Abstract: OBJECTIVE: A putative regulatory intronic polymorphism (PD1.3) in the programmed death 1 (PD-1) gene, a negative regulator of T cells involved in peripheral tolerance, is associated with increased risk for systemic lupus erythematosus (SLE). We undertook this study to determine the expression and function of PD-1 in SLE patients. METHODS: We genotyped 289 SLE patients and 256 matched healthy controls for PD1.3 by polymerase chain reaction-restriction fragment length polymorphism analysis. Expression of PD-1 and its ligand, PDL-1, was determined in peripheral blood lymphocytes and in renal biopsy samples by flow cytometry and immunohistochemistry. A crosslinker of PD-1 was used to assess its effects on anti-CD3/anti-CD28-induced T cell proliferation and cytokine production. RESULTS: SLE patients had an increased frequency of the PD1.3 polymorphism (30.1%, versus 18.4% in controls; P=0.006), with the risk A allele conferring decreased transcriptional activity in transfected Jurkat cells. Patients homozygous for PD1.3-but not patients heterozygous for PD1.3-had reduced basal and induced PD-1 expression on activated CD4+ T cells. In autologous mixed lymphocyte reactions (AMLRs), SLE patients had defective PD-1 induction on activated CD4+ cells; abnormalities were more pronounced among homozygotes. PD-1 was detected within the glomeruli and renal tubules of lupus nephritis patients, while PDL-1 was expressed by the renal tubules of both patients and controls. PD-1 crosslinking suppressed proliferation and cytokine production in both normal and lupus T cells; addition of serum from patients with active SLE significantly ameliorated this effect on proliferation. CONCLUSION: SLE patients display aberrant expression and function of PD-1 attributed to both direct and indirect effects. The expression of PD-1/PDL-1 in renal tissue and during AMLRs suggests an important role in regulating peripheral T cell tolerance.
Abstract: Current classifications of diabetes distinguish between type 1 diabetes (T1D) and type 2 diabetes (T2D), however recent evidence highlights overlap between T1D and T2D. Earlier studies have suggested altered nitric oxide (NO) metabolism in both T1D and T2D. In the present case-control study, we investigated whether the endothelial NO synthase gene intron 4 a/b polymorphism is associated with T1D and T2D in the island of Crete, a well-defined area with genetically homogeneous population. Mutated allele "a" was more common in individuals with both T1D and T2D than in controls (odds ratio [OR] = 1.71, 95% confidence interval [CI] = 1.06-2.77, p = 0.013; and OR = 1.50, 95% CI = 0.930-2.42, p = 0.047, respectively). Mutated genotype (a/a or a/b) was more common in individuals with T1D than in nondiabetic individuals (OR = 1.93, 95% CI = 1.12-3.32, p = 0.008); this increased frequency was also observed for T2D, although not at a significant level (OR = 1.38, 95% CI = 0.802-2.37). No difference was found in the frequency of mutated allele a or mutated genotype (a/a or a/b) between T1D and T2D populations. In conclusion, our results indicate that allele a of the intron 4 endothelial NO synthase gene is associated with susceptibility to both T1D and T2D and may represent a common genetic factor for diabetes.
Abstract: Familial Mediterranean Fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent and short attacks of fever with serosal inflammation that are caused by mutations in MEFV gene that encodes pyrin protein. To date, more than 70 disease-associated mutations have been identified, almost all of them representing missense nucleotide changes. FMF is very common among patients with Mediterranean ancestry, although the exact prevalence is not yet known, Greeks are considered to be at 'intermediate risk'. In the present study, we studied FMF patients in natives of Crete, a population sharing a common genetic and cultural background. The spectrum of MEFV gene mutations in 71 patients as well as 158 healthy controls was studied by performing a molecular analysis focused on the 12 most frequent FMF-associated mutations. We found that 59 of 71 (83.1%) FMF patients had at least one MEFV mutation, five patients were homozygotes and 54 heterozygotes for FMF-associated mutations. No mutations were detected in 12 patients (16.9%). As in high-risk populations, common MEFV mutations were found in Cretan FMF patients, with the M694V being the most penetrant. M694V and M694I mutations were associated with severe phenotypes, with many patients presenting with uncommon clinical manifestations such as erysipelas-like erythema or renal disturbances. Of interest, 20 (37%) of our heterozygous FMF patients presented with a severe phenotype. Population genetics analysis showed an FMF carrier frequency in healthy Cretan population of approximately 6% (1:17) and places Cretans closer to the Western rather than Eastern populations of the Mediterranean basin. Finally, we constructed a three-dimensional model showing the interaction of the PRYSPRY domain of pyrin with caspase-1 onto which we mapped MEFV mutations, classified according to disease severity. In this model, the 'flexible loops' of caspase-1 appear to have no access to some positions that have been previously associated with mild disease, suggesting that alternative pathogenic pathways leading to FMF need to be explored.
Abstract: INTRODUCTION: Mannose-binding lectin (MBL) is involved in host's response to several infections including hepatitis B but little is known about MBL and hepatitis C virus (HCV) infection. The present study attempts to investigate whether MBL2 genotype and serum MBL levels affect the course of HCV infection. RESULTS AND DISCUSSIONS: We investigated the variant alleles in MBL2 gene promoter and exon-1 regions in 80 Caucasian HCV-infected patients. Mutations in MBL2 were determined by polymerase chain reaction and restriction fragment length polymorphisms analysis. Serum MBL levels were measured by ELISA. Polymorphism homozygosity in exon-1 region was significantly related to lower serum MBL levels (p < 0.001), to liver inflammation (p = 0.034, OR = 11.7) and, in a lesser degree, to fibrosis. Polymorphisms in promoter sites -221nt and -550nt were not shown to be related with serum MBL levels or progress to liver inflammation and fibrosis. Serum MBL levels were adversely associated with progression to fibrosis (p = 0.037). Response to antiviral treatment was related to hepatitis C virus genotype (p < 0.001, OR = 10.9), but not to MBL2 genotype or serum MBL levels. CONCLUSION: These findings suggest that polymorphisms in MBL2 gene exon-1 region are related to low serum MBL levels and progression of HCV infection to liver inflammation and fibrosis.
Abstract: Rheumatoid arthritis (RA) is a multifactorial disease that is increasing in incidence worldwide. It is associated with a complex mode of inheritance, with many genes being involved in the development and progression of the disease. Genome-wide association studies in different populations have recently revealed a significant association between a TRAF1/C5 and a STAT4 polymorphism and the development of RA. In the present study we performed a case-control study in the population of the island of Crete, Greece, aiming to replicate the former findings in a genetically homogeneous cohort of patients. We found that mutated allele A or genotypes A/A and G/A of the TRAF1/C5 rs10818488 SNP were more common in individuals with RA than in control individuals (odds ratio [OR]=1.7, 95% confidence interval [CI]=1.35-2.15, and OR=2.22, 95% CI=1.61-3.05, respectively). Similarly, mutated allele T or genotypes T/T and G/T of the STAT4 rs7574865 SNP were also associated with susceptibility to RA (OR=1.9, 95% CI=1.46-2.50, and OR=2.37, 95% CI 1.73-2.25, respectively). Thus, we conclude that mutant alleles or genotypes of both polymorphisms examined are associated with the development of RA in our population.
Abstract: We have previously shown that laboratory populations of the olive fruitfly Bactrocera oleae come to equilibrium with allele frequencies at the 6-phosphogluconate dehydrogenase (6-PGD) locus markedly different from those of wild populations. In this study, we present new evidence from perturbation experiments in support of the notion that the locus is under selective pressure under laboratory conditions. Eleven populations were started with frequencies at the 6-PGD locus different from the laboratory equilibrium. Over 12 generations, the populations showed a return to the previous equilibrium, indicating a direct and powerful selection pressure on the naturally occurring allozymes of this locus. That is, a marked increase of the F allele followed by a compensatory decrease of allele I. Populations were set up to minimize the effects of associative overdominance, and we discuss the possible influence of this factor. Nucleotide sequence for the 6-PGD F and I alleles revealed two missense mutations at positions 501 and 730 leading to different amino acids among the two alleles.
Abstract: Increasing evidence is emerging on genetic factors affecting host's response to infection in the middle ear. This review summarizes current knowledge on the field and on the contribution of nonspecific barriers, innate, and adaptive immunity. Better understanding of susceptibility to this very common disease will facilitate identification of high-risk individuals and optimization of prevention and treatment.
Abstract: Genes and mechanisms involved in autoimmune diseases, affecting approximately 5% of human population, remain still obscure but there is accumulating evidence that common genetic factors might predispose to multiple autoimmune disorders. STAT4, a transcription factor transmitting signals induced by several key cytokines, has recently been identified as a genetic risk factor for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren's disease (SD), thus indicating that multiple autoimmune diseases may share common biochemical pathways that lead to immune deregulation. Here we demonstrate for the first time, in a genetically homogeneous population, the association of the STAT4 rs7574865 G/T polymorphism, which has been shown to be associated with these autoimmune diseases, with susceptibility to type 1 diabetes (T1D). The susceptibility is associated with a significant increase of the frequency of the T allele (p = 0.0012, two-tailed chi(2), OR = 1.94, 95% CI = 1.29-2.91) in this single-nucleotide polymorphism (SNP). We also present an indication for association with Wegener's granulomatosis. These findings suggest that this variant form of STAT4 may have a putative key role in the development of a variety of autoimmune diseases, probably because of signaling defects that it causes in the IL-12 pathway.
Abstract: The realisation that the production of inflammatory cytokines in inflammatory rheumatic diseases may be induced by non-infectious endogenous signals has encouraged researchers to explore mechanisms of innate immunity and their contribution to the pathogenesis of these diseases. The nucleotide-binding and oligomerisation domain (NOD)-like receptors (NLRs) sense pathogens, products of damaged cells or endogenous metabolites and could potentially be involved in the initiation, amplification and progression of the inflammatory response in rheumatic diseases. NLRs are involved in the regulation of innate immune responses with some of them promoting the activation of inflammatory caspases within multiprotein complexes, called inflammasomes. A typical inflammasome consists of a sensor, an NLR protein, an adaptor protein such as ASC (for apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) and an effector protein that is a caspase that activates pro-inflammatory cytokines such as interleukin (IL)1beta and IL18. Recent data suggest a role of the inflammasome in the pathogenesis of autoinflammatory as well as inflammatory rheumatic diseases such as juvenile chronic arthritis, adult onset Still disease, rheumatoid arthritis and gout. Modulation of these pathways may be a potential therapeutic target for inflammatory rheumatic diseases.
Abstract: Nitric oxide (NO), a short-lived gaseous free radical, synthesized from L-arginine by NO synthases (NOS), is a potent mediator of biologic responses involved in the pathogenesis of autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Most biological necessary NO is produced by the family of three NOS. To date, several functionally relevant genetic polymorphisms in the eNOS gene have been associated with various vascular, infectious and autoimmune diseases. To our knowledge, no study has explored these polymorphisms for both SLE and RA in the same population. The objective of this study was to investigate the influence of the eNOS gene intron 4 a/b VNTR polymorphism (a 27-base-pair tandem repeat-based polymorphism) on susceptibility to SLE and RA in patients living in the island of Crete, a genetically homogeneous population. A group of 145 healthy subjects and 190 SLE patients were included in this study. Similarly, a second group of 235 healthy controls and 202 RA patients were analysed. In both cases, patients and controls were sex- and age-matched. Herein we report that the presence of a/b genotype of the eNOS gene may act as a risk factor not for the presence of SLE but for the development of glomerulonephritis (OR 2.71, 95% CI: 1.4-5.2), while it may be a susceptibility gene for RA (OR: 2.005, 95% CI: 1.31-3.07). Thus, in our population, the a/b genotype of the eNOS gene represents a severity rather than a susceptibility genotype for SLE.
Abstract: The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO(2) and the reduction of NADP. We have determined the genomic sequences of 6PGD of two species of Tephritidae, the medfly Ceratitis capitata and olive fruit fly Bactrocera oleae, and constructed a three-dimensional model of 6PGD of C. capitata based on the homologous known sheep structure. In a comparative study of 6PGD sequences from seven species, all the conserved and variable sites of the enzyme were analyzed and the regions of functional importance were localized, an attempt promoted also by the direct involvement of the enzyme in various human diseases. The enzymes between the two species of Tephritidae have a very high homology and further examination of the variable positions with respect to the highly conserved binding site residues enabled their grouping in three distinct categories, with possible association to dimer formation, functional specificity, and antigenicity. Moreover, placement of sequence differences on the 3-D model suggests probable sites accommodating variations appearing at the allozymic variants of both species.
Abstract: Familial Mediterranean fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent fever and serositis that affects mainly patients of the Mediterranean basin. The gene responsible for FMF, named MEFV, was cloned and several missense mutations were found to be responsible for the disease. Based on a recent molecular analysis of MEFV gene mutations in 43 patients from Crete aiming to correlate specific genotypes and clinical manifestations of FMF, we were prompted to construct a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin. The majority of the known MEFV mutations located on this domain have been classified, according to disease severity, and localized on this 3-D model. The functional consequences of these mutations and their implications on disease severity are discussed. Moreover, we report a putative novel missense mutation, S702C, which we identified in exon 10 of the MEFV gene and localized on the constructed 3-D model.
Abstract: Alcohol dehydrogenase is considered a very important enzyme in insect metabolism because it is involved (in its homodimeric form) in the catalysis of the reversible conversion of various alcohols in larval feeding sites to their corresponding aldehydes and ketones, thus contributing to detoxification and metabolic purposes. Using 14 amino acid ADH sequences recently determined in our laboratory, we constructed a three-dimensional (3D) model of olive fruit fly Bactrocera oleae ADH1 and ADH2, based on the known homologous Drosophila lebanonensis ADH structure, and the amino acid residues that have been proposed as being responsible for catalysis were located on it. Moreover, in a comparative study of the ADH sequences, the residues occupying characteristic positions in the ADH of species of the Bactrocera and Ceratitis genera (called genus-specific) as well as residues appearing only in ADH1 or ADH2 (called isozymic-specific) were defined and localized on the 3D model. All regions important for catalytic activity, such as those forming the substrate- and coenzyme-binding sites, are highly conserved in all tephritid species examined. Genus-specific amino acids are located on the outside of the protein, on loops and regions predicted to be antigenic. The higher percentage of genus-specific amino acid variation seems to be centered in the NAD adenine-binding site, located near the surface of the protein molecule. Nine of 12 isozymic-specific positions are lined along an "arc" on the surface of the protein, thus linking the two "monomer bases" of the dimer via the C-terminal interacting loops. Furthermore, the distribution of isozymic- and genus-specific amino acids on the monomer-monomer interface may have some evolutionary significance. Most amino acids predicted to be antigenic are positioned in peripheral regions of nonfunctional importance, but surprisingly, an additional antigenic region is contained within the (highly conserved in tephritids) C-terminal tail.
Abstract: The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO2 and the reduction of NADP. We have determined the amino acid sequence of 6PGD of Bactrocera oleae and constructed a three-dimensional model based on the homologous known sheep structure. In a comparative study of 6PGD sequences from numerous species, all the conserved and variable regions of the enzyme were analyzed and the regions of functional importance were localized, in an attempt promoted also by the direct involvement of the enzyme in various human diseases. Thus, analysis of amino acid variability of 37 6PGD sequences revealed that all regions important for the catalytic activity, such as those forming the substrate and coenzyme binding sites, are highly conserved in all species examined. Moreover, several amino acid residues responsible for substrate and coenzyme specificity were also found to be identical in all species examined. The higher percentage of protein divergence is observed at two regions that accumulate mutations, located at the distant parts of the two domains of the enzyme with respect to their interface. These peripheral regions of non-functional importance are highly variable and are predicted as antigenic, thus reflecting possible regions for antibody recognition. Furthermore, locating the differences between diptera 6PGD sequences on the three-dimensional model suggests probable positions of different amino acid residues appearing at B. oleae fast, intermediate, and slow allozymic variants.
Abstract: Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.
Abstract: In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.
Abstract: The phylogenetic relationships among the Drosophila melanogaster group species were analyzed using approximately 1700 nucleotide-long sequences of the mitochondrial DNA. Phylogenetic analysis was performed using this region consisting of a part of the cytochrome b (cytb) coding gene, the entire coding sequences of tRNA-Leu, tRNA-Ser and the first subunit of NADH dehydrogenase (NADH1), and a part of the 16S-rRNA gene. The study of these sequences showed that this region of mtDNA is very invariable, as regards with the type of the genes that it contains, as well as the order that they are located on it. The resulting phylogenetic trees reveal a topology that separates the species into three main ancestral lines, leading to the following subgroups: (a) ananassae subgroup, (b) montium subgroup, and (c) melanogaster and Oriental subgroups. The inferred topology complements and generally agrees with previously proposed classifications based on morphological and molecular data.
Abstract: In the olive fruit fly Bactrocera oleae and the med fly Ceratitis capitata previous studies have shown the existence of two Adh genes in each species. This observation, in combination with the former finding that various Drosophila species of virilis and repleta group encode two isozymes of ADH which are the result of a gene duplication, challenged us to address a scenario dealing with the evolutionary history of the Adh gene duplication in Tephritidae. In our lab we proceeded to the cloning and sequence analysis of Adh genes from more tephritid species, a prerequisite for further study of this issue. Here we show that phylogenetic trees produced from either the nucleotide or the amino acid sequences of 14 tephritid Adh genes consisted of two main clusters, with Adh sequences of the same "type" grouping together (i.e., Adh1 sequences form a cluster and Adh2 sequences form a second one), as expected if there was one duplication event before speciation within the family Tephritidae. We used the amount of divergence between the two isozymic forms of Adh of the species carrying both Adh1 and Adh2 genes to obtain an estimate of the age of the duplication event. Interestingly, our data again support the hypothesis that the duplication of an ancestral Adh single gene in the family Tephritidae occurred before the emergence of the genera Bactrocera and Ceratitis, thus suggesting that Adh duplication was based on a prespeciation rather than a postspeciation event that might have involved two independent duplication events, one in each of the two genera.
Abstract: The actin genes of five nearctic species of the Drosophila obscura group were mapped by in situ hybridization, using the 5C actin gene of D. melanogaster as a probe. In all species but D. azteca eight actin loci were observed variously dispersed over all five (A- E) chromosomal elements. In D. azteca ten actin hybridization sites were found; four of which most probably originated by duplications or by transposition events. Although the five nearctic species differ from all other Drosophila species of the D. obscura group so far studied in the number of loci as well as in the chromosomal distribution and location of the actin loci, the uniformity of the main pattern with six actin loci throughout the genus Drosophila reinforces the hypothesis that the chromosomal elements have maintained their essential identities during the course of evolution. Our findings are in accordance with the conclusion that the nearctic D. obscura species have differentiated from a common ancestor of the palearctic species and that they belong to two distinct subgroups, the pseudoobscura and the affinis subgroups.
Abstract: The purpose of this study is to demonstrate a clear connection between the presence of acetone in larval diet and alcohol dehydrogenase (ADH) activity in laboratory raised populations of Bactrocera oleae. ADH activity of B. oleae is depressed in acetone-impregnated diets. At the same time the change of activity is accompanied by a change in the relative proportions of the multiple forms of ADH. The bulk of activity in the most cathodally migrating form is lost, and all the activity becomes localized in the less cathodally migrating forms of the enzyme. Moreover, ADH activity, expressed in vivo, appears to drop after exposure to acetone, as shown by the fact that larvae become less sensitive to pentenol poisoning. Our results show clear selective differences imposed by acetone on three homozygous genotypes involving the ADH alleles F, S and I in B. oleae. The directions of these differences were found to vary with the fitness component under test. Acetone treatment seems to affect developmental time and larva's viability as well as allele frequencies of ADH under artificial rearing. The effect of acetone on the maintenance of ADH polymorphism in artificially reared populations of B. oleae is further discussed.
Abstract: The phylogenetic relationships among the Drosophila melanogaster subgroup species were analyzed using approximately 1550-nucleotide-long sequences of the Cu,Zn SOD gene. Phylogenetic analysis was performed using separately the whole region and the intron sequences of the gene. The resulting phylogenetic trees reveal virtually the same topology, separating the species into distinct clusters. The inferred topology generally agrees with previously proposed classifications based on morphological and molecular data. The amino acid sequences of the Cu,Zn SOD of the D. melanogaster subgroup species reveal a high-conservation pattern. Only 3.9% of the total amino acid sites are variable, and none affects the major structural elements. Comparison of the Drosophila Cu,Zn SOD amino acid sequences with the Cu,Zn SOD of Bos taurus and Xenopus laevis (whose three-dimensional structure has been elucidated) reveals conservation of all the protein's functionally important amino acids and no substitutions that dramatically change the charge or the polarity of the amino acids.
Abstract: We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera.
Abstract: Chromosomal homologies among the four palearctic Drosophila obscura group species D. ambigua, D. tristis, D. obscura, and D. subsilvestris and the "trans-palearctic" species D. bifasciata were established by in situ hybridization using the 5C actin gene of D. melanogaster as a probe. In all species two labeling sites were detected in each of chromosomal elements C and E and one in each of chromosomal elements A and D. In addition one labeling site was detected on element B for the species D. subsilvestris and D. bifasciata. The conservative distribution pattern of the genes of the actin multigene family, the similarities of the locations of the actin genes in the chromosomes of the five species studied, together with the concordant evidence of synteny of visible and other genetic markers as well as the similarities in banding patterns, all agree with the conclusion that the chromosomal elements have retained their essential identity throughout the evolution of these species. Using in situ hybridization detailed information of some homologous regions of chromosomes can also be established.
Abstract: Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.
Abstract: For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to 'mainstream' IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.
Abstract: The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.
Abstract: Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.
Abstract: Glutamate dehydrogenase (GLUD) is a key metabolic enzyme of the mitochondrion, playing an important role in mammalian neuronal transmission. GLUD deficiency has been associated with certain forms of neurodegeneration in the human cerebellum. Genomic DNA blot hybridization analysis and identification of a large number of GLUD-specific genomic clones have suggested that human GLUD is encoded by a multigene family consisting of at least six members. A functional GLUD gene, GLUD1, has been mapped to chromosome 10q22.3-23 and a full-length "processed" GLUD gene, GLUDP1, to chromosome Xq22-23. In the context of studing the structure, the role, and the chromosomal organization of the other family members, we have analysed in detail, a cosmid clone solely reactive with the 3' region of the GLUD cDNA. Structure and expression analysis of its GLUD-specific region suggests that it represents a truncated "processed" GLUD pseudogene. Fluorescence in situ hybridization using the entire cosmid as a probe, mapped this GLUD gene locus, termed GLUDP5, to chromosome 10p11.2.
Abstract: Drosophila melanogaster cMdh allozymic variants (MdhF MdhF/S, MdhS) were subjected to heat shock (33 degrees C/30 min----40 degrees C/30 min). This stress increases differentially MDH specific activity, with the cMdhF strain showing greater response as compared with the cMdhS one; heterozygotes exhibited generally intermediate values. Correlative differences were also revealed for some catalytic properties (Vmax, Vmax/Km ratio, thermostability) of the cMDH; this is not true for the mMDH. The catalytic behavior of the enzyme is correlated with the differential survival of the cMdh variants, with the cMdhF showing again higher survival than the cMdhS one, a fact which seems to contribute to temperature adaptation of D. melanogaster.
