Endochondral bone formation in toothless (osteopetrotic) rats: failures of chondrocyte patterning and type X collagen expression

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.     

Genotype-environment interaction for quantitative trait loci affecting life span in Drosophila melanogaster

The nature of genetic variation for Drosophila longevity in a population of recombinant inbred lines was investigated by estimating quantitative genetic parameters and mapping quantitative trait loci (QTL) for adult life span in five environments: standard culture conditions, high and low temperature, and heat-shock and starvation stress. There was highly significant genetic variation for life span within each sex and environment. In the analysis of variance of life span pooled over sexes and environments, however, the significant genetic variation appeared in the genotype x sex and genotype x environment interaction terms. The genetic correlation of longevity across the sexes and environments was not significantly different from zero in these lines. We estimated map positions and effects of QTL affecting life span by linkage to highly polymorphic roo transposable element markers, using a multiple-trait composite interval mapping procedure. A minimum of 17 QTL were detected; all were sex and/or environment-specific. Ten of the QTL had sexually antagonistic or antagonistic pleiotropic effects in different environments. These data provide support for the pleiotropy theory of senescence and the hypothesis that variation for longevity might be maintained by opposing selection pressures in males and females and variable environments. Further work is necessary to assess the generality of these results, using different strains, to determine heterozygous effects and to map the life span QTL to the level of genetic loci.     

Quantitative trait loci for life span in Drosophila melanogaster: interactions with genetic background and larval density

The genetic architecture of variation in adult life span was examined for a population of recombinant inbred lines, each of which had been crossed to both inbred parental strains from which the lines were derived, after emergence from both high and low larval density. QTL affecting life span were mapped within each sex and larval density treatment by linkage to highly polymorphic roo-transposable element markers, using a composite interval mapping method. We detected a total of six QTL affecting life span; the additive effects and degrees of dominance for all were highly sex- and larval environment-specific. There were significant epistatic interactions between five of the life span QTL, the effects of which also differed according to genetic background, sex, and larval density. Five additional QTL were identified that contributed to differences among lines in their sensitivity to variation in larval density. Further fine-scale mapping is necessary to determine whether candidate genes within the regions to which the QTL map are actually responsible for the observed variation in life span.     

Vaccination with inactivated virus but not viral DNA reduces virus load following challenge with a heterologous and virulent isolate of feline immunodeficiency virus

It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIV(PET)) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIV(AM6) and FIV(GL8). This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIV(GL8)-infected cats. In contrast, DNA vaccines based on either FIV(PET) or FIV(GL8), which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIV(PET) but had no measurable effect on virus load when the infecting virus was FIV(GL8). These results indicate that the more virulent FIV(GL8) is intrinsically more resistant to vaccinal immunity than the FIV(PET) strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.     

Quantitative genetics of ovariole number in Drosophila melanogaster. II. Mutational variation and genotype-environment interaction.

The rare alleles model of mutation-selection balance (MSB) hypothesis for the maintenance of genetic variation was evaluated for two quantitative traits, ovariole number and body size. Mutational variances (VM) for these traits, estimated from mutation accumulation lines, were 4.75 and 1.97 x 10(-4) times the environmental variance (VE), respectively. The mutation accumulation lines were studied in three environments to test for genotype x environment interaction (GEI) of new mutations; significant mutational GEI was found for both traits. Mutations for ovariole number have a quadratic relationship with competitive fitness, suggesting stabilizing selection for the trait; there is no significant correlation between mutations for body size and competitive fitness. Under MSB, the ratio of segregating genetic variance, VG, to mutational variance, VM, estimates the inverse of the selection coefficient against a heterozygote for a new mutation. Estimates of VG/VM for ovariole number and body size were both approximately 1.1 x 10(4). Thus, MSB can explain the level of variation, if mutations affecting these traits are under very weak selection, which is inconsistent with the empirical observation of stabilizing selection, or if the estimate of VM is biased downward by two orders of magnitude. GEI is a possible alternative explanation.