the reciprocal staining patterns compared to those obtained by all other BUdR-Giemsa procedures where BUdR-substituted regions appear pale staining. This method may be more convenient than pre-existing techniques for demonstrating late replicating chromosome regions, and may provide a higher degree of resolution of the late replicating regions. The finding that BUdR-substituted regions can be made to stain either intensely or palely with Giemsa, depending on the pH of the pretreatment NaH₂PO₄ solution, may have important implications concerning the mechanism of BUdR-induced chromosome differentiation.     

The genetic control of sporulation in Saccharomyces. II. Dominance and complementation of mutants of meiosis and spore formation

Summary Heat-sensitive sporulation-deficient (spo) mutants ofS. cerevisiae may be either dominant or recessive. The number of loci which can mutate to thespo phenotype has been estimated to be 48±27 from complementation studies. Comparison of the wild type and mutants by light microscopy after exposure to sporulation medium at the restrictive temperature and Giemsa staining has shown that mutant populations can not complete the meiotic nuclear divisions.Supported by NSF grants GB-8564 and GB-27688, and the Wallace C. and Clara Abbott Memorial Fund from the University of Chicago.     

Chromosome banding techniques for morphologically classified cells

This report describes staining techniques for chromosome banding and sister chromatid exchanges (SCEs) suited to a method that allows simultaneous analysis of cell morphology and karyotype. Mitotic cells are first identified by either cytochemical staining or immunologic methods. The preparations are then destained and treated with acid fixative. For G- and C-banding, the cells are incubated overnight at room temperature in S?orensen buffer and then stained with Giemsa. To demonstrate SCEs, the cells are fluorescent stained before being stained with Giemsa.     

Karyotypes,C-banding and nucleolar numbers inGuizotia (Compositae)

Karyotypes, constitutive heterochromatin and nucleolar numbers of five recognized taxa and two systematically new populations ofGuizotia have been studied using Giemsa or aceto-orcein staining, C-banding and silver nitrate staining. All accessions have 2n = 30 chromosomes, but satellite chromosome number and nucleolar number varied from four to eight. Centromere positions varied from predominantly median to submedian and subterminal in different materials. The satellites and an interstitial region in the short arm of one chromosome pair were C-banded in all materials. Telomeric and centromeric C-bands were also observed. The material could be classified into three groups, indicating possible phylogenetic relationships.     

Epidemiological study of malaria in north Sulawesi, Indonesia by fluorescence and Giemsa staining.

An epidemiological study of malaria infection was conducted in the Likupang District, Minahasa Regency, North Sulawesi Province, Indonesia, during August 2-15, 1991. In this study, 510 people of six villages, representing ages between 1 month to 84 years cooperated voluntarily. Blood smears stained with Giemsa and acridine orange (AO), revealed 33 and 83 malaria parasite positives respectively. This significant difference was due particularly to the fact that AO staining examined under either a daylight- or halogen-illuminated microscope equipped with interference filters was sensitive to detect low-density parasitemia in many subjects previously diagnosed negative by Giemsa staining in the field. The low malaria prevalence obtained by Giemsa staining may have been attributable to the lack of standard-quality diagnostic tools in the field or inadequate observation of the slides. In both staining methods, Plasmodium falciparum was found to be the predominant species, while the remainings were P. vivax or a mixture of both. Subjects infected with P. vivax revealed higher density of parasitemia and gametocytemia than those with P. falciparum.     

Selective staining of Y chromosomal loops in Drosophila hydei,D. neohydei,and D. eohydei

Modified Giemsa procedures have been developed which elicit differential and highly selective staining of individual Y chromosomal lamp-brush loops in spermatocyte nuclei of Drosophila hydei, D. neohydei, and D. eohydei. In all three species the Y loop pair known as the clubs stains a brilliant dark red with Giemsa at pH 10. With the same treatment other loop pairs either remain unstained, e.g. the threads, or show a differentiation between light blue and pink staining matrical material, e.g. pseudonucleolus and cones in D. hydei and D. eohydei. With eosin at pH 2.8 the threads in D. hydei can be stained intensely, as well as one matrical component of the pseudonucleolus. Pretreatment with RNase or TCA removes all stainability from the Y loops with Giemsa at pH 10. TCA treatment enhances eosin staining at pH 2.8. These and other variations of Giemsa may be utilized to establish homologies between Y loops in different species. The molecular basis of the staining reactions remains to be elucidated.     

Cytogenetic investigations in rye,wheat and triticale

Summary Chromosomes of tetra- and hexaploid wheat have been individually characterized by Giemsa and/or Leishman C-banding techniques. Appropriate methodological modifications resulted in almost identical staining of chromosomes of tetraploid wheat with Giemsa and Leishman solutions. Additionally comparison of Giemsa banded chromosomes of the A- and B-genome of Triticum turgidum 34 and Triticum aestivum cv Jubilar reveals similar or corresponding patterns in all homologous chromosomes with the exception of chromosome 7B. Apart from this intervarietal variation in certain homologous chromosomes of both wheat cultivars, intravarietal polymorphism is verified.     

Molecular characterization of “inverted” pericentromeric heterochromatin of chromosome 3

Summary Inversion of the pericentromeric region of human chromosome 3 [inv (3) (p11q11.2)] is a rare event. Initially, this inversion was identified with staining for Q-bands by fluorescence using quinacrine (QFQ) and later characterized with staining for C-bands by CBG technique. The molecular methods of fluorescence in situ hybridization (FISH) and AluI/Giemsa and TaqI/Giemsa techniques were utilized. The findings suggest that the variable band q11.2 on chromosome 3 contains alphoid DNA sequences, which appear to be similar to those identified by conventional methods in the centromeric region (band p11).