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.     

Relationship between relative dry mass and average band width in regions of polytene chromosomes of Drosophila

Whole-mounted polytene chromosomes from Drosophila melanogaster were prepared for high-voltage electron microscopy. Relative dry mass of chromosome regions was estimated by densitometry of electron microscopic negatives. Comparison of dry mass of regions of the male X chromosome with that of regions of associated autosomes established that dry mass values are proportional to DNA content. Relative dry mass values of regions of polytene chromosomes from salivary glands, fat body, and malpighian tubules were correlated with the average diameter of bands in these regions: as mass doubled, band width increased by a factor of approximately 2. To provide a standard for estimating absolute levels of polyteny, band widths were measured for chromosomes representing one major polytene class, 256n. These chromosomes were observed to have an average band width of 0.9 m — These observations provide limits to models of chromatin organization in bands. For each chromatid, this area can accommodate up to five chromatin fibers of 250 Å diameter. This value may represent the extent of folding of a chromatin fiber in an average band. Alternatively, a chromatin fiber of higher-order structure could have a maximum diameter of 560 Å in an average band.     

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

The arrangement of chromosomes in nuclei of sperm from plethodontid salamanders

Plethodontid salamanders have n = 13 or 14 large metacentric or sub-metacentric chromosomes. Sperm nuclei from Plethodon cinereus measure 72×1 m. The nucleoprotein of spermatids is at first finely granular. In elongate spermatids it clumps into larger granules, which then fuse to form the compact nucleoprotein of the mature sperm. The nuclei of mature sperm are negatively birefringent with respect to their length. — ³H RNA complementary to high-density satellite DNA of centromeric heterochromatin in P. cinereus has been hybridized in-situ to spermatids and sperm, and its site of binding to these cells has been examined by autoradiography. Labelling of round spermatid nuclei is localized in a single patch. Elongate spermatid nuclei are labelled only over the rear quarter of the nucleus. Label over the nuclei of mature sperm is localized in a region extending 10–20 m forwards from the rear of the nucleus. — In P. cinereus the ribosomal genes are located near the centromere on the short arm of chromosome 7. ³H ribosomal RNA hybridizes to a single patch in round spermatid nuclei. Elongate spermatid nuclei show label over a short segment of the rear half of the nucleus. In spermatids nearing maturity the labelled region is never more than 20 m long. — These results indicate that in P. cinereus each chromosome is arranged in a U formation with its centromere at the base of the sperm nucleus, and its arms extended forwards along the length of the nucleus. — Among plethodontids, increase in C value and corresponding increase in chromosome size is accompanied by increase in the length rather than the width of the sperm nucleus. — ³H ribosomal RNA hybridizes to a short segment in spermatid and sperm nuclei from Xenopus and Triturus. In these animals, the position of the labelled segment varies from sperm to sperm.     

The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting

The speed and ease of human cytogenetic analysis has been greatly enhanced by the technique of fluorescence in situ hybridization (FISH). Non-radioactive fluorescently tagged complex DNA probes specific for individual chromosomes can be hybridized to conventionally obtained metaphase chromosome spreads. Several chromosomes may be painted concurrently by using combinations of different labeled probes. Surveys of chromosome breakage and rearrangement may be performed very quickly by avoiding the time consuming process of GTG-banding. The application of FISH to mouse cytogenetics would allow large scale molecular toxicology studies to be conducted on the effects of such environmental insults as potential carcinogens, mutagens and radiation. Progress has been hampered, however, as the Mus musculus karyotype consists of 40 acrocentric chromosomes of approximately the same size, making the recognition and separation of individual chromosomes very difficult. We now describe the successful production and application of chromosome-specific composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robertsonian translocated chromosomes were isolated on a flow sorter and their DNA subsequently amplified by degenerate oligonucleotide primer (DOP) PCR. Small pools (300 copies) of each chromosome were denatured at 94° C then annealed with the primer at 30°C for 15 cycles. This was followed by 20 cycles at an annealing temperature of 62° C. Additional amplification was performed at an annealing temperature of 62° C. The chromosome-specific DNA was labeled with biotin 11-dUTP by nick translation and used for FISH. The usefulness of the technique for translocation detection is demonstrated by analyzing chromosome exchanges induced in mice irradiated with ¹³⁷Cs rays.     

Transgene Delivery with a Cationic Lipid in the Presence of Amyloid β (βAP) Peptide

The ability of a cationic lipid to deliver plasmid DNA (pDNA) in presence of the neurotoxic fragment of amyloid -peptide was evaluated. Pre-treatment of cells with AP (25–35) peptide resulted in a modest increase in transgene expression. When AP (25–35) peptide was mixed with the pDNA/liposome complex and used, the complexes lost their ability to transfect. However, the reverse sequenced AP (35–25) peptide demonstrated no significant differences in transgene expression in pre-treated cells, and in cells where AP (35–25) peptide was mixed with pDNA/liposome complexes and transfected. The amount of pDNA delivered to the cells was decreased in presence of AP (25–35) as measured with flow cytometry using fluorescently labeled liposomes. The decreased endocytosis may be due to their rod-like structure formation as demonstrated by electron microscopy and atomic force microscopy (AFM). These results demonstrate that AP (25–35) peptide may interfere with gene delivery with cationic systems.     

The DNA content of the individual chromosomes of rye

Summary The relative DNA content of individual chromosomes of Secale cereale L. was determined in 25 cells by microdensitometry of Feulgen stained preparations. The correlation value between relative DNA content and relative chromosome length was r=0.61 for all 328 chromosomes measured. However, the correlation coefficients calculated for individual cells as well as for mean values always approached 1. Taking into account the structure of rye chromosomes, this indicates that microdensitometric results may not be accurate when large quantities of heterochromatic DNA sequences are present in analyzed material.     

Structural paradox of polytene chromosomes

The observation of thick chromatin fibers in interbands of Dipteran polytene chromosomes suggests that there should be 5 to 10 times more mass and DNA in interbands than is commonly thought to be present. To resolve this paradox, the chromatin content of interbands was estimated, using whole-mounted polytene chromosomes from Drosophila melanogster. Densitometry of high voltage electron microscopic negatives provides an estimate of less than 4:1 for the average ratio of cross-sectional dry mass (or mass per unit chromosome length) of bands relative to interbands. This ratio, combined with an estimate of the length of chromosome composed of interbands, indicates that at least 26% of chromosome mass is contributed by interband chromatin. Since DNA comprises a similar proportion of chromatin mass in bands and interbands (Laird et al., 1980b), these data imply that DNA sequences in interbands represent at least 26% of the euchromatic genome of D. melanogaster. This result calls for reinterpretation of some of the genetic and molecular data from Diptera. The discrepancy between this higher estimate of interband mass and DNA, and previous estimates of 3-5%, is discussed. One possibility is that previous measurements were made on prominent interbands, which are proposed here to be in regions that are delayed in DNA replication. Such interbands would be reduced in polyteny and DNA content compared with the average interband region. The concept of local variations in polyteny is also used here to explain major differences in the cross-sectional mass of bands. This leads to a revised model of polytene chromosomes in which at least three levels of polyteny, rather than one or two levels, can be present within one euchromatic region.     

Replication in Drosophila chromosomes

DNA fibre autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons. Type I replicons are longer (mean size=64 m), have a very high rate of fork migration (average rate=0.95 m/min) and generally occur in large arrays often extending over several thousand m. In contrast, the type II replicons are smaller (mean size= 20 m), slow replicating (average rate=0.07 m/min) and occur in short arrays containing only a few closely spaced active replicons. Evidence is presented that type I replicons are active in the early S and type II in the late S. Observations on autoradiographic labelling of partially lysed polytene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateral polytene strands; these observations also suggest local variations in levels of polyteny within a chromosome. On the basis of this and other available information on replication in polytene chromosomes the possible roles of the two replicon types in the generation of the different ³H-thymidine labelling patterns of polytene chromosomes are discussed.We take pleasure in dedicating this paper to our inspiring teacher Prof. S.P. Ray Chaudhuri on his completing 75 years of fruitful life     

Chromosomal unit fibers in Drosophila

Chromosomal unit fibers consisting of long, regular fibers of about 0.40 m diameter were obtained from disintegrated, isolated chromosomes of two Drosophila melanogaster cell lines. In one cell line with an essentially normal karyotype, three clearly defined size classes of 15, 13, and 11 m length were observed corresponding to the three larger chromosomes of Drosophila. In a cell line carrying an additional translocation between the two largest chromosomes a 19 m fiber derived from the translocation chromosome was observed. Direct determinations of the DNA content per m length of Drosophila unit fibers show that DNA is contracted by a factor of about 1400x in agreement with calculations based on the length of the unit fibers and the known DNA content of the individual Drosophila chromosomes. These findings support our previously proposed model for the unit fiber sub-structure of chromosomes as being derived by a hierarchy of coiling with the corresponding contraction ratios being 7 (100 Å string of nucleosomes), 5 to 6 (250–300 Å thick nucleohistone fiber), and about 40 (unit fiber), resulting in a total contraction of DNA in unit fibers in the order of 1400x.     

Incidence of chromosome 18 disomy in human sperm nuclei as detected by nonisotopic in situ hybridization

Nonradioactive in situ hybridization with an -satellite DNA probe specific for chromosome 18 was performed on human interphase sperm nuclei to detect the frequency of sperm cells disomic for chromosome 18. A total of 16127 sperm heads from eight healthy donors, aged 23–57 years, was investigated, and a minimum of 2000 sperm nuclei per proband was analyzed. The disomy rate ranged from 0.25% to 0.5%, with an average of 0.36%. This frequency does not differ significantly from that determined for other chromosomes.     

Karyotyp und DNS-Gehalt von Bufo bufo,B. viridis,B. bufo × B. viridis und B. calamita (Amphibia,Anura)

The karyotypes in spermatogonial and leukocyte metaphases of the toads Bufo bufo, B. viridis and B. calamita (all 2n=22) were analysed and the DNA content of colchicine treated and Feulgen stained spermatogonial metaphase chromosomes measured microspectrophotometrically. The toad species possess similar karyotypes, but the chromosomes of B. bufo are somewhat longer than the chromosomes of B. viridis and B. calamita. All chromosomes of B. bufo contain significantly more than, but in no case twice as much DNA as their homologues in the other two species. Eight chromosomes of B. bufo contain 30–40%, three about 50% more DNA than their homologues in B. viridis. Exactly the same DNA-differences between both sets of chromosomes were found in B. bufo × B. viridis hybrids. Significant differences in the DNA amount of B. viridis and B. calamita exist only between the large chromosomes of these species. The ratio of the total DNA amount of the genomes in the three species is 1.49∶1.07∶1. These DNA-differences between the three toad species are confirmed by microspectrophotometric DNA measurements of their erythrocyte nuclei. It is supposed that these interspecific differences in DNA content of the toads are not a consequence of differential polyteny but are caused during the evolution process by local increase in DNA in all chromosomes of B. bufo and in the large chromosomes of B. viridis.     

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes

Summary Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (XqterXp22.2::Yq11Y qter) contained in the Chinese hamster x human hybrid cell line 445 x 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.     

A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

Weitere Untersuchungen über Chromosomenverhältnisse und DNS-Gehalt bei Anuren (Amphibia)

The karyotypes of the toad Bufo marinus L. (2n=22) and the frogs Limnodynastes tasmaniensis Gthr. (2n=24), Rana temporaria L., R. esculenta L. (both 2n=26) and R. arvalis Nills. (2n=24) were analysed in colchicine treated leukocyte and spermatogonial metaphases and/or embryonic and larval mitoses. The DNA content of Feulgen stained erythrocyte nuclei was measured microspectrophotometrically. Heteromorphic sex chromosomes are absent in all species. L. tasmaniensis has the lowest DNA content among these species. The south American toad B. marinus shows a karyotype similar to the other known toad species and contains the same amount of DNA as the European species B. calamita with the lowest DNA amount among the European toads. In southern German populations of R. temporaria besides animals with the “standard”-karyotype (2n=26) individuals with 1 or 2, in rare cases with 3 or 4 supernumerary chromosomes have been found. The supernumeraries are heterochromatic and smaller than the smallest chromosome of the “standard”-karyotype. If only 1 or 2 supernumerary chromosomes are present, they seem to show normal mendelian inheritance as a rule. The observation of a few tadpoles with intraindividual different numbers of supernumeraries points to the occurrence of unequal distribution of these chromosomes in individuals containing a higher number of supernumerary chromosomes. The karyotype of R. esculenta is very similar to the “standard”-karyotype of R. temporaria, but the chromosomes of R. esculenta are somewhat longer than those of R. temporaria. R. esculenta contains about 54% more DNA than R. temporaria in the erythrocyte nuclei, so that it must be assumed that all chromosomes of R. esculenta contain more DNA than their homologues in R. temporaria. R. arvalis possesses about 28% more DNA than R. temporaria. It is supposed that these interspecific differences in DNA content of the Rana species — as observed earlier in Bufo species — are not a consequence of differential polyteny but are caused during evolutionary processes by local increase in DNA in the chromosomes of R. esculenta and R. arvalis.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK₁ cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In early restoration cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK₁ cells in g₁ and those in G₂/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1–2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.     

Local variation in diapause characteristics of Tetranychus urticae Koch (Acarina: Tetranychidae)

Summary Diapause characteristics of nine local populations of Tetranychus urticae Koch (Acarina: Tetranychidae) occurring on rose in central Japan were investigated. The percentage of females that entered diapause at 18° C and 9L: 15D photoperiod ranged between 0 and 77%. In addition, both photoperiodic and temperature responses varied widely among these populations. We suggest that temperature response might be a better trait than photoperiodic response for selection among local populations. Females in diapause survived –24° C better than those that were not. However, no difference in cold hardiness among these populations was found. The mite responded rapidly to artificial selection for low and high diapause percentage. The response was asymmetrical, being easier in the direction of low diapause percentage. Reciprocal and back crosses using selected low and high diapause percentage cultures showed that the genetic control of diapause was complicated. Neither the lowdiapause nor the high-diapause trait dominated over the other. However, the low-diapause trait seemed to be stronger. The results of this study suggest that variation in diapause response is maintained by (1) adaptation to local environments and (2) the complexity of the genetics of diapause. Such variation may provide the genetic raw material for natural selection and is a prerequisite for the extension of ecological range.     

Estimation of the sizes of polymorphic C-bands in man by measurement of DNA content of whole chromosomes.

A method is proposed for estimating the sizes of polymorphic C-bands of human chromosomes by microdensitometric measurement of the DNA content of whole chromosomes. The method requires measurements of: a chromosome known not to by polymorphic, as a standard against which to normalize all measurements; the polymorphic chromosomes of interest; and a homologous polymorphic chromosome lacking the C-band. In an example studies here, the C-band of chromosome 9 was estimated to contain about 0.0296 pg of DNA.     

Complete characterization of a large marker chromosome by reverse and forward chromosome painting

Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the degenerate oligonucleotide primed — polymerase chain reaction, we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (reverse chromosome painting) and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome (forward chromosome painting) and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.