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L Alphey

Publications and source records attributed to L Alphey.

17 recordsLinked to original sources
PubMed2006

High efficiency site-specific genetic engineering of the mosquito genome.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Aedes↗
PubMed2001

Biochemical characterization of recombinant Drosophila type 1 serine/threonine protein phosphatase (PP1c) produced in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express Drosophila melanogaster type 1beta serine/threonine phosphoprotein phosphatase catalytic subunit (PP1beta9C). A construct encoding PP1beta9C with a short NH(2)-terminal fusion including six histidine residues was introduced into the X-33 and KM71H strains of P. pastoris by homologous recombination. Recombinant protein was purified from cell free extracts 24 h after methanol induction. PP1beta9C was purified to a specific activity of 12,077 mU/mg by a three-step purification method comprising (NH(4))(2)SO(4)-ethanol precipitation followed by Ni(2+)-agarose affinity chromatography and Mono Q anion-exchange chromatography. This purification scheme yielded approximately 80 microg of active, soluble PP1beta9C per 1 L of culture. In contrast to recombinant PP1beta9C overexpressed in bacteria, which differs from native PP1c in several biochemical criteria including the requirement for divalent cations, sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity, recombinant PP1beta9C produced in P. pastoris has native-like properties. P. pastoris thus provides a reliable and convenient system for the production of active, native-like recombinant PP1beta9C.

Animals↗
PubMed2001

Vectors for the expression of tagged proteins in Drosophila.

Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

Amino Acid Sequence↗
PubMed2000

Protein phosphatase 1beta is required for the maintenance of muscle attachments.

Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1] [2] [3] [4] [5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3] [6] [7]; each has one beta and several alpha isoform genes (alpha(1), alpha(2), alpha(3) in flies, alpha and gamma in humans; mammalian PP1beta is also known as PP1delta). The alpha/beta subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila beta isoform has a distinct biological role. We show that PP1beta9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.

Alleles↗
PubMed1999

The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue.

Phosphatase inhibitor-2 (I-2) is a mammalian phosphoprotein that binds to the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1c) and inhibits its activity in vitro. Recombinant PP1c differs from native PP1c in several biochemical criteria, including the requirement for Mn(2+), sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity. I-2 can convert recombinant PP1c into a native-like activity in vitro. It has therefore been suggested that I-2 may act as a molecular chaperone for PP1 in vivo. We have identified a Drosophila homologue (I-2Dm) in a two-hybrid screen for PP1c-binding proteins. The sequence of I-2Dm is 35% identical with that of I-2, whereas the catalytic subunits themselves are >85% identical in flies and humans; however, we show that many biochemical properties of I-2 are conserved. Like I-2, I-2Dm can convert recombinant PP1c to a native-like activity. This strongly suggests that this ability is an essential, conserved role of I-2 and I-2Dm.

Amino Acid Sequence↗
PubMed1998

A Drosophila homologue of oxysterol binding protein (OSBP)--implications for the role of OSBP.

The identification of a Drosophila homologue (OSBP-Dm) of mammalian oxysterol binding protein (OSBP) is reported. OSBP-Dm was identified by its ability to overcome the cell cycle arrest induced by over-expression of Wee1p in fission yeast. OSBP-Dm has an overall sequence identity of 52% with mammalian OSBP, and shows a number of highly conserved regions of functional significance. Insects are unable to biosynthesize the steroid core, relying instead on dietary sterols to satisfy their requirements. It is therefore unlikely that OSBP-Dm is involved in feedback inhibition of the mevalonate pathway, as has previously been suggested for its mammalian homologues.

Amino Acid Sequence↗
PubMed1997

KLP38B: a mitotic kinesin-related protein that binds PP1.

We have identified a new member of the kinesin superfamily in Drosophila, KLP38B (kinesin-like protein at 38B). KLP38B was isolated through its two-hybrid interaction with the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1). We demonstrate that recombinant KLP38B and PP1 associate in vitro. This is the first demonstration of direct binding of a kinesin-related protein to a regulatory enzyme. Though most closely related to the Unc-104 subfamily of kinesin-related proteins, KLP38B is expressed only in proliferating cells. KLP38B mutants show cell proliferation defects in many tissues. KLP38B is required for normal chromatin condensation as embryos from KLP38B mutant mothers have undercondensed chromatin at metaphase and anaphase. This is the first time that a kinesin-related protein has been shown to have such a role. Incomplete lethality of a strong KLP38B allele suggests partial redundancy with one or more additional kinesin-related proteins.

Amino Acid Sequence↗
PubMed1997

PCR-based method for isolation of full-length clones and splice variants from cDNA libraries.

Most cDNA library screening procedures do not distinguish between full-length and incomplete clones and therefore may yield incomplete cDNA fragments. Thus, there is a widespread need for a method allowing the efficient selection of full-length clones. I present a rapid, PCR-based method that allows the simultaneous screening of > 10(6) cDNAs. The longest cDNA is identified in the first step so that incomplete clones may be eliminated from study at this stage to save time. The method also facilitates the identification and isolation of rare splice variants from a background of a more abundant variant.

Animals↗
PubMed1995

Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster.

The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin-dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.

Amino Acid Sequence↗
PubMed1993

The cdc25 homologue twine is required for only some aspects of the entry into meiosis in Drosophila.

The twineHB5 mutation prevents spindle formation during the entry into meiosis in Drosophila males, but chromosome condensation and nuclear envelope breakdown both still occur. This suggests the possibility that this particular cdc25 homologue is required to activate a p34cdc2 kinase required for only some of the events of this G2-M transition. In contrast, meiotic spindles do form in twineHB5 females, although these appear abnormal. However, the female meiotic divisions do not arrest at metaphase I as in wild type, but continue repeatedly, leading to gross non-disjunction. Small chromatin masses, corresponding in size to the fourth chromosomes, often segregate properly to the spindle poles. These can persist into the embryos derived from twineHB5 females, where they appear to participate in mitotic divisions on thin spindles. In addition, these embryos contain a small number of large chromatin masses that are not associated with spindles.

Animals↗
PubMed1992

twine, a cdc25 homolog that functions in the male and female germline of Drosophila.

twine is the second homolog of the fission yeast gene cdc25 to be found in Drosophila. Both string and twine cDNAs can rescue a temperature-sensitive cdc25 mutation in fission yeast, but not a deletion. We detect the expression of string but not twine transcripts in the proliferating cells of newly cellularized embryos, in third instar larval brains, and in imaginal discs. Both genes are abundantly expressed in nurse cells during oogenesis, the maternal transcripts persisting throughout the syncytial stage of embryonic development. In the testis, twine transcripts are seen in the growing stage of premeiotic cysts. Analysis of a twine mutant suggests a requirement for the gene during oogenesis, during syncytial embryonic development, and for male meiosis. Meiosis does not occur in homozygous twine males, which produce cysts containing 16 rather than 64 spermatids.

Amino Acid Sequence↗
PubMed1990

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1.

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombrádi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.

Amino Acid Sequence↗
PubMed1990

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1.

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left arm of the second chromosome at position 28D2-4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2A alpha or PP2A beta, indicating that PP2A may be the most conserved of all known enzymes.

Amino Acid Sequence↗
PubMed1990

Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string.

We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25-22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.

Amino Acid Sequence↗
PubMed1989

Mitosis in Drosophila development.

Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.

Animals↗