Anastassios C. Mintzas

In the present study, we characterized a 3320‐bp genomic DNA fragment encoding two medfly ( Ceratitis capitata ) homologues of the Drosophila melanogaster heat shock protein 23 ( hsp23 ) gene, named Cchsp23‐α and ‐β . The two medfly hsp23 genes are transcribed in opposite directions and encode two almost identical proteins. Furthermore, the two genes exhibit a very high degree of similarity in their 5′ untranslated and proximal promoter regions. Phylogenetic analysis indicated that the CcHsp23 proteins are orthologous to Drosophila Hsp23 and Sarcophaga crassipalpis Hsp23. Structural analysis of the 5′ flanking regions of the Cchsp23 genes revealed the presence of several putative heat shock elements. Both CcHsp23 genes are induced by heat in a similar manner. In addition to heat‐induction, the Cchsp23 genes are expressed at several stages of normal development as well as in ovaries and testes. In general, the developmental expression patterns of the medfly genes are similar, suggesting that they are under similar regulatory mechanisms. However, the expression of the Cchsp23 genes differs significantly from the expression of the Drosophila hsp23 gene in certain embryonic and larval stages, suggesting differential regulation of the hsp23 genes in the two dipteran species. The expression of both Cchsp23 genes in adult flies is increased with age, especially in males.

In the present study, a genomic DNA clone encoding the medfly homolog of Drosophila melanogaster hsp27 gene, named Cchsp27 , was isolated. We sequenced a part of the clone containing the coding region, the 5′ untranslated region and approximately 2.8 Kb of the 5′ flanking region of the gene. Phylogenetic analysis of several insect small heat shock proteins, suggested that Cc Hsp 27 is orthologous to Drosophila Hsp 27 and Sarcophaga crassipalpis Hsp 25. The Cchsp27 gene was mapped at the 81A division of the sixth chromosome which coincides with one of the major heat shock puffs of medfly. Structural analysis of the 5′ flanking region of the Cchsp27 gene revealed the presence of five putative heat shock elements and one putative ecdysone response element. In addition to heat induction, the Cchsp27 gene was expressed at several stages of normal medfly development. In general, the developmental expression pattern of the Cchsp27 gene was similar to the respective pattern of Drosophila hsp27 gene. However, there were some important differences in certain developmental stages suggesting differential regulation of the hsp27 gene in the two dipterans species. Salivary gland culture experiments showed that the Cchsp27 gene is regulated by 20‐hydroxyecdysone.

In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome α5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome α5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome α5 subunits from different organisms indicated that the medfly 20S proteasome α5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis. Arch. Insect Biochem. Physiol.

In order to define the regulatory elements responsible for the expression of the medfly hsp83 (Cchsp83) gene, we determined the sequence of a genomic region of the gene that included 3,536 bp upstream of the transcription initiation site, the first untranslated exon of 144 bp, a 275‐bp intron, and 516 bp of the second coding exon. Structural analysis of the 5′ flanking region revealed the presence of a typical TATA box, 28 bp upstream of the transcription start site, and seven putative heat shock elements (HSEs) further upstream. The 5′ untranslated region of the Cchsp83 mRNA was found to contain extensive secondary structure in the first 126 nucleotides. We carried out deletion functional analysis of the proximal promoter region (–380/+139) in vivo by germ line transformation using the lacZ as a reporter gene. We found that sequences in the –380/–86 region are essential for the constitutive expression of the Cchsp83 gene. Under normal conditions, the –380/+139 region was able to drive significant levels of transgene expression in all developmental stages of the medfly as well as in the ovaries and testis. In most stages, the temporal expression pattern of the reporter gene was similar to the respective pattern of the endogenous Cchsp83 gene. Although the –380/+139 promoter region contained two putative HSEs, it was found unable to confer any heat‐induced expression in the reporter gene. Arch. Insect Biochem. Physiol. 2007.

This report presents the cDNA cloning, heat shock regulation and developmental expression of the hsp90 gene homologue of the Mediterranean fruit fly Ceratitis capitata (medfly). The isolated cDNA contained the coding region, the 3′UTR and most of the 5′UTR of the medfly hsp90 homologue, which was named Cchsp83 . The deduced CcHSP83 polypeptide contained all the highly conserved amino acid segments that characterize the cytosolic members of the HSP90 family. Genomic analysis showed that the Cchsp83 gene is unique and was mapped at the 94C division of the sixth polytene chromosome. The size of the Cchsp83 mRNA was found to be approximately 2.7 kb. The predicted molecular mass of the CcHSP83 protein was 81.4 kDa, while the apparent molecular weight estimated by SDS‐PAGE was approximately 90 kDa. Phylogenetic analysis based on 14 insect HSP90 amino acid sequences was consistent with the known phylogeny at low taxonomic level. The Cchsp83 gene is constitutively expressed in all stages of medfly development and is induced from a low level to several‐fold by heat, depending on the developmental stage. Heat shock induction begins at 30 °C, reaching a maximum between 35 and 41 °C. Cchsp83 RNA expression is highly regulated during embryonic development; however, the temporal fluctuations in RNA levels during embryogenesis were not followed by similar fluctuations in the levels of the protein.

The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp 70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos ‐mediated germ‐line transformation. The activity of each promoter was evaluated in 15 transformed lines by β‐galactosidase quantitative assays. The heat‐inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The −263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the −263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo .

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. Arch. Insect Biochem. Physiol. 56:133–142, 2004.

Using 5′ RACE with specific primers for the ecdysone receptor B1 isoform of the Mediterranean fruit fly (medfly), Ceratitis capitata, we isolated a cDNA clone encoding the specific region of the medfly ecdysone receptor A isoform (CcEcR‐A). The CcEcR‐A‐specific region was very similar to the EcR‐A‐specific region of Drosophila melanogaster and less similar to the EcR‐A‐specific regions of Lepidoptera. The developmental expression of both CcEcR‐A and CcEcR‐B1 mRNAs was studied in whole animals, salivary glands and ovaries by RT‐PCR, using isoform‐specific primers. Both CcEcR mRNAs are present in very early embryos, decrease to very low levels during the first hours of embryogenesis and are highly expressed in all consequent embryonic stages. During metamorphosis both isoforms are present showing two peaks; the first at the larval–prepupal transition and the second during the second half of prepupal development. These peaks are correlated with the two puffing cycles and the two major 20‐hydroxyecdysone (20E) increases that occur during medfly metamorphosis. CcEcR‐B1 mRNA was directly induced in larval salivary glands in vitro by 20E, even at very low concentrations of the hormone, while CcEcR‐A mRNA was slightly induced only by high 20E concentrations and in the absence of a protein synthesis inhibitor. During oogenesis, the CcEcR mRNAs were expressed synchronously, peaking at the beginning of both previtellogenic and vitellogenic phases.