Cloning and characterization of the ribosomal protein CcP0 of the medfly Ceratitis capitata.

The gene of the ribosomal protein CcP0, the third member of the ribosomal P-protein family of the medfly Ceratitis capitata, was identified by genomic and cDNA sequence analysis. It codes for a polypeptide of 317 amino acids and its predicted amino acid sequence shows great similarity to the P0 proteins of other eukaryotic organisms. The CcP0 gene was expressed in Escherichia coli and the 34-kDa recombinant protein was identical to the P0 protein of purified medfly ribosomes. Both proteins reacted positively with a specific monoclonal antibody against the highly conserved C terminus of eukaryotic ribosomal P proteins. Interestingly, the medfly CcP0 seems to be the only P0 protein of higher eukaryotic organisms with basic character (pI 8.5), as shown by electrofocusing of purified ribosomes.

The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae.

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1alpha/YP2betapair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1alphaand YP2alpha proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1alpha/betadefective strain, where a helping effect on the binding of the YP2alphaand YP2betaproteins on the ribo-somes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.

Isolation and expression of the genes encoding the acidic ribosomal phosphoproteins P1 and P2 of the medfly Ceratitis capitata.

The genes of the acidic ribosomal proteins P1 and P2 (CcP1 and CcP2) of the medfly Ceratitis capitata were isolated from a genomic library using homologue DNA probes prepared by PCR. Sequencing and characterization of the two genes revealed strong similarities of the encoded amino acid sequence to the homologous proteins of Drosophila melanogaster and other eukaryotic species. The predicted amino acid sequences of the CcP1 and CcP2 proteins shared an almost identical carboxyl terminal sequence of 10 amino acids common to most known acidic ribosomal proteins. The CcP2 gene lacked intervening sequences in contrast to the CcP1 gene, which was interrupted by an intron of 188 nucleotides. Both genes were cloned in expression pT7 vectors and were expressed in Esherichia coli. The 17- and 15-kDa recombinant proteins reacted with a monoclonal antibody specific to the highly conserved carboxyl terminus of eukaryotic acidic ribosomal proteins, confirming their equivalence to these ribosomal components. Both recombinant proteins were electrophoretically identical to acidic proteins extracted from purified ribosomes of C. capitata.

Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata.

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0-2 days old embryos. The two protein groups correspond to multi-phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.

Ribosome-associated ribonucleases from six-day larvae of the insect Ceratitis capitata.

A 34 kDa, poly(U) and poly(C)-specific ribonuclease, is shown to be tightly bound on purified polysomes from six-day-old larvae of the insect Ceratitis capitata. High salt treatment (400 mM KCl) is necessary to release it completely from the polysomes. Removal of the RNase does not disrupt the structure of the ribosomes, as shown by centrifugation on sucrose gradients and poly U directed polyphenylalanine synthesis.

A temperature-sensitive mutant affecting the process of chorion gene amplification in Drosophila melanogaster.

K575 is a temperature-sensitive female sterile mutant which shows abnormal chorion structure and subnormal amounts of the major chorion proteins at the restrictive temperature. These phenotypes apparently result from a temperature-sensitive defect in amplification. Both clusters of chorion genes are affected, indicating that the gene operates in trans.

Comparison of native and KCl treated 40S ribosomal subunits from the silkmoth Antherea pernyi and mammals.

Native 40S ribosomal subunits and 18S ribosomal RNA from ovarian follicles of the silkmoth A. pernyi showed a lower sedimentation coefficient in comparison to ascites cells, in contrast to the KCl treated 40S ribosomal subunits where no difference was observed in both tissues. Moreover the silkmoth native 40S ribosomal subunits--in contrast to the KCl treated ones--could not reassociate with radioactive ascites cell 60S ribosomal subunits. These results, combined with the great similarities in the two dimensional electrophoretic patterns of 40S ribosomal proteins from silkmoth follicles and other mammalian cells lead to the possibility of the existence of a specific RNase associated with the 40S ribosomal subunit.

Developmental and evolutionary comparisons of proteins from purified ribosomal subunits in two silkmoths.

Developmental comparisons of silkmoth ribosomal proteins were performed by parallel two-dimensional electrophoretic analyses of proteins derived from developing adult wings and from ovarian follicles at the prechoriogenic and choriogenic stages of development (before and during secretion of the chorion, respectively). Proteins of 40S and 60S subunits were prepared and analyzed separately. A single major developmental difference was observed, exclusively in choriogenic follicles: the majority of a 40S subunit protein, S6, was shifted to a more acidic form, possibly as a result of phosphorylation. A less prominent change was apparently due to quantitative variation between two forms of one large subunit protein. The developmental comparisons were performed in two species of the genus Antheraea, with consistent results. Comparisons between the two species revealed remarkable evolutionary conservation of the ribosomal protein patterns, sharply contrasting with the evolutionary diversification of chorion structural proteins in the same two species. The only detectable interspecies differences in ribosomal components were (a) slightly more acidic behaviour of one small-subunit protein and more basic behaviour of one large-subunit protein in Antheraea polyphemus as compared to Antheraea pernyi, and (b) presence of an apparently species-specific 60 S subunit protein in A. pernyi, which in A. polyphemus probably co-migrates with a neighbouring protein.