Statistical Analysis of Nondisjunction Assays in Drosophila

Many advances in the understanding of meiosis have been made by measuring how often errors in chromosome segregation occur. This process of nondisjunction can be studied by counting experimental progeny, but direct measurement of nondisjunction rates is complicated by not all classes of nondisjunctional progeny being viable. For X chromosome nondisjunction in Drosophila female meiosis, all of the normal progeny survive, while nondisjunctional eggs produce viable progeny only if fertilized by sperm that carry the appropriate sex chromosome. The rate of nondisjunction has traditionally been estimated by assuming a binomial process and doubling the number of observed nondisjunctional progeny, to account for the inviable classes. However, the correct way to derive statistics (such as confidence intervals or hypothesis testing) by this approach is far from clear. Instead, we use the multinomial-Poisson hierarchy model and demonstrate that the old estimator is in fact the maximum-likelihood estimator (MLE). Under more general assumptions, we derive asymptotic normality of this estimator and construct confidence interval and hypothesis testing formulae. Confidence intervals under this framework are always larger than under the binomial framework, and application to published data shows that use of the multinomial approach can avoid an apparent type 1 error made by use of the binomial assumption. The current study provides guidance for researchers designing genetic experiments on nondisjunction and improves several methods for the analysis of genetic data.MEIOSIS is a specialized cell division, where a diploid cell undergoes a single round of replication followed by two rounds of segregation to produce four haploid gametes. During this segregation, chromosomes must correctly separate (or disjoin) from their homologs at meiosis I, followed by sister chromatids disjoining at meiosis II. When chromosomes fail to disjoin from their partners, the resultant nondisjunction produces aneuploid gametes with the wrong number of chromosomes. The study of meiotic nondisjunction in Drosophila has a long and distinguished history of publication in genetics, with the inaugural article published in this journal being Calvin Bridges'' use of nondisjunction to prove the chromosome theory of heredity (Bridges 1916). The first study that screened variants isolated from natural populations used nondisjunction to identify meiotic mutants (Sandler et al. 1968), as did the first EMS-induced mutant screen (Baker and Carpenter 1972). Subsequent screens using new mutagens or techniques have also relied on measuring nondisjunction to identify mutants of interest (Sekelsky et al. 1999). Indeed, much of the progress that has been made in the study of meiosis would not have been possible without the use of nondisjunction to identify new mutations that are defective at some step in chromosome segregation.However, one difficulty in estimating nondisjunction rates is that in most instances the resulting aneuploid progeny cannot survive. Fortunately, in Drosophila it is possible to design crosses to recover them. Sex determination in flies is based on the number of X chromosomes, rather than a masculinizing Y chromosome as in mammals. This means that XO flies are viable (but sterile) males, while XXY flies are viable females. Therefore, it is possible to recover both normal and nondisjunctional progeny, as a nullo-X egg fertilized by an X-bearing sperm will survive as an XO male, while a diplo-X egg fertilized by a sperm lacking an X will be female (XXY). By using visible markers on the sex chromosomes, these exceptional progeny are straightforward to identify. However, if those eggs are fertilized by the other class of sperm, the resulting OY or XXX progeny are inviable. Therefore, the nondisjunction rate that occurs during meiosis is not equal to the proportion of nondisjunctional progeny, as only 50% of nondisjunctional eggs receive sperm compatible with viability, while all normal eggs are viable.Given this experimental limitation, what is the correct method to calculate the error rate during meiosis? For this discussion, let N be the total number of progeny produced in an experiment, let X₁ be the number of inviable nondisjunctional progeny (OY and XXX), let X₂ be the number of viable nondisjunctional progeny (XO and XXY), and let X₃ be the number of normal progeny (XY and XX), such that N = X₁ + X₂ + X₃. If all progeny could be counted, then the nondisjunction rate would simply be (X₁ + X₂)/N.However, only flies that survive to adulthood can be counted, and therefore both X₁ and N are unknown. As X- and Y-bearing sperm are produced in equal numbers, live and dead nondisjunctional progeny are also expected in equal numbers. Therefore, K.W. Cooper (Cooper 1948) proposed the widely used estimator for the X chromosome nondisjunction rate, where X₂ is substituted for X₁ in the above formula, giving the rate as:(1)While this estimator works, the statistical properties of this estimator are not clear. Instead of following the early literature to combine X₁ and X₂ and use a binomial distribution, we go back to the three original categories and model the process as a multinomial distribution with latent number of progeny N, considering all three possible phenotypes for each progeny (nondisjunctional dead, nondisjunctional living, and normal). Whether a nondisjunctional oocyte becomes a nondisjunctional dead or nondisjunctional living progeny depends on the sex chromosome content of the sperm that fertilized it. As X- and Y-bearing sperm are produced in equal numbers during male meiosis, the usual genetic expectation for the rates of nondisjunctional dead and living progeny will be . However, even assuming that the rates of nondisjunctional dead and living progeny are different, with a Poisson assumption of N, we can derive the maximum-likelihood estimators (MLEs) for the nondisjunctional dead and nondisjunctional living rates. Under the usual genetic expectation of equality, the MLE of the nondisjunctional rate coincides with Cooper''s estimator, and we furthermore derive the exact distribution of . Under another set of reasonable assumptions, we show the consistency and asymptotic normality of Cooper''s estimator, and derive asymptotic results when comparing two nondisjunction rates. All these distributional results enable us to develop confidence interval and hypothesis testing related to p, or p_x − p_y in the case of comparing two nondisjunction rates from populations x and y.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

Effects of transmission reduction by insecticide-treated bed nets (ITNs) on parasite genetics population structure: I. The genetic diversity of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> parasites by microsatellite markers in western Kenya

Background

Insecticide-treated bed nets (ITNs) reduce malaria transmission and are an important prevention tool. However, there are still information gaps on how the reduction in malaria transmission by ITNs affects parasite genetics population structure. This study examined the relationship between transmission reduction from ITN use and the population genetic diversity of Plasmodium falciparum in an area of high ITN coverage in western Kenya.

Methods

Parasite genetic diversity was assessed by scoring eight single copy neutral multilocus microsatellite (MS) markers in samples collected from P. falciparum- infected children (< five years) before introduction of ITNs (1996, baseline, n = 69) and five years after intervention (2001, follow-up, n = 74).

Results

There were no significant changes in overall high mixed infections and unbiased expected heterozygosity between baseline (%M_A = 94% and H_e = 0.75) and follow up (%M_A = 95% and H_e = 0.79) years. However, locus specific analysis detected significant differences for some individual loci between the two time points. Pfg377 loci, a gametocyte-specific MS marker showed significant increase in mixed infections and H_e in the follow up survey (%M_A = 53% and H_e = 0.57) compared to the baseline (%M_A = 30% and H_e = 0.29). An opposite trend was observed in the erythrocyte binding protein (EBP) MS marker. There was moderate genetic differentiation at the Pfg377 and TAA60 loci (F_ST = 0.117 and 0.137 respectively) between the baseline and post-ITN parasite populations. Further analysis revealed linkage disequilibrium (LD) of the microsatellites in the baseline (14 significant pair-wise tests and I ^S _A = 0.016) that was broken in the follow up parasite population (6 significant pairs and I ^S _A = 0.0003). The locus specific change in H_e, the moderate population differentiation and break in LD between the baseline and follow up years suggest an underlying change in population sub-structure despite the stability in the overall genetic diversity and multiple infection levels.

Conclusions

The results from this study suggest that although P. falciparum population maintained an overall stability in genetic diversity after five years of high ITN coverage, there was significant locus specific change associated with gametocytes, marking these for further investigation.

     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule.     

BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.     

The spindle-associated transmembrane protein Axs identifies a membranous structure ensheathing the meiotic spindle

Mutations in the aberrant X segragation (Axs) gene disrupt the segregation of achiasmate chromosomes during female meiosis in Drosophila melanogaster. We show that Axs encodes the founding member of an eukaryotic family of transmembrane proteins. Axs protein colocalizes with components of the endoplasmic reticulum and is present within a structure ensheathing the meiotic spindle. In both meiotic and mitotic cells, Axs is recruited to the microtubules of assembling spindles. We propose that Axs and the sheath represent novel mediators of meiotic spindle assembly and chromosome segregation.