ABSTRACT
Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.
Subject(s)
Fluorescent Dyes , Fluorescent Dyes/chemistry , Viscosity , FluorescenceABSTRACT
Introduction: Extracted from gamboge resin, gambogic acid (GBA) is a natural product that displays a complex caged xanthone structure and exhibits promising antitumor properties. However, efforts to advance this compound to clinical applications have been thwarted by its limited availability that in turn, restricts its pharmacological optimization. Methods: We report here an efficient method that allows multigram scale isolation of GBA in greater than 97% diastereomeric purity from various sources of commercially available gamboge. The overall process includes: (a) isolation of organic components from the resin; (b) separation of GBA from the organic components via crystallization as its pyridinium salt; and (c) acidification of the salt to isolate the free GBA. Results and Discussion: We found that GBA is susceptible to epimerization at the C2 center that produces epi-gambogic acid ( epi-GBA), a common contaminant of all commercial sources of this compound. Mechanistic studies indicate that this epimerization proceeds via an ortho-quinone methide intermediate. Although the observed stereochemical erosion accounts for the chemical fragility of GBA, it does not significantly affect its biological activity especially as it relates to cancer cell cytotoxicity. Specifically, we measured similar levels of cytotoxicity for either pure GBA or an equilibrated mixture of GBA/ epi-GBA in MBA-MB-231 cells with IC50 values at submicromolar concentration and induction of apoptosis after 12 hours of incubation. The results validate the pharmacological promise of gambogic acid and, combined with the multigram-scale isolation, should enable drug design and development studies.
ABSTRACT
Racemic resolution of (+/-)-MAD28, a representative caged xanthone, was accomplished using (1S, 4R)-(-)-camphanic chloride as the chiral agent. Selective crystallization of the resulting diastereomers in acetonitrile produced, after hydrolysis, the pure enantiomers. Screening of racemic MAD28 and both enantiomers across a broad spectrum of breast cancer cell lines revealed that they: (a) are equipotent in each of the breast cancer subtypes examined; and (b) exhibit a higher degree of cytotoxicity against breast cancer cell lines of basal-like subtype and triple negative receptor status. The results support the notion that MAD28 and related caged xanthones are promising drug leads against chemoresistant and metastatic cancers.
Subject(s)
Antineoplastic Agents/chemistry , Xanthones/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Female , Humans , Molecular Conformation , Stereoisomerism , Xanthones/chemical synthesis , Xanthones/pharmacologyABSTRACT
Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and related caged xanthones in spheroidsMARY-X, an in vitro preclinical IBC model, we constructed a library of synthetic analogs and performed structure-activity relationship studies. The studies revealed that functionalizing the A-ring of the caged xanthone framework can significantly affect potency. Specifically, introduction of hydroxyl or fluorine groups at discrete positions of the A-ring leads to enhanced cytotoxicity at submicromolar concentrations. These compounds induce complete dissolution of spheroidsMARY-X with subsequent apoptosis of both the peripherally- and centrally-located cells, proliferative and quiescent-prone (e.g. hypoxic), respectively. These results highlight the structural flexibility and pharmacological potential of the caged xanthone motif for the design of IBC-targeting therapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Inflammatory Breast Neoplasms/drug therapy , Xanthones/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Inflammatory Breast Neoplasms/pathology , Molecular Structure , Structure-Activity Relationship , Xanthones/chemical synthesis , Xanthones/chemistryABSTRACT
The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 µM for gambogic acid and 0.66 +/-0.02 µM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 µM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore.
Subject(s)
Antineoplastic Agents/chemistry , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , Neoplasms/drug therapy , Spheroids, Cellular/metabolism , Amino Acid Motifs , Animals , Apoptosis , Cell Line, Tumor , Garcinia/chemistry , Humans , Immunohistochemistry , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Microscopy, Confocal , Microscopy, Fluorescence , Neoplasm Transplantation , Tumor Cells, Cultured/drug effects , Xanthones/chemistryABSTRACT
The UBR1 ubiquitin ligase promotes degradation of proteins via the N-end rule and by another mechanism that detects a misfolded conformation. Although UBR1 was shown recently to act on protein kinases whose misfolding was promoted by inhibition of Hsp90, it was unknown whether this ubiquitin ligase targeted other client types of the chaperone. We analyzed the role of UBR1 in the degradation of nuclear receptors that are classical clients of Hsp90. Our results showed that UBR1 deletion results in impaired degradation of the glucocorticoid receptor and the androgen receptor but not the estrogen receptor α. These findings demonstrate specificity in the actions of the UBR1 ubiquitin ligase in the degradation of Hsp90 clients in the presence of small molecule inhibitors that promote client misfolding.
ABSTRACT
Natural products of the caged Garcinia xanthones (CGX) family are characterized by a unique chemical structure, potent bioactivities and promising pharmacological profiles. We have developed a Claisen/Diels-Alder reaction cascade that, in combination with a Pd(0)-catalyzed reverse prenylation, provides rapid and efficient access to the CGX pharmacophore, represented by the structure of cluvenone. To further explore this pharmacophore, we have synthesized various A-ring oxygenated analogues of cluvenone and have evaluated their bioactivities in terms of growth inhibition, mitochondrial fragmentation, induction of mitochondrial-dependent cell death and Hsp90 client inhibition. We found that installation of an oxygen functionality at various positions of the A-ring influences significantly both the site-selectivity of the Claisen/Diels-Alder reaction and the bioactivity of these compounds, due to remote electronic effects.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Garcinia/chemistry , Oxygen/chemistry , Xanthones/chemical synthesis , Xanthones/pharmacology , Antineoplastic Agents, Phytogenic/chemical synthesis , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Mitochondria/drug effects , Molecular Structure , Structure-Activity Relationship , Xanthones/chemistryABSTRACT
Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.
Subject(s)
Gene Regulatory Networks , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Ubiquitin-Protein Ligases/genetics , Azetidinecarboxylic Acid/pharmacology , Benzoquinones/pharmacology , Blotting, Western , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Lactams, Macrocyclic/pharmacology , MAP Kinase Kinase Kinases/chemistry , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Microbial Viability/drug effects , Microbial Viability/genetics , Microscopy, Fluorescence , Mutation , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Folding , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.
Subject(s)
Benzoquinones/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival/drug effects , Cells, Cultured , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Ubiquitin-Protein Ligases/deficiencyABSTRACT
Quality control processes regulate the proteome by determining whether a protein is to be folded or degraded. Hsp90 is a hub in the network of molecular chaperones that maintain this process because it promotes both folding and degradation, in addition to regulating expression of other quality control components. The significance of Hsp90's role in quality control is enhanced by the function of its clients, which include protein kinases and transcription factors, in cellular signaling. The inhibition of Hsp90 with small molecules results in the rapid degradation of such clients via the ubiquitin/proteasome pathway, and also in the induction of the Hsp70 molecular chaperone. These two events result in markedly different outcomes depending on cell type. For tumor cells there is a profound loss of signaling in growth promoting pathways. By contrast, increased amounts of Hsp70 in neuronal cells ameliorate the toxicity that is associated with the formation of aggregates observed in neurodegenerative conditions. In this review we discuss the mechanisms underlying these differential effects of Hsp90 inhibition on the quality control of distinct client proteins. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).
Subject(s)
HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , Protein Folding , Proteolysis , Signal TransductionABSTRACT
Molecular chaperones promote polypeptide folding in cells by protecting newly made and otherwise misfolded proteins against aggregation or degradation by the ubiquitin proteasome pathway. The roles of Saccharomyces cerevisiae Cdc37 and Ydj1 molecular chaperones are described in this chapter. We focus on biogenesis of protein kinases that require several different molecular chaperones for their proper folding. Specific among these is Cdc37, which binds directly to its kinase clients either during or shortly after translation and protects them against rapid proteasomal degradation. Ydj1 has a similar role, but is less specific for protein kinases in its role as a molecular chaperone. The method that we describe uses pulse chase and immunoprecipitation to analyze the fate of newly made proteins. Two kinetically distinct pathways of degradation can be discerned using this methodology that is dependent on the presence of an Hsp90 inhibitor or occurs in mutants of the molecular chaperones under study. The first is "zero-point" degradation that occurs either during or immediately after translation. The second is a slower pathway, where the half-life of kinase is approximately 20 min after translation.
Subject(s)
Molecular Chaperones/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , HSP40 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Protein Folding , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/geneticsABSTRACT
Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.
Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Unfolded Protein Response/physiology , Animals , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , HSP70 Heat-Shock Proteins/metabolism , Luciferases, Firefly/metabolism , Peptides/chemistry , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Conformation , Protein Denaturation , Protein Folding , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Genetic Complementation Test , HSP70 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/genetics , Immunoblotting , Molecular Chaperones/genetics , Mutation , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin/metabolismABSTRACT
Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.
Subject(s)
Benzoquinones/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase 4/metabolism , Half-Life , Mice , Molecular Chaperones/metabolism , Organ Specificity/drug effects , Protein Binding/drug effects , Protein Kinases/metabolism , Protein Processing, Post-Translational/drug effects , Protein-Tyrosine Kinases/metabolism , Time FactorsABSTRACT
The Hsp90-Cdc37 chaperone pair has special responsibility for folding of protein kinases. This function has made Hsp90 a target for new chemotherapeutic approaches, and several compounds are currently being tested for their ability to inhibit many different kinases simultaneously. Not all kinases are sensitive to these inhibitors, however, and this difference might depend on how each kinase interacts with Hsp90 and Cdc37 during folding of the nascent chain and thereafter. Indeed, several kinases require the persistent presence of both chaperones after initial folding and some of these kinases seem to be particularly sensitive to Hsp90 inhibitors. This requirement might relate to conformational changes that take place during the protein kinase activity cycle.
Subject(s)
Cell Cycle Proteins/physiology , Chaperonins/physiology , HSP90 Heat-Shock Proteins/physiology , Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Benzoquinones/pharmacology , Catalytic Domain , Enzyme Activation , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Hydrolysis , Lactams, Macrocyclic/pharmacology , Mammals/metabolism , Models, Molecular , Protein Conformation , Protein Folding , Protein Interaction Mapping , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Protein Structure, Tertiary , Structure-Activity Relationship , Yeasts/metabolismABSTRACT
Ribosomal protein S21 (RpS21) belongs to a small group of ribosomal or ribosome-associated proteins. Mutations in the RpS21 gene cause dominant Minute and recessive lethal tumorous phenotypes in Drosophila melanogaster. Studies in several organisms suggest that RpS21 is involved in the regulation of protein synthesis and cell growth. In this report, we used an RT-PCR fragment of D. melanogaster RpS21 mRNA to clone a RpS21 cDNA from the Mediterranean fruit fly, Ceratitis capitata. The isolated cDNA contained both 5' and 3' untranslated regions, and encoded a polypeptide of 83 amino acids with a predicted molecular mass of 9.1 kDa. The deduced protein sequence showed 91% amino acid identity to D. melanogaster RpS21 and strong homology with all known ribosomal S21 proteins. DNA blot hybridization indicated the existence of a single RpS21 gene in the Ceratitis capitata genome. Analysis of the 5' untranslated region revealed the occurrence of a major oligopyrimidine tract at the 5' end, which characterizes most mRNAs undergoing a growth-dependent translational control. Study of the mRNA patterns during development suggested that the expression of Ceratitis RpS21 is temporally regulated at the level of transcription.
Subject(s)
Ceratitis capitata/growth & development , Ceratitis capitata/genetics , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Ceratitis capitata/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Drosophila melanogaster/genetics , Female , Gene Expression Regulation, Developmental , Genome , Larva/genetics , Larva/growth & development , Larva/metabolism , Male , Molecular Sequence Data , Phylogeny , Protein Isoforms/genetics , Pupa/genetics , Pupa/growth & development , Pupa/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
This review summarizes structural and functional studies on medfly promoters and regulatory elements that can be used for driving sex-specific, conditional and constitutive gene expression in this species. Sex-specific and conditional promoters are important for generating transgenic sexing strains that could increase the performance of the Sterile Insect Technique while strong constitutive promoters are necessary for developing sensitive transgenic marker systems. The review focuses on the functional analysis of the promoters of two male-specific and heat shock medfly genes. A special emphasis is put on the potential utility of these promoters for developing transgenic sexing strains.