The LSP1-α gene is not dosage compensated in the Drosophila melanogaster species subgroup

The X-linked subunit of larval serum protein 1 (LSP1-) is shown to lack dosage compensation in six members of the melanogaster species subgroup, viz., Drosophila melanogaster, D. simulans, D. mauritiana, D. erecta, D. yakuba, and D. teissieri, by quantitative filter hybridization and by electrophoretic and autoradiographic analyses of fat body proteins. These results support the hypothesis that there is little selection pressure on the LSP1- gene to acquire dosage compensation.H.W.B. acknowledges a Commonwealth Scholarship. This work was supported by an SRC grant to D.B.R.     

Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carboxylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the α-Esterase cluster. Much in contrast to the vital importance attributed to carboxylesterases in general, the in vivo function of individual α-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila melanogaster fat body. One of the fat body carboxylesterases, α-Esterase-7, was selected for mutational analysis by gene targeting to generate a deletion mutant fly. Phenotypic characterization of α-Esterase-7 null mutants and transgenic flies, which overexpress a chimeric α-Esterase-7:EGFP gene, reveals important functions of α-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented first deletion mutant of any α-Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of α-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.     

The existence of LSP-1βS in Drosophila melanogaster natural populations in two northern states

LSP-1^S is present in Michigan and Massachusetts Drosophila melanogaster natural populations. Its frequency, 10%, is significantly higher in an East Jordan, Mich. (latitude, 45.10° N), population than in East Lansing, Mich. (latitude 42.44° N), or Hadley, Mass. (latitude, 42.21° N), populations, where it averages 3% at each location. The average frequency of LSP-2^S is more comparable, 6, 5, and 7% at East Jordan, East Lansing, and Hadley, respectively. LSP-1^F variants are also present. A total of 342 single third-instar larvae was scored for LSP-1 autosomal variants, and 323 for LSP-2 variants. Each larva represented a newly established isofemale line from collections at East Jordan in 1981 and 1983, East Lansing in 1982, and Hadley in 1981, 1982, and 1983. Within localities, frequencies of hemolymph protein variants did not differ significantly between years. Proteins 9, 10, 11, and 15 correspond to the LSP-1, , and triplet and LSP-2 polypeptide in D. melanogaster. Our results together with those of Singh and Coulthart [(1982). Genetics 102:437] indicate that D. melanogaster populations in north temperate climates maintain considerable genetic heterogeneity for the larval hemolymph proteins.     

The circadian basis of ovarian diapause regulation in Drosophila melanogaster: is the period gene causally involved in photoperiodic time measurement?

Females of a wild-type strain of Drosophila melanogaster (Canton-S), and of several clock mutants (period), were able to discriminate between diapause-inducing short days and diapause-averting long days with a well-defined critical daylength. The critical daylengths of a short-period mutant (pers) and a long-period mutant (perL2) were almost identical, both to each other and to that of Canton-S. The critical daylength of an arrhythmic mutant (perol), however, was about 3 hr shorter than that of Canton-S, and that of per- was about 5 hr shorter. Exposure of Canton-S females to Nanda-Hamner experiments, consisting of a 10-hr photophase coupled to a dark phase varying between 4 and 74 hr, showed (1) that the photoperiodic clock in D. melanogaster measures nightlength rather than daylength, and (2) that photoperiodic time measurement is somehow based on (or affected by) constituent oscillators in the circadian system. Nanda-Hamner results for the period mutants all showed similar profiles regardless of genotype, or the presence or absence of per locus DNA. These results suggest that photoperiodic induction and locomotor activity do not share a common pacemaker in D. melanogaster, and that the per gene is not causally involved in nightlength measurement by the photoperiodic clock, although flies in which the per locus is missing (per-) or defective (perol) show an altered critical value.     

The clock gene period of the housefly,Musca domestica,rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.     

New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism but not in suckling

The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism.     

DCg1 αIV collagen chain of Drosophila melanogaster is synthesized during embryonic organogenesis by mesenchymal cells and is deposited in muscle basement membranes

We report the purification and characterization of three sequence-specific polyclonal antibodies raised against specific portions of the Drosophila αIV collagen chain produced from the gene DCg1. These antibodies were used for immunolocalization experiments on tissue sections from embryonic organogenesis stages (13–17) and first larval stages. This analysis was paralleled by in situ hybridization experiments with a labeled fragment of the gene DCg1. We demonstrated that, by late embryogenesis, the DCg1 αIV chain was synthesized by individual mesoblasts and deposited in basement membranes of skeletal and visceral muscles. These sites of αIV collagen deposition were the same, by first and second instars, but the protein was then synthesized by fat body cells. Our results were reminiscent of those obtained for vertebrate in vitro myogenesis, they suggested, moreover, a tissue-specific composition of basement membranes in Drosophila melanogaster.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

The calcitonin/calcitonin gene related peptide-α gene is not required for 1α,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), can suppress disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Calcium appears to be a critical component of 1,25(OH)₂D₃-mediated suppression of EAE, as complete disease prevention only occurs with a concomitant increase in serum calcium levels. Calcitonin (CT) is a peptide hormone released in response to acute increases in serum calcium, which led us to explore its importance in 1,25(OH)₂D₃-mediated suppression of EAE. Previously, we discovered that co-administration of pharmacological doses of CT enhanced the suppressive effect of 1,25(OH)₂D₃ on EAE, suggesting CT may play a role in 1,25(OH)₂D₃-mediated suppression of EAE. To determine the importance of CT in EAE we have utilized a mouse strain in which the gene encoding CT and its alternative splice product, calcitonin gene related peptide-α (CGRP), have been deleted. Deletion of the CT/CGRP gene had no effect on EAE progression. Furthermore, treatment with 1,25(OH)₂D₃ suppressed EAE in CT/CGRP knock-out mice equal to that in wild type mice. Therefore, we conclude that CT is not necessary for 1,25(OH)₂D₃-mediated suppression of EAE.     

The effects of different training modalities on monocarboxylate transporters MCT1 and MCT4, hypoxia inducible factor-1α (HIF-1α), and PGC-1α gene expression in rat skeletal muscles

Molecular Biology Reports - The research literature suggests that different training modalities cause various patterns in training-induced genes expression. This study aimed to investigate the...     

The increase in rat ventricular automaticity induced by salbutamol is mediated through β(1)- but not β(2)-adrenoceptors: role of phosphodiesterases

AimsWhile β₂-adrenoceptor (AR) agonists are useful bronchodilators, they also produce cardiac arrhythmias. These agents are not fully selective and also activate β₁-AR, but the involvement of β₁-AR and β₂-AR in the observed pro-arrhythmic effect has not been established. We studied the effect of β₁-AR and β₂-AR activation on ventricular automaticity and the role of phosphodiesterases (PDE) in regulating this effect.Main methodsExperiments were performed in the spontaneously beating isolated right ventricle of the rat heart. We also measured cAMP production in this tissue.Key findingsThe β₂-AR agonist salbutamol (1-100 μM) produced a concentration-dependent increase in ventricular automaticity that was not affected by 50 nM of the β₂-AR antagonist ICI 118551. This effect was enhanced by the non-selective PDE inhibitor theophylline (100 μM) and by the selective PDE4 inhibitors rolipram (1 μM) and Ro 201724 (2 μM), but not modified by the selective PDE3 inhibitors cilostamide (0.3 μM) or milrinone (0.2 μM). The effects of salbutamol alone and in the presence of either theophylline or rolipram were virtually abolished by 0.1 μM β₁-AR antagonist CGP20712A. Salbutamol (10 μM) increased the cAMP concentration, and this effect was abolished by CGP 20712A (0.1 μM) but enhanced by theophylline (100 μM) or rolipram (1 μM). Cilostamide (0.3 μM) failed to modify the effect of salbutamol on cAMP concentration.SignificanceThese results indicate that the increase of ventricular automaticity elicited by salbutamol was exclusively mediated through β₁-AR and enhanced by non-selective PDE inhibition with theophylline or selective PDE4 inhibition. However, PDE3 did not appear to regulate this effect.     

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.