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.     

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.     

Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels.

For the detection of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Coomassie blue is used commonly on account of its simplicity and reliability. In this report we show that enhanced heat, in addition to dramatically decreasing the time required for staining and destaining, also significantly increased the detection sensitivity. For a 1.5 mm gel, the staining time was 5 min at 55, 62.5 or 70 degrees C while the destaining time was 45, 45 and 20 min respectively. For a 0.8 mm gel, the staining time could be reduced to 1 min at 65 degrees C compared to 2 min at 60 degrees C and 5 min at 55 degrees C. The destaining time required was 8, 15 and 20 min at the respective temperatures.     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

Observations on the selective demonstration of nucleolar material by protein staining techniques in epon thick sections.

The effect of different protein stainings on thick sections of Epon embedded salivary glands of Chironomus pallidivittatus is described. Pretreatment of sections with several metallic salts affected the affinity for aniline blue black in different structures. The uranyl and aluminium ions followed by aniline blue black resulted in a highly selective nucleolar staining which proved also efficient in Allium cepa meristematic cells and which seems useful for the preferential demonstration of nucleolar material in thick sections.     

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.     

Selective protection of specific DNA sequences in the heterochromatin of C-banded human Y chromosomes.

The molecular basis of C-banding was investigated by in situ hybridization of human Y chromosome-derived repeated sequences, DYZ1 and DYZ2, to untreated or to alkaline-treated metaphases. Autoradiography of G-banded metaphases showed that both probes hybridized to the long arm of Y. Alkaline hydrolysis significantly reduced grain number for DYZ2 (58%-82%; P less than .05) but not for DYZ1 (P greater than .05). Similar results were observed for interphase nuclei. These findings demonstrated that the heterochromatin of the long arm contains at least two repetitive DNA fractions having two different sensitivities to alkaline hydrolysis. These observations support the notion that DYZ2 maps terminally on the Yq arm and may be nonheterochromatic.     

Sequence instability and functional inactivation of murine Y chromosomes can occur on a specific genetic background.

When the Y chromosome from Mus. poschiavinus (YPos) is backcrossed onto the C57BL/6J laboratory strain, testicular dysfunction occurs at high frequencies. When five different multicopy probes from the recombinationally suppressed region of the Y chromosome were used, genomic DNAs from sibling female progeny of C57BL/6J YPos males were found to contain YPos-specific sequences ranging from trace levels to levels consistent with an intact Y chromosome. Females with a high copy number of YPos-specific sequences had a karyotype of XYPos and were sterile. Females with trace levels of these sequences were XX and fertile. Repeated sequences in the testis-determining-region (Sxr) of inactive YPos chromosomes were unstable relative to sequences in non-Sxr regions. In contrast, the YPos chromosome was stable and functioned normally in other inbred laboratory strains such as 129/Sv. The frequency and extent of YPos chromosome instability increased with successive backcrosses from stable (129/Sv) to unstable (C57BL/6J) genetic backgrounds. Traces of YPos-specific sequences were first detected in N2 female offspring of F1 males. Therefore, sequences were deleted from YPos chromosomes in the F1 male germ line and were transmitted to N2 females; inactive YPos chromosomes (XYPos females) were first detected in the N3 generation. The mouse line being derived by backcrossing the YPos chromosome onto C57BL/6J inbred strains ended in the N7 generation, since all XYPos offspring were sterile. Even stable repeated sequences from the non-Sxr regions of their inactive YPos chromosomes were precisely rearranged in these N7 offspring at high frequencies. These data are consistent with hybrid dysgenesis in mammals.     

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.     

Observations on the selective demonstration of nucleolar material by protein staining techniques in epon thick sections

Summary The effect of different protein stainings on thick sections of Epon embedded salivary glands of Chironomus pallidivittatus is described. Pretreatment of sections with several metallic salts affected the affinity for aniline blue black in different structures. The uranyl and aluminium ions followed by aniline blue black resulted in a highly selective nucleolar staining which proved also efficient in Allium cepa meristematic cells and which seems useful for the preferential demonstration of nucleolar material in thick sections.This work was supported by grants from Deutscher Akademischer Austauschdients to S.A.B. and from Alexander von Humboldt-Stiftung to J.C.S.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

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.