Est-6 Gene Research Articles

Molecular Cloning and Characterization of a New Family VI Esterase from an Activated Sludge Metagenome.

A new esterase gene, est6, was discovered in an activated sludge metagenomic library. The 729-bp gene encodes a 242-amino acid protein (designated Est6) with a molecular mass of 26.1 kDa. Est6 shared only a moderate identity to a putative hydrolase with the highest BLASTP analysis score. Most of the closely related proteins are uncharacterized and are predicted from genome sequencing data of microorganisms or metagenomic DNA sequences. The phylogenetic analysis of Est6 showed that the protein was assigned to family VI esterases/lipases. The catalytic triad of Est6 was predicted to be Ser135, Asp188, and His219, with Ser135 in a typically conserved pentapeptide (GFSQG) of family VI members, which was further confirmed by site-directed mutagenesis. The est6 gene was overexpressed successfully in its soluble form in Escherichia coli and then purified to its tag-free form and homogeneity by affinity chromatography. The purified Est6 in pH 8.0 buffer was active as a monomer. The optimal conditions for Est6 activity were at a temperature of 45 °C and pH of 8.0 when using p-nitrophenyl acetate as a substrate. The enzyme was stable over wide temperature and pH ranges, and it exhibited activity in the presence of organic solvents, metal cations, or detergents. Furthermore, the enzyme showed significant regioselectivity in the spectrophotometric analysis. In conclusion, Est6 might have the potential for applications in biotechnological processes.

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

BackgroundInsects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme.ResultsWe first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation.ConclusionsOur study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction.

Identification and characterization of novel esterases from a deep-sea sediment metagenome

A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.

Entropy and GC Content in the β-esterase Gene Cluster of the Drosophila melanogaster Subgroup

We perform spectral entropy and GC content analyses in the beta-esterase gene cluster, including the Est-6 gene and the psiEst-6 putative pseudogene, in seven species of the Drosophila melanogaster species subgroup. psiEst-6 combines features of functional and nonfunctional genes. The spectral entropies show distinctly lower structural ordering for psiEst-6 than for Est-6 in all species studied. Our observations agree with previous results for D. melanogaster and provide additional support to our hypothesis that after the duplication event Est-6 retained the esterase-coding function and its role during copulation, while psiEst-6 lost that function but now operates in conjunction with Est-6 as an intergene. Entropy accumulation is not a completely random process for either gene. Structural entropy is nucleotide dependent. The relative normalized deviations for structural entropy are higher for G than for C nucleotides. The entropy values are similar for Est-6 and psiEst-6 in the case of A and T but are lower for Est-6 in the case of G and C. The GC content in synonymous positions is uniformly higher in Est-6 than in psiEst-6, which agrees with the reduced GC content generally observed in pseudogenes and nonfunctional sequences. The observed differences in entropy and GC content reflect an evolutionary shift associated with the process of pseudogenization and subsequent functional divergence of psiEst-6 and Est-6 after the duplication event.

The beta-esterase gene cluster of drosophila melanogaster: is psiEst-6 a pseudogene, a functional gene, or both?

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.

Molecular population genetics of the beta-esterase gene cluster of Drosophila melanogaster.

We have investigated nucleotide polymorphism at the beta-esterase gene cluster including the Est-6 gene and psiEst-6 putative pseudogene in four samples of Drosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in both Est-6 and psiEst-6. Total nucleotide diversity is twice in psiEst-6 as in Est-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within the beta-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected within Est-6 and, to a much greater extent, within psiEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in the beta-esterase gene cluster. However there are some 'footprints' of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection between Est-6 and psiEst-6 may play an important role in the evolution of the beta-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene. Est-6 and psiEst-6 may represent an indivisible intergenic complex ('intergene') in which each single component (Est-6 or psiEst-6) cannot separately carry out the full functional role.

DNA polymorphism in the beta-Esterase gene cluster of Drosophila melanogaster.

We have analyzed nucleotide polymorphism within a 5.3-kb region encompassing the functional Est-6 gene and the psiEst-6 putative pseudogene in 28 strains of Drosophila melanogaster and one of D. simulans. Two divergent sequence types were detected, which are not perfectly associated with Est-6 allozyme variation. The level of variation (pi) is very close in the 5'-flanking region (0.0059) and Est-6 gene (0.0057), but significantly higher in the intergenic region (0.0141) and putative pseudogene (0.0122). The variation in the 3'-flanking region is intermediate (0.0083). These observations may reflect different levels of purifying selection in the different regions. Strong linkage disequilibrium occurs within the region studied, with the largest values revealed in the putative pseudogene and 3'-flanking region. Moreover, recombination is restricted within psiEst-6. Gene conversion is detected both within and (to a lesser extent) between Est-6 and psiEst-6. The data indicate that psiEst-6 exhibits some characteristics that are typical of nonfunctional genes, while other characteristics are typically attributed to functional genes; the same situation has been observed in other pseudogenes (including Drosophila). The results of structural entropy analysis demonstrate higher structural ordering in Est-6 than in psiEst-6, in accordance with expectations if psiEst-6 is indeed a pseudogene. Taking into account that the function of psiEst-6 is not known (but could exist) and following the terminology of J. Brosius and S. J. Gould, we suggest that the term "potogene" may be appropriate for psiEst-6, indicating that it is a potential gene that may have acquired some distinctive but unknown function.

Molecular evolution of the Est-6 gene in Drosophila melanogaster: contrasting patterns of DNA variability in adjacent functional regions

We have investigated nucleotide polymorphism at the esterase 6 gene ( Est-6) gene, including the complete coding region (1686 bp), as well as the 5′-flanking (1183 bp) and 3′-flanking (193 bp) regions of the gene, in 30 strains of Drosophila melanogaster and in one strain of Drosophila simulans. The level of silent variation is similar in the coding and in the 3′-flanking region, but smaller in the 5′-flanking region. Strong linkage disequilibrium occurs within each region; and also, although less pronounced, between the 5′-flanking region and the rest of the gene, including the 3′-flanking region. We suggest that the pattern of nucleotide polymorphism of Est-6 may be shaped by: (1) directional and balancing selection acting on the promoter and the coding region; and (2) interactions between the two regions that involve variable degrees of hitchhiking. The patterns of linkage disequilibrium, as well as the statistics Z nS (Genetics 146 (1997)1197) and B and Q (Genet. Res. 74 (1999) 65), may be interpreted as there being multiple targets of selection within the gene. The previously reported Est-6 allozyme latitudinal clines may be accounted for by the interaction between selective processes in the promoter and coding regions.

Cloning of the esterase-5 locus from Drosophila pseudoobscura and comparison with its homologue in D. melanogaster.

A clone of the esterase-5 (Est-5) gene from Drosophila pseudoobscura has been isolated by hybridization to the cloned Est-6 gene of D. melanogaster. Southern analysis and sequencing of the cloned DNA revealed three regions of similarity to Est-6 that have been tentatively identified as genes, Est-5A, Est-5B, and Est-5C. Introduction of each of the three genes separately into D. melanogaster by P-element transformation has demonstrated that Est-5B encodes an enzyme with the same physical properties as EST 5 in D. pseudoobscura. Sequence analysis indicates that Est-5B encodes a 545-amino-acid protein and is composed of two exons separated by a 55-bp intron in the same position as the 51-bp intron in Est-6. Comparison of the Est-5B coding region with that of Est-6 reveals an overall similarity (73% at both the nucleotide and amino acid levels) that is substantially lower than that for other genes sequenced in both of these species. Total nucleotide and nonsynonymous site differences between Est-6 and Est-5B are more abundant in the second exon than in the first, suggesting differential effects of selection or mutation on these two exons. Comparisons of the 5'-flanking DNA of Est-5B and Est-6 reveal four short conserved sequence elements, but the remaining upstream sequences show no significant similarity. Conservation in the 3'-flanking DNA is limited to the presence of two polyadenylation sites that may correlate with the existence of two transcripts from both Est-5B and Est-6. The patterns of nucleotide substitutions and amino acid replacements between Est-5B and Est-6 are consistent with the hypothesis that mutation and genetic drift are responsible for the differences between these two genes.

Genetic control of esterase-6 isozymes in hexaploid wheat and rye

The EST-6 leaf esterase phenotypes from euploid, nullisomic-tetrasomic and rye chromosome addition and substitution lines of common wheat were determined using polyacrylamide gel electrophoresis. Evidence is presented to demonstrate that Est-6 is a new set of genes, that are expressed in the leaf. The Est-6 gene set were clearly distinguished from the Est-5 genes which are expressed in the grain. The three homoeoallelic loci, Est-A6, Est-B6 and Est-D6, were located on chromosomes 3A, 3B and 3D. An Est-R6 gene was located on chromosome 6R is involved in rye. Some considerations concerning homoeology between homoeologous group 3 of wheat and the rye chromosome 6R are made.

Molecular analysis of duplicated esterase genes in Drosophila melanogaster.

Genomic clones containing sequences homologous to an esterase 6 (Est-6) cDNA clone were isolated from a library of Drosophila melanogaster DNA. Comparison of the genomic and cDNA sequences revealed that the Est-6 gene comprises two exons, one of 1,387 bp and one of 248 bp, separated by a short intron of 51 bp. Further sequencing revealed the presence of a tandem duplication of the Est-6 gene (denoted Est-P) which also has an exon of 1,387 bp and an exon of 248 bp, separated by a short intron of 56 bp. The two genes show similarities of 64% and 60% at the DNA and protein levels, respectively. The coding regions of the genes are 197 bases apart, and presumptive 5' regulatory sequences of Est-P overlap at least the 3' noncoding region of Est-6. Transcripts homologous to Est-P were detected in late larvae and adults of each sex, whereas Est-6 transcripts are present in all life stages but are predominant in adult males. This suggests different physiological functions for the products of the two genes. Southern and Northern blot hybridization analyses of the 20-kb region surrounding the Est-6/Est-P duplication failed to detect any other duplicated esterase genes, although this region is actively transcribed.

Amino acid polymorphisms for esterase-6 in Drosophila melanogaster.

High-resolution electrophoresis has revealed 10 allozymes of esterase-6 (EC 3.1.1.1) in Drosophila melanogaster. The sequences of 13 isolates of the Est6 gene covering all 10 allozymes were obtained and 52 nucleotide differences were found. Sixteen of these cause amino acid replacements, of which three result in charge differences whose size and direction are consistent with the electrophoretic mobilities of the allozymes in which they occur. The smeared electrophoretic phenotype of one allozyme can be explained by the loss of a cysteine residue involved in a disulfide bridge. Several minor mobility variants within the major F and S electrophoretic phenotypes differ by amino acid substitutions that are generally conservative for charge but not for some other properties (size, polarity, or hydrophobicity). Four amino acid differences are found among different isolates of the same allozymes and, overall, 12 amino acid haplotypes occur among the 13 isolates sequenced. Nevertheless, the most common variants within F and S are distinguished by only two amino acids (Asn/Asp at 237 and Thr/Ala at 247), and these are the most likely targets for the selection underlying complementary latitudinal clines in F and S frequencies.

Molecular population genetics of structural variants of esterase 6 in Drosophila melanogaster

Several lines of evidence indicate that natural selection operates between the major EST6-F and EST6-S allozymes of Drosophila melanogaster. In particular, consistent latitudinal clines and seasonal variation in their relative frequencies strongly suggest that they are not selectively equivalent in field populations. Several laboratory studies have found frequency-dependent fitness differences among the Est6-F and Est6-S genotypes. Moreover, the purified EST6-F and EST6-S allozymes differ in biochemical properties and the physiology of the enzyme, as a major component of the seminal fluid, suggests that these differences could affect reproductive aspects of fitness. However, molecular analyses reveal high levels of variation in the EST6 protein both within and between the EST6-F and EST6-S allozymes. Limited thermostability and more sensitive electrophoretic analyses reveal at least 17 variants of the two allozymes and sequence comparisons among 13 isolates of the Est6 gene reveal 16 nucleotide polymorphisms that would lead to amino acid differences. Two closely linked amino acid differences are strongly associated with the major difference between EST6-F and EST6-S; either or both of these are likely to cause the observed biochemical differences between EST6-F and EST6-S and may be the primary targets for the selection between these allozymes. The functional and adaptive significance of the other amino acid polymorphisms is unclear, although the data suggest that the EST6-8 haplotype within EST6-S has both arisen and proliferated relatively recently.

Latitudinal relationships of esterase-6 and phosphoglucomutase gene frequencies in Drosophila melanogaster.

Geographic variation in Esterase-6 (Est-6) and Phosphoglucomutase (Pgm) gene frequencies in Australasian populations of Drosophila melanogaster are compared with analogous data collated from 16 previous reports for North America and Europe/Asia. A large-scale latitudinal cline is found on all three zoogeographic zones for Est-6 and overall, Est-61.00 frequency increases from about 20 per cent around 20 degrees latitude to about 80 per cent approaching 50 degrees latitude. In contrast, there is no consistent evidence for the latitudinal cline in Pgm gene frequencies in any of the three zones with Pgm1.00 frequency generally about 85 per cent and Pgm1.20 and Pgm0.70 frequencies each between 5 per cent and 10 per cent. The consistent Est-6 clines are attributed to latitudinal selection gradients but not consistent correlations are found between Est-6 gene frequencies and maximum or minimum temperature or rainfall which might be associated with these gradients. The directions of the Est-6 clines in fact run counter to expectations based on the in vitro thermostabilities of the respective allozymes.