Genetics of the cell cycle: Adaptive modification of the <Emphasis Type="Italic">CycB</Emphasis><Superscript><Emphasis Type="Italic">2g</Emphasis></Superscript> mutation expression in dividing cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The effect of mutation CycB ^2g on mitosis in neural ganglia and imaginal disks was studied in third-instar larvae of Drosophila melanogaster. Chromosome condensation and segregation were shown to be impaired in dividing cells of mutant larvae. During the three-year period of maintenance of the mutation in heterozygote, frequencies of some defects decreased via cellular adaptive modification.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 312–319.Original Russian Text Copyright © 2005 by Lebedeva, Trunova, Omelyanchuk.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Developmental potencies of nuclei from cleavage,preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster

Summary Wild-type nuclei from eggs ofDrosophila melanogaster at various developmental stages and from different regions of the egg—cleavage nuclei, pole nuclei from preblastoderm, and lateral nuclei from syncytial blastoderm—were singly implanted into unfertilizedy w sn ³ lz _50e eggs to determine their developmental potencies.All three types of transplanted nuclei were almost equally effective in initiating development of unfertilized eggs. Development was arrested in one of five critieal embryonic stages or in one of the three larval instars. The frequency of individuals reaching a distinct stage was approximately the same for all three types of donor nuclei. The stage-specific pattern of defects was independent of the type of nucleus transplanted.The deviations from normal development were broadly similar to those seen in controls developing from fertilized eggs which had only been punctured or into which cytoplasm had been injected. Many defective embryos also occurred in these control experiments. These and other observations indicate that a large proportion of irregularly developed individuals found after nuclear transfer can be ascribed to loss of egg material, disturbances in the internal organization of the egg during nuclear implantation, and the difficulty the implanted nucleus has in adjusting to the autonomous processes within the egg, such as the formation and migration of cytoplasmic islands.Some of the defective embryos and larvae originating from nuclear transfer were implanted into adult hosts. After culture for 14 days the early embryonic stages had formed several larval structures, and the late embryonic and larval stages had developed all larval organs. The proliferated imaginal primordia of thesein vivo cultured embryos and larvae, as well as the imaginal disks of the third instar larva, were then implanted into larval hosts with which they passed through metamorphosis and differentiated into imaginal structures. All three types of donor nuclei were capable of producing all adult structures derivedin situ from imaginal disks. The phenotype of these structures waswild-type, thus demonstrating their origin from the transplanted nuclei.The problem as to why not all transplanted nuclei initiated development, and why development after nuclear transplantation was arrested at the third larval instar, at the latest, is discussed.This article is dedicated to Professor Friedrich Seidel on the occasion of his 75th birthday.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G₂-early prophase. The 250–400 nm thick compact chromatin threads in G₂ nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1–1.5 m) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120–250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.     

Effects of Hoechst 33258 on condensation patterns of hetero- and euchromatin in mitotic and interphase nuclei of Drosophila nasuta

Embryonic and third instar larval brain cells of D. nasuta were cultured in vitro in the presence of Hoechst 33258 (H) and H + 5-bromodeoxyuridine (BUdR) for periods varying from 2 to 24 h at 24 °C. Air-dried chromosome preparations were made with and without hypotonic pretreatment and stained with Giemsa. Metaphase chromosomes from H-treated (2 h) embryonic preparations show typical inhibition of condensation of the A-T-rich heterochromatin as in mouse. Presence of BUdR with H causes inhibition of condensation in fewer embryonic metaphase cells, but in the affected metaphases the degree of inhibition is more severe. In larval brains, however, even a 24 h H or H + BUdR treatment does not cause any significant inhibition of heterochromatin condensation. It is suggested that the differences in H effect on metaphase chromosomes of embryos and larval brains is related to differences in chromosome organization in the two cell types. Exposure of H-treated embryonic as well as larval brain cells to a hypotonic salt solution prior to fixation causes a ‘supercondensation’ of the heterochromatic chromocentre in most interphase nuclei. Presence of BUdR along with H reduces the frequency of cells showing such ‘supercondensed’ chromocentre. The euchromatin region in H-treated interphase nuclei is, on the other hand, slightly more diffuse than in control nuclei. Apparently, H-binding to DNA affects the nucleoprotein organization in hetero- and euchromatic regions of interphase nuclei in specific ways.     

Gene transfer in Drosophila melanogaster: cytological alterations in the salivary chromosomes of transformed stocks

Cytological abnormalities are observed in salivary chromosomes of stocks of Drosophila melanogaster possessing DNA-induced, v ⁺ transformations mapping either at 10.0 or 133.0. In initial studies, the untransformed control stock and six transformed stocks were assigned code numbers prior to cytological examination. Salivary chromosome regions 1B9 · 10—1C2 · 3 and 10A1 · 2—10B1 · 2 were carefully examined in each of the coded stocks. (Band 1B11 is tentatively identified as the site of the suppressor-of-sable locus at 10.0 and band 10A1 is the site of the vermilion locus at 133.0.) When cytological studies were complete each of the stocks was identified. In every transformed stock examined, anomalies had been scored in association with the chromosome region corresponding to the map position of the DNA-induced alteration. In the control stock, anomalies were observed at neither position. — Approximately 10–15% of the nuclei in transformed stocks exhibit significant departure from normality in the pertinent chromosome region. The perturbations range from minor alterations of banding pattern to apparent pieces of extra chromatin in the most extreme cases. In stocks with v⁺ transformations mapping at 133.0, apparent extra chromatin is observed with frequencies varying from 0.05 to 0.02. In these cases the abnormal structures are associated with salivary band 10A1 · 2, forming a band-like structure, frequently an almost perfect doublet (open or closed), often lying in an abnormal position, and with fine chromatin threads connecting to the chromosomal doublet. In stocks with v⁺ transformations mapping at 10.0, apparent extra chromatin is observed with a frequency of about 0.001. These abnormal structures are frequently thread-like, lying to the side or off the tip of the chromosome, with compact regions which sometimes resemble chromomeres, and with fine threads connecting to the chromosomal 1B11 region.     

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster

Summary The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [³H]thymidine autoradiography on 2-m plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

Chromatin organization in the male germ line of Drosophila hydei

The chromatin organization in developing germ cells of Drosophila hydei males was studied with the highly sensitive DNA stain DAPI (4, 6-diamidino-2-phenylindole dichloride). The prophase of meiosis I is characterized by decondensed chromosomes and only late during this stage do they condense rapidly. The sex chromosomes show allocycly. During postmeiotic development the final condensation of chromatin is preceded by a cycle of condensation and subsequent decondensation. Meiotic chromosomes were studied in more detail after orcein staining. Pairing sites of the sex chromosomes could be localized in the distal end of the heterochromatic arm of the X chromosome and distally in both arms of the Y chromosome. The various heterochromatic parts of the genome condense differentially in meiosis. Chromatin reorganization was studied cytochemically with antibodies raised against histones H1 and H2A of D. melanogaster. The core histone H2A is present in spermatid nuclei until the late elongation stage. However, histone H1 is not found in the chromatin later than the early primary spermatocyte stage. Thus, chromatin reorganization during spermatogenesis in D. hydei is complex. The process is discussed with regard to possible functions.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

Different hybrid effects in reciprocal crosses betweenChironomus thummi thummi andCh. th. piger including spontaneous chromsome aberrations and sterility

Hybrids from reciprocal erosses between twoChironomus thummi thummi andCh. th. piger laboratory stocks show four abnormalities in comparison to the parental stocks. One cross direction (Ch. th. thummi x Ch. th. piger ) is characterized by chromosome aberrations, reduced hatchability and malformations, whereas in reciprocal hybrids both sexes are sterile. Sterility is the consequence of rudimentary or non developed gonads.InCh. th. thummi x Ch. th. piger crosses chromosome aberrations were analysed in salivary gland nuclei. These aberrations are all somatic in origin, and they are induced during the first 40 h of embryonic development, prior to the onset of polytenization. The chromosomes of both subspecies are equally affected. In all four chromosomes breaks occur preferentially at specific regions. Reduced hatchability and malformations are presumably caused by chromosome mutations because within egg-masses a correlation exists between the rate of salivary gland chromosome aberrations and the rates of hatchability and malformations.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae

Summary At 26° C temperature-sensitivetu(1) Sz ^ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ^ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ^ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ^ts.The discriminatory ability oftu-Sz ^ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between self and not self by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.     

Molecular flexibility of extended and compacted polynucleosomes

We have studied the effects of Na⁺ (5–120 mM) and Mg²⁺ (0–6 mM) on the internal and overall flexibility of polynucleosome fragments from nucleasesolubilized chromatin from Ehrlich ascites cells. The mobility was monitored by the steady-state fluorescence polarization of the intercalated ethidium cation. The internal polynucleosome flexibility decreases continuously as the extended chromatin fragments are being compacted at increasing salt concentrations, and it can be further suppressed at ionic strengths above those where the 30 nm fiber is formed. The effect may be visualized as an initial formation of a loose 30 nm fiber that is further compacted at increasing ionic strengths. We observe several differences in the effects of Na⁺ and Mg²⁺ upon chromatin compaction. First, chromatin compacted by Mg²⁺ is less flexible than that compacted by Na⁺, suggesting a tighter chromatin structure with Mg²⁺. Second, Mg²⁺ affects the internal mobility in polynucleosome fragments shorter than 6–7 nucleosomes, which are too short to be compacted with Na⁺. Third, Mg²⁺ causes extensive macroscopic aggregation at concentrations above 0.2–0.3 mM, but the aggregation is uncorrelated with the intramolecular compaction. A quantitative evaluation of the overall polynucleosome tumbling mobility indicates that the compacted fragments possess more internal flexibility than do corresponding high molecular weight chromatin fibers. Finally, we note a correlation between the ethidium binding constant and the internal chromatin flexibility, possibly arising from lower torsional and unwinding flexibility of the linker DNA segments of compacted chromatin fibers.Abbreviations FPA fluorescence polarization anisotropy - CT calf thymus - HMW high molecular weight - ARF amplitude reduction factor - kbp kilobasepairs This project is supported by the Swedish Natural Research Council. P.E.N. is the recipient of a Hallas-Mølle Fellowship through the NOVO Foundataion     

Conditions for open-air outdoor culture of Dunaliella salina in southern Spain

The optimization of the operation, under the climatic conditions of southern Spain, of an experimental plant for -carotene production by Dunaliella has been pursued. The effects of mixing, culture depth, cell density and dilution cycles on -carotene and biomass productivity were studied under a semicontinuous culture regime in open tanks outdoors. Using 3 m²-surface containers, the highest productivity values, for both -carotene and biomass, were recorded with a flow rate of 0.55 m s^–1; 10 cm depth; 0.7 – 0.9 × 10⁶cell ml^–1, population density; and dilution cycles of two days. An average annual productivity of 1.65 g (dry wt) m^–2 d^–1 was estimated for Dunaliella biomass, being that for -carotene of about 0.1 g m^–2 d^–1. Under these optimized conditions, experiments have been carried out at the Cadiz Bay with 20 m²-surface tanks during a whole-year cycle. The results obtained have validated this location and the operating conditions established as being most appropriate for efficient mass production of -carotene rich D. salina.     

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

Key message

PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used.

Abstract

Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).