Banding patterns in newt chromosomes by the giemsa stain

Specific banding patterns can be produced on the mitotic chromosomes of the newt species Triturus vulgaris meridionalis and T. italicus by using the Giemsa stain technique. These bands are most useful cytogenetic markers in karyotyping, since they facilitate identification of the individual elements of the complements. Evaluation of the shape of chromosomes as well as of the banding patterns produced by the Giemsa stain indicates that the karyotypes of T. vulgaris meridionalis and T. italicus are differentiated: hence the specific distinction of the two Salamandrids, still debated by taxonomists, appears supported by chromosome evidence. — Most of the bands seem to correspond to the heterochromatic tracts observable on mitotic chromosomes from embryos and larvae either untreated or submitted to cold treatment. Besides, the comparison of mitotic karyotypes and lampbrush maps shows that the bands located near the centromeric regions of mitotic chromosomes probably correspond to the so-called bars visible on either side of centromeres of lampbrush chromosomes, while some of the subterminal bands may correspond to the sphere.This work was financially supported by C. N. R., Roma.     

Heated giemsa solution for producing more consistent bands on mammalian chromosomes

Summary Highly consistent results were obtained in banding of chromosomes with the 2xSSC method of Sumner et al. modified by the use of preheated Giemsa staining solution at 40–45°C. A 4xSSC method modified in the same way turned out to give the same consistent banding on flame-dried chromosomal specimens. Temperature seems to be a decisive factor in the band-producing capacity of a given Giemsa dye.

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Zusammenfassung Beständig gute Ergebnisse bei der Bandenmusterfärbung der Chromosomen erzielten wir mit der 2xSSC-Methode von Sumner et al., modifiziert durch den Gebrauch von auf 40–45°C erwärmter Giemsalösung. Die auf ähnliche Weise modifizierte 4xSSC-Methode ergab ebenso beständige Bandenmuster bei flammengetrockneten Chromosomenpräparaten. Die Temperatur scheint also ein grundlegender Faktor bei der Bandmusterung auslösenden Fähigkeit einer gegebenen Giemsalösung zu sein.

     

Cytochemical study of pseudoisocyanine stained human chromosomes

Summary Human meiotic and mitotic chromosomes were studied with N-N diethyl pseudoisocyanine stain. Following methylation and oxydation, the staining allowed microscopic observation of slides with both monochromatic light and fluorescence. In addition, stained preparations can be permanently conserved.Preceeded by diverse methods of chromosome denaturation or 5-BUDR incorporation, PIC lends itself to a large number of banding techniques.Cytochemical study of stained chromosomes demonstrated a certain PIC affinity for DNA although tests performed do not exclude the possibility of PIC reaction with certain proteins.This investigation was supported by INSERM (ATP no. 8-74-29).     

The role of trypsin in the pre-treatment of chromosomes for giemsa banding

Summary The role of trypsin in the elicitation of G-banding on human chromosomes was studied in two separate laboratories. Enzyme activity and ability of trypsin to chelate calcium were manipulated by dilution of the treatment solution, and by inhibition with diisopropylphosphofluoridate, diphenylcarbamyl chloride, or soybean trypsin inhibitor. In all cases, chromosomes were affected in proportion to the enzyme activity of the treatment solution rather than the ability of the solution to bind calcium. It is concluded that calcium chelation is not sufficient to explain G-banding by trypsin, but that proteolytic activity is required.     

The giemsa banding pattern of canine chromosomes, using a cell synchronization technique

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on the Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.     

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.     

Identification of rye chromosomes in triticales by giemsa staining in relation to grain shrivelling

In hexaploid triticale, cultivar UPT 72142 having highly shrivelled grains, there were only five pairs of rye chromosomes, whereas cultivars UPT 78268 and UPT 79245 having medium grains had six and five pairs, respectively. Plump grained cultivars UPT 79347 and UPT 79339 had 2 pairs of rye chromosomes. A decrease in the number of rye chromosomes in triticales is con-elated with grain improvement.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

Differential giemsa staining of kinetochores and nucleolus organizer heterochromatin in mitotic chromosomes of higher plants

Differential Giemsa staining techniques have been used to stain kinetochores and nucleolus organizer heterochromatin in four species of higher plants. Using these techniques it has been possible to follow developmental changes of kinetochores through mitosis. In addition, these same techniques also have allowed the determination of the number and sites of nucleolus organizers in the various chromosome complements studied.     

Observations on specific giemsa staining of the Y and on selective oil destaining of the chromosomes.

The human Y chromosome can be differentially stained with Giemsa using simple procedures. This phenomenon is strikingly to that observed with quinacrine fluorescence. The specific Giemsa-Y stain may be selectively removed by the action of an oil. The same oil, under certain conditions, selectively removes Giemsa stain from all chromosomes, resulting in R- and T-banding patterns. These bands, which are obtained through subtraction of dye from Giemsa-stained chromosomes, allow slides to be further processed.     

Mechanisms of giemsa banding of chromosomes. I. Giemsa-11 banding with azure and eosin.

Two components of Giemsa are necessary to obtain Giemsa-11 banding. These are an azure (either azure A or B) and eosin Y. The conditions under which azure and eosin interact to differentiate 9qh and other magenta-colored regions involve: (1) the absolute concentrations and ratio of the two dyes; (2) the pH and, to a lesser extent (3) the buffer composition of the staining solution. Differentiation is accompanied by the presence of magenta-colored precipitate, the formation of which is altered by any of the above-mentioned conditions. The absorption spectra of magentacolored and adjacent pale blue regions, measured in situ, show a significant change from those of dye mixtures and dye components in solution. These changes suggest the formation of an azure-eosinate complex. At neutral pH, differentiation of magenta-colored regions is not successful under conditions which denature DNA; e.g. (1) high temperatures; or (2) incubation in formamide. At alkaline pH (11.6), neither moderately high temperature nor fixation of chromosomes with formalin appears to affect Giemsa-11 banding. Thus, differential denaturation of DNA does not appear to play a key role.     

Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes

Constitutive heterochromatin of chromosomes can be visualized utilizing a new differential staining technique which was originally developed by Gall and Pardue (1971). The method facilitates the more certain identification of specific chromosomes within and between cell populations of different origins. Marker chromosomes can be identified in established cell lines over many months of serial passage. Chromosomes of similar morphology within karyotypes of man and mouse can be distinguished in a number of instances. For example, the Y chromosomes of both mouse and man can now be easily detected. The hetero-chromatic staining method also permits discrimination between mouse and human chromosomes in somatic cell hybrids, thus facilitating the assignment of gene markers to chromosomes in somatic cell genetics systems. Instances of translocation of centric heterochromatin to other parts of chromosomes in established tissue culture cell lines are described. An instance of the inheritance of a polymorphic variation in autosomal heterochromatin in man is reported. It is postulated that polymorphisms in the centric heterochromatin may account largely for small heritable chromosome length variations previously described in human populations and termed minor chromosome variants.     

2-colored fluorescence of differentially condensed chromosomes stained with acridine orange]

Metaphase chromosomes of the Chinese hamster differentially-condensed under the influence of a) 5-bromdeoxyuridine, b) colcemide, and c) cold, were stained with acridine-orange (AO) in concentrations of 1.5 X 10(-7) to 3 X 10(-5) g/ml at pH 4.1 to 8.5. It was found that stretched chromosomal segments fluoresced in the orange/red part of the spectrum, whereas normally condensed ones--were green. The colour distribution along the chromosome depended mainly on the AO concentration and the exposure in the UV-light, and was independent of pH and molarity of the buffer. Apparently this phenomenon cannot be attributed to the uneven denaturation of the chromosomal DNA, but rather depends on structural and/or chemical differences between euchromatin and heterochromatin.