Coupling caspase cleavage and ubiquitin-proteasome-dependent degradation of SSRP1 during apoptosis.

Structure-specific recognition protein (SSRP1) is an 87 kDa protein that heterodimerizes with Spt16 to form FACT, a complex initially shown to facilitate chromatin transcription. Despite its crucial roles in transcription and replication, little is known about the dynamics of FACT turnover in vivo. Here, we show that SSRP1 is cleaved during apoptosis by caspase 3 and/or 7 at the DQHD(450) site. Analysis of the resulting fragments suggests that cleavage of SSRP1 generates a truncated, chromatin-associated form of FACT. Furthermore, the N-terminal product is stabilized by proteasome inhibitors and ubiquitylated in cells, suggesting degradation through the ubiquitin-proteasome pathway. These results demonstrate that SSRP1 degradation during apoptosis is a two-step process coupling caspase cleavage and ubiquitin-dependent proteolysis.

Annotation pattern of ESTs from Spodoptera frugiperda Sf9 cells and analysis of the ribosomal protein genes reveal insect-specific features and unexpectedly low codon usage bias.

MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda.

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

Characterization of a highly conserved satellite DNA from the parasitoid wasp Trichogramma brassicae.

An EcoRI satellite DNA has been isolated, cloned and sequenced from Trichogramma brassicae, a minute parasitic wasp. This repeated family represents 16% of the genome. The monomer is 385 base pairs (bp) long and has an A+T content of 64.5%. The average nucleotide sequence variability among 12 randomly chosen monomers is extremely low (0.5%), suggesting that the amplification of the monomer into a high-copy-number family occurred recently. An EcoRI satellite DNA probe has been developed and used, at high stringency, as an identification tool to unambiguously discriminate T. brassicae from nine other Trichogramma species. However, at a lower stringency, a hybridization signal can be detected in two closely related Trichogramma species, and, using PCR assay, the presence of the T. brassicae EcoRI monomer has been detected in several other species of Trichogramma. These results argue in favor of the 'library' model of satellite DNA evolution that predicts that related species share a number of low-copy satellite sequences, some of which could be amplified into a major satellite family in each of the species. Furthermore, this T. brassicae EcoRI satellite DNA sequence exhibits particular internal features such as a long inverted repeat that can form a dyad structure. Such sequence motifs seem to be a common characteristic of satellite DNAs, suggesting that they could result from selective forces acting on repetitive DNA.