bluuebunny/crossref_metadata_2025 huggingface dataset:
Conservation of an Inhibitor of the Epidermal Growth Factor Receptor, Kekkon1, in Dipterans
Authors: Derheimer Frederick A , MacLaren Christina M , Weasner Brandon P , Alvarado Diego , Duffy Joseph B
Published: August 2004

Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1's function and evolution, we identified kek1 orthologs within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, we find that the cytoplasmic domain contains a region with the highest degree of conservation, which we have implicated in EGFR inhibition and dubbed the Kek tail (KT) box.
Genetic Dissection of itpr Gene Function Reveals a Vital Requirement in Aminergic Cells of Drosophila Larvae
Authors: Joshi Rohit , Venkatesh K , Srinivas R , Nair Shalima , Hasan Gaiti
Published: August 2004

Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus we have sequenced and identified single point mutations in seven mutant chromosomes. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with our earlier observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae. Here we show that the focus of this critical function lies in aminergic cells by rescue with UAS-itpr and DdCGAL4. However, this function does not require synaptic activity, suggesting that InsP3-mediated Ca2+ release regulates the neurohormonal action of serotonin.
Pleiotropic Effect of Disrupting a Conserved Sequence Involved in a Long-Range Compensatory Interaction in the Drosophila Adh Gene
Authors: Baines John F , Parsch John , Stephan Wolfgang
Published: August 2004

Recent advances in experimental analyses of the evolution of RNA secondary structures suggest a more complex scenario than that typically considered by Kimura's classical model of compensatory evolution. In this study, we examine one such case in more detail. Previous experimental analysis of long-range compensatory interactions between the two ends of Drosophila Adh mRNA failed to fit the classical model of compensatory evolution. To further investigate and verify long-range pairing in Drosophila Adh with respect to models of compensatory evolution and its potential functional role, we introduced site-directed mutations in the Drosophila melanogaster Adh gene. We explore two alternative hypotheses for why previous analysis of long-range compensatory interactions failed to fit the classical model. Specifically, we investigate whether the disruption of a conserved short-range pairing within Adh exon 2 has an effect on Adh expression or if there is a dual functional role of a conserved sequence in the 3′-UTR in both long-range pairing and the negative regulation of Adh expression. We find that a classical result was not observed due to the pleiotropic effect of changing a nucleotide involved in both long-range base pairing and the negative regulation of gene expression.
Evolution of Autosomal Suppression of the Sex-Ratio Trait in Drosophila
Authors: Vaz Suzana Casaccia , Carvalho Antonio Bernardo
Published: August 2004

The sex-ratio trait is the production of female-biased progenies due to X-linked meiotic drive in males of several Drosophila species. The driving X chromosome (called SR) is not fixed due to at least two stabilizing factors: natural selection (favoring ST, the nondriving standard X) and drive suppression by either Y-linked or autosomal genes. The evolution of autosomal suppression is explained by Fisher's principle, a mechanism of natural selection that leads to equal proportion of males and females in a sexually reproducing population. In fact, sex-ratio expression is partially suppressed by autosomal genes in at least three Drosophila species. The population genetics of this system is not completely understood. In this article we develop a mathematical model for the evolution of autosomal suppressors of SR (sup alleles) and show that: (i) an autosomal suppressor cannot invade when SR is very deleterious in males (c < ⅓, where c is the fitness of SR/Y males); (ii) “SR/ST, sup/+” polymorphisms occur when SR is partially deleterious (∼0.3 < c < 1); while (iii) SR neutrality (c = 1) results in sup fixation and thus in total abolishment of drive. So, surprisingly, as long as there is any selection against SR/Y males, neutral autosomal suppressors will not be fixed. In that case, when a polymorphic equilibrium exists, the average female proportion in SR/Y males' progeny is given approximately by (ac+1−a+a2(c+1)2+1−4ac)∕4ac, where a is the fitness of SR/ST females.
The Drosophila GAGA Factor Is Required for Dosage Compensation in Males and for the Formation of the Male-Specific-Lethal Complex Chromatin Entry Site at 12DE
Authors: Greenberg Anthony J , Yanowitz Judith L , Schedl Paul
Published: August 2004

Drosophila melanogaster males have one X chromosome, while females have two. To compensate for the resulting disparity in X-linked gene expression between the two sexes, most genes from the male X chromosome are hyperactivated by a special dosage compensation system. Dosage compensation is achieved by a complex of at least six proteins and two noncoding RNAs that specifically associate with the male X. A central question is how the X chromosome is recognized. According to a current model, complexes initially assemble at ∼35 chromatin entry sites on the X and then spread bidirectionally along the chromosome where they occupy hundreds of sites. Here, we report that mutations in Trithorax-like (Trl) lead to the loss of a single chromatin entry site on the X, male lethality, and mislocalization of dosage compensation complexes.
Chl1p, a DNA Helicase-Like Protein in Budding Yeast, Functions in Sister-Chromatid Cohesion
Authors: Skibbens Robert V
Published: August 2004

From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sisterchromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated.
Velvet, a Dominant Egfr Mutation That Causes Wavy Hair and Defective Eyelid Development in Mice
Authors: Du Xin , Tabeta Koichi , Hoebe Kasper , Liu Haiquan , Mann Navjiwan , Mudd Suzanne , Crozat Karine , Sovath Sosathya , Gong Xiaohua , Beutler Bruce
Published: August 2004

In the course of a large-scale program of ENU mutagenesis, we isolated a dominant mutation, called Velvet. The mutation was found to be uniformly lethal to homozygotes, which do not survive E13.5. Mice heterozygous for the Velvet mutation are born with eyelids open and demonstrate a wavy coat and curly vibrissae. The mutation was mapped to the proximal end of chromosome 11 by genome-wide linkage analysis. On 249 meioses, the locus was confined to a 2.7-Mb region, which included the epidermal growth factor receptor gene (Egfr). An A → G transition in the Egfr coding region of Velvet mice was identified, causing the amino acid substitution D833G. This substitution alters an essential triad of amino acids (DFG → GFG) that is normally required for coordination of the ATP substrate. As such, kinase activity is at least mostly abolished, but quaternary structure of the receptor is presumably maintained, accounting for the dominant effect. Velvet is the first known dominant representative of the Egfr allelic series that is fully viable, a fact that makes it particularly useful for developmental studies.
Multilocus Analysis of Variation and Speciation in the Closely Related Species Arabidopsis halleri and A. lyrata
Authors: Ramos-Onsins Sebastián E , Stranger Barbara E , Mitchell-Olds Thomas , Aguadé Montserrat
Published: August 2004

Nucleotide variation in eight effectively unlinked genes was surveyed in species-wide samples of the closely related outbreeding species Arabidopsis halleri and A. lyrata ssp. petraea and in three of these genes in A. lyrata ssp. lyrata and A. thaliana. Significant genetic differentiation was observed more frequently in A. l. petraea than in A. halleri. Average estimates of nucleotide variation were highest in A. l. petraea and lowest in A. l. lyrata, reflecting differences among species in effective population size. The low level of variation in A. l. lyrata is concordant with a bottleneck effect associated with its origin. The A. halleri/A. l. petraea speciation process was studied, considering the orthologous sequences of an outgroup species (A. thaliana). The high number of ancestral mutations relative to exclusive polymorphisms detected in A. halleri and A. l. petraea, the significant results of the multilocus Fay and Wu H tests, and haplotype sharing between the species indicate introgression subsequent to speciation. Average among-population variation in A. halleri and A. l. petraea was ∼1.5- and 3-fold higher than that in the inbreeder A. thaliana. The detected reduction of variation in A. thaliana is less than that expected from differences in mating system alone, and therefore from selective processes related to differences in the effective recombination rate, but could be explained by differences in population structure.
Population Genetics of the Wild Yeast Saccharomyces paradoxus
Authors: Johnson Louise J , Koufopanou Vassiliki , Goddard Matthew R , Hetherington Richard , Schäfer Stefanie M , Burt Austin
Published: August 2004

Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km2 sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is ∼0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species.
The Dominance of the Herbicide Resistance Cost in Several Arabidopsis thaliana Mutant Lines
Authors: Roux Fabrice , Gasquez Jacques , Reboud Xavier
Published: August 2004

Resistance evolution depends upon the balance between advantage and disadvantage (cost) conferred in treated and untreated areas. By analyzing morphological characters and simple fitness components, the cost associated with each of eight herbicide resistance alleles (acetolactate synthase, cellulose synthase, and auxin-induced target genes) was studied in the model plant Arabidopsis thaliana. The use of allele-specific PCR to discriminate between heterozygous and homozygous plants was used to provide insights into the dominance of the resistance cost, a parameter rarely described. Morphological characters appear more sensitive than fitness (seed production) because 6 vs. 4 differences between resistant and sensitive homozygous plants were detected, respectively. Dominance levels for the fitness cost ranged from recessivity (csr1-1, ixr1-2, and axr1-3) to dominance (axr2-1) to underdominance (aux1-7). Furthermore, the dominance level of the herbicide resistance trait did not predict the dominance level of the cost of resistance. The relationship of our results to theoretical predictions of dominance and the consequences of fitness cost and its dominance in resistance management are discussed.
Identification of Wheat Chromosomal Regions Containing Expressed Resistance Genes
Authors: Dilbirligi Muharrem , Erayman Mustafa , Sandhu Devinder , Sidhu Deepak , Gill Kulvinder S
Published: August 2004

The objectives of this study were to isolate and physically localize expressed resistance (R) genes on wheat chromosomes. Irrespective of the host or pest type, most of the 46 cloned R genes from 12 plant species share a strong sequence similarity, especially for protein domains and motifs. By utilizing this structural similarity to perform modified RNA fingerprinting and data mining, we identified 184 putative expressed R genes of wheat. These include 87 NB/LRR types, 16 receptor-like kinases, and 13 Pto-like kinases. The remaining were seven Hm1 and two Hs1pro-1 homologs, 17 pathogenicity related, and 42 unique NB/kinases. About 76% of the expressed R-gene candidates were rare transcripts, including 42 novel sequences. Physical mapping of 121 candidate R-gene sequences using 339 deletion lines localized 310 loci to 26 chromosomal regions encompassing ∼16% of the wheat genome. Five major R-gene clusters that spanned only ∼3% of the wheat genome but contained ∼47% of the candidate R genes were observed. Comparative mapping localized 91% (82 of 90) of the phenotypically characterized R genes to 18 regions where 118 of the R-gene sequences mapped.
Linkage Disequilibrium Testing When Linkage Phase Is Unknown
Authors: Schaid Daniel J
Published: August 2004

Linkage disequilibrium, the nonrandom association of alleles from different loci, can provide valuable information on the structure of haplotypes in the human genome and is often the basis for evaluating the association of genomic variation with human traits among unrelated subjects. But, linkage phase of genetic markers measured on unrelated subjects is typically unknown, and so measurement of linkage disequilibrium, and testing whether it differs significantly from the null value of zero, requires statistical methods that can account for the ambiguity of unobserved haplotypes. A common method to test whether linkage disequilibrium differs significantly from zero is the likelihood-ratio statistic, which assumes Hardy-Weinberg equilibrium of the marker phenotype proportions. We show, by simulations, that this approach can be grossly biased, with either extremely conservative or liberal type I error rates. In contrast, we use simulations to show that a composite statistic, proposed by Weir and Cockerham, maintains the correct type I error rates, and, when comparisons are appropriate, has similar power as the likelihood-ratio statistic. We extend the composite statistic to allow for more than two alleles per locus, providing a global composite statistic, which is a strong competitor to the usual likelihood-ratio statistic.
Developmental Associations Between Traits: Covariance and Beyond
Authors: Rice Sean H
Published: August 2004

Statistical associations between phenotypic traits often result from shared developmental processes and include both covariation between the trait values and more complex associations between higher moments of the joint distribution of traits. In this article, an analytical technique for calculating the covariance between traits is presented on the basis of (1) the distribution of underlying genetic and environmental variation that jointly influences the traits and (2) the mechanics of how these underlying factors influence the development of each trait. It is shown that epistasis can produce patterns of covariation between traits that are not seen in additive models. Applying this approach to a trait in parents and the same trait in their offspring allows us to study the consequences of epistasis for the evolution of additive genetic variance and heritability. This analysis is then extended to the study of more complicated associations between traits. It is shown that even traits that are not correlated may exhibit developmental associations that influence their joint evolution.
Impact of Nonrandom Mating on Genetic Variance and Gene Flow in Populations With Mass Selection
Authors: Sánchez Leopoldo , Woolliams John A
Published: August 2004

The mechanisms by which nonrandom mating affects selected populations are not completely understood and remain a subject of scientific debate in the development of tractable predictors of population characteristics. The main objective of this study was to provide a predictive model for the genetic variance and covariance among mates for traits subjected to directional selection in populations with nonrandom mating based on the pedigree. Stochastic simulations were used to check the validity of this model. Our predictions indicate that the positive covariance among mates that is expected to result with preferential mating of relatives can be severely overpredicted from neutral expectations. The covariance expected from neutral theory is offset by an opposing covariance between the genetic mean of an individual's family and the Mendelian sampling term of its mate. This mechanism was able to predict the reduction in covariance among mates that we observed in the simulated populations and, in consequence, the equilibrium genetic variance and expected long-term genetic contributions. Additionally, this study provided confirmatory evidence on the postulated relationships of long-term genetic contributions with both the rate of genetic gain and the rate of inbreeding (ΔF) with nonrandom mating. The coefficient of variation of the expected gene flow among individuals and ΔF was sensitive to nonrandom mating when heritability was low, but less so as heritability increased, and the theory developed in the study was sufficient to explain this phenomenon.
The Yeast Splicing Factor Prp40p Contains Functional Leucine-Rich Nuclear Export Signals That Are Essential for Splicing
Authors: Murphy Mark W , Olson Brian L , Siliciano Paul G
Published: August 2004

To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.
Estimating Effective Population Size or Mutation Rate With Microsatellites
Authors: Xu Hongyan , Fu Yun-Xin
Published: August 2004

Microsatellites are short tandem repeats that are widely dispersed among eukaryotic genomes. Many of them are highly polymorphic; they have been used widely in genetic studies. Statistical properties of all measures of genetic variation at microsatellites critically depend upon the composite parameter θ = 4Nμ, where N is the effective population size and μ is mutation rate per locus per generation. Since mutation leads to expansion or contraction of a repeat number in a stepwise fashion, the stepwise mutation model has been widely used to study the dynamics of these loci. We developed an estimator of θ, θ^F, on the basis of sample homozygosity under the single-step stepwise mutation model. The estimator is unbiased and is much more efficient than the variance-based estimator under the single-step stepwise mutation model. It also has smaller bias and mean square error (MSE) than the variance-based estimator when the mutation follows the multistep generalized stepwise mutation model. Compared with the maximum-likelihood estimator θ^L by Nielsen (1997), θ^F has less bias and smaller MSE in general. θ^L has a slight advantage when θ is small, but in such a situation the bias in θ^L may be more of a concern.
A Bivalent Polyploid Model for Mapping Quantitative Trait Loci in Outcrossing Tetraploids
Authors: Wu Rongling , Ma Chang-Xing , Casella George
Published: August 2004

Two major aspects have made the genetic and genomic study of polyploids extremely difficult. First, increased allelic or nonallelic combinations due to multiple alleles result in complex gene actions and interactions for quantitative trait loci (QTL) in polyploids. Second, meiotic configurations in polyploids undergo a complex biological process including either bivalent or multivalent formation, or both. For bivalent polyploids, different degrees of preferential chromosome pairings may occur during meiosis. In this article, we develop a maximum-likelihood-based model for mapping QTL in tetraploids by considering the quantitative inheritance and meiotic mechanism of bivalent polyploids. This bivalent polyploid model is implemented with the EM algorithm to simultaneously estimate QTL position, QTL effects, and QTL-marker linkage phases by incorporating the impact of a cytological parameter determining bivalent chromosome pairings (the preferential pairing factor). Simulation studies are performed to investigate the performance and robustness of our statistical method for parameter estimation. The implication and extension of the bivalent polyploid model are discussed.
Influence of Dominance, Leptokurtosis and Pleiotropy of Deleterious Mutations on Quantitative Genetic Variation at Mutation-Selection Balance
Authors: Zhang Xu-Sheng , Wang Jinliang , Hill William G
Published: August 2004

In models of maintenance of genetic variance (VG) it has often been assumed that mutant alleles act additively. However, experimental data show that the dominance coefficient varies among mutant alleles and those of large effect tend to be recessive. On the basis of empirical knowledge of mutations, a joint-effect model of pleiotropic and real stabilizing selection that includes dominance is constructed and analyzed. It is shown that dominance can dramatically alter the prediction of equilibrium VG. Analysis indicates that for the situations where mutations are more recessive for fitness than for a quantitative trait, as supported by the available data, the joint-effect model predicts a significantly higher VG than does an additive model. Importantly, for what seem to be realistic distributions of mutational effects (i.e., many mutants may not affect the quantitative trait substantially but are likely to affect fitness), the observed high levels of genetic variation in the quantitative trait under strong apparent stabilizing selection can be generated. This investigation supports the hypothesis that most VG comes from the alleles nearly neutral for fitness in heterozygotes while apparent stabilizing selection is contributed mainly by the alleles of large effect on the quantitative trait. Thus considerations of dominance coefficients of mutations lend further support to our previous conclusion that mutation-selection balance is a plausible mechanism of the maintenance of the genetic variance in natural populations.
Bayesian Estimation of Genomic Distance
Authors: Durrett Richard , Nielsen Rasmus , York Thomas L
Published: August 2004

We present a Bayesian approach to the problem of inferring the number of inversions and translocations separating two species. The main reason for developing this method is that it will allow us to test hypotheses about the underlying mechanisms, such as the distribution of inversion track lengths or rate constancy among lineages. Here, we apply these methods to comparative maps of eggplant and tomato, human and cat, and human and cattle with 170, 269, and 422 markers, respectively. In the first case the most likely number of events is larger than the parsimony value. In the last two cases the parsimony solutions have very small probability.
On the Metropolis-Hastings Acceptance Probability to Add or Drop a Quantitative Trait Locus in Markov Chain Monte Carlo-Based Bayesian Analyses
Authors: Jannink Jean-Luc , Fernando Rohan L
Published: August 2004

The Metropolis-Hastings algorithm used in analyses that estimate the number of QTL segregating in a mapping population requires the calculation of an acceptance probability to add or drop a QTL from the model. Expressions for this acceptance probability need to recognize that sets of QTL are unordered such that the number of equivalent sets increases with the factorial of the QTL number. Here, we show how accounting for this fact affects the acceptance probability and review expressions found in the literature.
Marker-Based Cloning of the Region Containing the UhAvr1 Avirulence Gene From the Basidiomycete Barley Pathogen Ustilago hordei
Authors: Linning R , Lin D , Lee N , Abdennadher M , Gaudet D , Thomas P , Mills D , Kronstad J W , Bakkeren G
Published: August 2004

Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940s. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain 54 progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a nonrepetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an ∼80-kb region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.
A dnaC Mutation in Escherichia coli That Affects Copy Number of ColE1-Like Plasmids and the PriA-PriB (but Not Rep-PriC)Pathway of Chromosomal Replication Restart
Authors: Harinarayanan R , Gowrishankar J
Published: August 2004

Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells. In this study, a mutation [dna-C1331(A84T)] in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype. The mutation also reduced the copy number of the plasmids in otherwise wild-type strains. In comparison with the isogenic dnaC+ strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins. However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability. A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks. We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom– ColE1-like plasmids).
The Identification of Pcl1-Interacting Proteins That Genetically Interact With Cla4 May Indicate a Link Between G1 Progression and Mitotic Exit
Authors: Keniry Megan E , Kemp Hilary A , Rivers David M , Sprague George F
Published: August 2004

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4Δ strain. These mutations define >60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G1 cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a two-hybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1•Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G1 Pcl1•Pho85 complex regulates mitotic exit machinery.
PaASK1, a Mitogen-Activated Protein Kinase Kinase Kinase That Controls Cell Degeneration and Cell Differentiation in Podospora anserina
Authors: Kicka Sébastien , Silar Philippe
Published: August 2004

MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation.
Differential Effects of Drosophila Mastermind on Asymmetric Cell Fate Specification and Neuroblast Formation
Authors: Yedvobnick Barry , Kumar Anumeha , Chaudhury Padmashree , Opraseuth Jonathan , Mortimer Nathan , Bhat Krishna Moorthi
Published: August 2004

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 → GMC-1 → RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.
Evidence for a piwi-Dependent RNA Silencing of the gypsy Endogenous Retrovirus by the Drosophila melanogaster flamenco Gene
Authors: Sarot Emeline , Payen-Groschêne Geneviève , Bucheton Alain , Pélisson Alain
Published: August 2004

In Drosophila melanogaster, the endogenous retrovirus gypsy is repressed by the functional alleles (restrictive) of an as-yet-uncloned heterochromatic gene called flamenco. Using gypsy-lacZ transcriptional fusions, we show here that this repression takes place not only in the follicle cells of restrictive ovaries, as was previously observed, but also in restrictive larval female gonads. Analyses of the role of gypsy cis-regulatory sequences in the control of gypsy expression are also presented. They rule out the hypothesis that gypsy would contain a single binding region for a putative Flamenco repressor. Indeed, the ovarian expression of a chimeric yp3-lacZ construct was shown to become sensitive to the Flamenco regulation when any of three different 5′-UTR gypsy sequences (ranging from 59 to 647 nucleotides) was incorporated into the heterologous yp3-lacZ transcript. The piwi mutation, which is known to affect RNA-mediated homology-dependent transgene silencing, was also shown to impede the repression of gypsy in restrictive female gonads. Finally, a RNA-silencing model is also supported by the finding in ovaries of short RNAs (25–27 nucleotides long) homologous to sequences from within the gypsy 5′-UTR.
Linkage Disequilibrium in the Domesticated Pig
Authors: Nsengimana Jérémie , Baret Philippe , Haley Chris S , Visscher Peter M
Published: August 2004

This study investigated the extent of linkage disequilibrium (LD) in two genomic regions (on chromosomes 4 and 7) in five populations of domesticated pigs. LD was measured with D′ and tested for significance with the Fisher exact test. Effects of genetic (linkage) distance, chromosome, population, and their interactions on D′ were tested both through a linear model analysis of covariance and by a theoretical nonlinear model. The overall result was that (1) the distance explained most of the variability of D′, (2) the effect of chromosome was significant, and (3) the effect of population was significant. The significance of the chromosome effect may have resulted from selection and the significance of the population effect illustrates the effects of population structures and effective population sizes on LD. These results suggest that mapping methods based on LD may be valuable even with only moderately dense marker spacing in pigs.
Functional Divergence in Tandemly Duplicated Arabidopsis thaliana Trypsin Inhibitor Genes
Authors: Clauss M J , Mitchell-Olds T
Published: August 2004

In multigene families, variation among loci and alleles can contribute to trait evolution. We explored patterns of functional and genetic variation in six duplicated Arabidopsis thaliana trypsin inhibitor (ATTI) loci. We demonstrate significant variation in constitutive and herbivore-induced transcription among ATTI loci that show, on average, 65% sequence divergence. Significant variation in ATTI expression was also found between two molecularly defined haplotype classes. Population genetic analyses for 17 accessions of A. thaliana showed that six ATTI loci arranged in tandem within 10 kb varied 10-fold in nucleotide diversity, from 0.0009 to 0.0110, and identified a minimum of six recombination events throughout the tandem array. We observed a significant peak in nucleotide and indel polymorphism spanning ATTI loci in the interior of the array, due primarily to divergence between the two haplotype classes. Significant deviation from the neutral equilibrium model for individual genes was interpreted within the context of intergene linkage disequilibrium and correlated patterns of functional differentiation. In contrast to the outcrosser Arabidopsis lyrata for which recombination is observed even within ATTI loci, our data suggest that response to selection was slowed in the inbreeding, annual A. thaliana because of interference among functionally divergent ATTI loci.
Multiple Loci and Epistases Control Genetic Variation for Seed Dormancy in Weedy Rice (Oryza sativa)
Authors: Gu Xing-You , Kianian Shahryar F , Foley Michael E
Published: August 2004

Weedy rice has much stronger seed dormancy than cultivated rice. A wild-like weedy strain SS18-2 was selected to investigate the genetic architecture underlying seed dormancy, a critical adaptive trait in plants. A framework genetic map covering the rice genome was constructed on the basis of 156 BC1 [EM93-1 (nondormant breeding line)//EM93-1/SS18-2] individuals. The mapping population was replicated using a split-tiller technique to control and better estimate the environmental variation. Dormancy was determined by germination of seeds after 1, 11, and 21 days of after-ripening (DAR). Six dormancy QTL, designated as qSDS-4, -6, -7-1, -7-2, -8, and -12, were identified. The locus qSDS-7-1 was tightly linked to the red pericarp color gene Rc. A QTL × DAR interaction was detected for qSDS-12, the locus with the largest main effect at 1, 11, and 21 DAR (R2 = 0.14, 0.24, and 0.20, respectively). Two, three, and four orders of epistases were detected with four, six, and six QTL, respectively. The higher-order epistases strongly suggest the presence of genetically complex networks in the regulation of variation for seed dormancy in natural populations and make it critical to select for a favorable combination of alleles at multiple loci in positional cloning of a target dormancy gene.
Stochastic Tunnels in Evolutionary Dynamics
Authors: Iwasa Yoh , Michor Franziska , Nowak Martin A
Published: August 2004

We study a situation that arises in the somatic evolution of cancer. Consider a finite population of replicating cells and a sequence of mutations: type 0 can mutate to type 1, which can mutate to type 2. There is no back mutation. We start with a homogeneous population of type 0. Mutants of type 1 emerge and either become extinct or reach fixation. In both cases, they can generate type 2, which also can become extinct or reach fixation. If mutation rates are small compared to the inverse of the population size, then the stochastic dynamics can be described by transitions between homogeneous populations. A “stochastic tunnel” arises, when the population moves from all 0 to all 2 without ever being all 1. We calculate the exact rate of stochastic tunneling for the case when type 1 is as fit as type 0 or less fit. Type 2 has the highest fitness. We discuss implications for the elimination of tumor suppressor genes and the activation of genetic instability. Although our theory is developed for cancer genetics, stochastic tunnels are general phenomena that could arise in many circumstances.
Intron Size Correlates Positively With Recombination Rate in Caenorhabditis elegans
Authors: Prachumwat Anuphap , DeVincentis Laura , Palopoli Michael F
Published: August 2004

A negative correlation between intron size and recombination rate has been reported for the Drosophila melanogaster and human genomes. Population-genetic models suggest that this pattern could be caused by an interaction between recombination rate and the efficacy of natural selection. To test this idea, we examined variation in intron size and recombination rate across the genome of the nematode Caenorhabditis elegans. Interestingly, we found that intron size correlated positively with recombination rate in this species.
A Genetic Selection for Circadian Output Pathway Mutations in Neurospora crassa
Authors: Vitalini Michael W , Morgan Louis W , March Irene J , Bell-Pedersen Deborah
Published: August 2004

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.
Properties of Unpaired DNA Required For Efficient Silencing in Neurospora crassa
Authors: Lee Dong W , Seong Kye-Yong , Pratt Robert J , Baker Kevin , Aramayo Rodolfo
Published: August 2004

The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be “unpaired” to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.
Altering a Gene Involved in Nuclear Distribution Increases the Repeat-Induced Point Mutation Process in the Fungus Podospora anserina
Authors: Bouhouche Khaled , Zickler Denise , Debuchy Robert , Arnaise Sylvie
Published: August 2004

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.