The Effect of Isogenization on the Phenotypic Manifestation of Quantitative Traits in Drosophila melanogaster

A comparative analysis of the phenotypic values of the proximal and distal fragments of the radial wing vein was carried out in heterogeneous lines of Drosophila melanogaster and in isogenic lines derived from them with the help of a balancer line. The mean values of the traits in the isogenic lines were shown to significantly differ from the corresponding values in the parental heterogeneous lines. Apparently, the change in the trait values was caused by a double recombination exchange between the inverted and the normal chromosomes, which suggests partial crossing over suppression in the balancer lines.     

Comparative analysis of MGE 412 patterns in 18 isogenic lines of Drosophila melanogaster

Comparative analysis of patterns of mobile genetic element 412 was conducted in 18 isogenic lines of Drosophila melanogaster isolated in three isogenic experiments in 1987 through 1999. Twelve "extra-hot" isogenization sites (in 15-18 lines) and 23 "hot" isogenization sites (> or = 10 lines) were found; of these, 19 occurred in the original heterogeneous line. These sites virtually do not overlap with hot induction sites of transposition. Sites of the latter group generally retain their positions during isogenization. It was shown that no more than 20% of the new sites were brought from the balancer by double recombination, while inbreeding and outbreeding caused 80% of them. Different factors were shown to have different hot isogenization sites. A similarity tree was constructed for the patterns of 18 isogenic lines. The maximum peak of the tree was very low (< 0.25), i.e., the isogenic lines are more similar to than different from one another. The tree was subdivided into subtrees. The division was in good agreement with the isogenization groups corresponding to individual isogenization experiments. Significant correlation was found between the total fragment length and the number of new sites per lines.     

Comparative Analysis of MGE 412 Patterns in 18 Isogenic Lines of Drosophila melanogaster

Comparative analysis of patterns of mobile genetic element 412 was conducted in 18 isogenic lines of Drosophila melanogaster isolated in three isogenic experiments in 1987 through 1999. Twelve extra-hot isogenization sites (in 15–18 lines) and 23 hot isogenization sites (10 lines) were found; of these, 19 occurred in the original heterogeneous line. These sites virtually do not overlap with hot induction sites of transposition. Sites of the latter group generally retain their positions during isogenization. It was shown that no more than 20% of the new sites were brought from the balancer by double recombination, while inbreeding and outbreeding caused 80% of them. Different factors were shown to have different hot isogenization sites. A similarity tree was constructed for the patterns of 18 isogenic lines. The maximum peak of the tree was very low (< 0.25), i.e., the isogenic lines are more similar to than different from one another. The tree was subdivided into subtrees. The division was in good agreement with the isogenization groups corresponding to individual isogenization experiments. Significant correlation was found between the total fragment length and the number of new sites per lines.     

Comparative contribution of different genetic factors in induction of MGE transpositions under isogenization

Localization patterns of mobile genetic element 412 in polytene chromosomes of larvae from the control (riC) line, the balancer line, the F1 and F2 generations of the isogenization scheme, and 10 final isogenic lines were obtained and compared. The contributions of the recombination transfer of mobile genetic element copies from the balancer line, the outbreeding of control and balancer lines, and the inbreeding of isogenized lines to the rate of transposition were determined and estimated. These constituted < 0.187, < 0.30, and > 0.207 events per initial mobile genetic element copy per isogenized haploid genome per isogenization, respectively. During consecutive steps of isogenization (F1-F2-isogenic lines), the total transposition rate decreased: 2.09, 1.78, and 0.69. This was explained in terms of the existence of large selective and random losses in the variability of mobile genetic elements within the sites of their patterns during isogenization. The existence of a recombination transfer does not change the main conclusions and estimates regarding isogenization-induced transpositions.     

Analysis of MGE Dm412 localization pattern in Drosophila melanogaster lines undergoing long-term selection for a quantitative character]

The subpopulation composed of the mixture of Drosophila isogenic lines with interrupted wing radial vein (mutation radius incompletus, ri) was subjected to long-term selection in different directions for increase or decrease in expression of the ri gene. As a result, the lines with contrasting different values of mean character phenotype were developed. The isogenic lines of mean character phenotype were developed. The isogenic lines and F2 from their crosses with selected lines were analysed by the pattern of copia-like MGE DM412 localization. The isogenic lines were shown to have individual pattern, the selected lines differing strongly from them. Selection led to the loss of Dm412 localization sites during negative selection, while positive selection results both in loss and acquisition of sites. Correlation between the phenotype of the quantitative character and the pattern of MGE Dm412 was revealed.     

Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks

Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.     

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. III. Genetic Variability of Inducing Factors of Amylase and Fitness

"Inducibility" of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme's specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster . Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r_g = 0.63 ± 0.2), the enzyme activities in the normal medium were not (r_g = 0.12 ± 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms.     

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.     

Novel lethal mouse mutants produced in balancer chromosome screens

Mutagenesis screens are a valuable method to identify genes that are required for normal development. Previous mouse mutagenesis screens for lethal mutations were targeted at specific time points or for developmental processes. Here we present the results of lethal mutant isolation from two mutagenesis screens that use balancer chromosomes. One screen was localized to mouse chromosome 4, between the STS markers D4Mit281 and D4Mit51. The second screen covered the region between Trp53 and Wnt3 on mouse chromosome 11. These screens identified all lethal mutations in the balancer regions, without bias towards any phenotype or stage of death. We have isolated 19 lethal lines on mouse chromosome 4, and 59 lethal lines on chromosome 11, many of which are distinct from previous mutants that map to these regions of the genome. We have characterized the mutant lines to determine the time of death, and performed a pair-wise complementation cross to determine if the mutations are allelic. Our data suggest that the majority of mouse lethal mutations die during mid-gestation, after uterine implantation, with a variety of defects in gastrulation, heart, neural tube, vascular, or placental development. This initial group of mutants provides a functional annotation of mouse chromosomes 4 and 11, and indicates that many novel developmental phenotypes can be quickly isolated in defined genomic intervals through balancer chromosome mutagenesis screens.     

利用两种方法构建的近等基因系对水稻两个多效区段遗传效应进行评价

水稻每穗颖花数是水稻产量的重要构成因子之一。适当的抽穗期和株高对水稻高产是非常必要的。依据珍汕97和HR5衍生的重组自交系初步定位的结果, 利用高世代回交的方法构建了第7染色体同时控制抽穗期、株高和每穗颖花数的靶区段近等基因系(BC4F2); 利用基于重组自交系群体的杂合区段自交的方法构建了第8染色体同时控制抽穗期、株高和每穗颖花数的靶区段近等基因系, 并利用两个近等基因系对这两个多效区段的遗传效应进行了准确的评价。两个近等基因系的QTL分析结果表明, 3个性状都是由一个QTL或紧密连锁的QTL控制, 而且加性效应和显性效应的方向均相同; 同时3个性状在各自的近等基因系中呈现典型的双峰分布或不连续分布,这些结果暗示3个性状可能是一因多效的结果。文章还对抽穗期和株高与水稻产量的关系、3个性状显著正相关在育种中的应用及两种构建近等基因系方法的优缺点也进行了讨论。     

Effect of differing degrees of genetic variability on the toxic effects of aflatoxin B1 among resistant strains of Drosophila melanogaster (Diptera)

Toxicity studies, using outbred lines with much genetic variability and isogenic lines with no genetic variability from two strains of Drosophila melanogaster, Lausanne-S and Oregon-R, are reported. In both of these wild-type strains, larval and pupal development are known to be relatively resistant to the toxic effects of media containing aflatoxin B₁, a mycotoxin with carcinogenic and mutagenic properties. To eliminate genetic variability, each strain was made “isogenic” by a standard chromosomal substitution technique. Each isogenic strain, in comparison to the appropriate outbred control strain, showed a significant decrease in egg-to-adult viability when offspring were allowed to develop from the egg stage on media containing 1.0 ppm aflatoxin B₁. However, the resistance levels shown by the offspring of crosses between the two isogenic strains were not significantly different in viability than those of the appropriate controls. The relationship of these results with the level of genetic variability prossessed by the outbred and isogenic lines is discussed.     

Transposable elements and the penetrance of quantitative characters in Drosophila melanogaster

We wanted to determine whether there is a correlation between the quantitative character, the penetrance of the loss of humeral bristles in scute lines, and the distribution of transposable genetic elements in their genomes. We derived 18 isogenic lines with penetrance ranging between 2.8% and 92.0% from six mutant lines. The localization of the transposable elements (TEs) P, mdg1, Dm412, copia, gypsy and B104 was determined in all isogenic derivatives by in situ hybridization. The total number of the TE sites over all lines was 180. A comparison of the distribution of the TEs in the isogenic lines revealed the location of sites typical of lines with similar penetrance, no matter which parental line was involved. The results obtained suggest that such typical sites appear to tag the genome regions where the polygenes affecting the character in question are most likely to be found.     

Micro-spatial population differentiation in activity of glycerol-3-phosphate oxidase (GPO) from mitochondria of Drosophila melanogaster

Replicate mass-bred laboratory populations of D. melanogaster were derived from females collected in the Tahbilk winery cellar and from females collected outside but from within two kilometres of the cellar. When mitochondrial extracts from larvae were assayed for specific activity of glycerol-3-phosphate oxidase the cellar populations had levels only 50% of those from the outside area, confirming an earlier report of such a difference among isofemale lines derived from these same areas. This micro-spatial differentiation occurred when larvae were raised on a medium supplemented with both sucrose (5% w/v) and ethanol (4% v/v), known to effect high GPO activity, but was not detected when the larvae were raised on unsupplemented medium.A heritable basis for larval GPO activity variation was confirmed in a set of 32 isogenic second chromosome substitution lines and measured in a subset of 4 of these lines about 25 generations later. A reciprocal cross using two isogenic substitution lines with the highest and lowest activities suggested the difference was attributable to genes acting additively and that there were no maternal or paternal effects. The detection of a collection site difference in GPO enzyme activity in the isogenic lines suggests that polymorphic variation on the second chromosome is responsible for the differentiation at the winery.Variation in adult GPO activity did not show a dependence on the winery location from where the isogenic lines were derived nor was there an effect of line. Adult GPO activity was significantly higher than that detected in larval tissues and did not show a dependence on the sugar/ethanol level in the growth medium.     

The Effect of Level of Heterozygosity on the Performance of Hybrids between Isogenic Lines of Barley

Sixteen "isogenic" lines of Atlas 46 barley differing in one to four short chromosome segments, and 16 heterozygotes obtained by crossing these lines to male-sterile Atlas, were used to study the effect of level of heterozygosity on performance. In field tests conducted in four environments (two planting dates in two years) significant differences were found among the homozygous isogenic lines for the traits seed yield, kernel weight, tiller number, plant height, and heading time; thus each of the marked chromosome segments carries genes which, when homozygous, affect these quantitative characters. It was also found that heterozygotes produced more and heavier kernels and were taller and earlier than homozygotes but there was no clear indication that the degree of heterosis increased as the number of heterozygous segments increased from one to five. Degree of heterosis was, however, strongly affected by the environment, by allelic state at each segment (especially the segment marked by the two-row, six-row spike locus), and also by genotype for other marked segments. These results indicate that heterosis in barley has a more complex structure than can be adequately represented by simple models, such as the multiplicative model in which fitnesses are the product of fitnesses at individual loci, or threshold models in which optimum fitness is approached asymptotically as the number of heterozygous loci increases.     

Increased Selection Response in Larger Populations. II. Selection for Ethanol Vapor Resistance in Drosophila Melanogaster at Two Population Sizes

The effect of large population size on selection response was investigated using Drosophila melanogaster, with four "small" lines of 160 selected parents/generation compared to two "large" lines of 1,600 selected parents/generation. All lines were selected under similar conditions at a selection intensity of approximately 0.55 standard deviations, for 65 generations, for increased ethanol vapor resistance (measured in minutes required to become anesthetized). Two unselected control lines of 320 parents/generation were also maintained. A significant effect of population size was found. The final treatment means and standard errors were: 27.91 +/- 1.28 min (two "large" lines); 19.40 +/- 1.54 min (four "small" lines); and 4.98 +/- 0.35 min (two control lines). To estimate the mutation rate for the trait, two isogenic lines of about 400 selected parents were selected for 29 generations. The mean increase in additive genetic variance per generation was 0.0009 times the initial environmental variance of the outbred lines. This is comparable to other reported mutation rates. Mutation can explain part of the difference in evolved resistance between treatments, but it appears that even at rather large population sizes, a large difference in long-term response can be obtained in larger outbred lines, from more complete utilization of the initial genetic variation.     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK _m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee.     

Retention of induced mutations in a Drosophila reverse-genetic resource

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.     

Associations among inbred lines of maize using electrophoretic,chromatographic, and pedigree data

Summary Associations among 18 Lancaster Sure Crop derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the degree of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) and starch gel electrophoresis of allozymes and to compare associations among lines revealed by biochemical and pedigree data. RP-HPLC revealed 11 different chromatograms that uniquely identified 79% of lines that differed by more than isogenic or near isogenic segments. Allozymic data for 21 loci provided unique discrimination among 93% of non-isogenic lines. Chromatographic and allozymic data together provided unique characterization of all non-isogenic lines. Cluster and multivariate analyses of biochemical data associated lines into three groups that would have been expected on the basis of pedigree breeding records. More detailed associations were dependent upon the data set employed. Multivariate and cluster analysis of chromatographic, electrophoretic, and pedigree data could be useful in revealing more detailed associations among elite germplasm than hitherto available, thus providing data pertinent to line and hybrid development, plant variety protection, and germplasm security.     

The Effect of Sequence Homozygosity on the Frequency of X-Chromosomal Exchange in Drosophila Melanogaster Females

The repair of mismatched heteroduplex DNA has been implicated in the normal resolution of meiotic exchange events. Although sequence microheterogeneity over defined intervals of homologous chromosomes has been correlated with local effects on recombination, this correlation has not previously been extended to effects on chromosomal levels of exchange. In order to determine the role of microheterogeneity in normal exchange between homologs, a system was devised for monitoring exchange between isogenic X chromosomes. Lack of microheterogeneity did not significantly alter the frequency of exchange along the isogenic X chromosomes relative to controls or to previously reported values. There were, however, characteristic levels of exchange intrinsic to the cloned X chromosomes in each of the lines tested.