Nucleolus Organizers in MUS MUSCULUS Subspecies and in the Rag Mouse Cell Line

Silver staining has been used to detect active nucleolus organizer regions (NOR's). By this criterion six mouse chromosomes, numbers 12, 15, 16, 17, 18 and 19, can have an NOR. The number and distribution of chromosomes with NOR's vary among inbred strains of Mus musculus musculus (C57BL/6J, BALB/cJ, C3H/HeJ and C3H/StCr1BR) and in M. musculus molossinus. In a musculus x molossinus F₁ hybrid, nucleolus organizers from each parent are silver stained.—Chromosomes which have NOR's in diploid cells also show them in tetraploid cells and in established cell lines. The BALB/cJ strain shows Ag-staining of NOR's on chromosomes 12, 15, 18 and occasionally 16. In the RAG cell line, which was derived from BALB/c, active NOR's are seen on 12, 15 and 18, even after these chromosomes have undergone structural rearrangements in the cell line. Some correlation exists between the amount of Ag-stain and the size of a secondary construction region, with a large amount of Ag-stain present on a chromosome which has a prominent secondary constriction. There is no correlation between the amount of Ag-stain and the presence or absence of C-band material.     

Human chromosome variation: the discriminatory power of Q-band heteromorphism (variant) analysis in distinguishing between individuals, with specific application to cases of questionable paternity.

The chromosomes from 57 persons were analyzed by means of quinacrine fluorescent staining in order to assess the amount of variation and the discriminatory power of Q-band heteromorphism analysis. Chromosomes 3, 4, 13, 14, 15, 21, 22, and Y of each person were visually compared to those of 56 others, for a total of 1,596 comparisons. No two persons were found to have the same set of variants. The number of differences between chromosomes for each comparison ranged from 2 to 12 out of a possible total of 14 for females and 15 for males. Relatives were also distinguishable, and differences ranged from two to seven. We used the frequency with which each chromosome was useful for telling two people apart, and estimated the probability of finding two persons with the same set of quinacrine variants as .0003. Distinctly different heteromorphisms were found in the 39 unrelated persons for each of the chromosomes examined. In this small population, the number of different sets of variants observed for chromosomes 3, 4, 13, 14, 15, 21, 22, and Y were six, seven, 27, 16, 20, 15, 24, and five, respectively, for a total number of possible combinations of 1.14 X 10(15). As a test of the usefulness of chromosome heteromorphisms in paternity cases, 12 father-mother-child trios of virtually certain paternity, owing to the father-child segregation of a rare structural rearrangement, were coded and recombined at random to produce 120 cases of uncertain paternity. When the code was broken, 108 "alleged fathers" had been excluded correctly and the 12 biological fathers had been included correctly.     

The chromosomal localisation of human satellite DNA I

The major concentrations of human satellite DNA I (1.688 g/ml) have been localised on human chromosome preparations by the technique of in situ hybridisation using radioactive complementary RNA synthesised in vitro. Chromosomes were identified by prior study using quinacrine fluorescence microscopy. The satellite DNA is concentrated, mainly in centromeric constitutive heterochromatin, on many chromosomes but is especially obvious in the fluorescent distal segment of the Y chromosome.     

Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis

Summary The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22) is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine in relation to Q-banding on CHO chromosomes has been investigated using X-ray microanalysis. Technical problems involved in this type of experiment were studied in detail. It was necessary to use a solution of quinacrine acetate in acetic acid to ensure that the only chlorine detectable in quinacrine-stained chromosomes was in the quinacrine molecule. Electron irradiation during analysis rapidly destroys quinacrine fluorescence, but the chlorine is not lost from the chromosomes, and there are several reasons for supposing that a reliable distribution of quinacrine on the chromosome can be obtained by the method. — Small variations along the chromosome in the amounts of chlorine (representing quinacrine) and of phosphorus (mainly DNA) occur. The distribution patterns for chlorine and phosphorus show a good resemblance to each other for each homologous chromosome; quinacrine fluorescence patterns (Q-bands) do not resemble chlorine distribution patterns, however. The results of this study therefore support the view that Q-bands result from the differential quenching of fluorescence along chromosomes to which the quinacrine is essentially uniformly bound, and do not reflect differential binding of quinacrine along the chromosome.With an Appendix by A. D. Carothers and D. Rutovitz     

Chromosome Markers in MUS MUSCULUS: Strain Differences in C-Banding

The mitotic chromosomes of several inbred strains of mice and a series of F(1) hybrids have been analyzed by quinacrine staining and further characterized by the centromeric heterochromatin banding (C-banding). Inbred strains had the same amount of C-banding material on homologous chromosomes but showed variation in the amount on different chromosomes. F(1) hybrids showed characteristics of each parent and it appears that the amount of C-banding on each chromosome is a simple inherited polymorphism. In this study 12 different chromosomes could be distinguished by their C-banding, and these can be used as normal chromosome markers.     

Evidence for the existence of satellite DNA-containing connection between metaphase chromosomes

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA-protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G(2) phase; (e) RNA helicase p68 and CENP-B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other.     

Parental origin of chromosomes in Down's syndrome

Summary The number of 21 chromosomes of 15 individuals with Down's syndrome and their parents were examined in an attempt to determine the parental origin of the extra number 21 chromosome and the stage of meiosis at which nondisjunction occurred. Chromosomes were stained with quinacrine hydrochloride and photographed; serial prints were made ranging from underexposed to overexposed. Twelve of the 15 families (80%) were informative: nondisjunction occurred in maternal meiosis I in eight (66.7%) families, in paternal meiosis I in two (16.7%) families, and in paternal meiosis II in two (16.7%) families. The production of serial exposures of chromosomes at the time of printing proved to be a valuable method of enhancing slight differences in short arm and satellite structure of the number 21 chromosomes and thereby increasing the number of informative families.     

Relationship between the number and function of human ribosomal genes

Summary The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of ³H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.     

Human tumor and rodent-human hybrid cells with an increased number of active human NORs.

A human fibrosarcoma line, HT1080-6TG, with a near diploid number of chromosomes, has an average of 7.3 chromosomes with an Ag-stained nucleolus organizer region (NOR). Cells of this line with an increased number of chromosomes have an increased number of Ag-stained NORs. This cell line has been used as the human parent in constructing mouse-human and rat-human hybrids that segregate rodent chromosomes. The hybrid ccell lines, which have 100 or more chromosomes per cell, show a proportionate increase in the number of Ag-stained NORs (means, 11.4--16.8). The frequency of association of acrocentric chromosomes increases in a similar fashion. There is no evidence of inactivation of human NORs in these cells.     

Chromosomal localisation of DNA sequences in condensed and dispersed human chromatin.

The DNAs purified from condensed and dispersed human chromatin were used as templates for the in vitro synthesis of ³H-labelled complementary RNAs (cRNAs). These cRNAs were hybridised in situ to preparations of fixed human metaphase chromosomes which had previously been stained with quinacrine and photographed with fluorescent (UV) light. Autoradiographs of the hybridised chromosomes were stained and photographed and the results analysed by comparison of the fluorescence photographs with the autoradiographs. This method allowed positive identification of every chromosomal site of hybridisation and quantitative analysis of grain distribution over a number of metaphase spreads. The cRNA transcribed from condensed chromatin DNA (cRNA_C) hybridised mainly to a limited number of sites close to or including centromeric heterochromatin (C-bands) and also to the brightly fluorescent regions of the Y chromosome. Many of these C-band regions are known to contain satellite DNAs, indicating that the repeated DNA in the condensed chromatin fraction consists largely, if not entirely, of satellite sequences. The cRNA transcribed from dispersed chromatin DNA (cRNA_D) does not contain satellite DNAs and hybridised more generally over the chromosome arms. However, the main sites of hybridisation with cRNA_D included the C-bands in the Y chromosome and autosomes, i.e. those regions which bound cRNA_C. This suggests that nonsatellite repeated DNA sequences may be associated with satellite DNAs in the chromosomes. No general correlation between the distribution of either kind of cRNA and the overall level of quinacrine fluorescence in chromosomes or chromosome arms was detectable, nor could the dispersed fraction be equated with cytological euchromatin, since it hybridised in many sites which appear heterochromatic. However, there was a suggestion that some non-fluorescing Q-bands bound cRNA_D preferentially. The differences which were found between the distribution of the cRNAs from the two chromatin fractions may be associated with differences in genetic activity.     

Cytogenetics of bisexual/unisexual species of Poecilia. III. The karyotype of Poecilia formosa, a gynogenetic species of hybrid origin.

Chromosomes of the Amazon molly, Poecilia formosa, a unisexual species of hybrid origin, were investigated by C-banding, silver staining, and fluorescent staining with DAPI, quinacrine dihydrochloride, and chromomycin A3. Analysis of heterochromatin distribution indicates that chromosomes similar to the W chromosome of P. latipinna are not present in the unisexual species. Therefore, morphologically differentiated sex chromosomes do not form the basis of the unisexuality in P. formosa. The number and location of nucleolar organizer regions vary in P. formosa and do not correlate well with those of the parental species.     

Zonal Fractionation of Mammalian Metaphase Chromosomes and Determination of Their DNA Content

Chinese hamster cells in culture were synchronized, collected at metaphase, homogenized to release the chromosomes, and the chromosomes fractionated in a sucrose gradient using a zonal centrifuge with an A12 zonal rotor. Chromosomes in the separated fractions as well as in control metaphase spreads were quantitatively classified into five easily distinguished groups, according to individual measurements of length and centromeric index. For each zonal fraction, chemical determinations were made of the amount of DNA per average chromosome. Using the group compositional data for each fraction, the amount of DNA per average chromosome in each of the groups was then calculated to be: Group I (chromosomes 1, 2)= 1.00 ± 0.14 pgm/chromatid; Group II (chromosomes 4, X, 5) -0.39 ± 0.05 pgm/chromatid; Group III (chromosomes Y, 6, 7, 8)=0.24 ± 0.04 pgm/chromatid; Group IV (chromosomes 9, 10, 11)=0.13 ± 0.004 pgm/ chromatid; and Group V (a small marker in this cell line)=0.06 pgm/ chromatid. These values are in good agreement with the literature values for relative chromosomal DNA content derived from cytospectrophotometric measurements of fuelgen stained hamster metaphase spreads. They indicate that unlike the case for human chromatids the amount of DNA found in hamster chromatids is not directly proportional to the chromatid length.

The larger chromosomes contain more DNA per unit length than smaller chromosomes. The magnitude of this effect is considerably greater than that which may be ascribable to centromeric constriction.     

Ag-staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity

The Ag-staining of the nucleolus organizer regions (NORs) of prematurely condensed chromosomes (PCCs) of human cells showing different degrees of rRNA-gene activity clearly indicates a close correlation between the positive Ag-staining of NORs and the activity of rRNA genes. The Ag-stain, however, seems insensitive to low rates of rRNA synthesis and obviously follows a threshold reaction. Furthermore it was found that the frequency of Ag-positive chromosomes involved in satellite associations in interphase does not differ from that in metaphase.     

Conservation of relative chromosome positioning in normal and cancer cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.     

Banded chromosomes of the owl monkey, Aotus trivirgatus.

Chromosomes of the owl monkey, Aotus trivirgatus, with 2n=54, 53, or 52, have been stained to show quinacrine (Q-) and Giemsa (G-) bands, and a karyotypic arrangement has been proposed based on lengths, centrometric index, and banding pattern. C-bands were present at the centromeric region of every chromosome and over the entire short arm of certain acrocentric chromosomes; 5-methylcytosine was concentrated in the same regions. Bright Q-bands at the telomeric ends of the short arms of some chromosomes probably represent a second type of repetitive DNA. Ag-staining showed that only the chromosomes bearing a secondary constriction are nucleolus organizer chromosomes.     

Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

Immunological visualization of 5-methylcytosine-rich regions in Indian Muntjac metaphase chromosomes

Regions rich in 5-methylcytosine were localized in male metaphase chromosomes of the Indian muntjac deer (Muntiakus muntjak). Chromosomes were ultraviolet irradiated and subsequently photooxidized in the presence of methylene blue to induce maximum DNA denaturation. Following treatment with anti 5-methylcytosine antibody (anti 5-MeC), regions of antibody binding were visualized by an immunofluorescence or immunopreoxidase staining procedure. All chromosomes showed some level of antibody binding along their length and at centromeric regions, with intense binding evident in the centromere of chromosome 3 and the elongated centromeric "neck" of chromosome 3-X. The Y chromosome displayed low levels of antibody binding. The banding pattern observed with anti 5-MeC is the reverse of that obtained by quinacrine staining.     

Nucleolus organizer activity and the origin of Robertsonian translocations.

Chromosomes with active nucleolus organizer regions (NOR's) were identified by combined Q-banding (in some cases), and silver staining in mouse cell lines. NOR-bearing chromosomes were overrepresented among the chromosomes involved in Robertsonian translocations in LM(TK-), A9, and RAG cell lines. Usually only one NOR-bearing chromosome was seen in any biarmed chromosome; relatively few contained two NOR-bearing chromosomes. Thus the nucleolus plays an important role, but nucleolar fusion is relatively unimportant, in the origin of Robertsonian translocations in the mouse.