rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila

Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgn^G4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgn^G4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgn^G4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila.     

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms

In the Netherlands, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteria are highly prevalent in poultry, and chicken meat has been implicated as a source of ESBL-producing E. coli present in the human population. The current study describes the isolation of ESBL-producing E. coli from house flies and blow flies caught at two poultry farms, offering a potential alternative route of transmission of ESBL-producing E. coli from poultry to humans. Overall, 87 flies were analyzed in 19 pools. ESBL-producing E. coli bacteria were detected in two fly pools (10.5%): a pool of three blow flies from a broiler farm and a pool of eight house flies from a laying-hen farm. From each positive fly pool, six isolates were characterized and compared with isolates obtained from manure (n = 53) sampled at both farms and rinse water (n = 10) from the broiler farm. Among six fly isolates from the broiler farm, four different types were detected with respect to phylogenetic group, sequence type (ST), and ESBL genotype: A₀/ST3519/SHV-12, A₁/ST10/SHV-12, A₁/ST58/SHV-12, and B1/ST448/CTX-M-1. These types, as well as six additional types, were also present in manure and/or rinse water at the same farm. At the laying-hen farm, all fly and manure isolates were identical, carrying bla_TEM-52 in an A₁/ST48 genetic background. The data imply that flies acquire ESBL-producing E. coli at poultry farms, warranting further evaluation of the contribution of flies to dissemination of ESBL-producing E. coli in the community.     

DNA (cytosine-5)-methyltransferase 1 as a mediator of mutant p53-determined p16<Superscript>ink4A</Superscript> down-regulation

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16^ink4A protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene expression as well as hypermethylation of the p16 ^ink4A promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16^ink4A protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16^ink4A protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ^ink4A promoter and subsequently down-regulates p16^ink4A protein. Z. Guo and M.-H. Tsai contributed equally to this work.     

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis

In eukaryotes, the ubiquitin–proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 ^+/+ (wild-type) and Dmp53 ^?/? Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to “counterbalance” Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.     

High photosynthetic rate of a chlorophyll mutant of cotton

In a chlorophyll mutant (virescent) and wild-type cotton (Gossypium hirsutum L.), a number of photosynthetic parameters have been measured and compared with those published for other chlorophyll mutants. (a) The photosynthetic rates at 230 w/m² (400-700 nm) from a tungsten lamp were 36.8 mg CO₂ fixed/dm²·hr (virescent) and 39.5 mg CO₂ fixed/dm²·hr (wild-type). On a chlorphyll basis, the photosynthetic rates were 36.8 and 12.1 mg CO₂ fixed/mg chl·hr, respectively. (b) The photosynthetic rates at 13 w/m² (400-700 nm) from a tungsten source were 7.1 mg CO₂ fixed/dm²·hr (virescent) and 7.4 mg CO₂ fixed/dm²·hr (wild-type). On a chlorophyll basis, the photosynthetic rates were 6.0 and 1.4 mg CO₂ fixed/mg chl·hr, respectively. (c) The chlorophyll a/b ratios of the virescent and wild-type leaves were 3.3 and 4.1 (d) The chlorophyll/carotenoid ratios for the virescent and wild-type leaves were 3.2 and 7.3, respectively. (e) The photosynthetic carbon metabolism of the chlorophyll mutant was through the reductive pentose phosphate cycle. (f) The CO₂ compensation points for the virescent and wild-type plants were similar. (g) The mutant and wild-type leaves have the same quantum yield in the red part of the visible spectrum, but the virescent leaves have a lower quantum yield in the blue part of the spectrum. (h) Virescent and wild-type leaves contain similar levels on a protein basis of several reductive pentose phosphate cycle enzymes.     

Structural Insights into Conformational Stability of Wild-Type and Mutant β1-Adrenergic Receptor

Recent experiments to derive a thermally stable mutant of turkey beta-1-adrenergic receptor (β₁AR) have shown that a combination of six single point mutations resulted in a 20°C increase in thermal stability in mutant β₁AR. Here we have used the all-atom force-field energy function to calculate a stability score to detect stabilizing point mutations in G-protein coupled receptors. The calculated stability score shows good correlation with the measured thermal stability for 76 single point mutations and 22 multiple mutants in β₁AR. We have demonstrated that conformational sampling of the receptor for various mutants improve the prediction of thermal stability by 50%. Point mutations Y227A^5.58, V230A^5.61, and F338M^7.48 in the thermally stable mutant m23-β₁AR stabilizes key microdomains of the receptor in the inactive conformation. The Y227A^5.58 and V230A^5.61 mutations stabilize the ionic lock between R139^3.50 on transmembrane helix3 and E285^6.30 on transmembrane helix6. The mutation F338M^7.48 on TM7 alters the interaction of the conserved motif NPxxY(x)_5,6F with helix8 and hence modulates the interaction of TM2-TM7-helix8 microdomain. The D186-R317 salt bridge (in extracellular loops 2 and 3) is stabilized in the cyanopindolol-bound wild-type β₁AR, whereas the salt bridge between D184-R317 is preferred in the mutant m23. We propose that this could be the surrogate to a similar salt bridge found between the extracellular loop 2 and TM7 in β₂AR reported recently. We show that the binding energy difference between the inactive and active states is less in m23 compared to the wild-type, which explains the activation of m23 at higher norepinephrine concentration compared to the wild-type. Results from this work throw light into the mechanism behind stabilizing mutations. The computational scheme proposed in this work could be used to design stabilizing mutations for other G-protein coupled receptors.     

SPAK-Sensitive Regulation of Glucose Transporter SGLT1

The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na⁺-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active ^T233ESPAK, WNK-insensitive ^T233ASPAK or catalytically inactive ^D212ASPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak ^wt/wt) and from gene-targeted mice carrying WNK-insensitive SPAK (spak ^tg/tg). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I _g) was significantly decreased following coexpression of wild-type SPAK and ^T233ESPAK, but not by coexpression of ^T233ASPAK or ^D212ASPAK. Kinetic analysis revealed that SPAK decreased maximal I _g without significantly modifying the glucose concentration required for halfmaximal I _g (K _m). According to the chemiluminescence experiments, wild-type SPAK but not ^D212ASPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I _g, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak ^wt/wt than in spak ^tg/tg mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.     

Mechanism of suppression in Drosophila: a change in tyrosine transfer RNA

The mechanism of suppression of the vermilion locus in Drosophila melanogaster is examined. The suppressor locus, su(s)², is shown to control directly the amount of a specific tyrosine transfer RNA which occurs in the adult fly. Wild-type flies have three chromatographic forms of tyrosine tRNA but flies that are homozygous for the suppressor gene su(s)² contain little or none of the second chromatographic form. The isoacceptor patterns of tRNA for leucine, phenylalanine and serine are identical in the suppressor mutant and wild-type fly. Genetic data show that the phenotypic expression of su(s)² and the altered chromatographic pattern of tyrosine tRNA are recessive and that both map at the same position on the left tip of the X chromosome. Furthermore, another suppressor of vermilion was induced by ethyl methane sulfate, su(s)^e1, that is at the same locus as su(s)² and that produces the same change in tyrosine tRNA as su(s)².     

Utilization of U-14C amino acids or U-14C protein by adult Glossina morsitans during in utero development of larva

Administration of U¹⁴C protein hydrolysate in the diet of adult female Glossina morsitans at different times throughout the second reproductive cycle was followed by analysis of the distribution of radioactivity between the adult flies, their excreta, and the fully grown third instar larvae produced by these flies. A constant proportion of the total administered label was recoverable independently of the time lapse between administration and assay. Peak incorporation of labelled material occurred in the larva between the seventh and eighth day of a 9 or 10 day interlarval period, indicating that the larva feeds avidly on recently synthesized maternal uterine gland secretion at this time. Haemocoelic injection of U¹⁴C protein hydrolysate into similar adult females, between feeds, resulted in continued incorporation of labelled material by the larva to within 12 hr of parturition. Results are consistent with the hypothesis that uterine gland secretion and larval feeding continue throughout the intrauterine life of the larva.A constant and low proportion of detectable label remained in the adult fly while increased incorporation by the larva was paralleled by a reduction of detectable label in the adult excreta. This indicates direct competition between the uterine gland cells and those of the Malpighian tubules for free amino acids in the haemolymph.Administration of U¹⁴C protein in the adult diet did not result in incorporation of label by the developing larva, and the bulk was excreted as protein by the adult fly. Apparently the midgut trypsin of G. morsitans is incapable of splitting this labelled protein.Analysis of urine and haemolymph samples from flies in early pregnancy, recently fed on a diet containing U¹⁴C protein hydrolysate or U¹⁴C protein, shows that free labelled amino acids in the diet enter the adult haemolymph almost immediately after feeding, and are excreted along with dietary water during initial diuresis. The labelled protein used in these experiments was not taken up by the haemolymph and consequently did not appear in the urine.Implications are that the adult female G. morsitans possesses little storage capacity for substances in the diet which are destined to provide nutrients for the developing larva. Assuming a 48 hr digestion time, the digestive products of a blood meal ingested on day 5 or 6 of a 9 day interlarval period will provide the bulk of nutrients for larval growth. It is therefore significant that blood meals ingested at this time are larger than those ingested earlier or later in the cycle.     

The mechanism of the effect of apterous 56f mutation on the reproductive function of Drosophila melanogaster

The effects of L-dihydroxyphenylalanine (L-DOPA) and 20-hydroxyecdysone (20E) were studied with respect to the content of dopamine (DA), intensity of the juvenile hormone (JH) degradation, and fecundity of the wild-type flies (Canton S) and JH-deficient apterous ^56f mutants (in young females, carrying this mutation, the levels of DA and 20E production were strongly increased). Fly feeding with L-DOPA proved to increase the level of DA in a dose-dependent manner and reduce JH degradation in 2-day-old females of both strains. Feeding with 20E produced the same effect. Treating the wild-type flies with 2.5 mg L-DOPA caused a 24-h delay in beginning of oviposition and reduction in fecundity throughout the experiment. An L-DOPA dose of 1 mg caused no such changes. An experimental increase in 20E titer led to reduced fecundity of the wild-type flies, though no delay in oviposition was observed. In mutant flies, an increase in DA and 20E levels accelerated beginning of oviposition and increased fecundity of young females, though the latter parameter was reduced in mature individuals. Thus, an increase in endogenous DA and 20E characteristic of young apterous ^56f females is assumed to be a compensatory response that leads to a higher JH titer in order to induce vitellogenesis.     

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.     

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Accumulation of Mutant p53 Modulates the Growth, Clonogenicity, and Radiochemosensitivity of Malignant Glioma Cells Independent of Endogenous p53 Status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53^V143A in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53^V143A modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53^V143A inhibited the camptothecin-induced accumulation of p21^WAF1/CIP1 in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53^V143A. Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53^V143A. Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53^V143A-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

Digestive processes of haematophagous insects: control of trypsin secretion in Glossina morsitans

Glossina morsitans females were fed upon goats or components of beef blood through an Agar/Parafilm membrane and for each fly the following were determined: fly weight, meal weight, posterior midgut trypsin, posterior midgut protein, anterior midgut trypsin, and anterior midgut protein. Secretion of trypsin was stimulated by feeding flies upon goats, defibrinated beef blood, beef serum, haemolysed beef erythrocytes but not washed beef erythrocytes. There was a significant correlation between posterior midgut trypsin and the amount of protein in the posterior midgut, and the slope of the regression of trypsin upon protein content was significantly different from zero. There was a significant correlation between posterior midgut trypsin and meal size for flies 0 to 24 hr after emergence, but not those 24 to 48 hr old when fed upon a goat. For unfed flies there was a significant correlation between posterior midgut trypsin and fly weight.     

TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRAS^mut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53^−/−, TP53^+/−, TP53^mut/+, and TP53^mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRAS^mut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (^−/− or ^−/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.     

Membrane reconstitution in chl-r mutants of Escherichia Coli K 12: VIII. Purification and properties of the FA factor,the product of the chl B gene

The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10 % sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named “F_A factor”, does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified F_A protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the “complementation reaction”, i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. F_A protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that F_A factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein F_A can be released from either of these membranes (native or reconstituted) by removing Mg²⁺ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of F_A protein does not exert any effect on the level of the nitrate reductase activity.     

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage¹. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions^2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony^5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus^10-12. There is significant interest in F. arisanus biology^13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts^14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade¹⁸. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach¹². The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.