A chicken c-Rel-estrogen receptor chimeric protein shows conditional nuclear localization, DNA binding, transformation and transcriptional activation.

In this report, we characterize the biological and biochemical properties of a conditional protein containing chicken c-Rel fused to the hormone-binding domain of the human estrogen receptor. This chimeric c-RelER protein causes estrogen-dependent, but otherwise c-Rel-specific, transformation of avian fibroblasts in vitro. Our results demonstrate that c-RelER heterodimerizes with wild-type c-Rel and forms specific complexes with IkappaB-alpha. Estrogen causes translocation of c-RelER to the nucleus and stabilizes its binding to DNA. Hormone-activated c-RelER induces transcription of at least four cellular genes that are constitutively active in wild-type c-Rel-transformed fibroblasts. Two distinct cell populations were examined that differed with respect to their growth phenotypes. The growth of fibroblasts with moderate expression levels of c-RelER was stimulated by estrogen. In contrast, the addition of estrogen to cells with high cRelER expression levels resulted in inhibition of cytokinesis and the arrest of growth. The carboxy terminal transactivation domain of c-Rel was required for the induction of these effects since neither v-Rel nor c-Rel deletion mutants were able to induce similar changes. Taken together, our results demonstrate that high levels of c-Rel expression can affect cell cycle control and/or cytokinesis. Furthermore, they also indicate that the biological properties of c-Rel in cell growth and differentiation will potentially differ depending on the level of expression.

Characterization of the P25 silk gene and associated insertion elements in Galleria mellonella.

Insect silk genes attract attention by their precise territorial and developmental regulations and extremely high expression rates. Our present investigations demonstrated that the P25 silk gene of Galleria mellonella is down-regulated by ecdysteroid hormones. The gene was identified within 5217 nucleotides (nt) of two genomic clones. In contrast to other silk genes, Galleria P25 lacks the canonical TATA box. Transcription is initiated within a region of three nucleotides that lie at the end of a capsite initiator sequence ACAGT and about 90 nt downstream from a CAAT box. A stretch of 32 nt with a core sequence CTTTT was detected in the 5' region of Galleria P25 as well as in the presumptive regulatory regions of all other silk genes that are expressed in the posterior silk gland. However, consensus sequences reported for the regulatory regions of Bombyx silk genes are not obvious in Galleria P25. The coding sequence of this gene included 654 nt, is interrupted by 4 introns, and ends in position +3369; a potential polyadenylation signal starts at +4382. The gene contains 3 copies of a short interspersed nuclear element (SINE), which are located in the upstream region (-833 to -579) and in the first (+542 to +840) and second (+2259 to +2556) introns. The repeat, which was named Gm1, occurs in some other Galleria genes and exhibits homology to Bm1 SINE of the silkworm and to a similar element of a spider. Another insertion of at least 150 nt and with loosely defined borders is present in the 3' untranslated region (UTR) of Galleria P25. It includes a box (+3453 to +3552) of 99 nt that is tentatively called Lep1 because it was disclosed also in some other Lepidoptera. Lep1 seems to represent the core region of insertion elements that occur in the genomes of lepidopteran insects in various species specific and region specific modifications.

Use of two transcription starts in the G6PD gene of the bark beetle Ips typographus.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived from a gene that includes eight exons and spans over 7100 nucleotides (nt). By means of two transcription starts, the gene generates two mRNA isoforms that are present in similar amounts in the larvae, pupae and adults. The A isoform includes exon IA of 115 nt, which is followed by intron 1a extending to position 3457 of the gene. The B mRNA isoform begins with exon IB (100 nt) that occupies positions 3291-3390 within the 1a intron. Exons II to VII are included in both mRNA isoforms. The gene contains 31.6% (36.5% in the translated region) of the GC nucleotides. Two transcription starts and the exon/intron organization distinguish bark beetle G6PD from the homologous genes known in other insects. Two enzyme variants were detected in the protein extracts of individual bark beetles but their relationship to the A and B mRNA isoforms is uncertain.

Molecular phylogeny of Calyptratae (Diptera: Brachycera): the evolution of 18S and 16S ribosomal rDNAs in higher dipterans and their use in phylogenetic inference.

Sequences for nearly complete 18S rRNA and partial 16S rRNA genes were determined for sixteen species representing twelve calyptrate families. Two unique insertions are present in expansion regions of the 18S rDNA in nycteribiids. Alignments containing other dipteran rRNA genes provided good resolution at higher taxonomic level: monophyly of Calyptratae is well supported. While both 16S and 18S rDNA matrices produce unstable topologies within Calyptratae when analysed separately, their combination results in a tree with several robust and well supported nodes. Of three superfamilies recognized in recent classifications, the Hippoboscoidea is well supported by 16S rDNA and by combined matrices. The representatives of Muscoidea, Musca sp. and Antipoda sp., display a tendency to cluster within Oestroidea. The comparison of secondary structures of two variable regions indicates that Sarcophagidae are related to Calliphoridae rather than to Tachinidae.

Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor.

Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteins, and contains a few small peptides of hitherto unknown functions. We demonstrate that two of these peptides inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These 'silk proteinase inhibitors' 1 and 2 (SPI 1 and 2) are produced in the middle section of the silk-secreting glands prior to cocoon spinning and their production is controlled at transcription level. The full length cDNA of pre-SPI 1 contains 443 nucleotides and encodes a peptide of 76 amino-acid residues, of which 20 make up a signal sequence. The mature SPI 1 (6056.7 Da, 56 residues) is a typical thermostable Kunitz-type proteinase inhibitor with Arg in P1 position. The cDNA of pre-SPI 2 consists of 260 nucleotides and yields a putative secretory peptide of 58 amino-acid residues. The functional SPI 2 (3993 Da, 36 residues) is a single-domain Kazal-type proteinase inhibitor with unique structural features: free segment of the N-terminus is reduced to a single amino-acid residue, lack of CysI and CysV precludes formation of the A-ring and provides increased flexibility to the C-ring, and absence of several residues around the normal position of CysV shortens and changes the alpha helix segment of the protein. The structure reveals that the length and arrangement of the B-ring, including exposure of the P1 residue, and the position of the C-terminus relative to the B-loop, are essential for the activity of the Kazal-type inhibitors.

Identification of a novel type of silk protein and regulation of its expression.

The silk of lepidopteran insects has been studied extensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the middle section. We now describe a third category that is named seroins to accentuate the fact that both the sericin- and the fibroin-producing cells participate in seroin secretion. Using a probe derived from the N-terminal sequences of a 23-kDa components of Galleria mellonella silk, we isolated silk gland-specific cDNA encoding 167 amino acids, of which 17 constitute the signal peptide. The following 14 residues match the N-terminal sequences of the 23- and 22.5-kDa silk proteines. The reaction of these proteins with concanavalin A and the presence of two glycosylation sites in the seroin peptide sequence indicate that seroin is secreted in two forms that both contain a mannose-rich sugar moiety. Seroin is distinguished from other silk proteins by high proline content (34 residues or 20.26% by weight), lack of cysteines, and the presence of two kinds of short amino acid repeats. The seroin gene is expressed in both the posterior and middle silk gland sections. The expression fluctuates during development in correlation with the feeding regime and the changes in hormone titers: seroin mRNA is high in the silk glands of feeding larvae, declines at ecdysis, reaches a maximum during cocoon spinning, and thereafter rapidly drops to an undetectable level. In vivo and in vitro experiments showed that the drop is caused by ecdysteroid hormones and is prevented by juvenile hormones. N-terminal sequencing of several silk proteins of Bombyx mori revealed that the 8- and 13-kDa proteins share 5 or 6 out of 10 identified amino acids with the N terminus of Galleria seroin and obviously represent seroin homologues. The result suggests that seroin-type proteins are a general component of lepidopteran silk.

Identification of four small molecular mass proteins in the silk of Bombyx mori.

This paper describes cDNAs of four small-size proteins that occur in the cocoon silk of Bombyxmori. Two of them (9.9 and 10.3 kDa), which have the N-terminal sequences and the spacing of a few amino acids at C-termini similar to the seroin of Galleria mellonella, are called seroin 1 and seroin 2. The corresponding genes are expressed in the middle, and to a small extent also in the posterior silk gland sections. The seroin 1 and less conspicuously the seroin 2 mRNAs accumulate in the course of the last larval instar to a maximum in postspinning larvae. Two other proteins (6 kDa and 4.7 kDa) of B. mori cocoons were identified as a typical Kunitz-type and a somewhat unusual Kazal-type proteinase inhibitors, and named BmSPI 1 and BmSPI 2, respectively. Their genes are expressed in the middle, and the BmSPI 1 gene slightly also in the posterior silk gland sections. The expression ensues a few days after the last larval ecdysis and increases until the cocoon spinning. Post-spinning larvae still contain high amounts of the BmSPI 1 but no BmSPI 2 transcripts. It is assumed that seroins and proteinase inhibitors are involved in cocoon protection against predators and microbes.

Light-chain fibroin of Galleria mellonella L.

The posterior section of Galleria mellonella silk glands contains two abundant mRNAs that are identical except for the non-coding tail, which includes either two (1.1 kb mRNA) or three (1.2 kb mRNA) consensus sequences for polyadenylation sites. The transcripts are 40% homologous in the coding as well as non-coding regions with the mRNA encoding light-chain fibroin (L-fibroin) in Bombyx mori; the deduced translation product shows 43% identity with the Bombyx L-fibroin peptide, with all three cysteines conserved. Amino acid analysis of the N-termini of Galleria silk proteins revealed that L-fibroin (25 kDa) occurs in two isoforms, the shorter one lacking the Ala-Pro dipeptide residue at its N-terminus. The 29 and 30 kDa Galleria silk proteins appear to be homologs of Bombyx silk component P25. The results suggest that evolutionary diversification of Galleria and Bombyx L-fibroins involves alternative polyadenylation and proteolytic processing sites.

The P25 component of Galleria silk.

The water-insoluble core of lepidopteran silk is composed of four major proteins, but only three genes have been identified. This study demonstrates that the 29- and 30-kDa components of Galleria mellonella silk are derived from a single gene designated P25. The gene is expressed exclusively in the posterior section of the silk glands as a 2-kb mRNA, which accumulates in the feeding larvae and declines at molting. The mRNA encodes a peptide of 24,864 Da that exhibits 51% identity with the putative product of the P25 gene of Bombyx. The conservation of several amino acid stretches, including the relative positions of all 8 cysteines in the mature polypeptide, implies that the P25 proteins play similar, and apparently significant roles in silk formation in the two species. A Galleria P25 cDNA yields a peptide of about 25 kDa when translated in vitro; the 29- and 30-kDa forms present in the silk are derived from this primary translation product by differential glycosylation.