Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

The identification of human chromosomes by quinacrine fluorescence after hybridisation in situ

Summary A method is described for producing fluorescent bands on human chromosomes by staining with quinacrine after hybridisation in situ. The advantages of the method include the elimination of artefacts arising from staining before hybridisation, the fact that there is no reduction in sample number between staining and autoradiography, the ease with which autoradiographic grains can be identified and counted, and the reduction in exposure time.Offprint requests to: S.S. Lawrie     

The sites of radiation induced-breakage in human lymphocyte chromosomes, determined by quinacrine fluorescence

We have examined the distribution of 298 radiation-induced breakpoints in human chromosomes by quinacrine fluorescence. Very little damage was discovered which would not have been detectable with simple staining methods. The data suggest an excessive number of breaks in chromosomes 3 and a deficit in No. 16. The participation of chromosomes in rearrangements appears to be proportional to chromosome length. Within chromosome arms, breakpoints are selectively placed in telomeric regions.     

Ultrastructure of sheep metaphase chromosomes identified by study of total preparations of metaphase plates]

The data obtained suggest wide possibilities of using the elaborated method of the whole metaphase plates investigation under the electron microscope to disclose the specific characters of the structure of individual chromosomes. Each chromosome has a definite number of bands of condensed DNP material. The number and disposition of bands are essentially the same in homologous chromosomes. The Giemsa-positive disks which could be seen after differential chromosome staining correspond to the bands of condensed material.     

The possibility to distinguish chromosomes of Petunia hybrida by quinacrine fluorescence

A karyotype analysis of a diploid inbred line of Petunia hybrida stained with aceto-orcein is given. Five of the seven pairs of chromosomes can be identified by their relative lengths and arm ratios. The two remaining pairs stained with quinacrine show different fluorescence patterns.     

Simultaneous observation of quinacrine bands and silver grains on radiolabeled metaphase chromosomes

A procedure is described for quinacrine banding of radiolabeled metaphase chromosomes for autoradiography. The chromosomes can be labeled either in vivo or by in situ hybridization. The banding procedure involves treating the slides with RNase and formamide and staining in quinacrine. The slides are then processed for autoradiography. After development of the photoemulsion, the chromosomes can be karyotyped with UV light by their fluorescent banding patterns and the silver grains overlaying the chromosomes can be demonstrated by the addition of tungsten light. It is possible by careful manipulation of the visible light to simultaneously observe both fluorescent bands and silver grains. This technique should significantly increase the accuracy of chromosome identification after autoradiography and decrease the time and effort required for such analysis.     

Separation of human metaphase chromosomes at neutral pH by velocity sedimentation at 20 times gravity

A method for the separation of large quantities of human metaphase chromosomes at neutral pH is described. The separation was performed in a specially designed sedimentation chamber which was placed in a bucket of a speed-controlled centrifuge. Flow deflectors in the chamber allowed both the undisturbed introduction of gradient and chromosomes, as well as the undisturbed fractionation of the content of the chamber. The centrifuge was run at 20 g for 1 h taking care of slow acceleration and deceleration. The slow morphological changes of the chromosomes due to ageing at neutral pH do not affect the resolving power of the sedimentation technique within this hour. This in contrast to separation of chromosomes at neutral pH by velocity sedimentation at unit gravity during 20 h, as described before. The main advantage of chromosome sorting at neutral pH is that the fractionated chromosomes are more suitable for gene transfer and gene mapping experiments.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Isolation of specific human metaphase chromosomes

The human karyotype can be subdivided into seven fractions containing specific chromosomes to provide material for recombinant DNA research. The isolated metaphase chromosomes are sorted according to size by velocity zonal centrifugation, and specific chromosome groups are further purified by electrostatic deflection in a flow microfluorometer. Rapid improvements in technology should soon provide preparations of single chromosomes.     

Filter combinations and light sources for fluorescence microscopy of quinacrine mustard or quinacrine stained chromosomes

Summary The efficiency of various combinations of primary and secondary filters, and light sources for the fluorescence microscopy of chromosomes stained with quinacrine mustard or quinacrine has been studied quantitatively. Using epi-illumination, strong fluorescence could be obtained with a mercury or xenon lamp in combination with two KP 490 short-wave pass interference filters (tilted to an angle of 60° with the excitation beam) as primary filter, and a K 490 as a secondary filter. The combination of a mercury lamp and a narrow band interference filter with a maximal transmission at about 436 nm as a primary filter together with a K 490 secondary filter results in a good visual image contrast, sufficiently strong fluorescence, and a relatively slow rate of fading.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

Summary The mechanism has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns.A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNa, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.In honour of Prof. P. van Duijn