The effect of clupein on anisotropy and basophilia of polytene chromosomes.

When polytene chromosomes are subjected to a clupein treatment, their properties of basophilia and anisotropy are affected. The basophilia is deeply reduced, except in the nucleolar zones, puffs and sites of RNA accumulation. On the other hand, the chromosome birefringence increases. The phenomenon of anomalous dispersion of birefringence usually observed on polytene chromosomes stained with toluidine blue solutions turns into a normal negative dispersion of birefringence, when staining is preceded by clupein treatment. It is concluded that the clupein molecules attach orderly and preferentially to sequential DNA phosphates unbound to chromosome proteins, accentuating DNA anisotropic characteristics. The clupein molecules appear not attaching to RNA phosphates.     

Positive and negative birefringence in chromosomes

By using the optical properties of birefringence of DNA, the arrangement of these molecules has been studied in Dinoflagellate chromosomes and Dipteran polytene chromosomes. These latter are used, here, as a reference material. These observations have been made under a polarizing microscope on intact and stretched chromosomes. — Intact Dinoflagellate chromosomes show a positive birefringence, in contrast with polytene chromosomes bands which are negatively birefringent. From these observations one can deduce the preferential orientation of DNA filaments, in Dinoflagellates, normal to the chromosome axis, and in polytene chromosomes parallel to the same axis. — After stretching, these two kinds of chromosomes are negatively birefringent. In both cases, DNA molecules have been aligned along the stretch axis. — In Dinoflagellate chromosomes the passage from a positive to a negative birefringence is realized without any isotropic stage. The intermediary state presents a biaxial structure.     

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (2⁶–2⁷ chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 Å in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.     

Transcription of intercalary heterochromatin regions in Drosophila melanogaster cell culture

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of D. melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to the chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologue only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al., 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in a cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "'asymmetrical" type of label distribution is observed after hybridization with cell RNA. - These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.     

A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Dynamic inorganic ion-protein interactions in structural organization of DNA of living cell nuclei.

Staining polarization optical techniques showed differences in the structural organization of DNA of chromatin in interphase nuclei and in mitotic chromosomes. The DNA was non-birefringent in intact interphase cell nuclei, but birefringent in chromosomes and in isolated nuclei incubated in a physiological electrolyte solution. The birefringence of DNA appears to be related to an unfolding of DNA filaments induced by free cations and to the oriented binding of dye molecules to DNA phosphates. We propose that the actual concentration of free cations inside the living cell nuclei is regulated by a dynamic interaction between nuclear proteins and ions.     

Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.     

Replication of heterochromatin and structure of polytene chromosomes

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.     

Double-stranded DNA with the size expected of replicons can be released from Chironomus polytene chromosomes

The effect of the drug 5-fluorodeoxyuridine on DNA synthesis in Chironomus polytene chromosomes was investigated. The DNA was labelled by injection of radioactive precursor into living animals pre-treated with the drug, extracted with a neutral non-denaturing buffer at 25 ° C and then characterized by gel electrophoresis. After a short pulse a heterogeneous double-stranded DNA population is released from the polytene chromosome. These fragments are later joined together to produce a double-stranded DNA with a size ranging between 8–13 × 10⁶ D. The release of both types of fragments from the polytene chromosome is prevented by lysing the cells at 0 ° C instead of at 25 ° C. The larger double-stranded DNA has the size expected of replicons in Chironomus. The results are discussed in relation to the organization of replication in polytene chromosomes.     

Fluorescence microscopic and autoradiographic analyses of the differential reaction of various regions of polytene chromosomes in Chironomus to prolonged exposure to 7-amino-actinomycin D and 3H-actinomycin D

Dynamics of binding of a fluorescent analogue of actinomycin D -- 7-amino-actinomycin D -- and 3H-actinomycin D with polytene chromosomes of Ch. thummi was studied. Biological effects of AMD, 7-amino-AMD, and 3H-AMD on polytene chromosomes were found to be similar. These ligands provoke the reduction of the nucleolus and Balbiani rings and the appearance of giant pseudo-puffs in heterochromatic centromere regions of polytene chromosomes. There was no intermediate binding of 7-amino-AMD to DNA in vivo both after a longterm treatment of larvae with fluorochrome and in chase experiments. It was found that a loosening of chromatin in centromere regions accompanied by a weakening of its fluorescence took place in the formation of pseudo-puffs. Possible mechanisms of pseudo-puff formation under the influence of AMD and 7-amino-AMD are discussed. Essential factors may be peculiarities of DNA nucleotide composition in centromere regions, DNA packing, alteration of physico-chemical properties of DNA in the complex with AMD (despiralizations and elongation), and an inhibition of RNA synthesis necessary for the maintenance of normal structure of polytene chromosomes.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Evidence for the uninemy of eukaryotic chromatids

Polytene chromosomes of Chironomus thummi were stretched to their rupture in a pronase solution. A 176±26-fold elongation was achieved. The DNA compaction ratio, defined as the ratio of DNA length in a haploid set (85±5 mm) to the length of a polytene chromosome set (520±40 m), was 164±22. Closeness of these two values demonstrates the uninemy of the chromatids of Chironomus chromosomes. The effect of ethidium bromide on the elastic properties of chromosomes prestretched in a pronase solution and the lengthening of these chromosomes after ethidium staining suggest that DNA molecules are double-stranded and supercoiled to the moment of the chromosome rupture. It is concluded that a Chironomus chromatid consists of a single DNA molecule (or of a single chain of linked DNA molecules) both ends of which are located in the telomeres.To the memory of Prof. Vera V. Khvostova     

Large scale synchronous mating and the study of macronuclear development in Euplotes crassus

After conjugation in hypotrichous ciliates, a new macronucleus is produced from a copy of the micronucleus. This transformation involves large-scale reorganization of DNA, with conversion of the chromosomal micronuclear genome into short, gene-sized DNA molecules in the macronucleus. To study directly the changes that occur during this process, we have developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus. Electron microscope studies show that the micronuclear chromosomes are polytenized during the first 20 h of macronuclear development. The polytene chromosomes lack the band-interband organization observed in other hypotrichs and in the Diptera. Polytenization is followed by transectioning of the chromosomes. We isolated DNA at various times of macronuclear development and found that the average molecular weight of the DNA decreases at the time of chromosome transectioning. In addition, we have shown that a small size group of macronuclear DNA molecules (450-550 base pairs) is excised from the chromosomal DNA approximately 10 h later in macronuclear development.     

Polyteny and endomitosis in supergiant trophoblast cells of the gray vole Microtus subarvalis

A cytomorphological study was made of peculiarly structured polytene chromosomes in supergiant trophoblast cells of Microtus subarvalis. The polyteny level was extremely high (over 1024C). The polytene chromosomes are characterized by a rather high degree of condensation of single chromosomes, and, as a consequence, close chromosome junctions and the typical disk pattern are lacking. The presence of complex nucleoli in the nuclei of these cells also testifies to a great detachment of chromonemes in polytene chromosomes of the studied supergiant trophoblast cells. Compared to other rodent species, a lower degree of chromoneme junction in the vole polytene chromosomes may cause their easy dissociation into single chromonemata, whose further condensation results in endomitotic chromosome formation. The chromosome depolytenization, earlier suggested from the analysis of interphase nucleus markers, has been traced here in detail. The process of polytene chromosome splitting was most obvious in the nucleolus-organizing chromosomes. A hony-combed nucleolus splits into numerous micronucleoli. The nucleus pattern becomes altered. Once in the polytene nucleus, chromosome bundles were located below the nuclear membrane and the central zone of the karyoplasm was not completely filled up. However, after dissociation of polytene chromosomes the whole karyoplasm was filled up with small nucleoli, and a thin layer of endomitotic chromosomes was seen beneath the nuclear membrane. The correlation between endomitosis and polyteny is discussed in terms of the dissociation of polytene chromosomes and formation of endomitotic chromosomes.