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Duplication and Divergence of the Genes of the α-Esterase Cluster of Drosophila melanogaster (1996)

Robin, Charles ; Russell, Robyn J. ; Medveczky, Kerrie M. ; [et al.]

Springer

Journal of molecular evolution 43 (1996), S. 241-252

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ISSN: 1432-1432

Keywords: Key words: Esterase — Gene cluster — Pseudogene —Drosophila melanogaster

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract. The α-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/PL00006083

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2

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Duplication and divergence of the genes of the α-esterase cluster ofDrosophila melanogaster (1996)

Robin, Charles ; Russell, Robyn J. ; Medveczky, Kerrie M. ; [et al.]

Springer

Journal of molecular evolution 43 (1996), S. 241-252

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ISSN: 1432-1432

Keywords: Esterase ; Gene cluster ; Pseudogene ; Drosophila melanogaster

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The α-esterase cluster ofD. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF02338832

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3

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Characterization of the EstP Protein in Drosophila melanogaster and Its Conservation in Drosophilids (1997)

Dumancic, Mira M. ; Oakeshott, John G. ; Russell, Robyn J. ; [et al.]

Springer

Biochemical genetics 35 (1997), S. 251-271

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ISSN: 1573-4927

Keywords: EstP ; EST7 ; gene duplication ; gene regulation ; carboxylesterase

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract The β-esterase cluster of D. melanogaster comprises two tandemly duplicated genes. Est6 encodes the well-characterized 5′ gene, but the product of the second gene, denoted EstP, had not previously been identified. Here we show that the EstP gene encodes the carboxylesterase EST7. Expression of EstP using the Baculovirus system led to production of a carboxylesterase biochemically indistinguishable from EST7. Furthermore, a naturally occurring EstP variant produces greatly reduced amounts of EstP mRNA and no detectable EST7 protein. Finally, introduction of a wild-type copy of EstP by germline transformation into the variant strain confers the wild-type EST7 phenotype. We show that EST7 differs from EST6 in its substrate and inhibitor specificities and tissue distribution. Germline transformation experiments show that EstP expression is controlled by sequences located between 192 bp 5′ and 609 bp 3′ of the EstP coding region. Data comparisons with other drosophilid esterases suggest that the site of expression, and hence the function, of EST7 has been conserved across lineages in both the subgenera Drosophila and Sophophora.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1021897016276

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4

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Biochemistry of Esterases Associated with Organophosphate Resistance in Lucilia cuprina with Comparisons to Putative Orthologues in Other Diptera (1997)

Campbell, Peter M. ; Trott, Josephine F. ; Claudianos, Charles ; [et al.]

Springer

Biochemical genetics 35 (1997), S. 17-40

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ISSN: 1573-4927

Keywords: organophosphorus insecticide resistance ; esterase ; Lucilia cuprina ; Musca domestica

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis (“ali-esterase”) in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OPs have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1022256412623

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5

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Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster (1996)

Myers, Mark A. ; Healy, Marion J. ; Oakeshott, John G.

Springer

Biochemical genetics 34 (1996), S. 201-218

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ISSN: 1573-4927

Keywords: Drosophila ; esterase 6 ; glycosylation ; mutagenesis ; reproduction

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00553667

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6

Electronic Resource

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster (1996)

Myers, Mark A. ; Healy, Marion J. ; Oakeshott, John G.

Springer

Biochemical genetics 34 (1996), S. 201-218

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ISSN: 1573-4927

Keywords: Drosophila ; esterase 6 ; glycosylation ; mutagenesis ; reproduction

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF02407020

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