Protozoans as indicators of sequential batch processes for phenol treatment; an autoecological approach.

The objective of this study was the investigation of the potential use of protistan species as quality indicators of the activated sludge performance in sequential batch processes receiving toxic compounds. Two laboratory scale sequential batch reactors (SBR) were used, a conventional one and a system with plastic biofilm carriers (SBBR), treating wastewater containing phenol at concentrations ranging from 1 up to 40 mg/L. Physicochemical analyses of the samples included the determination of MLSS, effluent suspended solids, BOD5, nitrogen-ammonia, nitrogen-nitrate and phenol. The activated sludge protistan community was identified and enumerated in each reactor. Statistical analyses included Canonical Correspondence Analysis and Indicator Species Analysis of the collected experimental data. Canonical Correspondence Analysis showed inversely proportional relationships between the protozoa and the physicochemical parameters of the effluent as well as protozoan species competition. Indicator species analysis revealed the presence and the prevalence of different species under various phenol influent concentrations. No indicator species were observed for the period of operation under 5 mg/L influent phenol in both reactors, while no indicator species were observed for 20 mg/L influent phenol in the SBR reactor. Carchesium and Epistylis sp. showed the higher values for 1 mg/L phenol in the SBR, while Holophrya sp. showed lower indicator values for the same period in the SBBR. Although several species showed a good correlation to the treatment efficiency of the reactors, Blepharisma sp., could be used as the primary indicator species in both reactors for the operation period under 40 mg/L phenol, as deduced by statistical analysis.

alpha1-Acid glycoprotein production in rat dorsal air pouch in response to inflammatory stimuli, dexamethasone and honey bee venom.

This study shows the rapid and differential production of the 40-43 kDa and the 70-90 kDa alpha1-acid glycoprotein (AGP) fucosylated glycoforms after treatment of the dorsal air pouch with bacterial lipopolysaccharide (LPS), HgCl(2) or Freund's complete adjuvant (FCA). The 40-43 kDa and the 70-90 kDa AGP production is peaked 1-3 h post-LPS treatment. We observed that the responses to LPS and FCA are similar in that both AGP isoforms are induced whereas they differ in that the FCA exhibits a 6 h lag period. The response to HgCl(2,) however, exhibits the specific biphasic induction only of the 40-43 kDa AGP. The serum 40-43 kDa AGP glycoform gradually increases in response to all of the above stimulants and peaks by 24 h post- treatment. The increase of the 70-90 kDa AGP levels in the air pouch occurs in association with the accumulation of polymorphonuclear (PMN) cells while dexamethasone (DEX) increases only the 40-43 kDa AGP production in the absence of PMN accumulation. Macrophage-monocyte lineage cells forming the air pouch lining tissue may potentially be the cells that secrete the 40-43 kDa AGP while polymorphonuclear cells that infiltrate the air pouch secrete the 70-90 kDa AGP. The 40-43 kDa and 70-90 kDa AGP production induced by LPS in the air pouch precedes that of interleukin-1 (IL-1) or interleukin-6 (IL-6) while the 40-43 kDa AGP glycoform potentially increases IL-6 production by air pouch PMN exudate cells. These significant differences suggest a local pro-inflammatory role of AGP. Honeybee venom suppressed arthritis development and exhibited differential local or systemic regulation of AGP in serum vs. air pouch exudate or synovial fluid. This study with the air pouch model of facsimile synovium tissue suggests that local alpha1-acid glycoprotein (AGP) production may contribute to pro-inflammatory and anti-inflammatory activities during the local acute phase response or during chronic inflammatory stress as in arthritis.

Immunostimulatory activity of potential probiotic yeast strains in the dorsal air pouch system and the gut mucosa.

AIMS: To determine the immunostimulatory activity of 15 presumptive probiotic yeast strains in the dorsal air pouch system in comparison with their activity in the gut mucosa. METHODS AND RESULTS: Presumptive probiotic yeast strains previously isolated from human gastrointestinal tract and Feta cheese were further characterized genotypically and biochemically. The Saccharomyces cerevisiae 982, Saccharomyces boulardii KK1 and Kluyveromyces lactis 630 strains exhibited in the air pouch increased polymorphonuclear cell influx and phagocytic activity as well as cytokine production with similar potency as the probiotics Ultra levure S. boulardii and Lactobacillus acidophilus NCFB 1748. Oral administration of these strains in mice results in differential activation of small intestine immune responses concerning IgA and cytokine production as well as Toll-like receptor expression. CONCLUSION: Besides the Saccharomyces strains 982 and KK1, the K. lactis 630 strain could also be considered as a candidate probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: The air pouch model may be used as an alternative and rapid method for the discrimination and selection of potential probiotic yeast strains.

Validation of the dorsal air pouch model to predict and examine immunostimulatory responses in the gut.

AIMS: To validate the use of the air pouch system to predict and examine early immune responses induced by the presumptive probiotics Lactobacillus paracasei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut mucosa. METHODS AND RESULTS: Only the DC412 strain interacted strongly with the cells forming the air pouch lining tissue and induced early innate immune responses such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour necrosis factor alpha (TNF-alpha) production that equal the respective responses induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains exhibiting strong immunoregulatory activity in the air pouch also interacted strongly with the gut-associated lymphoid tissue (GALT). The strain DC412 exerts its effect on the intestine through stimulation of Toll-like receptor (TLR)2/TLR4-mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-gamma), TNF-alpha, interleukin (IL)-6 and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the same cytokine profile in addition to IL-12B, and this response is potentially mediated by the synergy of TLR2 and TLR9. CONCLUSION: The strain DC412 possesses the in vitro and in vivo characteristics of a probiotic micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The dorsal mouse or rat air pouch may be used as an alternative and rapid method for the initial discrimination and selection of potential probiotic Lactobacillus strains.

Effectiveness of ozonation and chlorination on municipal wastewater treatment evaluated by a battery of bioassays and biomarkers.

A battery of bioassays, including biological toxicity as well as in vitro mouse spleen lymphoproliferative responses and cytokine production, was conducted to compare the effectiveness of tertiary treatment methods such as coagulation (Coag) and absorption on granular activated carbon (GAC) and disinfection processes such as chlorination and ozonation in removing toxic or stress inducing agents from reclaimed wastewater. Whole effluent toxicity (WET) testing of secondary treated (ST) wastewater using as test species Vibrio fischeri, Daphnia magna and Tetrahymena thermophila as well as phytotoxicity revealed moderate toxicity effects that depend on the organism used. All bioassays exhibited decrease of the ecotoxicological responses after tertiary treatment. However, mitogenic responses were proved to be more sensitive. Endotoxin present in ST samples may be responsible for the increased strong lymphoproliferative activity as well as interleukin-1 (IL-1) production by mouse splenocytes. Tertiary treatment of ST with coagulation and/or adsorption on granular activated carbon (GAC) in combination with ozonation reduced WET to control levels. Ozonation alone or in combination with any other treatment removed endotoxin more efficiently than chlorination and thus reduced spleen lymphoproliferative responses and IL-1 production.

Major histocompatibility complex class II (DRB1*, DQA1*, and DQB1*) and DRB1*04 subtypes' associations of Hashimoto's thyroiditis in a Greek population.

Hashimoto's thyroiditis (HT) is an autoimmune disease resulting from complex interactions between genetic and environmental factors. The disease is associated with certain human leukocyte antigen (HLA) class II alleles in various populations. We aimed to determine in this study, for the first time in a Greek population, the association of HLA-DRB1*, -DQA1*, and -DQB1* alleles with HT. HLA-DRB1*, -DQA1*, and -DQB1* alleles' and -DRB1*04 subtypes' distribution was evaluated in 125 patients with HT and in 500 healthy control individuals by using a DNA-based sequence-specific primer method. Chi(_)squared tests and Bonferroni correction method were applied in the statistical analysis of the data. Significantly higher frequency of DRB1*04 (24.8% vs 7.7%, P < 0.0001) was observed in HT patients, while HLA-DRB1*07 was significantly decreased (2.8% vs 7.9%, P < 0.05). HLA-DRB1*04 subtyping showed a significant increase of DRB1*0405 (21% vs 7.8%, P < 0.0001) in HT patients. Also significant high frequencies of DQB1*0201 (14.8% vs 8.2%, P < 0.001), DQB1*0302 (18.8% vs 7.0%, P < 0.0001), and DQA1*0301 (25.6% vs 7.8%, P < 0.0001) were recorded in the patient group. Conducting the first research of this kind in a Greek population, our study tries to provide an evaluation of the prevalence of HT relating to HLA-DRB1*0405, and we report a relative risk of 2.7 for HT in a Greek population.

Characterization of vanA-type Enterococcus faecium isolates from urban and hospital wastewater and pigs.

AIMS: In this study we analysed urban, hospital wastewater and pig faeces samples to investigate the presence of vancomycin-resistant Enterococcus faecium strains (VREF) and to determine potential links among the strains originating from the above sources and VREF strains causing clinical infections. METHODS AND RESULTS: Urban, hospital wastewater and pig faeces exhibited high VREF prevalence of 52%, 87% and 85%, respectively. Pulsed field gel electrophoresis (PFGE) clustering of VREF genotypes as well as discriminant analysis of antibiotic resistance patterns of VREF strains revealed their source specificity while strains isolated from hospitalized humans were genetically distinct. CONCLUSIONS: PFGE genotypes and antimicrobial resistance patterns in VREF isolates are distinguishable by each sample origin. The observed high genetic diversity of VREF suggests horizontal transfer of genetic elements among VREF. Phenotypic and genotypic data indicate that VREF isolates of hospital-treated wastewater might pass to the urban wastewater system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information to understand the origin and the mechanism of circulation of vancomycin resistance in food animals and wastewater treatment plants for minimizing the risk of transmission of VRE in human population.

Effect of hexavalent chromium on the activated sludge process and on the sludge protozoan community.

The objectives of this study were the determination of chromium effects to the performance of an activated sludge unit and the investigation of the response of the activated sludge protozoan community to Cr(VI). Two bench scale activated sludge reactors were supplied with synthetic sewage containing Cr(VI), at concentrations from 1 up to 50 mg L(-1). Protozoan species were identified and were related to the system efficiency. Variations in the abundance and diversity of the protozoan species were observed under various chromium concentrations. High removal rates of organics and nutrients were observed after the acclimatization of the activated sludge, which were related to the initial chromium(VI) concentration. Chromium(VI) removal efficiency was high in all cases. The protistan community was affected by the influent chromium content. Dominance of sessile species was observed in the reactor receiving 5 mg L(-1) influent chromium, whereas co-dominance of sessile and carnivorous species was observed in the reactors receiving higher chromium concentrations.

Bioassays and biomarkers for ecotoxicological assessment of reclaimed municipal wastewater.

The aim of this work was to examine the ecotoxicity of reclaimed wastewater by the use of bioassays and the determination of immunological parameters. Secondary and tertiary mucicipal wastewater samples were examined for their physicochemical and microbiological characteristics as well as for their endotoxin concentrations. The ecotoxicological characteristics were assessed by a battery of bioassays, using Vibrio fischeri, Daphnia magna and Tetrahymena thermophilla as test species and phytotoxicity. The mitogenic responses of mouse splenocytes were as well used as bioassay. The cytokines of IL-1, IL-2, IL-10, IFNgamma and TNFalpha, were also determined in the supernatant of splenocyte cultures and served as molecular biomarkers. All bioassays exhibited decrease of the ecotoxicological responses after tertiary treatment. However, mitogenic responses were proved to be more sensitive. IL-1 increased, while IL-2 production was unaffected. The fact that IL-10 production increased in response to secondary treated effluents in conjunction with the increased endotoxin levels, suggest Th2 type immune responses. Although results obtained from the toxicity bioassays after the tertiary treatment showed comparable results to those of controls, cytokine levels indicated the induction of immune response even after tertiary treatment. Consequently, cytokine production could be used as a sensitive biomarker for the evaluation of treatment efficiency of the reclaimed wastewaters intended for reuse.

Changes in numbers and kinds of bacteria during a chickpea submerged fermentation used as a leavening agent for bread production.

The microflora developed during a submerged fermentation of coarsely ground chickpea (Cicer arietinum L.) in water (primary starter) and during raising a dough from wheat flour (adapted starter) was studied. In the fermenting liquid, only populations of Bacillus and Clostridium developed. Bacilli increased their loads significantly (p<0.05) during fermentation for 8-12 h and then remained constant. Clostridia developed (p<0.05) subsequently to levels of 10(7) cfu/ml at 18 h, when the pH of the fermenting liquid had decreased (p<0.05) to approximately 5.4. It also seems that the rise of the adapted starter within 2 h was caused by enzymes present in the primary starter and those liberated after cell death by the declining populations of bacilli and clostridia. The principal groups of isolates in all fermentation experiments (with chickpea seeds from five different areas) seemed to have the phenotypic characteristics of Bacillus cereus group and Clostridium perfringens. SDS-PAGE of whole-cell proteins elucidated the taxonomic position of the B. cereus group of strains as B. cereus and Bacillus thuringiensis and confirmed the phenotypic allocation of C. perfringens isolates. Strains phenotypically characterized as Bacillus licheniformis and Clostridium beijerinckii were also found to belong to these same species by SDS-PAGE. In addition, results showed that the fermenting broth was not toxic to mice when inoculated intraperitoneally and the product can thus be considered as safe for consumption.

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese.

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.

Experimental hyperlipidemia and the effect of NSAIDs.

The effects of ip administration of NSAIDs in experimentally induced hyperlipidemia in rats was studied. An isotonic solution of Triton WR1339 (tyloxapol) was administered ip to rats one hour after ip administration of the examined anti-inflammatory drug. After 24 h, blood was collected for the determination of plasma total cholesterol (TC), LDL and trigluceride (TG) concentrations. The NSAIDs used in our experimental model are selective or non selective COX-1 inhibitors as well as one non selective COX-2 inhibitor. Most of the drugs significantly reduced the TC, TG and LDL concentrations in the plasma of hyperlipidemic rats. While studies link atheromatosis to inflammation, our results potentially also link anti-inflammatory activity with hypolipidemia. Thus, NSAIDs not only may address the inflammatory aspect of atherosclerosis but also may contribute directly by inducing hypolipidemia.

Activation of soil respiration and shift of the microbial population balance in soil as a response to Lavandula stoechas essential oil.

Lavandula stoechas, a native plant of Greece, is rich in essential oil and fenchone is its major constituent. We examined the effect of the essential oil and its main constituents on soil metabolism and microbial growth. Addition of the essential oil or fenchone to soil samples induced a remarkable increase in soil respiration. This was accompanied by an increase in the soil bacterial population of three orders of magnitude. This sizable population was not qualitatively similar to that of the control soil samples. One bacterial strain dominated soil samples treated with L. stoechas essential oil or fenchone. By use of the disk diffusion assay, we evaluated the capacity of three bacterial strains that we isolated from the soil samples, as well as Escherichia coli and Bacillus subtilis (reference strains), to grow in the presence of the essential oil and three of its main constituents (fenchone, cineol, alpha-pinene). The substances tested did not inhibit the growth of the strain found to dominate the bacterial populations of treated soil samples; they severely inhibited B. subtilis. The other two isolated strains could also grow in liquid cultures in the presence of different quantities of essential oil or fenchone. Addition of fenchone at the end of the exponential phase increased the cell numbers of the strain that dominated the bacterial populations of treated soil samples, indicating use of the substrate added. On the basis of these results, we propose a scheme of successional stages during the decomposition process of the rich-in-essential-oil litter of aromatic plants that abound in the Mediterranean environment.

Effects of mercuric chloride on the regulation of expression of the acute phase response components alpha(1)-acid glycoprotein and C/EBP transcription factors.

We have previously shown that in response to treatment with HgCl(2), the adult mouse liver exhibits both transcriptional and translational regulation of the acute phase response genes. In this study we asked whether the heavy metal treatment affects the regulation of the C/EBP transcription factors which play a key role in regulation of the acute phase response gene. Our studies have shown that the AGP gene is transcriptionally activated while transcription of the CCAAT/enhancer-binding trans-activating protein (C/EBP)alpha gene is slightly down-regulated and that of the C/EBPbeta gene does not respond. Both the C/EBPalpha and C/EBPbeta mRNAs produce multiple isoforms possibly by alternative translation initiation (ATI) of multiple internal AUG initiation sites. The C/EBPbeta mRNA appears to be stabilized. Although similar regulatory processes occur in response HgCl(2) vs. LPS, our data suggest that the translational processes (ATI) are differentially affected. In addition, a major difference lies in the fact that the C/EBPbeta gene is not transcriptionally activated by HgCl(2). Our data show decreased binding activity and pool levels of the C/EBPalpha isoform (p42(C/EBPalpha)) and increased binding activity and pool levels of C/EBPbeta isoform (p35(C/EBPbeta)) in response to HgCl(2). We propose that this isoform may be involved in the regulation of AGP gene expression in response to heavy metals and that there is a significant difference between the HgCl(2)-mediated and LPS-mediated inflammatory response.

Anti-inflammatory properties of diclofenac transition metalloelement complexes.

As part of our research into understanding drug-metalloelement interactions, we have prepared complexes of Cu(II), Co(II), Ni(II), Mn(II), Fe(II), Fe(III), and Pd(II) with Diclofenac, in order to investigate their anti-inflammatory activity. Their inhibitory effects on rat or mouse paw edema induced by Carrageenan, Con-A, Nystatin, and Baker's yeast were compared with those of Diclofenac. Furthermore, the action of Diclofenac's metalloelement complexes on phagocytosis of yeast by rat peritoneal cells, as well as the capacity of some of the metalloelement complexes to inhibit lipid peroxidation of liver microsomal membranes was also investigated. These complexes exhibited a strong inhibitory effect on Carrageenan-, ConA-, and Nystatin-induced edemas (35-80% inhibition) comparable to the inhibition caused by Diclofenac (61-76% inhibition). Furthermore, complexes with Co(II), Ni(II), Pd(II), and Mn(II) were found to have an anti-inflammatory profile (35-50% inhibition) superior to diclofenac (17% inhibition) when inhibiting inflammations due to Baker's yeast, the mechanism of which involves mainly the activation of lipoxygenase and/or complement system. Complexes of Ni(II) and Pd(II), which showed significant inhibition of induced-edemas in rats, were also tested in mice at lower and higher doses and showed a significant dose-dependent inhibition of edemas in mice. Some of these complexes also interfere with in vitro phagocytosis. The most active anti-inflammatory complexes Co(II), Pd(II), and Ni(II), also offered significant protection against lipid peroxidation in vitro, acting as antioxidant compounds, properties that are not demonstrated by Diclofenac. Finally, it is noted that almost all metalloelement complexes of Diclofenac showed high anti-inflammatory activity at molecular concentrations much lower than that of Diclofenac. From the present study it is suggested that the anti-inflammatory activity of Diclofenac is enhanced by the formation of coordination complexes with transition metalloelements.

Induction of a subgroup of acute phase protein genes in mouse liver by hyperthermia.

We have demonstrated that two members of the acute phase reactant family of positively regulated genes, alpha 1-acid glycoprotein (AGP-1 and AGP-2) and C-reactive protein (CRP) are induced by hyperthermia, while two others, the serum amyloid A (SAA) and alpha 1-antitrypsin (AT) genes, are not. Albumin (ALB), a negative acute phase reactant gene, is also induced by hyperthermia. The AGP-1, AGP-2, and CRP genes require glucocorticoids, but not IL-6, IL-1 beta or TNF alpha in response to hyperthermia. As with LPS, the C/EBP beta mRNA levels increased, while the C/EBP alpha mRNA levels decreased in response to LPS. In contrast to the LPS response, C/EBP delta was unchanged. Protein pool levels and DNA-binding activities of the 35 and 20 kDa C/EBP beta isoforms increase, whereas protein pool levels of the 42 kDa C/EBP alpha decrease and the 30kDa remained high. These studies suggest that the synthesis of specific C/EBP alpha and C/EBP beta isoforms is induced by hyperthermia, and that the regulation of the AGP-1 and AGP-2 genes during heat stress may involve one of these isoforms. The difference between the responses to hyperthermia and LPS is that the former, may not involve the participation of cytokines. Furthermore, since cis-acting heat shock elements (HSE) are located in the promoter regions of the ALB, CRP, and C/EBP beta genes, these regulatory sequences may be involved in the in vivo activation of these genes by hyperthermia.

Regulation of cytokine gene expression by adjuvants in vivo.

Antibody isotype affects biological activity of the antibodies and therefore should be considered in prevention of disease by vaccination. In previous reports, we demonstrated that adjuvants affect the antibody isotype switching process and favour the production of certain isotypes. The present study extends these findings and shows fundamental differences in the cytokine induction pattern according to the adjuvant used. Cytokine mRNA levels were determined by in situ RNA-RNA hybridization performed on splenocytes isolated from mice injected with different adjuvants. The results revealed that Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Al(OH)3 and QuilA administration results in a type-2 (humoral) response, increasing IL-4, IL-5 and IL-13 gene expression, while poly I:C exhibits a type-1 (cell-mediated) response, increasing the production of interferon-gamma (IFN-gamma), IL-2 and IL-6 mRNA. Finally, BeSO4 and poly A:U augment IL-5 and IL-6 mRNA production, while lipopolysaccharide (LPS) and LiCl augment IL-6 and tumour necrosis factor-alpha (TNF-alpha) mRNA production. Also, the adjuvants appear capable of overcoming the inherent IL-2/IFN-gamma and IL-4 dichotomy of C57B1/6 and BALB/c mice, respectively, in response to cellular antigens such as Leishmania and herpes simplex virus (HSV). The overall data suggest that adjuvants direct the isotype switching process via induction of certain cytokines, a finding that can be useful in selection of the most efficient isotype of protective antibodies for disease prevention by vaccination.

Heat shock gene expression during recovery after transient cold shock in Drosophila auraria (Diptera: Drosophilidae).

Newly synthesized polypeptides during recovery from prolonged cold treatment (0 degree to -1 degree C) of Drosophila auraria, a montium subgroup species, of the melanogaster species group, were analysed in denaturing polyacrylamide gels. In addition, during the cold shock recovery period, Northern analysis of the hsp83 mRNA was performed. A significant induction of two polypeptides, which exhibited electrophoretic mobilities, with the heat inducible 83 and 70 kD hsp83 and hsp70 was detected, but no such induction was evident in the so-called 'small' hsp genes. These results are compared and discussed with those observed in D. melanogaster.

A Drosophila hsp70 gene contains long, antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci.

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD(+)-dependent glutamate dehydrogenase (NAD(+)-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.

The beta-tubulin genes of Drosophila auraria are arranged in a cluster.

When the beta 1-, beta 2- and beta 3-tubulin-specific DNAs from Drosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes of Drosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a lambda EMBL3 genomic library of D. auraria, and they all contain beta-tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.