Quinacrine Mustard Staining Research Articles

Aneuploid Chromosome Constitutions of the Small Japanese Field Mouse, Apodemus argenteus, in the Western Mountainous Area of Kanagawa Prefecture, Honshu, Japan

Abstract. We studied an occurrence of chromosomal aneuploidy for ten individuals in the small Japanese field mouse, Apodemus argenteus, from the western mountainous area of Kanagawa Prefecture, Honshu, Japan. Eight of the individuals examined showed the standard 2n = 46 and FNa = 48 constitutions as the most frequent karyotype. However, of those eight individuals, seven carried an inter-cellular mosaicism with a few metaphases carrying a small-sized supernumerary element. In addition, one individual showed an aneuploid complement that included a small-sized bi-armed B chromosome (2n = 47). The B chromosome consisted of the C-heterochromatin and was characterized by delayed fluorescence by the quinacrine mustard staining, suggesting that its feature is identical to the B chromosomes previously confirmed in other localities. Moreover, an XO female showing a standard autosomal complement with only one X chromosome (2n = 45, XO) was also recognized. According to the present observations, these chromosomal aneuploidies are retained in wild populations of A. argenteus as constantly occurring phenomena.

The karyotype of the domestic sheep (Ovis aries) identified by quinacrine mustard staining and fluorescence microscopy.

The modal chromosome number of the domestic sheep (Ovis aries) is 54. It is possible to identify 25 out of the 27 pairs of chromosomes by means of quinacrine mustard staining and fluorescence microscopy. There is often a considerable difference in length between the homologues of the same pair. The submetacentric autosomes may be derived from the basic karyotype of the family Bovidae by Robertsonian fusion. Consequently, the karyotype of the sheep chromosomes has been arranged in another way than in the goat and cattle. The Q-band pattern of the sheep autosomes is identical with that of the goat autosomes, except the Robertsonian fusions, and with cattle autosomes, except Pair No. 16. In the sheep, the X chromosome is one of the largest chromosomes of the karyotype. Its relative length is about 0.5 per cent smaller than in the other two species studied. Nevertheless, the number of Q-bands in the X chromosome of the sheep seems to be the same as in the goat and cattle, but the sequence of the bands is different. The Y chromosome of the sheep is the smallest chromosome, and its fluorescence is identical with the Y chromosome of goat and cattle.

Nucleolus and chromosome relationships at pachynema in four Scarabaeoidea (Coleoptera) species with various combinations of NOR and sex chromosomes

Nucleolus organizer regions (NORs) and nucleolus locations were studied after silver staining in spermatocytes at pachynema from four beetle species selected for their various combinations of sex chromosomes. Their karyotypic formulae were: 18,neoXY (Dorcus parallelipipedus); 25,X (Passalus unicornis) and 20,Xyp (Cetonia aurata and Protaecia (Potosia) opaca). NORs were located in the short arms of a unique acrocentric autosome pair in the first three and in intercalary position in a sub-metacentric autosome pair in the last species. Silver staining gave remarkably more consistent results in pachytene than in mitotic spreads, enabling the detection of both NORs and nucleoli, and also better results in embryo than in spermatogonial metaphases. At pachynema the NORs were elongated, roughly in proportion to the number of nucleoli, which always remained associated with NOR. Nucleoli were not recurrently associated with sex chromosomes, except in P. unicornis, at late pachynema. In C. aurata and P. opaca the sex body was recurrently associated with acrocentric short arms and metacentric telomeres, respectively. Even in these simple situations, with NORs located in a single autosome pair, the number of nucleoli and their relationships with sex chromosomes varied strongly from species to species. These variations appear to be largely determined by the chromosome rearrangements which have occurred during evolution, which makes extrapolations and generalizations quite hazardous. In D. parallelipipedus pachytene cells a quasi-systematic and transient fusion between the terminal heterochromatin of two sub-metacentrics was detected. Other chromosome bivalents could also be occasionally associated, but not the NOR carrier one. A strong enhancement of DAPI or quinacrine mustard staining was observed at the fusion point.

Classification of 'Tosa-Buntan' Pummelo(Citurs grandis[L.] Osb.), 'Washington' Navel Orange(C. sinensis[L.] Osb.) and Trifoliate Orange(Poncirus trifoliata[L.] Raf.) Chromosomes Using Young Leaves.

Chromosome samples of young leaves of 'Tosa-Buntan' pummelo (Citrus grandis [L.] Osb.), 'Washington' navel orange (C. sinensis [L.] Osb.) and trifoliate orange (Poncirus trifoliata [L.] Raf.) were prepared by the enzymatic maceration method. The preparations were sequentially stained with Giemsa, quinacrine mustard (QM), chromomycin A3 (CMA), and 4'-6-diamidino-2-phenylindole (DAPI). The chromosome lengths and banding patterns among 4 staining methods were compared. The discrimination of centromeres was easier by QM staining than by Giemsa staining. By CMA staining, 5 characteristic banding patterns were observed. Therefore, 'Washington' chromosomes were divided into 4 groups, trifoliate orange chromosomes into 3 groups, and 'Tosa-Buntan' chromosomes into 5 groups. CMA staining enabled the confirmation of chromosome number as 18 in cells in which chromosome lengths were relatively long. Chromosome number in 'Washington' and 'Tosa-Buntan' were difficult to count by the Giemsa stain. We conclude that chromosome samples prepared by using young leaves and QM and CMA stainings are effective means to reveal chromosomes in citrus. Hence, a good possibility of karyotyping citrus in the future exists.

イヌにおけるFluorescent Body (F-body) の検出

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.

Separation of human X and Y spermatozoa by free-flow electrophoresis.

Free-flow electrophoresis is a fast and promising method for gamete separation. Pretreated seminal plasma-free human spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber flowing perpendicular to the forces of an electrical field, which separated the spermatozoa according to their differences in electrophoretic mobility. For characterization of the two classes of spermatozoa before and after separation, quinacrine mustard staining was used to identify the Y-chromosome-bearing spermatozoa carrying the fluorescent body (F-body). Human spermatozoa moved toward the anode and were separated into two main peaks. The faster moving fraction contained nearly 80% Y-bearing spermatozoa and the slower peak consisted mainly of pure X-bearing spermatozoa. Whereas sperm viability as determined by eosin staining was nearly unchanged, sperm motility was reduced after free-flow electrophoresis.

Are fluorescent bodies of Y-spermatozoa detectable in common with mammalan species?

An attempt was made to detect the fluorescent bodies (F-body), using Quinacrine mustard (Q-M) staining in the spermatozoa from eight mammalian species (human, bull, boar, dog, rabbit, rat, mouse, and mastomys) as well as in the cock (used as negative control). Sperm suspension, prepared after rinsing by repeated centrifugation with phosphate buffered saline (PBS), was either stained with Q-M for 24 h or treated with protease and then stained with Q-M for 60 min. The final concentration of Q-M in the mixed staining sperm suspension was 0.025 mg/ml. The examination using a reflecting fluorescent microscope revealed that the F-body found in human sperm was also present in the sperm of all the mammals but not in the cock after 24 h of staining. The enzyme-treated specimens showed higher incidences of F-bodies than specimens stained for 24 h without enzymatic digestion. These findings strongly suggest that the F-body is commonly present in the spermatozoa of many mammalian species.

In situ evaluation of the functional state of chromatin by means of Quinacrine Mustard staining and time-resolved fluorescence microscopy.

A pulsed laser microfluorometer with high spatial and temporal resolution was employed to study the functional state of chromatin, using Quinacrine Mustard as the fluorescent probe. Corresponding segments of polytene chromosomes of embryo suspensor cells of Phaseolus coccineus and parenchymal cells of Helianthus tuberosus, in phases not involving DNA synthesis, were selected as models. The fluorescence decay time turned out to be a discriminating parameter for the chromatin fractions of differing functional engagement. The results were interpreted on the basis of a different accessibility of the DNA to the intercalating agent, as a result of the different structural situation of the chromatin. This fact determines modifications of the energy transfer rates between dye molecules in different quantum efficiency conditions, which results in variations of fluorescence decay times.

Sex determination in nuclei of amnion fluid cells.

Hypotonic treatment of amnion fluid cell-nuclei was found to enhance demonstration of Y chromatin and of autosomal fluorescence in interphase cell-nuclei following quinacrine mustard staining. In contrast, visualization of the Barr body was not improved by this treatment. Nuclear sex determination on cellular material from 200 samples of amniotic fluid was unequivocal in 197 cases; in only 3 cases were the findings uncertain. There were no erroneous determinations. In one instance maternal cellular tissue was observed in smear preparations. Nuclear sex determination presents a simple method of supplementing and substantiating prenatal chromosomal diagnosis.

Inhibition of the development of Q-bands on human chromosomes by netropsin.

Netropsin, an oligopeptide-type basic antibiotic, having exclusively A-T-specific DNA-binding affinity and situating itself into the minor groove of the double helix, represses the development of Q-bands if human chromosome preparations are treated with it before quinacrine mustard staining. The most probable interpretation of this effect is that netropsin interferes with the intercalation of the dye molecules. It is assumed this phenomenon supports the hypothesis that quinacrine mustard binds preferentially to A-T-rich sequences of DNA in the metaphase chromosomes.

Localization and significance of the Q bands observed on mitotic chromosomes of the amphibian Pleurodeles waltlii Michah. after coloration by quinacrine mustare (author's transl)

Metaphase plates from tailbuds of Pleurodeles waltlii embryos (stage 34) with or without preceding cold treatment were obtained by squashing followed by quinacrine mustard staining. In both cases, caryograms were established and sites of Q bands located.--Most of the secondary constrictions exhibit very high fluorescence. In general, it is the same for most centromeric parts, but their fluorescence is quenched very strongly by cold treatment.--The proximal part of the long arm of chromosome VII and satellites of chromosomes III and XI exhibit dull fluorescence. All these sites are compared with heterochromatin localization. The relation between banding and base composition or non-histone proteins interactions is discussed.

Fluroescent characteristics of drumsticks, drumstick-like projections, and other nuclear bodies in human blood cells.

The fluorescent properties of drumsticks, drumstick-like appendages, and other nuclear bodies in the polymorphonuclear leukocytes from six human males and females were studied with the aid of the quinacrine-mustard staining technique. Both brightly and weakly fluorescent drumsticks (in females) and drumstick-like bodies (in males) were observed, and they were readily differentiated on the basis of size, shape and, usually, fluorescent intensity. An analysis of the correlation between the extent of nuclear lobulation of the polymorphs and the corresponding fluorescent patterns of the adjoining drumsticks and drumstick-like bodies indicated that a possible change in the state and/or condensation of chromatin in these nuclear bodies might occur with increasing age of the polymorphs. Although the brightly fluorescent regions of the nuclei usually corresponded to the areas darkly stained with Giemsa, much finer patterns of differential staining of drumsticks and other nuclear bodies were obtained only by the fluorescent method.

New techniques in the study of human chromosomes: methods and applications.

In 1968, Caspersson et al 10 demonstrated that the fluorescence of chromosomes after quinacrine mustard staining revealed a typical pattern of bands for each element. The importance of this breakthrough was apparently not grasped immediately by cytogeneticists. It was only when Caspersson et al.17,18 demonstrated that each human chromosome could be identified by this technique that banding patterns were studied the world over. A labeling of the pericentromeric regions was achieved by many authors.2,60,82 Very soon other methods derived from these findings were described by Sumner et al.,101 Drets and Shaw,27 and Schnedl.96 Before these developments, the controlled denaturation of chromosomes by heat was described by Dutrillaux and Lejeune,41 using a procedure derived from the experiments of Yunis et al 107 In mid-1971, another technique, proteolytic digestion, was discovered by Dutrillaux et al 36 and numerous modifications were then reported.47,97,105 Finally, modification of the pH of the Giemsa stain solution was described by Patil et al,84 and, in a very different way, Zakharov et al 108 demonstrated that treatment of living cells with 5-bromodeoxyuridine (BUdR) could reveal a specific pattern of localized uncoiling.

Chromosome changes revealed by the Q-band staining method during cell senescence of WI-38.

used to initiate separate aging experiments which were carried out serially over a three year period. In order to detect subtle, progressive changes in banding associated with cell age, we examined the chromosome constitution of WI-38 during early (passage 27) , middle (passages 27-40), and late (passage 40) phases of FL at weekly intervals. A total of 140 cells of high quality were randomly sampled during FL from experiments I and IV: 20 from early; 54 from middle; and 66 from late phase. The quinacrine mustard (QM) staining method (6, 7) was employed in this study. This technique permits the analysis of longitudinal differentiation along chromosome arms and allows the identification of each homologous chromosome pair. Moreover, this method reveals alterations in chromosome structure with a greater degree of discrimination than conventional chromosome staining procedures. Cultures were tested for pleuropneumonia-like organisms (PPLO) periodically by the Baltimore Biological Laboratories. No infections were detected. Results. Our observations revealed no detectable alteration in the banding pattern of diploid chromosome sets during FL. This indicates that in the majority of cells no progressive change in chromosome structure occurs during the so-called aging process. Nevertheless, it is known from earlier studies that generally a small proportion of cells in normal cultures undergo chromosomal rearrangement, even during the early in vitro period (3). We have used the QM technique to obtain greater insight into the nature, origin and fate of such chromosomal alterations. In this report, we have selected three chromosome modifications which are particularly instructive.

Prenatal cytogenetic analysis of women with high risk for genetic disorders.

165 prenatal cytogenetic analyses are reported. The culture and Giemsa or quinacrine mustard (QM) staining processes are described. Karyotypes from both Giemsa and QM metaphases were analyzed. The main indications for amniocentesis were: 1)previous child with Down's syndrome (65), 2)advanced maternal age (74), 3)D/G carrier (5), 4)Duchenne muscular dystrophy (5) or 6)previous indication of other chromosomal anomaly. In the advanced maternal age group, 4 G21 and 1 E18 trisomy fetuses were detected. No chromosomal abnormalities were seen in the group referred for a previous child with Down's syndrome, although one woman was found to have a 9/13 translocation herself. Another woman with 13/14 translocation gave birth to a healthy boy with a 13/14 translocation, as predicted. Of 5 women referred for D/G translocation carriers, 1 had a fetus with a 46, X,Y,-D + t(DqGq) karyotype. Sex determination for X-linked anomalies resulted in detection of 2 Duchenne's muscular dystrophy, 1 hemophilia, 1 Norrie's syndrome, and 1 Pelizaeus-Merzbacher's syndrome.

Trisomy of the short arm of chromosome 8: association with translocation between chromosomes 8 and 22 46,XY,22-,t(8p22q) plus.

The case of a retarded child with trisomy of the short arm of chromosome 8 associated with translocation between the short arm of chromosome 8 and the long arm of chromosome 22 is reported. Balanced translocation involving the same chromosomes was present in the mother and brother of the propositus. The specific chromosomes involved in the abnormality in this family were identified by use of fluorescence microscopy with quinacrine mustard staining, autoradiography and Giemsa banding. This appears to be the first case report of this anomaly, although trisomy of the short arm of chromosome 9 has been reported previously.

A system of nomenclature for band patterns of mouse chromosomes.

Mouse chromosomes banded by quinacrine mustard staining, by the ASG technique, or by Giemsa staining following trypsinization or chymotrypsinization are described in detail. Three hundred and twelve regions within the mouse karyotype can be distinguished and a simple system of nomenclature is proposed for naming these regions. This nomenclature is applied to discussion of the locations of the breakpoints of twenty translocations and of many specific gene loci.

Chromosome banding patterns in preimplantation mouse embryos.

The chromosomes of first cleavage mouse embryos show the same banding patterns after quinacrine mustard staining as do chromosomes of differentiated mouse cells. Whatever feature of chromosome structure the banding pattern reveals thus appears not to be altered during development and differentiation.

A study on fluorescent barr body (X-chromatin) and fluorescent body (Y-chromatin) staining with quinacrine

The author investigated the most suitable conditions in staining both fluorescent X-chromatin and Y-chromatin.1) Quinacrine mustard (Q. M.) staining was far superior to Quinacrine dihydrochloride (Q. D.) staining on the incidence of X and Y-chromatin.2) The time factor was found to be the most important for staining, for buccal smear as well as for blood smear.3) It was also revealed that blood smear without fixation showed high incidence in fluorescent body than that with fixation if the samples were observed within a few days.4) The incidence of fluorescent X and Y-chromatin was studied in normal males and females by applying the staining conditions described above. The results were as follows:a) The average incidence of fluorescent Y-chromatin in normal males was 39. 8% in buccal mucosal cells with 0.5% Q. D. and 54.0% with 0.005% Q. M. On the other hand in blood smears, the average incidence was 46.1% with Q. D. and 67.3% with Q. M.b) The average incidence of fluorescent X-chromatin in normal females was 12.0% in buccal mucosal cells with Q. D. and 17.3% Q. M. A low incidence was observed in normal males: 0.6% with Q. D. and 0.8% with Q. M. respectively.5) The average incidence of double structure in buccal smear was 3.0±1.1%.6) In the screening test by this fluorescent staining method for sex check up of 217 female participants in the international Winter Sports Festival held in Sapporo in 1972, one person was found to have sex chromosomal constitution of XY.7) X and Y-chromatin test by this fluorescent staining method was applied to 15 patients with sex chromosomal anomaly and it was confirmed that this staining method was very useful for inferring sex chromosomal constitution except for mosaicism.

The karyotype of the pig (Sus scrofa domestica), identified by quinacrine mustard staining and fluorescence microscopy.

The karyotype of the pig <i>(Sus scrofa domestica</i>) was characterized by means of quinacrine mustard staining and fluorescence microscopy. The material examined consisted of 15 pigs of the Danish Landrace, five boars and 10 sows, representing five boar lines and eight sow strains. Previously it had been possible to identify only six pairs of chromosomes and the Y chromosome among the 38 chromosomes of the pig. With the present method it was possible to identify both autosomes and sex chromosomes with great reliability. Fluorescence variants were not demonstrable.