Showing papers on "Metaphase published in 1973"
TL;DR: Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated.
Abstract: Fluorescence of the dye 33258 Hoechst bound to chromosomes is partially quenched by the introduction of BrdU into chromosomal DNA. This finding has allowed microfluorometric detection of DNA synthesis in human metaphase chromosomes. Incorporation, shortly before cell harvest, of a pulse of thymidine into chromosomes otherwise substituted with BrdU results in sharply defined foci of bright 33258 Hoechst fluorescence, corresponding to regions of late DNA replication. The latereplicating X chromosome can thereby be clearly identified, and the time course of appearance of its fluorescent bands can be compared with that of its earlier replicating homologue. Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated. This approach should constitute, in many instances, a convenient, high-resolution fluorometric alternative to autoradiography.
804 citations
TL;DR: Ch Chromosome-sized DNA molecules were obtained almost quantitatively from unsynchronized cultured cells, suggesting that the size of the chromosomal DNA is conserved throughout much of the cell cycle.
Abstract: Measurements of viscoelastic retardation times of detergent-Pronase lysates of Drosophila cells demonstrated the presence of large numbers of DNA molecules of a size commensurate with that of the chromosomes. The values estimated from the retardation times for the molecular weights of the largest molecules ranged from about 20×109 to 80×109 daltons depending on the species of Drosophila. The molecular weights of the DNA molecules were independent of the metaphase shapes (i.e., metacentric or submetacentric), but were proportional to the DNA contents of the chromosomes in the case of translocations or deletions. It was concluded, therefore, that the DNA molecules must run the length of the chromosome and cannot be discontinuous at the centromere. When compared with the values of the DNA contents of Drosophila chromosomes determined by other methods, the results were consistent with the model of one, or possibly two, DNA molecules per chromosome; the simplest conclusion, that there is only one DNA molecule per chromosome (for simple chromosomes), rests on a long extrapolation of an empirical relation between retardation time and molecular weight, but is also favored by indirect evidence. Further possibilities which could not be excluded were that the large DNA molecules contained Pronase-resistant, non-DNA links, or that a fraction of smaller DNA molecules might also have been present in the chromosomes. Chromosome-sized DNA molecules were obtained almost quantitatively from unsynchronized cultured cells, suggesting that the size of the chromosomal DNA is conserved throughout much of the cell cycle. The molecules were stable for periods of up to several days at 50° C in solutions containing detergent, Pronase, and EDTA.
249 citations
TL;DR: Transfer of genetic information from isolated mammalian chromosomes to recipient cells has been demonstrated and colonies of cells containing hypoxanthine phosphoribosyl transferase appeared to represent progeny of individual cells that had ingested chromosomes, replicated, and expressed the hprt gene.
Abstract: Transfer of genetic information from isolated mammalian chromosomes to recipient cells has been demonstrated. Metaphase chromosomes isolated from Chinese hamster fibroblasts were incubated with mouse A9 cells containing a mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus. Cells were plated in a selective medium, resulting in death of all unaltered parental A9 cells. However, colonies of cells containing hypoxanthine phosphoribosyl transferase (EC 2.4.2.8) appeared with a variable frequency of about 10-6 to 10-7. The enzyme from these cells was indistinguishable from that from Chinese hamster cells, as shown by DEAE-cellulose chromatography and gel electrophoresis, and differed clearly from the mouse enzyme. The colonies, thus, did not result from reversion of A9 parental cells to wild type, but appeared to represent progeny of individual cells that had ingested chromosomes, replicated, and expressed the hprt gene. These colonies differed from each other in stability of expression of the transferred gene.
214 citations
TL;DR: It is concluded that these treatments act by promoting the disruption of chromatin structure which is then reformed in the presence of the Giemsa stain, the Azure-B component of the stain acting in a manner similar to divalent cations.
Abstract: Bands have been observed in the fixed mitotic chromosomes of Mus musculus L cell with no further treatment. These bands, which are very similar to G bands, can be seen by phase contrast microscopy, ultraviolet microscopy and Gold/Palladium shadowing. This indicates that the cause of banding is a differential distribution of chromatin. Alterations in chromatin morphology can be induced by post fixation treatments of the fixed chromosome. A model is constructed on this evidence which unifies the apparent variety in the techniques which are thought to induce G bands and explains the action of Giemsa stain. It is concluded that these treatments act by promoting the disruption of chromatin structure which is then reformed in the presence of the Giemsa stain, the Azure-B component of the stain acting in a manner similar to divalent cations. A new technique for inducing C bands is reported. The denaturation and differential reassociation of DNA is not a suitable explanation of the mechanism of this technique. The hypothesis put forward here, explaining G bands, also explains the induction of C bands as a result of a differential destruction of chromatin morphology. The differential distribution of chromatin that gives G bands is thought to be disrupted by a technique that maintains the more resistant morphology of the centromeric heterochromatin, C bands.
101 citations
TL;DR: The four satellite DNAs from calf showed a strongly clumped distribution in interphase nuclei of both confluent and growing calf kidney cells in vitro, and preferentially situated at the centromeres of the autosomes.
93 citations
TL;DR: An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum, which consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein.
81 citations
TL;DR: The results are discussed with reference to the hypothesis that the control mechanism of meiotic chromosome pairing involves centromeric microtubules of the spindle that are responsible for the positional adjustment, during the last mitotic anaphase, of potential pairing partners.
Abstract: Injection of 0.5% colchicine into immature tillers of genotypes of Triticum aestivum, T. aestivum x Aegilops mutica and T. aestivum x Secale cereale hybrids induces asynapsis at first meiotic metaphase irrespective of the homologous or homoeologous nature of the potential pairing chromosomes. The induction of asynapsis occurs at a time during and immediately following the last premeiotic mitosis of pollen mother cells. No disruption of synapsis and chiasma formation occurs in anthers having pollen mother cells originally at leptotene or immediately prior to leptotene when cultured in White9s medium plus colchicine. Tetraploid and octaploid pollen mother cells resulting from the disruption of premeiotic spindles by colchicine show pairing of chromosomes only in bivalents, in genotypes normally having a degree of multivalent pairing configurations. The induction of multipolar mitotic spindles with 0.01% colchicine results in the development of pollen mother cell mosaics with different numbers of chromosomes. Such cells show high levels of chromosome pairing, including multivalents, in some genotypes that normally have very little chromosome pairing. The injection of 0.5% chloral hydrate during the last premeiotic mitosis of the archesporium causes no disturbances of meiotic pairing. The results are discussed with reference to the hypothesis that the control mechanism of meiotic chromosome pairing involves centromeric microtubules of the spindle (not affected by chloral hydrate) that are responsible for the positional adjustment, during the last mitotic anaphase, of potential pairing partners.
80 citations
TL;DR: Human metaphase chromosomes were photooxidized in the presence of methylene blue and the chromosome banding patterns appeared to reflect DNA base composition, indicating the feasibility of a combined chemical-immunochemical investigation of the chemical organization of chromosomes.
Abstract: Human metaphase chromosomes were photooxidized in the presence of methylene blue, a process that destroys guanine residues in DNA. Indirect immunofluorescence showed that such chromosomes reacted with a cytosine-specific antibody revealing a consistent fluorescent banding pattern by which each chromosome could be identified. The observed fluorescent patterns were the reverse of those produced in formamide-denatured chromosomes treated with an antibody specific for adenine and of the patterns obtained with quinacrine and with Giemsa staining by the G-banding techniques. The patterns were identical to Giemsa R-banding patterns. The chromosome banding patterns, therefore, appeared to reflect DNA base composition, indicating the feasibility of a combined chemical-immunochemical investigation of the chemical organization of chromosomes.
74 citations
70 citations
TL;DR: A karyotypic analysis showed that this new temperature sensitive cell line from the Chinese hamster line CCL39 has lost one of the medium‐sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental.
Abstract: The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium-sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental.
In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter.
Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G1/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G1 (release from serum starvation).
Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature-sensitive phenotype.
62 citations
TL;DR: The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy and confirmed Inoué's earlier contention that D2O treatment can modify the available spindle pool.
Abstract: The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoue. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55–65 kcal/mol and +197–233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoue and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoue, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D2O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D2O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoue's earlier contention that D2O treatment can modify the available spindle pool.
TL;DR: When metaphase chromosomes prelabeled with 3H-thymidine are treated with various C-banding procedures, autoradiographs show a significant amount of DNA loss in the euchromatic segments.
Abstract: When metaphase chromosomes prelabeled with 3H-thymidine are treated with various C-banding procedures, autoradiographs show a significant amount of DNA loss in the euchromatic segments. The
TL;DR: The results suggest that the infected cell has to pass through a critical stage situated in late G(1) or early S before SV40 DNA replication can eventually be initiated.
Abstract: The relationship between replication of simian virus 40 (SV40) DNA and the various periods of the host-cell cycle was investigated in synchronized CV1 cells. Cells synchronized through a double excess thymidine procedure were infected with SV40 at the beginning or the middle of S, or in G2. The first viral progeny DNA molecules were in all instances detected approximately 20 h after release from the thymidine block, independent of the time of infection. The length of the early, prereplicative phase of the virus growth cycle therefore depended upon the period of the cell cycle at which the cells were infected. Infection with SV40 was also performed on cells obtained in early G1 through selective detachment of cells in metaphase. As long as the cells were in G1 at the time of infection, the first viral progeny DNA molecules were detected during the S period immediately following, whereas if infection took place once the cells had entered S, no progeny DNA molecule could be detected until the S period of the next cell cycle. These results suggest that the infected cell has to pass through a critical stage situated in late G1 or early S before SV40 DNA replication can eventually be initiated.
TL;DR: In this article, HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy.
Abstract: When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole Both chromosomal and interpolar (pole-to-pole) microtubules were also present Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles
TL;DR: Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period and, in cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.
TL;DR: Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures to study DNA replication in diploid Chinese hamster cells.
Abstract: Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G1 between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.
TL;DR: Electrophoresis of well washed metaphase and interphase chromatin indicated their non-histone proteins were quite similar and even though some nuclear sap proteins appear to be readily washed off the chromatin this does not mean they have no gene regulatory function.
TL;DR: Evidence from a wide range of species suggests that the production of cytoplasmic nucleoloids during microsporogenesis is a general phenomenon among angiosperms, probably linked with the rapid build-up of ribosome numbers which follows upon the period of elimination in the meiotic prophase.
Abstract: The origin of the nucleolus-like bodies (“nucleoloids”) released into the cytoplasm during the meiotic divisions in pollen mother cells ofLilium has been traced. Chains of accessory nucleoli are formed at the nucleolus organising regions (NOR) of the nucleolar chromosomes during pachytene and diplotene while the parent-cell nucleolus is undergoing dissolution. Autoradiography using3H-uridine as a tracer shows that this involves the resumption of RNA synthesis at the NOR, although no new synthesis is associated with the parent-cell nucleolus. The accessory nucleoli are released from the NOR to become distributed throughout the nucleus in late prophase; there is no evidence that they contain DNA. In division phases, their material is probably held at the chromosome surfaces as part of the metaphase sheath. After the divisions, globuli are re-formed, and these eventually appear as the nucleoloids after detachment into the cytoplasm. It seems improbable that a gene amplification phase is associated with accessory nucleolus or nucleoloid formation. Evidence from a wide range of species suggests that the production of cytoplasmic nucleoloids during microsporogenesis is a general phenomenon among angiosperms, probably linked with the rapid build-up of ribosome numbers which follows upon the period of elimination in the meiotic prophase.
TL;DR: This chapter discusses new staining methods for chromosomes, and a new compound was found after a systematic search for agents that would stain adjacent regions of chromosomes differentially.
Abstract: Publisher Summary This chapter discusses new staining methods for chromosomes. Virtually every aspect of mammalian cytogenetics has been altered by the development of simple techniques for the differential staining of metaphase chromosomes. The ability to identify each chromosome, as well as small homolog differences, has brought cytogenetics to the brink of an exciting new phase. The difficulties encountered by many laboratories in obtaining consistent, satisfactory banding with these techniques have led to frustration and delay in the realization of much of this potential and have prompted this review. This compound was found after a systematic search for agents that would stain adjacent regions of chromosomes differentially. Differential staining along the arms as well as the centromeres was noted in the later experiments.
TL;DR: Methods are currently being explored which would permit the application of cytogenetic procedures such as metaphase and anaphase analyses to in vivo mammalian systems in order that due consideration may be given to metabolic activation or detoxification reactions which might affect the mutagenic or clastogenic potential of environmental agents under study.
Abstract: Methods are currently being explored which would permit the application of cytogenetic procedures such as metaphase and anaphase analyses to in vivo mammalian systems in order that due consideration may be given to metabolic activation or detoxification reactions which might affect the mutagenic or clastogenic (chromosome breaking) potential of environmental agents under study. It has been recommended (1) that such in vivo cytogenetic methods be employed in mutagenicity testingf programs which are being designed to evaluate mutagenic potentials and riskbenefit ratios of a wide variety of drugs and other chemical agents when human exposure is involved. Metaphase chromosome analysis has been adapted readily to in vivo testing of chemical mutagens (2-5). More recently, Palmer, Kelly-Garvert, and Legator (6) have described a potentially useful method for in vivo collection of anaphase figures from rat bone marrow cells which involves the application of a mitotic arresting agent (colcemide, CIBA, New Jersey) in vivo, followed by a short-term in vitro incubation (lll/' hr) in the absence of colcemide to reconstitute spindle fibers and initiate anaphase movement. Preliminary studies with the in vivo anaphase method in our laboratory have revealed a number of technical limita-
Journal Article•
TL;DR: The quinacrine fluorescence technique permits far more detailed characterization and comparison of heteroploid cell lines than any previous method, and A9HT, although it has a smaller average number of chromosomes per cell, appears to be more heterogeneous.
Abstract: SUMMARY The fluorescent banding patterns of quinacrine-stained metaphase chromosomes have been studied in 2 related mouse cell lines, Ao. and a malignant derivative of A9, A9HT. In both cell lines virtually every chromosome has a distinctive banding pattern which permits its recognition. More than three quarters of the chromosomes have structural rearrangements, but the origin of nearly two thirds of the chromosomes could be determined by their banding patterns. The quinacrine fluorescence technique permits far more detailed characterization and comparison of heteroploid cell lines than any previous method. A9 and A9HT are karyologically quite similar, with many of the same marker chromosomes. There are, however, characteristic differences. A9HT, although it has a smaller average number of chromosomes per cell, appears to be more heterogeneous.
TL;DR: A new modification of the commonly used air-drying technique for spreading chromosomes was employed using bone marrow cells and identified the heteropycnotic X-chromosome with high stainability in female mice, which was not observed in males.
TL;DR: The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy.
Abstract: The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy. The HS-β satellite DNA is located at the kinetochores of all but three chromosome pairs. The HD satellite is located predominantly in the short arms of the chromosomes containing HS-β and in the kinetochores of chromosome pairs that lack HS-β. The regions that contain the satellite DNA sequences can also be identified by the Giemsa staining technique, and can be visualized with phase contrast microscopy or following Feulgen staining of fixed chromosome preparations.
TL;DR: The C9 bodies observed in interphase with this cytological procedure are frequently associated with the nucleolus and this heterochromatin is less compact than previously shown and may represent repetitive DNA with a specific role.
Abstract: Using a specific technique which stains the secondary constriction of number 9 chromosomes in man, we observed that this segment frequently appears to be composed of multiple small units packed together in metaphase as well as in interphase. During prophase of the first meiotic division in man, this segment looks like a diffuse structure, suggesting division and multiplication of these units. On the other hand, the C9 bodies observed in interphase with this cytological procedure are frequently associated with the nucleolus. This heterochromatin is less compact than previously shown and may represent repetitive DNA with a specific role.
TL;DR: The prematurely condensed chromosomes are considerably longer and thinner than the regular metaphase chromosomes and their morphology reflects the stage of the cell cycle of this interphase nucleus; single chromatid for G1, double chromatids for G2 and unevenly condensed chromatin for S.
Abstract: WHEN two mammalian cells fuse and one of the partners is in mitosis while the other is in interphase, the former will induce premature chromosome condensation (PCC) of the latter1. The prematurely condensed chromosomes are considerably longer and thinner than the regular metaphase chromosomes and their morphology reflects the stage of the cell cycle of this interphase nucleus; single chromatid for G1, double chromatids for G2 and unevenly condensed chromatin for S.
Journal Article•
TL;DR: In this paper, the authors examined the rates of chromosome and pole motion during anaphase in HeLa cells using differential interference contrast and polarization optics, and they found that the mechanism of chromosome motion changes character when the chromosomes become spaced by the metaphase spindle length.
TL;DR: In this paper, the authors examined the rates of chromosome and pole motion during anaphase in HeLa cells using differential interference contrast and polarization optics, and they found that the mechanism of chromosome motion changes character when the chromosomes become spaced by the metaphase spindle length.
TL;DR: Mitosis in zoosporangia of the chytrid Phlyctochytrium irregulare is described from electron microscope observations and also from light microscope observations of both living and haematoxylin-stained thalli.
Abstract: Mitosis in zoosporangia of the chytrid Phlyctochytrium irregulare is described from electron microscope observations and also from light microscope observations of both living and haematoxylin-stained thalli. At the onset of prophase the centriole complex replicates, and the complexes migrate to polar positions. The semi-persistent nucleolus is appressed to the nuclear envelope as the nuclear pockets invaginate, finally rupturing to create polar fenestrae, through which spindle microtubules penetrate the nucleus from the region of the centrioles at prometaphase. Metaphase chromosomes form an equatorial plate. Initial separation at anaphase seems to be accomplished mainly by shortening of chromosome-to-pole microtubules; additional anaphase and telophase separation is accomplished by elongation of the nucleus. A system of perinuclear endoplasmic reticulum is formed during prophase and is completed by metaphase. It persists during all division stages after its formation. Features of this mitotic apparatus a...
TL;DR: Late 3H-thymidine incorporation patterns and quinacrine fluorescence banding patterns were analyzed in metaphase chromosomes prepared from human blood cultures and, in general, late-labeling regions correspond to the strongly fluorescent bands in the chromosomes.
Abstract: Late 3H-thymidine incorporation patterns and quinacrine fluorescence banding patterns were analyzed in metaphase chromosomes prepared from human blood cultures. In general, late-labeling regions correspond to the strongly fluorescent bands in the chromosomes. However, the dully fluorescent secondary constrictions of the chromosomes Nos. 1, 9 and 16 may show late replication in some instances. In contrast, the brilliantly fluorescent distal part of the Y chromosome is not labeled during the latest phase of the DNA replication. In the male X and in the isopycnotic X of the female the labeling pattern also agrees with the quinacrine fluorescence banding. The heteropycnotic X of the female is still more strikingly late-labeling. However, the pattern of its late replication agrees also with the quinacrine fluorescence bands.
TL;DR: In situ denaturation of metaphase chromosomes with alkali results in a shift from green to green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasingDenaturation of chromosomal DNA.
Abstract: In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.