Quinacrine Staining Research Articles

Cytological identification of sex in pig embryos at indifferent gonadal stages.

A reliable method of sex identification in pig embryos at sexually indifferent and early differentiated gonadal stages (age 21-26 days) was developed in association with ultrastructural studies on gonadal development and differentiation. The routine carbolfuchsin staining for the sex chromatin and the quinacrine mustard dihydrochloride staining for the fluorescent Y chromosome were not successful when applied to interphase cells. Therefore, it was necessary to develop a method for complete chromosome preparations. Head, heart and liver proved to have a sufficiently high spontaneous mitotic rate without any mitogenic stimulation. The tissues were suspended in the culture medium. Optimal conditions for colchicine treatment and hypotonization were tested and the details of the procedure are reported. This direct method yielded adequate metaphase plates for chromosome analyses in pig embryos. Because no culturing is used, the method is rapid and artefactual changes in the chromosomes during stimulated cell divisions in vitro are avoided.

Prenatal karyotyping of twins by ultrasonically guided amniocentesis

Twins were diagnosed with ultrasound in two women who had been referred because of a higher than normal risk of having malformed children. Amniocentesis was carried out under the guidance of ultrasound and amniotic fluid from both fetuses was removed for diagnostic procedures. Injection of Congo red proved that the amniocentesis needle was not re-entering the same amniotic cavity at repeated puncture. In both mothers the fetuses were found to be of different sexes. Mixture of maternal cells was excluded by means of quinacrine staining, allowing evaluation of chromosome markers.

Nichtzufällige Verteilung homologer Chromosomen (Nr. 9 und YY) in Interphasekernen Menschlicher Fibroblasten

The position of chromosomes No. 9 and of the Y chromosomes in interphase nuclei was observed by means of the Giemsa-11 staining and the quinacrine mustard fluorescence staining respectively. 3 fibroblast cultures from normal female persons and 1 culture from a person with the karyotype 47/XYY were used. The distance between the two homologous chromosomes was compared with the theoretical expected distance between two points which are randomly positioned in a circular area. The distance between the chromosomes No. 9 as well as between the two Y chromosomes is significantly smaller than expected with a random position. This tendency for a somatic association is stronger in the case of the two Y chromosomes than in the two chromosomes No. 9.

Leucocyte morphology and chromosome morphology.

The banding techniques currently employed in human cytogenetics for the identification of the individual chromosomes have been used to stain PHA lymphocytes and circulating leucocytes. The capacity of these techniques to localize singular chromosomes or chromosomal regions has been investigated. It has been observed that among the four major categories of bands (Q, G, E and R) only the quinacrine staining is informative in interphase nuclei, because of its peculiarity to stain the long arm of the Y chromosome and few other heterochromatic regions. Interphase nuclei treated according to the C-bands show the presence of several heterochromatic masses, corresponding to the centromeric areas of individual chromosomes, but as such they cannot be recognized accurately. More specific and selective techniques, like G-11 and G-Y protocols, appear to be suitable to localize the centromeric regions of chromosome no. 9 and and long arm of Y chromosome. Variation of the incubation time in the alkali-saline solutions and of pH values have proven to be appropriate for the demonstration of other heterochromatic regions in interphase nuclei and in circulating leucocytes. The "nuclear" approach to study of specific heterochromatic regions of human chromosomes may be of practical interest into the investigation of several biological problems and into the detection of individuals carrying chromosome variants.

Interphase chromosome arrangement in Anopheles atroparvus.

The arrangement of chromosomes in interphase nuclei of Anopheles atroparvus has been inferred from an analysis of: 1. The early stages of mitosis as seen following Quinacrine staining, and 2. The reversible effects on the chromatin pattern obtained following the treatment of living cells with various NaCl solutions, and the following conclusions have been reached: (a) The chromatin is connected to the nuclear membrane, (b) Homologous chromosomes show close side-by-side somatic pairing, (c) The long arms of the sex chromosomes form a fluorescent peripheral body, (d) The autosomes are strongly reflexed at the centromeres, (e) The autosomal centromeric regions are polarized towards the peripheral body, (f) The telomeric regions of all the autosomes are closely apposed.--A ring-shaped pattern of interphase chromatin is constantly and reversibly induced by NaCl 0.15 to 0.18 M solutions.--These relationships indicate a peripheral arrangement of the interphase somatic complement.--The distribution of the chromosomes in polytene nuclei and at the beginning of meiosis resembles that suggested above for somatic interphase cells. This distribution may apply more widely in the Diptera.

Biophysical Studies on the Mechanism of Quinacrine Staining of Chromosomes

Biophysical Studies on the Mechanism of Quinacrine Staining of Chromosomes

Heterochromatin and disproportionate chromosome replication in Anatopynia dyari (Diptera: Chironomidae).

Salivary gland chromosomes from four populations of Anatopynia dyari were examined together with mitotio and meiotic chromosomes from one of the sites. Both mitotic and meiotic cells possess large blocks of heterochromatin, some of which fluoresce brightly after quinacrine staining. Mitotic figures show twelve chromosomes consisting of a graded size series with 5 meta- and submetacentric pairs and one small telocentric pair. — Salivary gland chromosomes have a loose chromocentre and three distinct size classes of chromosomes. The size classes include 1 long metacentric, 4 medium acrocentrics and 1 very small telocentric which is also twice the thickness of the rest of the complement. Quinacrine staining produces bright fluorescence of the centromeric third of chromosome VI, some ectopically paired regions of the chromocentre, basal bands and the telomeres of some chromosomes. — The discrepancy between arm ratios and relative lengths of mitotic and polytene chromosomes is explained by under-replication of nonfluorescing heterochromatin in the latter case. Brightly fluorescing heterochromatin behaves in an anomalous manner suggesting that it is either over, or else not severely under-replicated in salivary glands. The extra thickness of chromosome VI also suggests that it undergoes an extra round of replication. — A common complex rearrangement was found in the long arm of chromosome III in three of the populations. In the one population tested it was in Hardy Weinberg equilibrium.

A ring chromosome, diagnosed by quinacrine fluorescence as No. 9, in a mentally retarded girl.

A ring chromosome No. 9 identified by quinacrine staining was found in a 22‐year‐old mentally retarded girl with low stature, fissure of the palate, protruding lower lip and valgus deformity of the knees. The patient showed heterozygosity (type II) for cystinuria. The ring was unstable and showed different configurations.

Replication pattern and quinacrine fluorescence of the chromosomes of a CV-1-derived African green monkey cell line

When tritiated thymidine is incorporated into cells of a heteroploid CV-1-derived line near the end of the DNA synthetic period (S), the chromosomes are preferentially labeled in the centromeric region. After staining with quinacrine, the centromeric region of every chromosome shows minimal fluorescence, whereas the remainder of most chromosomes shows fairly bright fluorescence with distinctive banding patterns. In contrast to the end-of-S pattern, the distribution of grains over chromosomes labeled during the middle of S is consistent with random labeling. A marker chromosome with a long secondary constriction in the long arm near the centromere possesses the same labeling pattern, with late replication limited to the centromeric region and perhaps the adjacent region of the short arm. The secondary constriction region itself is not late replicating, and it fails to fluoresce after quinacrine staining.

Fluorescence banding pattern of the chromosomes of Microtus agrestis with a benzimidazol derivative.

The fluorescent banding pattern of the chromosomes of Microtus agrestis following staining with a benzimidazol derivative (“Hoechst 33258”) has been studied. All chromosomes allow easy identification because of their characteristic longitudinal differentiation. The X chromosomes of both sexes show intense fluorescence of the long arm and of a small proximal segment of the short arm, while the rest of the short arm reveals banding patterns. In the Y chromosome, the very short arm is nonfluorescent and the entire long arm shows bright fluorescence. Examination of the interphase nuclei of cultured fibroblasts suggests that the facultative and the constitutive heterochromatin fluoresce intensely only when strongly condensed. In contrast to some other species, in which heterochromatic chromosomal segments show a characteristic staining behaviour, i.e. either positive or negative, with the fluorochrome benzimidazol derivative, this compound behaves rather indifferently in the case of the heterochromatic contents in Microtus agrestis. The staining effects of the benzimidazol dye have also been compared with the various Giemsa and the Quinacrine staining techniques.

Quinacrine fluorescence and Giemsa staining in plants.

THE application of fluorescent dyes such as quinacrine and its mustard to cytological studies1 has advanced the understanding of the linear differentiation of the chromosomes of various organisms. Vosa2,3 has shown that there are several types of heterochromatin in plants, as defined by allocyclic behaviour and these can be distinguished by their negative or positive sensitivity to cold and by their response to quinacrine staining. Recent advances in cytological techniques pioneered by Arrighi and Hsu4 and Pardue and Gall5 have further expanded knowledge of chromosome structure in man and other animals.

Klinefelter's syndrome in identical twins with the 46,XX chromosome constitution

Two monozygotic twins with male phenotype had a 46,XX karyotype in the buccal mucosa, blood, skin and testis. The data in fourteen previously described phenotypic males with the 46,XX karyotype are reviewed. Although they resemble patients with classic (47,XXY) Klinefelter's syndrome they tend to be better virilized and often have less severe seminiferous tubular damage. Quinacrine staining of interphase and metaphase nuclei of various tissues failed to demonstrate the presence of a Y chromosome. The reason for the development of testicular structures and a male phenotype in these patients remains speculative.

Fluorescent banding patterns of rat chromosomes in normal cells and primary hepatocellular carcinomas.

Quinacrine staining permits identification of rat chromosomes in metaphase that were formerly classified only in groups within the karyotype. This technique defined two types of abnormal chromosomes in cells of rat hepatomas.

Assignment of three human genes to chromosomes (LDH-A to 11, TK to 17, and IDH to 20) and evidence for translocation between human and mouse chromosomes in somatic cell hybrids (thymidine kinase-lactate dehydrogenase A-isocitrate dehydrogenase-C-11, E-17, and F-20 chromosomes).

Independently derived man-mouse somatic cell hybrids and their derivative subclones show a positive correlation between the expression of human lactate dehydrogenase A subunits and the occurrence of the human C-11 chromosome. Data are also presented that confirm the previously reported linkage of the thymidine kinase locus to the E-17 chromosome. A translocation of the E-17 chromosome provides presumptive evidence for the assignment of the thymidine kinase locus to the long arm segment of the E-17 chromosome. This translocation also provides evidence for translocation between man and mouse chromosomes in somatic cell hybrids. A presumptive association between the human phenotype for isocitrate dehydrogenase and the human F group is also described. Identification of specific human chromosomes was achieved by the application of several new cytological techniques: measurement of chromosome arm length, in situ annealing with mouse satellite complementary RNA, constitutive heterochromatin staining with Giemsa, and quinacrine mustard fluorochromatic staining.

Ectopic pairing of chromosome regions containing chemically similar DNA.

Using genetically controlled stocks ofDrosophila melanogaster we have compared the frequency of ectopic pairing in a line showing intense quinacrine fluorescence at two sites (81F and 83E) on chromosome 3 with one showing such fluorescence at only one of these sites (81F). The frequency of ectopic pairing is an order of magnitude greater in cells from the line showing intense fluorescence in both regions than in the line showing it in only one. These data indicate that ectopic pairing is dependent upon properties of discrete chromosome regions as small as individual bands. Since A: T-rich chromatin is known to fluoresce intensely after quinacrine staining, these data further suggest that ectopic pairing is dependent on similarities of the DNA of the discrete chromosome regions involved.

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.

Karyotype of the BHK 21 C13 cell line of the syrian hamster determined with the aid of quinacrine staining.

Data are presented on the chromosome number and karyotype of a population of BHK 21 C13 cells. By use of the quinacrine staining technique a precise classification of all but two pairs of C13 chromosomes is proposed. The mean relative lengths of metaphase chromosomes in a sample of conventionally stained cells are compared with those of a sample of quinacrine stained cells.

Chromosome variation within Drosophila simulans detected by quinacrine staining.

Chromosome variation within Drosophila simulans detected by quinacrine staining.

Quinacrine staining of chromosomes and evolutionary studies in Drosophila.

PARTICULAR chromosome regions have been found to fluoresce intensely after staining with quinacrine and related compounds1–16, and we have been studying the pattern of distribution of these regions in the chromosomes of various species and laboratory stocks from the genus Drosophila. We find that these patterns vary in interesting ways and that they are a characteristic of chromosomes that can be used in studies of evolutionary cytogenetics in much the same way as structural rearrangements17–19. The following are among our observations.

Differences in the quinacrine staining of the chromosomes of a pair of sibling species: Drosophila melanogaster and Drosophila simulans.

The patterns of intense fluorescence after staining with quinacrine dihydrochloride were studied in both the polytene chromosomes and mitotic chromosomes of a pair of sibling species, Drosophila melanogaster and D. simulans. Consistent differences between the two species were found in the pattern of fluorescence of both polytene and mitotic chromosomes. In addition, it was discovered that our stock of D. melanogaster (Oregon-R) is polymorphic at one autosomal position for the property of intense fluorescence after quinacrine staining. On the basis of these findings, the usefulness of quinacrine staining in the study of the cytogenetic structure of evolutionarily interesting populations is discussed.