Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling

Wingless (Wg) treatment of Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.     

A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Lactococcus lactis subsp. cremoris strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS905 element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS905 element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the oriC-terC symmetry of the chromosome and the local genetic environment.     

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.     

Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination

An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 x 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 has functions in addition to those of the Rad51/Rad52 protein complex.     

Mutations affecting intrachromosomal recombination and conversion in the binary y(2ns)sc(me) system in Drosophila melanogaster

The effect of mutations in various genes whose products are involved in chromosome organization on the mutagenesis in a double superunstable y2nsscme system was studied. Among these mutations were those in loci zeste, e(y) e(y) 1,2 and 3, responsible for long-distance interactions in chromosomes, and those in the loci E(z), Su(z), Scm, and Psc, whose protein products take part in chromatin compaction. Many of the mutations studied markedly affect the spectrum and the frequency of mutations in the system, in particular, the frequency of double events. The results suggest that the spatial organization of chromosomal domains plays a role in intrachromosomal recombination and gene conversion, since these processes were shown earlier to be dominant in the mutagenesis of a double superunstable system. Some of the mutations under study also modify the phenotypic manifestation of the y2nsscme allele.     

Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5' regions of polymerase chain reaction-amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5' emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5' emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation 'maps' onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.     

Failure of 2- and 10-meter radio waves to induce genetic damage in Drosophila melanogaster

Over the past 20 years, more than 300 patients have been anesthetized in the lateral sitting position during neurosurgical procedures in the posterior fossa and the cervical and upper thoracic spine. Since the patient can be placed quickly and easily in the horizontal position, the lateral sitting position has a number of advantages over the conventional sitting position, particularly in the treatment of arterial hypotension and venous air embolism. Furthermore, with the patient in the lateral horizontal position, the surgical procedure can be completed satisfactorily.     

A heterologous in vitro coculture system to study interaction between human bladder cancer cells and fibroblasts

Three-dimensional multicellular spheroids of two fibroblast cell lines (WI-38 and N1) and two differently differentiated bladder carcinoma cell lines (RT4 and J82) were used for cocultures of multicellular tumor spheroids with multicellular spheroids of fibroblasts. The aim of the study was the establishment and characterization of a standardized three-dimensional model for studies of tumor cell-fibroblast interaction as one aspect of tumor-stromal cell interactions of in vivo tumor tissue. Interaction of multicellular spheroids of both fibroblast cell types was analyzed by staining with antibodies against cytokeratin, vimentin and different extracellular matrix molecules. Further, proliferation assessment and phenotypic characterization of the cocultures are presented. Interactions varied with tumor cell type and fibroblast cell type, reflecting intrinsic properties of tumor cells and fibroblasts. The coculture of tumor cells with N1 reflected the in vivo situation the closest, since invasive properties of J82 as well as noninvasive properties of RT4 were characteristics seen in coculture.     

Adapting Eimeria tenella to grow in primary chicken kidney cells following repeated passages between cell culture and chickens

The resolving power of displacement chromatography using low-molecular-weight displacers was investigated using a model mixture containing bovine and horse heart cytochrome c. The linear and nonlinear adsorption behavior of these two proteins was examined in cation-exchange chromatography and shown to be quite similar. Furthermore, an analysis of the dynamic affinity of these proteins indicated extremely similar affinities under displacement conditions. Despite the extreme similarities in the adsorption behavior, displacement chromatography using a protected amino acid displacer resulted in excellent separation of the proteins with both high yields and purity. These results indicate that displacement chromatography may be efficacious for a wide variety of difficult protein separation problems.     

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria

We present three experiments which serve to identify carbon and proton sidechain resonances in 13C-labeled proteins. The first is an improvement on the previously published H(C)CH-COSY experiment and comprises the application of gradients for coherence selection and a reduction in the phase cycle. The second experiment is a new (H)CCH-COSY with two carbon dimensions. The (H)CCH-COSY presents several advantages over the H(C)CH-COSY experiment in terms of better sensitivity, improved resolution and easier identification of amino acid spins systems. The third experiment is a 2D proton-edited (H)C(C)H-COSY that allows suppression of methylene resonances. All three HCCH-COSY experiments show good sensitivity and excellent solvent suppression. The 2D version can be acquired in as little as 45 minutes and the 3D versions acquired overnight. The experiments are demonstrated on a 13C-labeled sample of the second PDZ domain from human phosphatase PTP1E in H2O solution.     

Sertoli cells in culture and mRNA differential display provide a sensitive early warning assay system to detect changes induced by xenobiotics

We have used cultured rat Sertoli cells as an "early warning system" to monitor for morphological and biochemical changes induced by two different xenobiotics-cadmium acetate and polychlorinated biphenyls (PCBs). Sertoli cells begin to round, become vacuolized, and detach from their substrate within 24 hours of culture in the presence of cadmium at concentrations of 0.5-1.0 microM. Similar results were obtained with a lower dose of cadmium (0.01 microM) after 72 hours. When Sertoli cells are cultured for 24 hours in the presence of a mixture of PCBs (3,3',4,4'-tetrachlorobiphenyl, 2,2',4,6,6'-pentachlorophenyl, and 2,2',3,3',4,5,5', 6,6'-nonachlorobiphenyl) at concentrations of 1.0-2.0 microM, they enlarge. After 72 hours, a lower dose of PCBs (0.01 microM) produces similar cellular enlargement. Despite their changes in morphology, no reduction in Sertoli cell viability was seen at any of the concentrations or time points studied for either toxicant. Using mRNA differential display, a number of novel cDNAs were detected when cells were cultured with either cadmium or the PCBs, demonstrating that changes in gene expression accompany the changes in Sertoli cell structure. We propose that Sertoli cells in culture and mRNA differential display provide a sensitive morphological and biochemical assay system to detect early direct effects of low concentrations of toxicants on mammalian Sertoli cells.     

Evidence for an inducible repair-recombination system in the female germ line of Drosophila melanogaster. III. Correlation between reactivity levels, crossover frequency and repair efficiency

We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system.     

A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors

We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.     

A system for mutation measurement in mammalian cells: application to gamma-irradiation

Monitoring of mutagenesis by environmental agents for the purpose of preventing genetic disease including cancer must include quantitation of cell killing, sensitive measurement of mutation production by appropriate doses of each agent, and assessment of mutation repair effects in mammalian cells. A four-step procedure, in the presence and absence of a repair suppressor, is proposed: (i) determination of the survival curve; (ii) measurement of the mitotic index in cells collected after treatment with colcemid; (iii) construction of a mutagenesis yield curve in the presence and absence of a repair suppressor, like caffeine; and (iv) assessment of the effect of test agents on the repair of mutations produced by other mutagens. The procedure is quantitative, reproducible, and reasonably rapid. It involves measurement of mutations causing visible chromosomal aberrations. Numerical parameters are proposed defining quantitatively mutation, cell killing, and mutation repair capacity. The procedure is applied to gamma-irradiation and can detect the effects of doses as low as 2-5 cGy. Theoretical analysis of the underlying processes is presented, using the concept of D(0)E, the effective dose of mutagen after repair mechanisms and neutralizing agents have acted. Microscopically visible chromosome aberrations are due to mutations that distort the process of mitotic chromosome condensation, with or without actual chromosome breakage.     

Correlation between cytotoxic lymphocytes and cells that synthesize the macrophage migration inhibitory factor in the H-2 system

High immunological specificity of the direct and indirect macrophage migration inhibition tests was demonstrated in the H-2 system. The capacity of immune lymphocytes for the MIF production was revealed under their incubation with splenic cells of the congenic or recombinant strains of mice sharing particular private or public H-2 specificities with the donor strains. Selective removal of cytotoxic fraction of lymphocytes resulting from their absorption on the corresponding target cells failed to reduce the capacity of the non-adherent cell population for the MIF production. A fraction of the MIF-producing lymphocytes was found to adhere to the target cells and thereafter to be eluated with the cytotoxic lymphocytes. MIF-producing and target-destroying T-lymphocyte populations are supposed to have antigen-binding receptors differing in their affinity and structural arrangement on the cell surface.