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).

Cloning and sequencing for the largest subunit of Chinese hamster RNA polymerase II gene: identification of a mutation related to abnormal induction of sister chromatid exchanges.

In order to analyze the mutation sites related to abnormal induction of sister chromatid exchanges (SCEs) in the RNA polymerase II largest subunit (RpII LS) gene of the Chinese hamster CHO-KI cell mutant, we have completely sequenced the whole region of the RpII LS cDNAs obtained from normal and mutant cells. By comparing both sequences, a mutation that results in an amino acid (aa) change in the RpII LS gene was found. This aa change was Pro (CCC) to Ser (TCC) at position 1006. Multiple alignment for aa sequences of RpII LS from various species revealed that this Pro residue was highly conserved throughout the eukaryotes. Considering the differences in physico-chemical properties between Pro and Ser residues, the Pro-->Ser substitution may alter the RpII LS structure.

Gene organization of human NOTCH4 and (CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.

The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.

Cloning and characterization of novel gene, DCRR1, expressed from Down's syndrome critical region of human chromosome 21q22.2.

The new gene, DCRR1, from the proximal part of the Down's syndrome critical region (DCR) was identified by the GRAIL analysis of the 97-kb nucleotide sequence of two P1 DNAs and the cDNA for DCRR1 gene was cloned. A 7.36-kb cDNA encodes the imcompleted open reading frame composed of 1941 amino acid residues (220.2 kDa). The deduced amino acid sequence contains the conserved domain for protein phosphatases at the N-terminus. The domain encoding the rod-like tail of a myosin heavy chain was also found near the C-terminal region besides the signature for an actin binding protein, profilin, suggesting its possible role as a microtuble-associated protein. Two different sizes (7.9 and 9.0 kb) of mRNAs were detected in the poly(A)+ RNA from abundant tissues by the Northern analysis. The smaller transcript was only transcribed at a high level in the testis. The imbalance of the DCRR1 gene dosage may contibute to the pathogenesis of Down's syndrome.