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1

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Molecular analysis of thelethal(1)B214 region at the base of theX chromosome ofDrosophila melanogaster (1992)

Russell, Robyn J. ; Healy, Marion J. ; Oakeshott, John G.

Springer

Chromosoma 101 (1992), S. 456-466

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ISSN: 1432-0886

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract Approximately 50 kb of genomic DNA was isolated from polytene chromosome bands 19F1 and 2 ofDrosophila melanogaster. Bands 19F1 and 2 are in the immediate vicinity of the β-heterochromatin at the base of theX chromosome and encompass thelittle flylike andlethal(1)B214 complementation groups. The cloned DNA consists of an approximately 21 kb stretch of unique or low copy number sequence that is bounded by repetitive elements interspersed with further unique sequences. The presence of repeated sequences is characteristic of regions within and adjacent to β-heterochromatin. At least part of a tRNA gene cluster is present within the 50 kb of cloned DNA. The cloned region also produces at least 18 discrete size classes of developmentally regulated poly(A)+ RNA species. A 2 kb EcoRI fragment (E10), which lies in the 21 kb stretch of unique sequence, generates seven of these transcripts (of sizes 3.5, 3.35, 2.1, 2.0, 1.5, 1.2 and 1.0 kb) in wild-type flies. However, a small deletion of approximately 75 bp in E10 in alethal(1)B214 mutant allele is associated with alterations in the production or processing of all seven of these transcripts. These data identify E10 sequences as belonging to thelethal(1)B214 gene and suggest that the wild-typelethal(1)B214 gene encodes multiple transcripts. Furthermore, no transcripts of the same size and having the same developmental profile as those generated by the wild-type E10 fragment were identified by probes covering the remainder of the cloned region. This suggests that at least the larger transcripts hybridizing to E10 are partly transcribed from sequences located outside the cloned region, which indicates that thelethal(1)B214 gene extends for more than 20 kb and contains other transcriptionally active sequences within it.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00582840

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2

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Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6 (1993)

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

Springer

Biochemical genetics 31 (1993), S. 259-278

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ISSN: 1573-4927

Keywords: serine esterase ; substrate interactions ; Drosophila ; acetylcholine

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb's free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00553170

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3

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A cluster of at least three esterase genes inLucilia cuprina includes malathion carboxylesterase and two other esterases implicated in resistance to organophosphates (1994)

Smyth, Kerrie-Ann ; Russell, Robyn J. ; Oakeshott, John G.

Springer

Biochemical genetics 32 (1994), S. 437-453

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ISSN: 1573-4927

Keywords: Lucilia cuprina ; malathion carboxylesterase ; organophosphate resistance

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract Three distinct malathion carboxylesterase (MCE) phenotypes have been identified among strains ofLucilia cuprina. The high-activity phenotype shows 1.6-and 33-fold more MCE specific activity than the intermediate- and low-activity phenotypes, respectively. Flies with high MCE activity are 1000-fold more resistant to malathion than flies with either low or intermediate MCE phenotypes, which are equally susceptible. High and low MCE specific activity are allelic and encoded by theRmal gene on chromosome 4.Rmal is clustered within one map unit of two other esterase genes,Rop1 andE9, which are implicated in resistance to other organophosphate insecticides. Intermediate MCE specific activity is also inherited within the cluster, although its allelism toRmal, Rop1, orE9 is unclear. The cluster does not contain the gene for the hemolymph esterase E4, which maps 6.1 map units fromRop1, on the other side of thebubbled wing marker. The cluster appears to be homologous to part of a tandem array of 11 esterase genes on chromosome 3R ofDrosophila melanogaster.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00566064

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4

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Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6 (1993)

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

Springer

Biochemical genetics 31 (1993), S. 259-278

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ISSN: 1573-4927

Keywords: serine esterase ; substrate interactions ; Drosophila ; acetylcholine

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF02401822

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5

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Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster (1991)

Healy, Marion J. ; Dumancic, Mira M. ; Oakeshott, John G.

Springer

Biochemical genetics 29 (1991), S. 365-388

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ISSN: 1573-4927

Keywords: Drosophila melanogaster ; esterases ; biochemical properties ; expression pattern

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00554144

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6

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A cluster of esterase genes on chromosome 3R ofDrosophila melanogaster includes homologues of esterase genes conferring insecticide resistance inLucilia cuprina (1994)

Spackman, Merrin E. ; Oakeshott, John G. ; Smyth, Kerrie-Ann ; [et al.]

Springer

Biochemical genetics 32 (1994), S. 39-62

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ISSN: 1573-4927

Keywords: esterases ; Drosophila melanogaster ; organophosphate resistance

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract We identify an esterase isozyme inDrosophila melanogaster, EST 23, which shares biochemical, physiological, and genetic properties with esterase E3, which is involved in resistance to organophosphate insecticides inLucilia cuprina. Like E3, theD. melanogaster EST 23 is a membrane-bound α-esterase which migrates slowly toward the anode at pH 6.8. Both enzymes have similar preferences for substrates with shorter acid side chain lengths. Furthermore, on the basis of their high sensitivity to inhibition by paraoxon and their insensitivity to inhibition by eserine sulfate, both enzymes were classified as subclass I carboxylesterases. The activity of each enzyme peaks early in development and, again, in the adult stage. Both enzymes are found in the male reproductive system and larval and adult digestive tissues, the latter being consistent with a role for these enzymes in organophosphate resistance. Fine structure deficiency mapping localizedEst 23 to cytological region 84D3 to E1-2 on the right arm of chromosome 3. Moreover, we show that the genes encoding three other esterase phenotypes also map to the same region; these phenotypes involve allozymic differences in EST 9 (formerly EST C), ali-esterase activity, defined by the hydrolysis of methyl butyrate, and malathion carboxylesterase activity, defined by hydrolysis of the organophosphate malathion. This cluster corresponds closely to that encompassing E3 and malathion carboxylesterase on chromosome 4 inL. cuprina, the homologue of chromosome 3R inD. melanogaster.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00557238

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7

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Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster (1994)

Saad, Marlene ; Game, Anne Y. ; Healy, Marion J. ; [et al.]

Springer

Genetica 94 (1994), S. 43-56

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ISSN: 1573-6857

Keywords: Drosophila ; esterase 6 ; reproduction

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01429219

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