Tom A. Grigliatti

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines.

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC; tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5–7, 72F1–2, 83F-84A; tRNA3Met, 46A1–2, 61D1–2, 70F1–2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84D3–4, 92B1–9; tRNA4Val, 56D3–7, 70BC.

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.

A discovery study of daunorubicin induced cardiotoxicity in a sample of acute myeloid leukemia patients prioritizes P450 oxidoreductase polymorphisms as a potential risk factor

Anthracyclines are very effective chemotherapeutic agents; however, their use is hampered by the treatment-induced cardiotoxicity. Genetic variants that help define patients sensitivity to anthracyclines will greatly improve the design of optimal chemotherapeutic regimens. However, identification of such variants is hampered by the lack of analytical approaches that address the complex, multi-genic character of anthracycline induced cardiotoxicity (AIC). Here, using a multi-SNP based approach, we examined 60 genes coding for proteins involved in drug metabolism and efflux and identified the P450 oxidoreductase (POR) gene to be most strongly associated with daunorubicin induced cardiotoxicity in a population of acute myeloid leukemia (AML) patients (FDR adjusted p-value of 0.15). In this sample of cancer patients, variation in the POR gene is estimated to account for some 11.6% of the variability in the drop of left ventricular ejection fraction (LVEF) after daunorubicin treatment, compared to the estimated 13.2% accounted for by the cumulative dose and ethnicity. In post-hoc analysis, this association was driven by 3 SNPs—the rs2868177, rs13240755, and rs4732513—through their linear interaction with cumulative daunorubicin dose. The unadjusted odds ratios (ORs) and confidence intervals (CIs) for rs2868177 and rs13240755 were estimated to be 1.89 (95% CI: 0.7435–4.819; p = 0.1756) and 3.18 (95% CI: 1.223–8.27; p = 0.01376), respectively. Although the contribution of POR variants is expected to be overestimated due to the multiple testing performed in this small pilot study, given that cumulative anthracycline dose is virtually the only factor used clinically to predict the risk of cardiotoxicity, the contribution that genetic analyses of POR can make to the assessment of this risk is worthy of follow up in future investigations.

Genes coding for valine transfer ribonucleic acid-3b in Drosophila melanogaster.

Abstract The genes for tRNA3bval were localized to 84D and 92B on the polytene chromosomes of Drosophila melanogaster with a possible minor site at 90B-C by hybridization in situ and autoradiography with 125I-labeled tRNA3bval. Flies carrying a duplication of the 84D region had increased amounts (30%) of tRNA3bval in proportion to the increased number of genes. While a proportional decrease in the amount of tRNAval3b in flies bearing a deletion of the same region was found, the total acceptance of valine remained at the level found in the wild type.

Single nucleotide polymorphisms in aldo-keto and carbonyl reductase genes are not associated with acute cardiotoxicity after daunorubicin chemotherapy.

Background: Evidence suggests that interpatient variability in anthracycline metabolic rate may contribute to the cardiotoxicity associated with anthracycline-based chemotherapy. Therefore, polymorphisms in the anthracycline metabolizing enzymes have been proposed as potential biomarkers of anthracycline-induced cardiotoxicity (AIC). Methods: We have previously shown that 13 of the naturally occurring nonsynonymous single-nucleotide polymorphisms (nsSNP) in the aldo–keto reductases (AKR) and carbonyl reductases (CBR) reduce anthracycline metabolic rate in vitro. Here, we test these SNPs individually and jointly for association with daunorubicin-induced cardiotoxicity in patients with acute myeloid leukemia (AML). Results: Five of the 13 nsSNPs exhibiting an in vitro effect on anthracycline metabolism were detected among the 185 patients with AML. No association was found between the SNPs and daunorubicin-induced cardiotoxicity in either individual or joint effect analyses. Conclusions: Despite the shown in vitro effect of nsSNPs in reductase genes on anthracycline metabolic rate, on their own these SNPs do not explain enough variability in cardiotoxicity to be useful markers of this adverse event. Impact: The results of this study provide important information for biomarker studies on side effects of anthracycline chemotherapy. Cancer Epidemiol Biomarkers Prev; 21(11); 2118–20. ©2012 AACR.

Hybridization of tRNAs of Drosophila melanogaster to the region of the 5S RNA genes of the polytene chromosomes

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled 125I-tRNAAsp2γ occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNAAsp2γ labeled by introducing 125I-5-iodocytidylyl residues into the 3′-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to its other sites. The hybridization of tRNALys2, tRNAGly3 and tRNAMet3 at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNALys2 no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNAAsp2γ in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.

Identification and analysis of Lydia, a LTR retrotransposon from Lymantria dispar

Degenerative PCR primers to conserved amino acid motifs were used to identify an LTR retrotransposon from Lymantria dispar. The isolated retrotransposon, Lydia, is 6655 base pairs (bp) in length and contains perfect 300 bp terminal repeats. The identified gag and pol related ORFs have a high degree of similarity to the corresponding regions of the retrotransposon Ted from Trichoplusia ni, although several reading frameshifts and missense mutations are evident. The high degree of similarity between Lydia and Ted LTRs lends support for a family of lepidopteran retrotransposons. Southern blot analysis of individuals from two geographically distinct gypsy moth populations demonstrates that Lydia is found in both populations and the position of this element within the genome of these isolated populations is variable.

Conservatism of sites of tRNA loci among the linkage groups of several Drosophila species.

SummaryThe sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.125I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA2Arg and tRNA2Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.

Informational redundancy of tRNASer4 and tRNASer7 genes in Drosophila melanogaster and evidence for intergenic recombination

Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.