Cytological and genetic analysis of the Y chromosome of Drosophila melanogaster

By applying quinacrine-, Hoechst- and N-banding techniques to neuroblast prometaphase chromosomes the Y chromosome of Drosophila melanogaster can be differentiated into 25 regions defined by the degree of fluorescence, the stainability after N-banding and the presence of constrictions. Thus these banding techniques provide an array of cytological landmarks along the Y chromosome that makes it comparable to a polytene chromosome for cytogenetic analysis. — 206 Y-autosome translocations (half of them carrying Y-linked sterile mutations) and 24 sterile y ⁺ Y chromosomes were carefully characterized by these banding techniques and used in extensive complementation analyses. The results of these experiments showed that: (1) there are four linearly ordered fertility factors in Y ^L and two fertility factors in Y ^S . (2) These fertility factors map to characteristic regions of the Y chromosome, specifically stained with the N-banding procedure. (3) The most extensively analyzed fertility factors are defined by a series of cytologically non-overlapping and genetically noncomplementing breaks and deficiencies distributed over large chromosome regions. For example, the breakpoints which inactivate the kl-5 and ks-1 loci are scattered along regions that contain about 3,000 kilobases (kb) DNA. Since these enormous regions formally define single genetic functions, the fertility genes of the Y chromosome have an as yet unappreciated physical dimension, being larger than euchromatic genes by two orders of magnitude.     

Meiosis in Male DROSOPHILA MELANOGASTER. II. Nonrandom Segregation of Compound-Second Chromosomes

The segregation of compound-second chromosomes in males from two different stocks has been examined. Segregation is random in males from the C(2L)RM4, dp; C(2R)RM4, px stock. Gametes containing only one of the two compound chromosomes comprise 50% of the gametes, and gametes containing either both elements or neither element make up the other 50% of the gametes.——In males from the C(2L)RM, b; C(2R)RM, cn stock, gametes containing either C(2L)RM, b or C(2R)RM, cn make up the majority of the gametes. Gametes containing both chromosomes or neither chromosome account for only 2-3% of the gametes. The nonrandom segregation is due to the C(2R)RM, cn chromosome.——Viability is reduced in flies carrying the C(2R)RM, cn chromosome. This includes larval lethality, delayed development and premature adult lethality. Cytologically, this chromosome contains a large duplication of 2L material, which includes material proximal to region 38 or 39. It is suggested that the viability and segregational properties associated with this chromosome are due to the duplicated 2L material.     

Mitotic Recombination in the Heterochromatin of the Sex Chromosomes of DROSOPHILA MELANOGASTER

The frequency of spontaneous and X-ray-induced mitotic recombination involving the Y chromosome has been studied in individuals with a marked Y chromosome arm and different XY compound chromosomes. The genotypes used include X chromosomes with different amounts of X heterochromatin and either or both arms of the Y chromosome attached to either side of the centromere. Individuals with two Y chromosomes have also been studied. The results show that the bulk of mitotic recombination takes place between homologous regions.     

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl‐dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C‐terminus to kleisin's N‐terminus. Our findings imply that proteolysis‐independent release of cohesin from chromatin is mediated by Wapl‐dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.     

Meiotic segregation of compound-3 chromosomes in Drosophila

Analysis of crossos between genetically marked stocks of Drosophila melanogaster showed, that the compound-3 chromosomes C(3L)RM and C(3R)RM segregate preterentially in female meiosis, and the following two types of eggs are formed predominantly: C(3L)RM; 0 and 0; C(3R)RM. In male meiosis segregation is almost random and four types of sperm are formed: 1. C(3L)RM; C(3R)RM, 2. 0; 0, 3. C(3L)RM; 0, 4. 0; C(3R)RM. The frequencies of these sperm types vary with the genotypes tested. In the stock C(3L)RM, st; C(3R)RM, p ^p, males produce 76.8% type 1 and 2, and 23.2% type 3 and 4; males of the stock C(3L)RM, ri; C(3R)RM, sr form 63.2% type 1 and 2, and 36.8% type 3 and 4.The segregational behaviour of compound-3 chromosomes found in female meiosis is expected according to the distributive pairing hypothesis. In the male however, where there is no distributive pairing, the stock-specific segregation of compound-3 chromosomes may be due to the presence of small homologous chromosome segments near the centromere which influence chromosome distribution.     

Distribution of 18+28S ribosomal genes in mammalian genomes

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

Regulation of the subcellular shuttling of Sgo1 between centromeres and chromosome arms by Aurora B-mediated phosphorylation

A minor fraction of cohesin complexes at chromosome arms is not removed by the prophase pathway, and maintained until metaphase and enriched at centromeres. Sgo1 localizes to chromosome arms from prophase to metaphase, and is indispensable for removing cohesin complexes from chromosome arms. However, it has not been established how the chromosome arm localization of Sgo1 leads to the establishment of cohesion on chromosomes. Here, we report that Aurora B kinase interacts with and phosphorylates Sgo1 in vitro and in vivo. Furthermore, the phosphorylation of Sgol by Aurora B kinase regulated the distribution of Sgo1 between centromeres and chromosome arms, and the expression of Aurora B kinase-dead mutants of Sgo1 caused mislocalization from centromeres to chromosome arms. These results suggest Aurora B kinase directly regulates the subcellular distribution of Sgo1 to facilitate the accurate separation of mitotic chromosomes     

Architecture of the Chinese hamster metaphase chromosome

The development of procedures for the isolation of unfixed metaphase chromosomes has made feasible a direct analysis of their morphology. Wholemount stereo electron microscopy was used to examine intact and partially disrupted chromosomes produced by physical shearing and extraction with salt and urea solutions. A model of chromosome architecture was developed to accommodate evidence from studies using both light and electron microscopy. In the proposed model the chromatid (anaphase chromosome) consists of two half-chromatids; each half-chromatid contains two deoxyribonucleoprotein ribbons wound into a single fiber (termed the core), with many loops of chromatin (termed epichromatin) attached along its length. The core ribbons are each about 50 Å thick by 4000 Å wide and are composed of many parallel deoxyribonucleoprotein strands. The epichromatin loops appear to be 250 Å supercoiled fibers containing about 75 per cent of the chromosomal DNA. The epichromatin can be selectively removed from the core fibers by extraction with 2.0 M NaCl or 6.0 M urea solutions.     

End-to-End Association of <Emphasis Type="Italic">X</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> Chromosomes in Mouse Meiosis

ACCORDING to the hypothesis of Crew and Koller¹ and Koller and Darlington², there are homologous segments in the X and Y chromosomes of the mouse and other mammals. The homologous regions in the mouse were believed to be localized in the extremely short arms proximal to the kinetochores. The end-to-end association at meiosis was thought to be the result of the formation of a chiasma between these homologous regions³. Electron microscopy revealed a short synaptonemal complex in mouse meiotic cells⁴. However, partial sex linkage has never been demonstrated in the mouse⁵ and other authors^6–10 believe that the X and Y chromosomes associate only by connexion between the chromosome ends furthest from the centromeres.     

Temperature-sensitive mutations in Drosophila melanogaster. XI. Male sterile mutants of the Y chromosome

Eight temperature-sensitive (ts) male sterile mutations have been induced by ethyl methanesulfonate treatment of Y chromosomes derived from a selected temperature-resistant Amherst wild-type stock of Drosophila melanogaster. Males carrying such mutated Y chromosomes (Y^ts) are sterile when raised at 29°C but fertile when reared at 22°C. Complementation tests of the mutants with Y chromosome fragments, deletions, and inter se localized all eight to the long arm of the chromosome in four different complementation groups.When Y^ts-bearing males, reared to adulthood at 22°C, were subjected to a 48-hr regimen at 29°C and mated to fresh virgin females daily, a significant reduction in fertility resulted 5 days after initiation of 29°C treatments. This period of sterility was transient (48–72-hr duration) and corresponded to a temperature-sensitive period (TSP) of spermatogenesis during the primary spermatocyte stage. A more precise definition of the TSP utilized exposure of subadult males to 29°C at selected developmental periods during which only certain germ cell stages are present. Upon eclosion adult males were subjected to a similar schedule of consecutive matings of 12-hr duration in order to detect any delay in the appearance of fertility. Different ts males could be distinguished by the resultant pattern of sterility, and the TSP of different mutations thus localized to either primary spermatocyte or immediately post-meiotic stage.Associated with Y^ts-mediated sterility, spermiogenesis is defective at restrictive temperature as evidenced by the production of nonmotile sperm and a failure to transfer such sperm to the female during copulation. In addition, electron microscopy detected a variety of ultrastructural abnormalities, including defects of axoneme formation, irregularities of Nebenkern derivative development, and failures of separation from the syncitial state or mature cyst with subsequent degeneration.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Studies on the Ribosomal RNA Cistrons in Interspecific Drosophila Hybrids. II. Heterochromatic Regions Mediating Nucleolar Dominance

Interspecific hybrids of D. melanogaster and D. simulans normally exhibit a secondary constriction only at the D. melanogaster nucleolus organizer (NO). This phenomenon, termed nucleolar dominance, occurs only when the NO-bearing sex chromosomes of both species are present in conjunction. Experiments were initiated to localize regions on the sex chromosomes of D. melanogaster involved in mediating this suppression. Sex chromosome heterochromatic rearrangements and deficiencies were introduced into F₁ hybrids and their corresponding effect on simulans NO constriction formation was examined in hybrid mitotic neuroblast tissue. Sex chromosomes deficient for both the D. melanogaster NO and adjacent heterochromatin were unable to restrict the formation of a constriction at the D. simulans NO. The presence of a D. melanogaster NO, however, was not sufficient for the establishment of nucleolar dominance. Results from an array of NO-bearing X and Y chromosome rearrangements and deficiencies indicate that at least one heterochromatic region, proximal to the NO on the D. melanogaster X and distal to the NO on the D. melanogaster Y, affects the induction of this interchromosomal phenomenon.     

Chromocentral nature of interchromosome connections coorienting nonhomologous chromosomes in meiosis of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> females

Data are presented in favor of universal significance of physical connections between pericentromeric regions of homologs in their orientation to the opposite poles of the first meiotic division in Drosophila melanogaster. Disturbances in the formation of such connections caused by structural or locus mutations are compensated for by the presence of pericentromeric chiasmata between homologs or (in the case of their absence) by chromocentral connections between nonhomologs being preserved up to the prometaphase. In the latter case, an interchromosome effect on chromosome disjunction and nonhomologous pairing is registered by genetic methods. Inhibition of the formation of the division spindle fibers during prometaphase of meiosis 1 by the long-term action of colcemide promotes the retention of connections between paired nonexchanged homologs and between nonhomologous chromosomes with abnormal homologous pairing because of heterozygosity for numerous inversions and transpositions (X and autosome 2). These connections are registered cytologically. Cytologically registered are also connections between normal X chromosomes and metacentric compounds by the arms of autosome 2 (C(2L)RM, C(2R)RM), which is the known case of the interchromosome effect on chromosome nondisjunction. It is supposed that cytologically detected associations between compounds are realized through a normal mechanism, as a result of interaction and formation of orienting connections between the homologous pericentromeric regions of these compounds. Cytological evidence is presented for colocation of compounds in the chromocentrally organized nucleus of somatic and germline cells.     

Locations of a Human Satellite DNA in Human Chromosomes

USING techniques for DNA/RNA or DNA/DNA hybridization in situ, Pardue and Gall¹ and Jones² made several significant discoveries on the chromosomal locations of the mouse satellite DNA: (1) this fraction of DNA is found in all chromosomes except the Y, (2) the cytological location of the satellite DNA is limited to the centromeric region of each chromosome and is probably absent in other regions and (3) the centromeric regions of all mouse chromosomes are hetero-chromatic.