Showing papers in "Chromosoma in 1975"

Visualization of nucleolar organizer regions im mammalian chromosomes using silver staining

Abstract: A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.

Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes.

Abstract: The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining.

Abstract: The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.

Electron microscopy of meiosis in Drosophila melanogaster females. I. Structure, arrangement, and temporal change of the synaptonemal complex in wild-type.

Abstract: Complete reconstruction of the synaptonemal complex in 12 pachytene (defined here as that stage in which the synaptonemal complex is continuous throughout the bivalents) nuclei from one wild-type germarium has permitted the following observations. 1) Drosophila melanogaster bivalents at pachytene exhibit a chromocentral arrangement; the pericentric heterochromatin of all bivalents lies in one region of the nucleus, the chromocenter. Telomeric ends do not appear to abutt the nuclear envelope. 2) Synaptonemal complex is present in the pericentric heterochromatin; however, it is morphologically distinct from that present in the euchromatic portion of thesynaptonemal complex of the bivalent arms is greatest at early pachytene; the synaptonemal complex then becomes progressively shorter. Minimum length is approximately one-half of the maximum. 4) Decrease in length of synaptonemal complex is accompanied by an increase in thickness. Reconstruction of 20 pachytene nuclei from an additional 8 germaria suggests that these observations are typical. Correlations between these cytological observations and genetic observations (e.g., patterns of crossing-over) are discussed.

Distribution of 18+28S ribosomal genes in mammalian genomes.

Abstract: In situ hybridization with 3H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). — The chromosomal sites which bind 3H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind 3H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. — Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.

Comparative aspects of DNA organization in metazoa

Abstract: Data on sequence organization in metazoa are reviewed and tabulated. It is shown that the features of sequence organization previously observed in Xenopus DNA are extremely widespread. At least 70% of DNA fragments 2,000–3,000 nucleotides long contain both single copy and repetitive sequence in all the organisms examined except Drosophila.

Mechanisms of chromosome banding. V. Quinacrine banding.

Abstract: A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10−6 to 2×10−5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs2SO4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.

Mechanisms of chromosome banding. VIII. Hoechst 33258-DNA interaction.

Abstract: The interaction of Hoechst 33258 with DNA has been examined to help clarify the mechanisms of banding. 1. In agreement with previous studies Hoechst fluorescence is enhanced to a greater degree in AT-rich compared to GC-rich DNA. 2. Hoechst causes an increase in the DNA Tm which is greater at the higher AT content of the DNA. 3. There is a decrease in extinction coefficient and shift in the adsorption spectra to a higher wavelength when Hoechst binds to DNA. 4. DNA is completely precipitated at a ratio of one dye molecular per base pair, and this precipitation is not affected by salt. 5. There is no increase in viscosity or change in the circular dichroism of DNA when bound to Hoechst. These findings suggest Hoechst does not bind to DNA by intercalation or by ionic interaction with the phosphate groups, but rather binds by an attachment to the outside of the double DNA helix by interacting with the base pairs. This type of binding allows greater sensitivity to the base composition than occurs with intercalating agents. In this respect its binding is similar to that of dibutyl proflavine (Muller et al., 1973).

Characterisation of the wheat genome by renaturation kinetics

Abstract: Reassociation kinetics of sheared hexaploid wheat DNA in 0.18 M Na+ at 60 °C show the presence of three major classes of nucleotide sequences; (1) a very rapidly reannealing fracton that comprises about 4 to 10% of the genome and reanneals virtually instantly. This fraction contains sequences which appear to be randomly distributed through at least 40% of the genome; they may be palindromic sequences; (2) a heterogeneous intermediate reannealing fraction comprising about 70–80% of the genome which consists of families with apparent repetition frequencies ranging from about 100–100000; and (3) a slow reannealing fraction. These fractions have been isolated and studied separately. Seventy percent of the slow reannealing fraction (12 to 20% of the genome) was found to contain sequences present in approximately six copies per hexaploid genome. The single copy sequences in the three constituent diploid genomes of hexaploid wheat appear to show near complete homology to one another.

Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique

Abstract: With an improved N-banding technique, the location of nucleolar organizing region was determined in 27 kinds of material including mammals, a marsupial, birds, amphibians, fishes, an insect and plants. In most cases the N-bands were clearly located on certain specific regions of chromosomes, such as the secondary constriction, satellite, centromere, telomere and heterochromatic segment, while in some species they were detected as very minute bodies distributed over many chromosomes. From the available cytological and biochemical data it was suggested that the N-bands represent certain structural non-histone proteins specifically linked to nucleolar organizers in various eukaryotic chromosomes.

Hoechst 33258 fluorescent staining of Drosophila chromosomes.

Abstract: Metaphase chromosomes of D. melanogaster, D. virilis and D. eohydei were sequentially stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophila's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation in the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differentiated by a combination of quinacrine and Hoechst staining. The distal block is always Q−H− while the proximal block is, for the various autosomes, either Q−H−, Q+H− or Q+H+. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H+ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H− heterochromatin. This affinity of fluorochrome similar heterochromatin was best seen in colcemid induced multiple somatic non-disjunctions where H+ chromosomes were distributed to one rosette and H− chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be a necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q+ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be “the most important factor responsible for quinacrine brightness”. All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single DNA molecule which is one heptanucleotide repeated two million times.

Sister chromatid exchanges in Vicia faba

Abstract: The relationship between chromosomal aberrations and sister chromatid exchanges (SCE's) after treatment of Vicia faba root tips with thiotepa, caffeine and 8-ethoxycaffeine (EOC) was studied by using a modified fluorescent plus Giemsa (EPG) technique. At concentrations which had little effect on the frequency of chromosomal aberrations, thiotepa strongly increased the frequency of SCE's, provided that the chromosomes were allowed to replicate between treatment and fixation. Frequently, the size of the exchanged material was smaller than the diameter of the chromatid. Post-treatments with caffeine of roots previously exposed to thiotepa strongly increased the frequency of aberrations, but had little effect on the frequency of SCE's. In contrast to thiotepa, EOC caused only a slight increase in the frequency of SCE's even at concentrations which produced a high frequency of chromosomal aberrations. Thus, there was not a close correlation between SCE's and chromosomal aberrations. Single-strand exchanges between the DNA double helices in sister chromatids were not detected.

A new method of in situ hybridization

Abstract: A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.

The synaptonemal complex and the spindle plaque during meiosis in yeast.

Abstract: Meiosis in Saccharomyces cerevisiae proceeds principally in the same manner as in other Ascomycetes. Leptotene is characterized by unpaired lateral components and pachytene by the presence of extensive synaptonemal complexes. The synaptonemal complex has the same dimensions and is similar in structure to those described for other organisms. Chromosome counts can now be made by reconstructing the synaptonemal complexes. Diplotene nuclei consistently contain a single polycomplex. The behaviour, doubling and the fine structure of the spindle plaque provide additional markers for the different stages of meiosis.

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac.

Abstract: The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles, SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.

DNA sequence organization in the genomes of five marine invertebrates.

Abstract: The arrangement of repetitive and non-repetitive sequence was studied in the genomic DNA of the oyster (Crassostrea virginica), the surf clam (Spisula solidissima), the horseshoe crab (Limulus polyphemus), a nemertean worm (Cerebratulus lacteus) and a jellyfish (Aurelia aurita). Except for the jellyfish these animals belong to the protostomial branch of animal evolution, for which little information regarding DNA sequence organization has previously been available. The reassociation kinetics of short (250–300 nucleotide) and long (2,000–3,000 nucleotide) DNA fragments was studied by the hydroxyapatite method. It was shown that in each case a major fraction of the DNA consists of single copy sequences less than about 3,000 nucleotides in length, interspersed with short repetitive sequences. The lengths of the repetitive sequences were estimated by optical hyperchromicity and S1 nuclease measurements made on renaturation products. All the genomes studied include a prominent fraction of interspersed repetitive sequences about 300 nucleotides in length, as well as longer repetitive sequence regions.

Causes and consequences of Robertsonian exchange.

Abstract: At least two types of Robertsonian exchange are now known in the acrocentric chromosomes of man. Both types involve breakage in the arms adjacent to the centromere. Evidence is presented for a third type of exchange, one involving breakage within the centromere itself, in the grasshopper Percassa rugifrons. In this species, which is regularly homozygous for a single fusion metacentric, the eighteen rod autosomes have small but pronounced granules at the centric end of the chromosome. When C-banded these granules show differential Giemsa staining and appear to represent centromeric chromomeres; these chromomeres are lacking in the metacentric fusion product. Equivalent fusions may have occurred in some mammal species too and possible examples of this are discussed in sheep and mice. The Percassa fusion has led to a modification in both the frequency and the distribution of chiasmata as judged by a comparison of these properties in the metacentric relative to the two next smallest rod equivalents. Comparable modifications are known to occur in other naturally occurring fusions but these changes are certainly not automatic consequences of fusion since they are not shown in at least some newly produced fusion mutants.

Differential replication of satellite DNA in polyploid tissues of Drosophila virilis.

Abstract: Satellite DNA amounts were examined in adult tissues of Drosophila virilis, a species whose DNA contains three prominent satellites. Satellite amounts in DNA from six of the seven tissues were lower than in DNA from diploid (adult brain) tissue. Satellite amounts in adult ovary DNA, however, were equivalent to or greater than diploid levels. When DNA from pupal ovaries was examined, a 30% increase in satellite amounts over diploid levels was found. An RNA-DNA hybridization experiment showed that the ribosomal RNA genes in pupal ovary DNA were under-replicated relative to diploid DNA levels.

Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine.

Abstract: Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5–50 μg) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.

The position of interphase chromosomes and late replicating DNA in centromere and telomere regions of Allium cepa L.

Abstract: Chromosomes in G1, S, G2 and early prophase of Allium cepa root tip nuclei are oriented in the same position as telophase chromosomes. The centromeric heteroehromatin is aggregated in a chromocenter at one side of the nucleus, the telomeres scattered at the opposite side. Telomeres appear to associate with other telomeres in interphase in a roughly two by two fashion. Telomere-centromere DNA is late replicating. These results support the conclusion that chromosomes in higher organisms frequently maintain their telophase orientation from the end of telophase, during interphase and well into the next prophase.

Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence.

Abstract: Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-or-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.

Location of genes coding for 18S and 28S ribosomal RNA within the genome of Mus musculus.

Abstract: Cytological detection of cistrons coding for 18S and 28S ribosomal RNA (rRNA) within the genome of Mus musculus inbred strain SEC/1ReJ was accomplished using the technique of in situ hybridization. Metaphase chromosome spreads prepared from cultured fetal mouse cells were stained with quinacrine-HCl and photographed. After destaining, they were hybridized to Xenopus laevis tritiated 18S and 28S rRNA, specific activity 7.5 X 10(6) dpm/mug. Silver grains clustered over specific chromosomes were readily apparent after 4 months of autoradiographic exposure. The identity of the labelled chromosomes was established by comparing the autoradiographs to quinacrine photographs showing characteristic fluorescent banding of the chromosomes in each metaphase spread. The 18S and 28S rRNA was found to hybridize to chromosomes 12, 18, and 16. Statistical analysis of the grain distribution over 26 spreads revealed that the three chromosomes were significantly labelled. Grains over these chromosomes were concentrated in an area immediately distal to the centromere, a region which in chromosomes 12 and 18 in this particular strain is the site of a secondary constriction. The relative size of the secondary constrictions, long and thus prominent on chromosome 12, obvious but shorter on 18, and indistinguishable on chromosome 16, correlated with the average number of grains observed over the centromeric region of these chromosomes, 2.5, 1.0, and 0.78, respectively.

Variation of C-bands in the chromosomes of several subspecies of Rattus rattus.

Abstract: All subspecies of black rats,(Rattus rattus) used in the present study are characterized by having large and clear C-bands at the centromeric region. The appearance of the bands, however, is different in the subspecies. Chromosome pair No. 1 in Asian type black rats (2n=42), which are characterized by an acrocentric and subtelocentric polymorphism, showed C-band polymorphism. In Philippine rats,(R. rattus mindanensis) the pair was subtelocentric with C-bands, but in Malayan black rats,(R. rattus diardii) it was usually acrocentric with C-bands. In Hong-Kong,(R. rattus flavipectus) and Japanese black rats,(R. rattus tanezumi) it was polymorphic with respect to the presence of acrocentrics with C-bands or subtelocentrics without C-bands. The other chromosome pairs showed clear C-bands, but in Hong-Kong black rats the pairs No. 2 and 5 were polymorphic with and without C-bands. In Japanese black rats, 6 chromosome pairs (No. 3, 4, 7, 9, 11 and 13) were polymorphic in regard to presence and absence of C-bands, but the other 5 chromosome pairs (No. 2, 5, 6, 8 and 10) showed always absence of C-bands. Only pair No. 12 usually showed C-bands. C-bands in small metacentric pairs (No. 14 to 20) in Asian type black rats were generally large in size, but those in the Oceanian (2n=38) and Ceylon type black rats (2n=40) were small. In the hybrids between Asian and Oceanian type rats, heteromorphic C-bands, one large and the other small, were observed. Based on the consideration of karyotype evolution in the black rats, the C-band is suggested to have a tendency toward the diminution as far as the related species are concerned.

3H-Uridine labelling patterns in the Drosophila melanogaster salivary gland chromosomes X, 2R and 3L

Abstract: RNA-synthesizing regions in the polytene chromosomes of D. melanogaster were located; the level of 3H-uridine incorporation in each of these regions was determined. — It was found that the number of RNA-synthesizing regions is much larger than the number of puffs: almost all the chromosome regions are labelled except dense bands. In most cases, “non-puffed” regions label 10 to even 100 times weaker than prominent puffs. Typical puffs, i.e. loci with increased chromosome diameter, together contain about 50% 3H-uridine incorporated into the chromosomes. — In general, there is a correlation between puff size and RNA synthetic activity, so that 3H-uridine incorporation rate increases directly with puff volume. However, within each class individual puffs differ in labelling intensity by many times. It is suggested that many regions of “small puffs”, including interbands, represent chromosome loci with constant transcriptional activity.

Mechanisms of chromosome banding. VII. Interaction of methylene blue with DNA and chromatin.

Abstract: The binding of methylene blue to DNA and chromatin treated in various ways was examined by equilibrium dialysis. The maximum r value (moles of bound dye/mole of nucleotide) was 1.0 for DNA, 0.6 for unfixed chromatin, and 0.83 for chromatin fixed in methanol-acetic acid. When fixed chromatin was treated with saline-citrate at 60 degrees C for 3 hours, as used for G-banding chromosomes, the r value decreased from 0.83 to 0.55. When unfixed chromatin was treated as for R-banding the r values also dropped. Equilibrium dialysis indicated there was no disproportionate increase of dye binding as the concentration of DNA increased. -- These results, and others, suggest that some of the Giemsa negative regions of G- and R-banded chromosomes are due to the denaturation of non-histone proteins so that they more effectively cover the DNA and prevent side binding of the thiazin dyes.

Mechanisms of chromosome banding. IV. Optical properties of the Giemsa dyes.

Abstract: A thorough understanding of the mechanisms of R-, C- and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral absorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. - Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not necessary component for banding. - The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their absorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.

The effects of growth in vitro on the chromosome complement of Daucus carota (L.) suspension cultures

Abstract: The chromosome number distributions and modal karyotypes of several suspension culture lines of Daucus carota L. have been analysed at various times after initiation. All lines had stable modal chromosome numbers and karyotypes, with small but significant variation about the modes. Some lines showed a predominance of diploid cells with a karyotype similar to the plant. Polyploid multiples of the modal chromosome number were present in all lines at low frequency. Variation of the 2,4-D concentration in the culture medium produced little alteration of the chromosome number distributions, but omission of 2,4-D produced a significant drop in the frequency of multipolar mitoses in those culture lines in which this treatment induced differentiation. There was no evidence of any direct effect of 2,4-D on general mitotic dynamics. Alteration of the frequency with which cultures were transferred to fresh medium showed that stationary phase was critical in the maintenance of the low frequency of tetraploids present in a predominantly diploid culture line. The results are explicable in terms of a competitive selection for cells with the dominant modal chromosome number in the presence of various mechanisms continuously producing polyploid, aneuploid and structurally altered karyotypes.

5-Methylcytosine in Heterochromatic Regions of Chromosomes: Chimpanzee and Gorilla Compared to the Human

Abstract: Fixed metaphase chromosomes of gorilla and chimpanzee were UV-irradiated to produce regions of single-stranded DNA and then treated with antibodies specific for the minor DNA base 5-methylcytosine (5 MeC). An indirect immunofluorescence technique was used to visualize sites of antibody binding. In the gorilla six pairs of autosomes contained major fluorescent regions, indicating localized regions of highly methylated DNA. These corresponded, with the exception of chromosome 19, to the major regions of constitutive heterochromatin as seen by C-banding. The Y chromosome also contained a highly fluorescent region which was located just proximal to the intense Q-band region. In the chimpanzee no comparable concentrations of highly methylated DNA were seen. Smaller regions of intense 5 MeC binding were present on perhaps six chimpanzee chromosomes, including the Y. Five of these corresponded to chromosomes which were highly methylated in the gorilla.--There is diversity among the human, gorilla and chimpanzee in both the size and location of concentrations of 5 MeC, supporting the idea that satellite DNA evolves more rapidly than DNA in the remainder of the chromosome.

Chromosome banding pattern conservatism in birds and nonhomology of chromosome banding patterns between birds, turtles, snakes and amphibians.

Abstract: The G-banded karyotypes of 4 species of birds representing the orders Galliformes, Columbiformes and Musophagiformes were compared. Banding pattern homology between orders was limited to 5 major chromosome arms and the Z chromosome. Even in these major chromosome arms pericentric and paracentric inversions produced alteration of the banding pattern sequences. Addition of constitutive heterochromatin was responsible for changes in banding pattern in the Z chromosome. The chromosome banding patterns of an emydid turtle, Terrepene Carolina, 5 species of boid snakes of the genera Liasis, Acrantophis, and Sanzinia and the African clawed-frog, Xenopus muelleri, were also compared to the bird chromosome banding patterns. No homology was observed between any of these major groups: bird, snake, turtle, amphibian. However, intergrouphomology was apparent. — The data obtained do not support reports of broad interordinal direct homology of the macrochromosomes of birds and refutes the idea of a primitive bird karyotype with 3 pairs of “A group” chromosomes and 3 pairs of “B group” chromosomes. — The major mechanisms responsible for chromosome evolution in birds appear to be centric and tandem fusions, paracentric and pericentric inversions, and addition or deletion of heterochromatin.