Karyotype evolution in cell lines of Drosophila melanogaster

The chromosomal changes occurred in two independent cell lines (GM₂ and GM₃) of Drosophila melanogaster maintained in medium supplemented with serum and in serum-free medium were compared. In both culture conditions and in both lines a chromosomal evolution was revealed. Structural and numerical variations were analysed. The breaks giving rise to rearrangements were at heterochromatic level. Moreover, a tetraploidisation followed by loss of chromosomes or of portions of chromosomes recalls an analogous cycle observed in human cells.This work was supported by a grant of the Consiglio Nazionale delle Ricerche, Roma.     

Chromosome changes in methotrexate-resistant cell lines of Drosophila melanogaster

Cells resistant to methotrexate (MTX) were obtained from two established cell lines of D. melanogaster and a karyological analysis was performed. No conspicuous changes in the frequencies of cell types were observed in MTX-resistant (MTX^R) subline 0.57, as compared with the original line, whose cells were mostly near-tetraploid. On the contrary, altered karyotypes were frequently noticed in the C1 82 MTX^R subline, as compared with the original line, whose cells were mostly diploid. The C1 82 MTX^R subline was characterized by mostly tetraploid cells and by the presence of chromosome fragments (in 48% of them). The mitotic segregation suggests the presence of a centromere in these fragments and the fluorescence pattern, after quinacrine staining, suggests that they may be derivatives of the X chromosome.     

Evolution of karyotype in haploid cell lines of Drosophila melanogaster

Seven continuous cell lines have been established in vitro from lethal embryos produced by the female sterile mutant mh 1182 of Drosophila melanogaster. Six lines show haploid metaphases. Karyotype analysis revealed a high level of aneuploid cells with frequent chromosome fragments. In three lines, haploid cells were quickly overgrown by diploid cells. Two lines were more stable but the proportion of haploid cells decreased with time. One line was stable, showing 80-90% of haploid cells for over 1 000 cell generations. Stable haploid clones have been isolated from two lines. Crossing of mh 1182/mh 1182 females with males bearing a ring X chromosome shows that the haploid genome retained in the cells is of maternal origin and that the diploid cells derive from pre-existing haploid cells. The appearance of the diploid cells and the conditions of karyotypic stability are analysed.     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.     

Derivation and characterization of three new human embryonic stem cell lines in Finland

Human embryonic stem cell (hESC) lines can be established from the preimplantation embryos. Due to their ability to differentiate into all three embryonic layers, hESC are of significant interest as a renewable source of cell material for different applications, especially for cell replacement therapy. Since the establishment of the first hESC lines in 1998, several studies have described the derivation and culture of new hESC lines using various derivation methods and culture conditions. Our group has currently established eight new hESC lines of which three of the latest ones are described in a more detailed way in this report. The described lines have been established using mechanical derivation methods for surplus bad quality embryos and culture conditions containing human foreskin fibroblast feeder cells and serum-free culture medium. All the new lines have a normal karyotype and typical hESC characteristics analyzed in vitro. The described hESC lines are available for research purposes upon request (www.regea.fi).     

Fish cell lines: Establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella)

Summary Three new cell lines were established from tissues of the grass carp,Ctenopharyngodon idella. Derived from the fin, snout, and swim bladder of two apparently healthy diploid fry, these cell lines have been designated GCF, GCS-2, and GCSB, respectively. The cells grew at temperatures between 24° and 36° C with optimal growth at 32° C and have been subcultured more than 50 times since their initiation in August 1986. Two of the lines remained diploid or pseudodiploid after 38 passages. The cells were tested for microbial contamination, and plating efficiencies were determined. The three cell lines were sensitive toRhabdovirus carpio (RVC), infectious hematopoietic necrosis virus (IHNV), golden shiner virus (GSV), chum salmon virus (CSV), and infectious pancreatic necrosis virus serotype VR299 IPNV). They were refractory to channel catfish virus (CCV), channel catfish reovirus (CRV), chinook salmon paramyxovirus (CSP), and an Ab serotype of IPNV. This work is a result of research sponsored by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under grant NA85AA-D-5G-095, and was undertaken while the first author was on leave from the Department of Fisheries, Huazhong Agricultural University, Wuhan, China. Salary support was provided by the People's Republic of China. Oregon Agricultural Experiment Station Technical Paper 8952.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

A method for establishing cell lines from Drosophila melanogaster embryos.

A simple method is presented for establishing continuous cell lines from Drosophila melanogaster embryos. Subculturing is performed after the first 8 weeks and at 2-week intervals thereafter. Initial plating densities of 5 x 10(4) to 5 x 10(5) cells per cm2 are required for maintaining the subcultures. Cell lines were established from wild-type embryos, from embryos bearing chromosomal rearrangements and from embryos bearing recessive mutations. Permanent lines have doubling times of 24 to 48 hr and have been maintained for as long as 13 months and 25 subcultures.     

The expression of PS integrins in Drosophila melanogaster imaginal disc cell lines

Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.     

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.     

Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.     

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.     

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.)

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28°C in Leibovitz‐15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Morphological and molecular characterization of new drosophila cell lines established from a strain permissive for Gypsy transposition

Summary The gypsy element of Drosophila melanogaster is the first retrovirus identified in invertebrates. Its transposition is controlled by a host gene called flamenco (flam): restrictive alleles of this gene maintain the retrovirus in a repressed state while permissive alleles allow high levels of transposition. To develop a cell system to study the gypsy element, we established four independent cell lines derived from the Drosophila strain SS, which contains a permissive allele of flamenco, and which is devoid of transposing copies of gypsy. The ultrastructural analysis of three SS cell lines revealed some remarkable characteristics, such as many nuclear virus-like particles, cytoplasmic dense particles, and massive cisternae filled with a fibrous material of unknown origin. Gypsy intragenomic distribution has been compared between the three cell lines and the original SS fly strain, and revealed in two of the cell lines an increase in copy number of a restriction fragment usually present in active gypsy elements. This multiplication seems to have occurred during the passage to the cell culture. Availability of SS cell lines should assist studies of gypsy transposition and infectivity and might be useful to produce high amounts of gypsy viral particles. These new lines already allowed us to show that the Envelope-like products of gypsy can be expressed as membrane proteins.     

Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster.

The three major yolk polypeptides of Drosophila melanogaster have been isolated from yolk spheres of early embryos. Their molecular weights, as determined by SDS-polyacrylamide electrophoresis, are 44,000, 45,000, and 46,000. A number of approaches have been used to show that each of these yolk polypeptides are different. They have different isoelectric points, they have different digestion products upon peptide mapping by limited proteolysis, and they show three different antigen-antibody systems when each polypeptide is reacted with an antisera made to a mixture of all three. Both the digestion with chymotrypsin and the immunoelectrophoresis studies indicate similarities between two of the polypeptides while the third appears unique. This is the first example of multiple yolk polypeptides of similar molecular weight.