Characterization of Drosophila heterochromatin

The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.     

Hoechst 33258 fluorescent staining of Drosophila chromosomes

Metaphase chromosomes of D. melanogaster, D. virilis and D. eopydei were sequentilly 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, Drosophia'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 by 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 differtiated 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 been seen in colcemide 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 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 a single DNA molecule which is one heptanucleotide repeated two million times.     

Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Molecular basis of chromosome banding

The effects of mouse satellite, main band and total DNA on the fluorescence intensity of quinacrine and of the bibenzimidazole derivative Hoechst 33258 were tested in solution. No significant differences were noticed between the double-stranded DNAs in spite of the 5% difference in AT-content between satellite and main band DNA. Single-stranded DNAs enhanced the fluorescence intensity of Hoechst 33258 far less than double-stranded DNAs. Having been denaturated and then reassociated the DNA fractions were intermediate in their enhancing effects on the fluorescence intensity of Hoechst 33258, the differences presumably being due to different degrees of reassociation. The effect of denatured and subsequently reassociated satellite DNA on the fluorescence intensity of quinacrine was similar to that of the native DNAs. Main band and total DNA quenched the fluorescence intensity of quinacrine more after denaturation-reassociation than it did when native. In the discussion the results are related to known cytological data.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

鱼类染色体的荧光显带研究

应用GC碱基特异性荧光染料色霉素A,辅以AT减基特异性荧光染料Hoechst33258,DAPI或喹吖因对鲤鱼,鲫鱼,大鳞副泥鳅和的有丝分裂染色体及黄鳝的有丝分裂和减数分裂染色体进行了荧光显带研究,结果发现,色霉素A3可以特异性地显示鱼类有丝分裂及减数分裂各个时期核仁组织区NORS的存在,Hoechst33258,DAPI或喹吖因则使这些区域（NORs)淡染,大鳞副泥鳞的染色体NORs 分布位置具有性别,根据实验结果,对有关鱼类染色体的荧光染色研究及其应用进行了讨论。     

Identical satellite DNA sequences in sibling species of Drosophila

The evolution of simple satellite DNAs was examined by DNA-DNA hybridization of ten Drosophila melanogaster satellite sequences to DNAs of the sibling species, Drosophila simulans and Drosophila erecta. Seven of these repeat types are present in tandem arrays in D. simulans and each of the ten sequences is repeated in D. erecta. In thermal melts, six of the seven satellite sequences in D. simulans and seven of the ten sequences in D. erecta melted within 1 deg.C of the corresponding values in D. melanogaster. The remaining sequences melted within 3 deg.C of the homologous hybrids. Therefore, there is little or no alteration in those satellite sequences held in common, despite a period of about ten million years since the divergence of D. melanogaster and D. simulans from a common ancestor. Simple satellite sequences appear to be more highly conserved than coding regions of the genome, on a per nucleotide basis. Since multiple copies of three satellite sequences could not be detected in D. simulans yet are present in D. erecta, a species more distantly related to D. melanogaster than is D. simulans, these sequences show discontinuities in evolution. There were major quantitative variations between species, showing that satellite DNAs are prone to massive amplification or diminution events over timespans as short as those separating sibling species. In D. melanogaster, these sequences amount to 21% of the genome but only 5% in D. simulans and 0.4% in D. erecta. There was a general trend of lower abundance with evolutionary distance for most satellites, suggesting that the amounts of different satellite sequences do not vary independently during evolution.     

Heterochromatin accumulation,disposition and diversity in Gibasis karwinskyana (Commelinaceae)

C-banding differences within Gibasis karwinskyana (Roem & Schult.) Rohw. were reassessed using dual fluorochrome staining. Pronounced differences in C-band pattern between two subspecies with identical basic karyotypes were due to different chromosomal locations of AT-rich and GC-rich heterochromatin. The AT-rich component had an equilocal distribution in the karyotype and has evidently been accumulated at telomeres, as shown by its prevalence in supernumerary segments and B chromosomes. The GC-rich component also varied in amount, but was limited to nucleolus organizing regions (NORs) and centromeres. Centromeres and telomeres are suggested to constitute separate, although perhaps interdependent, centres of heterochromatin amplification. The possible role of nuclear architecture in determining the accumulation, distribution and spread of these sequences is discussed.Abbreviations H Hoechst 33258 - CMA chromomycin A3 - NOR nucleolus organizing region - SS supernumerary segment - Q quinacrine dihydrochloride - H⁺ H etc. indicate enhanced (+) and quenched (-) fluorescence with the stated fluorochrome by H.C. Macgregor     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Satellite Ic: a possible link between the satellite DNAs of D. virilis and D. melanogaster.

In this study, we isolated and characterized a previously undetected cryptic satellite DNA comprising 0.1% of the total nuclear genome of D. virilis. This satellite is hidden from detection in neutral CsCl by satellite I and is therefore designated cryptic satellite I or Ic. Sequence analysis reveals that Ic is the repeating heptanucleotide [poly d(AATATAG): d(CTATATT)]. It is more closely related to the three simple sequence satellite DNAs of D. melanogaster, a distantly related species, than it is to any of the major D. virilis satellite DNA sequences. Ic may therefore be a link between the simple sequence satellites of D. virilis and D. melanogaster. As an extension of this theory, we have constructed a "family tree" linking the satellites of D. virilis and D. melanogaster by a series of "simple" operations. Only one intermediate required by this evolutionary scheme has not yet been identified.     

Cytological differentiation of constitutive heterochromatin

The constitutive heterochromatin, as demonstrated by the C band technique, may be subdivided into a number of categories when other characteristics are considered. The responses to fluorochromes QM and 33258 Hoechst, the behavior following G band staining, the repetitive DNA content, and many other criteria are useful for the classification of heterochromatin. The heterochromatin patterns of three mammalian species are presented to demonstrate that within each karyotype there may be several different types of C bands. In general, a correlation may also be made between GC-rich satellite DNA and dull (or negative) Q fluorescence, and between AT-rich satellite DNA and bright Q, fluorescence.On Sabbatical leave from Department of Biology, University of North Dakota, Grand Forks, North Dakota.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.     

Circular DNA Molecules in the Genus Drosophila

The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules.     

The effect of distamycin A on heterochromatin condensation of Drosophila chromosomes

Larval neuroblasts of four species of Drosophila (melanogaster, hydei, virilis and funebris) were treated with distamycin A, a DNA ligand which induces distinct undercondensation in AT-rich heterochromatin. For each species the patterns of undercondensation were correlated with distribution of quinacrine-bright regions and of satellite DNAs. An overlapping of distamycin A-sensitive and quinacrine-bright heterochromatic regions was demonstrated for D. melanogaster and D. virilis, but not for D. hydei and D. funebris. Distamycin A undercondensation is thus a further criterion for resolving heterochromatin into different parts and enables identification of the steps of the condensation process within heterochromatic regions.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Fine Mapping of Satellite DNA Sequences along the Y Chromosome of Drosophila Melanogaster: Relationships between Satellite Sequences and Fertility Factors

The entirely heterochromatic Y chromosome of Drosophila melanogaster contains a series of simple sequence satellite DNAs which together account for about 80% of its length. Molecular cloning of the three simple sequence satellite DNAs of D. melanogaster (1.672, 1.686 and 1.705 g/ml) revealed that each satellite comprises several distinct repeat sequences. Together 11 related sequences were identified and 9 of them were shown to be located on the Y chromosome. In the present study we have finely mapped 8 of these sequences along the Y by in situ hybridization on mitotic chromosome preparations. The hybridization experiments were performed on a series of cytologically determined rearrangements involving the Y chromosome. The breakpoints of these rearrangements provided an array of landmarks along the Y which have been used to localize each sequence on the various heterochromatic blocks defined by Hoechst and N-banding techniques. The results of this analysis indicate a good correlation between the N-banded regions and 1.705 repeats and between the Hoechst-bright regions and the 1.672 repeats. However, the molecular basis for banding does not appear to depend exclusively on DNA content, since heterochromatic blocks showing identical banding patterns often contain different combinations of satellite repeats. The distribution of satellite repeats has also been analyzed with respect to the male fertility factors of the Y chromosome. Both loop-forming (kl-5, kl-3 and ks-1) and non-loop-forming (kl-2 and ks-2) fertility genes contain substantial amounts of satellite DNAs. Moreover, each fertility region is characterized by a specific combination of satellite sequences rather than by an homogeneous array of a single type of repeat.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.