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Polygenic mutation in Drosophila melanogaster: Mapping spontaneous mutations affecting sensory bristle number.

Our ability to predict long-term responses to artificial and natural selection, and understand the mechanisms by which naturally occurring variation for quantitative traits is maintained, depends on detailed knowledge of the properties of spontaneous polygenic mutations, including the quantitative trait loci (QTL) at which mutations occur, mutation rates, and mutational effects. These parameters can be estimated by mapping QTL that cause divergence between mutation-accumulation lines that have been established from an inbred base population and selected for high and low trait values. Here, we have utilized quantitative complementation to deficiencies to map QTL at which spontaneous mutations affecting Drosophila abdominal and sternopleural bristle number have occurred in 11 replicate lines during 206 generations of divergent selection. Estimates of the numbers of mutations were consistent with diploid per-character mutation rates for bristle traits of 0.03. The ratio of the per-character mutation rate to total mutation rate (0.023) implies that >2% of the genome could affect just one bristle trait and that there must be extensive pleiotropy for quantitative phenotypes. The estimated mutational effects were not, however, additive and exhibited dependency on genetic background consistent with diminishing epistasis. However, these inferences must be tempered by the potential for epistatic interactions between spontaneous mutations and QTL affecting bristle number on the deficiency-bearing chromosomes, which could lead to overestimates in numbers of QTL and inaccurate inference of gene action.

Animals↗

Reaching the parts other methods cannot reach: an introduction to qualitative methods in health and health services research.

Qualitative research methods have a long history in the social sciences and deserve to be an essential component in health and health services research. Qualitative and quantitative approaches to research tend to be portrayed as antithetical; the aim of this series of papers is to show the value of a range of qualitative techniques and how they can complement quantitative research.

Health Services Research↗

A molecular defect in thrombasthenic platelets.

An IgG antibody found in the serum of a thrombasthenic patient reacted in complement fixation with platelets from 350 normal individuals but was nonreactive with platelets from eight other thrombasthenic patients. ADP-induced aggregation of normal platelets was inhibited by the patient's antibody. Family studies using the quantitative complement fixation test showed that healthy heterozygotes were easily distinguishable from normal or thrombasthenic individuals since their platelets had an intermediate amount of the reactive antigen. Indirect immunoprecipitation tests using this serum and soluble membrane antigens labeled with iodine-125 that had been extracted from normal platelets by the detergent Nonidet P-40 gave a single radioactive peak at 120,000 mol wt in sodium dodecyl sulfate polyacrylamide gel electrophoresis. A similar estimate of the molecular weight was obtained from Sephadex G-200 filtration of the soluble antigens extracted from normal platelets by spontaneous release or chaotropic agents and tested in complement fixation with the patient's serum. These findings strongly suggest that the molecule recognized by this antibody is absent or structurally modified in thrombasthenia cases and that it may be involved in platelet aggregation.

Adenosine Diphosphate↗

Gender, ethnicity and self-reported health: the case of African-Caribbean populations in London.

This paper explores quantitative and qualitative information on self-reported health, focusing especially on gender differences in the reporting of health problems by men and women. The research reported here particularly examines evidence relating to the African-Caribbean population in London, some of which suggests that there may be a distinctive pattern of reported illness in this ethnic group in Britain, which differentiates it from the average pattern for the majority population. Various population surveys using systematic measures have shown that women and men differ in terms of reported morbidity (particularly in the prevalence of self-reported illness and psychosocial health). This paper reviews the quantitative data available to investigate the gender differences in the African-Caribbean population, showing that the pattern seems to vary according to the measure of health used. We consider how qualitative material from research conducted in East London may complement quantitative survey data and provide possible explanations for the reported health of African-Caribbean women and men. We report on qualitative observations of the process of completing standardized questionnaire items and discussion of these by the informants. We also examine the understandings about health and illness expressed by African-Caribbean women and men during in-depth interviews.

Adolescent↗

Chemically-induced temperature sensitive mutants of dengue virus type 2. I. Isolation and partial characterization.

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.

Azacitidine↗

A human autoantibody to peroxisomes.

A complement fixing, non-organ specific IgG autoantibody is described in 29 patients. The autoantibody gives a highly characteristic, granular staining of liver cells, proximal kidney tubules and stomach surface epithelium. By studies with various subcellular fractions from rat liver, employing two different techniques (quantitative complement fixation, and absorption combined with indirect immunofluorescence) the autoantibody was shown to react with a peroxisomal antigen. No convincing clinical correlations were found.

Animals↗

[Studies on cross reactions of antisera against nucleotid-protein conjugates with denaturated desoxyribonucleic acids].

The reactivity of anti-nucleotide-sera (anti-UMP, anti-AMP, anti-GMP) with denatured DNA's of 10 different sources was investigated by means of quantitative complement fixation. The haptene specificity of the anti-nucleotide antibodies was shown by haptene inhibition of the reaction with DNA. Anti-UMP showed cross-reactions with other pyrimidine-derivatives, but to a lesser extent; anti-GMP cross-reacted with adenine structures. Quantitative differences were observed in the reaction of anti-UMP, anti-AMP and anti-GMP with DNA's of various (G + C)-contents. These differences could not be correlated in all cases with the overall base composition of the DNA.

Animals↗

Breakdown products of C 3 in human synovial fluids.

Activation of the complement sequence results in conversion of the third component of complement (C'3) to an inactive product (C'3i) and the elaboration of additional fragments of smaller molecular weights and faster electrophoretic mobilities. Immunoelectrophoretic analysis of fresh synovial fluids with an anti-human C'3 antiserum disclosed in some a variable degree of conversion of C'3 to C'3i, but a more striking finding was an additional line in the alpha-globulin region. This faster migrating protein gave a reaction of partial identity with C'3/C'3i. With this antiserum a similar pattern developed when fresh human serum was incubated with immune complexes, or aggregated gamma-globulin. The same breakdown product of C'3 was obtained by treatment of fresh human serum with Zymosan, ammonium, hydrazine, agar, or dextran. Heating serum at 56 degrees C for 1 hr destroys the breakdown product; aging of serum produces it. Breakdown products of C'3 were looked for in 49 synovial fluids from patients with a variety of joint diseases. A significant correlation was found between the demonstration of the fast migrating breakdown product of C'3 and the diagnosis of rheumatoid arthritis and the presence of rheumatoid factor. A similar immunoelectrophoretic pattern was not found in the serum of any of the patients studied. When human gamma-globulin, which has been reduced and alkylated, is heat aggregated it loses the ability to fix human complement but still reacts with rheumatoid factor. Addition of reduced, aggregated gamma-globulin to fresh normal human serum produced no conversion of C'3, but when incubated with serum containing a high titer of rheumatoid factor, there was conversion of C'3 and the appearance of a breakdown product. Quantitative complement fixation studies with fresh serum from normal subjects and patients with rheumatoid arthritis disclosed complement fixation by reduced, aggregated gamma-globulin. The per cent of complement fixation was proportional to the titer of rheumatoid factor present in the test serum. These findings were interpreted as showing that rheumatoid factor can fix complement.The possibility is discussed that the presence of breakdown products of C'3 in the synovial effusions of most patients with seropositive rheumatoid arthritis and the ability of rheumatoid factor to fix complement are related phenomena.

Animals↗

[The induction and disappearance of complement-fixing antibodies in the blood serum of experimental animals under the antigenic action of Trichomonas vaginalis, T. hominis and T. tenax].

For studying the dynamics of induction and disappearing of complement-fixing antibodies (CFA) specific to Trichomonas vaginalis, T. hominis and T. tenax we have immunized rabbits intravenously with live individuals of the protozoa as well as intravenously, intramuscularly and subcutaneously with individuals killed at 56 degrees C. Each method of immunization was carried out by 5 inoculations with intervals of 10 days. For carrying out quantitative complement fixation we have taken blood from the ear vein of all immunized rabbits immediately before the immunization and also after it, each 10th day during one year. The dynamics of rising and lowering of titres depended on the species of trichomonads and on the method of immunization. The antigens of T. vaginalis and T. hominis induced CFA in much higher titres as antigens of T. tenax. Titres of CFA specific for all three species of above-mentioned trichomonads rose most rapidly and to the highest level when the immunization of rabbits was carried out intravenously with live individuals of protozoa. Normalization of the titres of CFA has taken place during one year at least.

Animals↗

Detecting functional interactions in a gene and signaling network by time-resolved somatic complementation analysis.

Somatic complementation by fusion of two mutant cells and mixing of their cytoplasms occurs when the genetic defect of one fusion partner is cured by the functional gene product provided by the other. We have found that complementation of mutational defects in the network mediating stimulus-induced commitment and sporulation of Physarum polycephalum may reflect time-dependent changes in the signaling state of its molecular building blocks. Network perturbation by fusion of mutant plasmodial cells in different states of activation, and the time-resolved analysis of somatic complementation effects can be used to systematically probe network structure and dynamics. Time-resolved somatic complementation quantitatively detects regulatory interactions between the functional modules of a network, independent of their biochemical composition or subcellular localization, and without being limited to direct physical interactions.

Animals↗

Myeloid membrane markers. I. Expression of marker antigens specific for murine granulocytes and their precursor cells.

Bone marrow cells were investigated by immunoelectromicroscopy and by quantitative photometric immunoradioautography with a rabbit antiserum against murine bone marrow cells. The serum was absorbed with murine spleen, liver and thymus cells until it no longer reacted with thymocytes and lymph node cells in a quantitative complement fixation test. The antiserum stained granulopoietic but not erythropoietic or lymphopoietic cells. The density of the myeloid antigen on single cells increased with the differentiation from immature to mature granulopoietic cells. While the increase of label was statistically not significant at the level of differentiation from promyelocyte to myelocyte and metamyelocyte to band neutrophil, there was a remarkable gain of label from myeloblast to promyelocyte and from band neutrophil to segmented neutrophil. This was evident under the electron microscopy using peroxidase-labeled antibodies and could be measured quantitatively with photometric immunoradioautography using 125I-labeled antibodies.

Absorption↗

Health promotion through healthy public policy: the contribution of complementary research methods.

The focus of the "new public health" is moving from the level of individuals to the level of organizations, communities, and broader social policies. Traditional quantitative methods which are appropriate at the level of individual behaviour change, require adaptation and refinement when sociopolitical change becomes the mechanism for health promotion. Because of their training and experience, health services researchers and health educators, especially psychologists, are understandably resistant to making necessary methodologic changes. Well-designed and carefully conducted qualitative studies, using techniques such as ethnographic interviews, participant observation, case studies, or focus group activities, are required to complement quantitative approaches. These studies can fill gaps where quantitative techniques are suboptimal or even inappropriate. Hard qualitative techniques can also support soft quantitative methods. Their utility in process evaluation is now beyond dispute. Recent work at the New England Research Institute is used to illustrate the role of qualitative research in the evaluation of health promotion through planned sociopolitical change.

Health Policy↗

Genetic characteristics of conditional lethal mutants of vesicular stomatitis virus induced by 5-fluorouracil, 5-azacytidine, and ethyl methane sulfonate.

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.

Animals↗

The promotion of health through planned sociopolitical change: challenges for research and policy.

The focus of health promotion is moving from the level of individuals to organizations, communities and broader social policy. Traditional quantitative methods (e.g. social surveys and experimental designs) which are variously appropriate at the level of individual behavior change require adaptation and refinement when sociopolitical change becomes the mechanism for health promotion. Because of their training and experience health services researchers and health educators (especially psychologists) are understandably resistant to necessary methodologic changes. Well designed and carefully conducted qualitative studies (e.g. ethnographic interviewing, participant observation, case studies and focus group activities) are required to complement quantitative approaches, and can fill gaps where quantitative techniques are suboptimal or even inappropriate: hard qualitative techniques can support soft quantitative methods. Their utility in process evaluation is now beyond dispute. Recent work at the New England Research Institute (NERI) is used to illustrate the role of qualitative research in the evaluation of health promotion through planned sociopolitical change.

Health Behavior↗

Quantitative determination of antibody in idiopathic thrombocytopenic purpura. Correlation of serum and platelet-bound antibody with clinical response.

We studied the clinical applicability of a recently developed technic that determines antiplatelet antibody directly on the platelet surface or in serum. The technic is a quantitative complement lysis-inhibition assay. Normal platelets have less than 0.4 pg of surface IgG. All patients with idiopathic thrombocytopenic purpura who were studied had greater than that value. Surface IgG was increased in inverse proportion to the platelet count. Surface levels of greater than 1.1 pg correlated with failure to respond to prednisone therapy. Incubation of normal serums with normal platelets did not increase surface IgG of such platelets, but the incubation with thrombocytopenic serums increased their surface IgG 0.5 to 100 times. The degree of increase did not predict response to treatment. However, quantitation of surface IgG of thrombocytopenic platelets was useful in predicting response to treatment.

Absorption↗

Quantitative trait loci responsible for variation in sexually dimorphic traits in Drosophila melanogaster.

To understand the mechanisms of morphological evolution and species divergence, it is essential to elucidate the genetic basis of variation in natural populations. Sexually dimorphic characters, which evolve rapidly both within and among species, present attractive models for addressing these questions. In this report, we map quantitative trait loci (QTL) responsible for variation in sexually dimorphic traits (abdominal pigmentation and the number of ventral abdominal bristles and sex comb teeth) in a natural population of Drosophila melanogaster. To capture the pattern of genetic variation present in the wild, a panel of recombinant inbred lines was created from two heterozygous flies taken directly from nature. High-resolution mapping was made possible by cytological markers at the average density of one per 2 cM. We have used a new Bayesian algorithm that allows QTL mapping based on all markers simultaneously. With this approach, we were able to detect small-effect QTL that were not evident in single-marker analyses. Our results show that at least for some sexually dimorphic traits, a small number of QTL account for the majority of genetic variation. The three strongest QTL account for >60% of variation in the number of ventral abdominal bristles. Strikingly, a single QTL accounts for almost 60% of variation in female abdominal pigmentation. This QTL maps to the chromosomal region that Robertson et al. have found to affect female abdominal pigmentation in other populations of D. melanogaster. Using quantitative complementation tests, we demonstrate that this QTL is allelic to the bric a brac gene, whose expression has previously been shown to correlate with interspecific differences in pigmentation. Multiple bab alleles that confer distinct phenotypes appear to segregate in natural populations at appreciable frequencies, suggesting that intraspecific and interspecific variation in abdominal pigmentation may share a similar genetic basis.

Animals↗

Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.

Functional and genomic approaches can be integrated to screen efficiently for pathogenic alleles in founder populations. We applied such approaches to analysis of the cancer-associated cell cycle regulator CHEK2 in the Ashkenazi Jewish population. We first identified two extended haplotypes at CHEK2 that co-segregated with breast cancer in high-risk families. We sequenced CHEK2 in a case representing each haplotype and discovered two novel amino acid substitutions, CHEK2.S428F in the kinase domain and CHEK2.P85L in the N-terminal region. To assay these alleles for loss of CHEK2 function, we tested their capacity to complement Rad53 deletion in Saccharomyces cerevisiae. CHEK2.S428F failed to complement Rad53 and thus largely abrogates normal CHEK2 function, whereas CHEK2.P85L complemented Rad53 as well as did wild-type CHEK2. Epidemiologic analyses were concordant with the functional tests. Frequencies of CHEK2.S428F heterozygotes were 2.88% (47/1632) among female breast cancer patients not selected for family history or age at diagnosis and 1.37% (23/1673) among controls (OR=2.13, 95% CI [1.26, 3.69], P=0.004), whereas frequencies of CHEK2.P85L were 0.92% among cases and 0.83% among controls. On the basis of the experience of mothers, sisters and daughters of probands, breast cancer risk due to CHEK2.S428F was estimated as 0.17 (+/-0.08) by age 60. We conclude that CHEK2.S428F increases breast cancer risk approximately 2-fold among Ashkenazi Jewish women, whereas CHEK2.P85L is a neutral allele. In general, these results suggest that selecting probands with extended haplotypes that co-segregate with disease can improve the efficiency of resequencing efforts and that quantitative complementation tests in yeast can be used to evaluate variants in genes with highly conserved function.

Amino Acid Substitution↗