QUANTITATIVE GENETICS OF SPRINT RUNNING SPEED AND SWIMMING ENDURANCE IN LABORATORY HOUSE MICE (MUS DOMESTICUS)

We tested the hypothesis that locomotor speed and endurance show a negative genetic correlation using a genetically variable laboratory strain of house mice (Hsd:ICR: Mus domesticus). A negative genetic correlation would qualify as an evolutionary “constraint,” because both aspects of locomotor performance are generally expected to be under positive directional selection in wild populations. We also tested whether speed or endurance showed any genetic correlation with body mass. For all traits, residuals from multiple regression equations were computed to remove effects of possible confounding variables such as age at testing, measurement block, observer, and sex. Estimates of quantitative genetic parameters were then obtained using Shaw's (1987) restricted maximum-likelihood programs, modified to account for our breeding design, which incorporated cross-fostering. Both speed and endurance were measured on two consecutive trial days, and both were repeatable. We initially analyzed performances on each trial day and the maximal value. For endurance, the three estimates of narrow-sense heritabilities ranged from 0.17 to 0.33 (full ADCE model), and some were statistically significantly different from zero using likelihood ratio tests. The heritability estimate for sprint speed measured on trial day 1 was 0.17, but negative for all other measures. Moreover, the additive genetic covariance between speeds measured on the two days was near zero, indicating that the two measures are to some extent different traits. The additive genetic covariance between speed on trial day 1 and any of the four measures of endurance was negative, large, and always statistically significant. None of the measures of speed or endurance was significantly genetically correlated with body mass. Thus, we predict that artificial selection for increased locomotor speed in these mice would result in a decrease in endurance, but no change in body mass. Such experiments could lead to a better understanding of the physiological mechanisms leading to trade-offs in aspects of locomotor abilities.     

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.     

Genetics of seedling and adult plant resistance to net blotch (Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) in barley

Net blotch (caused by Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) are important foliar diseases of barley in the midwestern region of the USA. To determine the number and chromosomal location of Mendelian and quantitative trait loci (QTL) controlling resistance to these diseases, a doubled haploid population (Steptoe/Morex) was evaluated to the pathogens at the seedling stage in the greenhouse and at the adult plant stage in the field. Alleles at two or three unlinked loci were found to confer resistance to the net blotch pathogen at the seedling stage depending on how progeny exhibiting an intermediate infection response were classified. This result was corroborated in the quantitative analysis of the raw infection response data as 2 major QTL were identified on chromosomes 4 and 6M. A third QTL was also identified on chromosome 6P. Seven QTL were identified for net blotch resistance at the adult plant stage and mapped to chromosomes 1P, 2P, 3P, 3M, 4, 6P, and 7P. The 7 QTL collectively accounted for 67.6% of the phenotypic variance under a multiple QTL model. Resistance to the spot blotch pathogen was conferred by a single gene at the seedling stage. This gene was mapped to the distal region of chromosome 1P on the basis of both qualitative and quantitative data analyses. Two QTL were identified for spot blotch resistance at the adult plant stage: the largest QTL effect mapped to chromosome 5P and the other mapped to chromosome 1P near the seedling resistance locus. Together, the 2 QTL explained 70.1% of the phenotypic variance under a multiple QTL model. On the basis of the chromosomal locations of resistance alleles detected in this study, it should be feasible to combine high levels of resistance to both P. teres f. teres and C. sativus in barley cultivars.     

Use of the additive main effects and multiplicative interaction model in QTL mapping for adaptation in barley

The additive main effects and multiplicative interaction (AMMI) model has emerged as a powerful analytical tool for genotype x environment studies. The objective of the present study was to assess its value in quantitative trait locus (QTL) mapping. This was done through the analysis of a large two-way table of genotype-by-environment data of barley (Hordeum vulgare L.) grain yields, where the genotypes constituted a genetic population suitable for mapping studies. Grain yield data of 150 doubled haploid lines derived from the Steptoe x Morex cross, and the two parental lines, were taken by the North American Barley Genome Mapping Project (NABGMP) at 16 environments throughout the barley production areas of the USA and Canada. Four regions of the genome were responsible for most of the differential genotypic expression across environments. They accounted for approximately 50% of the genotypic main effect and 30% of the genotype x environment interaction (GE) sums of squares. The magnitude and sign of AMMI scores for genotypes and sites facilitate inferences about specific interactions. The parallel use of classification (cluster analysis of environments) and ordination (principal component analysis of GE matrix) techniques allowed most of the variation present in the genotype x environment matrix to be summarized in just a few dimensions, specifically four QTLs showing differential adaptation to four clusters of environments. Thus, AMMI genotypic scores, when the genotypes constituted a population suitable for QTL mapping, could provide an adequate way of resolving the magnitude and nature of QTL x environment interactions.Ignacio Romagosa was on sabbatical leave from the University of Lleida and the Institut de Recerca i Tecnologia Agroalimentàries, Lleida, Spain, when this study was conducted     

Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms.

Laboratory reactors operated under oligotrophic conditions were used to evaluate the importance of initial growth rate and substratum composition on the long-term persistence of coliforms in mixed-population biofilms. The inoculum growth rate had a dramatic effect on the ability of coliforms to remain on surfaces. The most slowly grown coliforms (mu = 0.05/h) survived at the highest cell concentration. Antibody staining revealed that Klebsiella pneumoniae existed primarily as discrete microcolonies on the surface. Both coliforms and heterotrophic plate count bacteria were supported in larger numbers on a reactive substratum, mild steel, than on polycarbonate.     

Sequence Variations in the Bovine Growth Hormone Gene Characterized by Single-Strand Conformation Polymorphism (Sscp) Analysis and Their Association with Milk Production Traits in Holsteins

Sequence variations in the bovine growth hormone (GH) gene were investigated by single strand conformation polymorphism (SSCP) analysis of seven amplified fragments covering almost the entire gene (2.7 kb). SSCPs were detected in four of these fragments and a total of six polymorphisms were found in a sample of 128 Holstein bulls. Two polymorphisms, a T->C transition in the third intron (designated GH4.1) and an A->C transversion in the fifth exon (designated GH6.2), were shown to be associated with milk production traits. GH4.1(c)/GH4.1(c) bulls had higher milk yield than GH4.1(c)/GH4.1(t) (P <= 0.005) and GH4.1(t)/GH4.1(t) (P <= 0.0022) bulls. GH4.1(c)/GH4.1(c) bulls had higher kg fat (P <= 0.0076) and protein (P <= 0.0018) than GH4.1(c)/GH4.1(t) bulls. Similar effects on milk production traits with the GH6.2 polymorphism were observed with the GH6.2(a) allele being the favorable allele. The average effects of the gene substitution for GH4.1 and GH6.2 are similar, with +/-300 kg for milk yield, +/-8 kg for fat content and +/-7 kg for protein content per lactation. The positive association of GH4.1(c) and GH6.2(a) with milk production traits may be useful for improving milk performance in dairy cattle.     

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live andin situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.     

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi.

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.     

Stimulatory and inhibitory effects of endogenous factors in head extracts of Formica polyctena (Hymenoptera) on the fluid secretion of Malpighian tubules

This study was designed to investigate the regulation of fluid secretion by the Malpighian tubules of the worker ant Formica polyctena (Hymenoptera). Different solvent systems were used to make crude head extracts and to determine the solubility of the diuretic factors. Surprisingly, when distilled water, acid acetone, methanol and 15% trifluoroacetic acid (TFA) were used as solvents, two consecutive significant stimulations of fluid secretion were obtained: the first, when adding the extract to the tubule and the second, when washing it out. Extract obtained with a fifth solvent, Ringer solution, gave an almost complete but reversible inhibition of fluid secretion. Extracts were prepurified by means of a disposable C₁₈ column by elution with 20, 40, 60 and 80% acetonitrile. When the fractions were kept apart the 40% acetonitrile fraction caused an inhibition of fluid secretion. The 20, 60 and 80% acetonitrile fractions on the other hand resulted in two consecutive stimulations as described above. The dose-response curve for 15% TFA extract was bell-shaped with a threshold concentration of 1 × 10⁻³ heads/μl Ringer. A maximum response (stimulation of fluid secretion by a factor of 3.3 ± 0.72, n = 10) was observed with a concentration of 5 × 10⁻² heads/μl Ringer. Higher concentrations resulted in small increases of fluid secretion rates and in the appearance of the second stimulation when the extract was washed out. The activity present in the heads of Formica was not destroyed by boiling or by proteolytic enzymes (trypsin, chymotrypsin, pronase E and proteinase K). Only immobilized aminopeptidase M, which destroys the activity of peptides with a free N-terminus, had a significant effect on the activity of a 15% TFA head extract. Various biogenic amines were tested for their ability to mimic the effect of the head extracts. Only octopamine and dopamine evoked a small and transient increase in secretion rate. Thus biogenic amines probably do not contribute to a large extent to the response of Formica tubules to the crude head extract. The possibility that both diuretic and antidiuretic factors are present in the extract is discussed.