Differential Giemsa staining in plants

Various modifications of reported banding techniques were performed using several cultivars of the genus Tulipa. Banding was obtained with Giemsa using a modified BSG technique and is reported for three cultivars. The chromosome banding noted in all cultivars was confined to terminal and interstitial regions; no banding was observed at the centromere. Complete banding patterns were established for two of the cultivars examined. The amount of banding per total chromosome complement of these cultivars was approximately 40% and 28%. The results demonstrated the existence of a wide range in the amount of constitutive heterochromatin as measured by the amount of banding between cultivars of similar and different species origins. The banding obtained is discussed with respect to the nature of the heterochromatin exhibited.     

An improved banding technique exemplified in the karyotype analysis of two strains of rat

Using an improved Giemsa banding technique karyotypes were prepared from cells of two strains of laboratory rat (AS and Hooded Lister). Slides, aged for 7 days at room temperature were incubated in 2 x SSO at 60 °C for 3 hours and then exposed to 1% trypsin for 90 seconds at 10 ° C. Following Giemsa staining, consistent banding patterns were found in both early and late metaphase cells without loss of chromosome morphology. No major differences were found in the Giemsa banding patterns of the rat strains studied. Some variability in the banding pattern was observed for the small subterminal autosome (B5).     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Q and G banding patterns of the chromosomes of some Primates

A study of the Q (quinacrine fluorescence) and G (Giemsa) banding patterns of the chromosomes of Pan troglodytes and Gorilla gorilla gorilla shows that they are almost identical. The differences include a pericentric inversion in pairs 5, 9, 19 and the X-chromosome, a possible translocation between pairs 7 and 17 of gorilla and a deletion of part of the long arms of the Y-chromosome in the chimpanzee. Several species of the genera Macaca, Papio and Cercocebus have the same karyotype and identical banding patterns. This suggests that speciation in this group may have taken place on purely genic grounds, without, involving any karyological changes.     

The distribution of repetitive DNA in the chromosomes of cultured cells of Drosophila melanogaster

The distribution patterns of different stains (orcein, quinacrine and Giemsa) in an established cell line of Drosophila melanogaster (GM₃ WS) were compared. Each chromosome stained both with quinacrine and with Giemsa shows up a specific banding pattern for heterochromatin. The comparison between the two patterns suggests a hypothesis concerning the significance of the fluorescence; moreover it permits the conclusion that heterochromatin in D. melanogaster mitotic chromosomes is all constitutive and that there is a correspondence between repetitive DNA and sections poor in mappable genes.This work was supported by a grant of the Consiglio Nazionale delle Ricerche Roma.     

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.     

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.     

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.     

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci. Received: 17 July 2000 / Accepted: 4 September 2000     

Chromosomal differentiation in two species of Aedes and their hybrids revealed by giemsa C-banding

Giemsa C-banding patterns in two species of mosquitoes, Aedes aegypti and Aedes mascarensis, their hybrids and backcross progeny revealed differences in the sex chromosome pair. In A. aegypti, the female determining or the m chromosome in both males and females shows a conspicuous band in the centromere region and another band in one arm. The male determining or the M chromosome is devoid of any bands. Progeny of crosses involving A. aegypti females and A. mascarensis males showed interesting albeit unexpected results. The intercalary band was suppressed in both sons and daughters. When such F₁ sons were backcrossed to A. aegypti females, a proportion of males developed into intersexes. These intersex progeny differed from the normal males in terms of their banding pattern. In the reciprocal cross (A. mascarensis female × A. aegypti male), the F₁ and the backcross progeny yielded the expected C-banding patterns. The implications of the reversible expression of the intercalary band on the A. aegypti m chromosome and its relevance to genetic regulation are discussed.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

小麦族披碱草属、鹅观草属和猬草属模式种的C带研究

采用改良的Giemsa C带技术,分析了小麦族披碱草属、鹅观草属和猬草属模式种的染色体C带带型。Elymus sibiricus、Roegneria caucasica和Hysrix patula的染色体在Giemsa C带带型上存在明显的差异,显示了这3个属模式种的物种特异性。3个模式种的Giemsa C带核型表明,C带带纹主要分布在染色体的末端和着丝粒附近,而中间带相对较少。对E.sibiricus、R.caucasica和H.patula的St、H、Y染色体组C带带型与其它物种的St、H、Y染色体组C带带型的差异进行了讨论。     

Giemsa Banding in Chromosomes of Douglas Fir Seedlings and Plantlets

Douglas fir plantlets have been produced by tissue culture.Karyotypes of seedlings and plantlets were prepared from roottip squash preparations using standard histological procedures.Adiploidchromosome number of 26 was common to both. The relative lengths of seedling and plantlet chromosomes werefound to be similar. The frequency of occurrences of secondaryconstrictions was found to be high in chromosomes three andten. Giemsa staining was successfully used to distinguish uniquechromosomal banding patterns in seedlings and plantlets. Pseradotsuga menziesii, Douglas fir, chromosomes, giemsa staining     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

Application of Giemsa banding to orchid karyotype analysis

A method for obtaining orchid chromosome squash preparations from ovular tissues and a Giemsa C-band technique are described. Jointly applied, they result in well-defined chromosome banding patterns. Preliminary tests with two species of the genusCephalanthera show that Giemsa banding is also well suited for orchids. Besides aiding in chromosome identification and karyotype analysis, it should prove valuable in studies of chromosomal variation and karyotype evolution of this large family.     

Similarity of giemsa banding patterns of chromosomes in several species of the Genus Rattus

Giemsa banding patterns of chromosomes in seven Rattus species were compared. Four species (R. rattus tanezumi, R. norvegicus, R. exulans and R. muelleri) had all 2n=42 and their karyotypes and banding patterns were similar, although slight differences were observed. Another subspecies (R. rattus rattus) and two other species (R. fuscipes and R. conatus) had fewer chromosomes than the above species by having large biarmed chromosomes developed probably by Robertsonian fusion. The origin of the arms of biarmed chromosomes was recognized by their characteristic banding patterns. The remaining species, R. sabanus, had a karyotype markedly different from the other species by having two small metacentrics although in the others their number was 7. Banding patterns of the chromosomes in this species, however, were also very similar to those of the other, and therefore the 7 small metacentrics seemed to have originated by pericentric inversion of small acrocentrics.Contribution No. 912 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan, Nos. 90183, 90375 and 744002.     

Karyotypic conservatism in five species of Prochilodus (Characiformes,Prochilodontidae) disclosed by cytogenetic markers

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.