Prediction of complex phenotypes using the Drosophila melanogaster metabolome

Understanding the genotype–phenotype map and how variation at different levels of biological organization is associated are central topics in modern biology. Fast developments in sequencing technologies and other molecular omic tools enable researchers to obtain detailed information on variation at DNA level and on intermediate endophenotypes, such as RNA, proteins and metabolites. This can facilitate our understanding of the link between genotypes and molecular and functional organismal phenotypes. Here, we use the Drosophila melanogaster Genetic Reference Panel and nuclear magnetic resonance (NMR) metabolomics to investigate the ability of the metabolome to predict organismal phenotypes. We performed NMR metabolomics on four replicate pools of male flies from each of 170 different isogenic lines. Our results show that metabolite profiles are variable among the investigated lines and that this variation is highly heritable. Second, we identify genes associated with metabolome variation. Third, using the metabolome gave better prediction accuracies than genomic information for four of five quantitative traits analyzed. Our comprehensive characterization of population-scale diversity of metabolomes and its genetic basis illustrates that metabolites have large potential as predictors of organismal phenotypes. This finding is of great importance, e.g., in human medicine, evolutionary biology and animal and plant breeding.Subject terms: Quantitative trait, Genetic association study     

Substrate specificities of family 1 UGTs gained by domain swapping

Family 1 glycosyltransferases are a group of enzymes known to embrace a large range of different substrates. This study devises a method to enhance the range of substrates even further by combining domains from different glycosyltransferases to gain improved substrate specificity and catalytic efficiency. Chimeric glycosyltransferases were made by combining domains from seven different family 1 glycosyltransferases, UGT71C1, UGT71C2, UGT71E1, UGT85C1, UGT85B1, UGT88B1 and UGT94B1. Twenty different chimeric glycosyltransferases were formed of which twelve were shown to be catalytically active. The chimeric enzymes of Arabidopsis thaliana UGT71C1 and UGT71C2 showed major changes in acceptor substrate specificity and were able to glycosylate etoposide significantly better than the parental UGT71C1 and UGT71C2 enzymes, with K_cat and efficiency coefficients 3.0 and 2.6 times higher, respectively. Chimeric glycosyltransferases of UGT71C1 combined with Stevia rebaudiana UGT71E1, also afforded enzymes with high catalytic efficiency, even though the two enzymes only display 38% amino acid sequence identity. These chimeras show a significantly altered regiospecificity towards especially trans-resveratrol, enabling the production of trans-resveratrol-β-4′-O-glucoside (resveratroloside). The study demonstrates that it is possible to obtain improved catalytic properties by combining domains from both closely as well as more distantly related glycosyltransferases. The substrate specificity gained by the chimeras is difficult to predict because factors determining the acceptor specificity reside in the N- terminal as well as the C-terminal domains.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by double-staining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with Mr approximately 46,000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia

The distribution of single-cell DNA content was investigated in biopsy specimens from the human cervix of 121 women suspected of having intraepithelial neoplasia. Comparison of the results of the histopathological examination with the ploidy level showed that all normal specimens were diploid. Thus, no false-positive results occurred. Most of the specimens classified as mild and moderate dysplasia were diploid as well. Aneuploid cell populations occurred in 78% of the lesions classified as severe dysplasia and carcinoma in situ. The ploidy level distribution permitted a natural division of the aneuploid cell populations into two groups with DNA indices either above or below 1.5. The importance of the aneuploidy in carcinogenesis is discussed.     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Preliminary evidence of segregation distortion in the SLA system

The segregation of 11 well-defined SLA haplotypes was investigated in 40 Land-race and 48 Large White Danish and Swiss litters. Among the 11 haplotypes, the segregation of one (SLA 20-8.2.11) deviated significantly from the expected 1:1 segregation ratio in back-cross families. Tests indicated that these families were homogeneous with respect to segregation distortion, although the distortion was more pronounced in litters sired by heterozygous Danish boars than by heterozygous Swiss boars and Danish and Swiss sows. The data presented do not allow any definite conclusion about the cause of the segregation distortion. The possibility of the distortion being caused either by a complex similar to the T/t-complex found in mouse and contemplated in man or directly by the SLA region is discussed.     

Effect of extracellular Ca2+ and Mg2+ on enzyme release from quiescent fibroblasts during various exposures

Release of enzymes from human fibroblasts, as a result of either ATP depletion caused by metabolic inhibitors or a direct cell membrane damage, was increased in the absence of extracellular Ca2+. The lack of extracellular Mg2+ resulted only in a small nonsignificant increase of enzyme release during ATP depletion. However, in a Ca2+-free medium the enzyme release was very much enhanced during ATP depletion if Mg2+ was absent concomitantly. The data show that extracellular calcium protects the cells from cell damage, and that the presence of extracellular magnesium in a calcium-free medium protects the cell against energy-dependent cell damage.     

Cloning of a gene from Bacillus cereus with homology to the mreB gene from Escherichia coli.

We have cloned and sequenced a gene coding for a putative shape-determining protein (MreB) highly homologous to the mreB gene product of Escherichia coli. The amino acid (aa) identity was 53% and the similarity 72%. The gene is expressed early in the logarithmic phase. The aa sequence comparison showed that the protein, like the E. coli MreB, has structural similarity to actin and heat-shock protein Hsc70 encoded by a new super-gene family.     

A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

Background  

Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide.