UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coil genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNA"rP gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.We recently have sequenced a part of the Mycoplasma capricolum ribosomal-protein gene cluster that codes for polypeptides highly homologous to the Escherichia coli ribosomal proteins S3 and L16. The sequence contains four UGA codons in the reading frames; three appear at the sites corresponding to tryptophan, and one, at a site corresponding to arginine in the E. coli proteins. No "universal" UGG codon for tryptophan has so far been found. We have also isolated a clone containing a pair of M. capricolum tRNA genes, the sequence of both of which resembles that of tRNATrp of E. coli. The anticodon sequence of one of these tRNA genes is 5'-T-C-A-3', which can base-pair with both opal codon UGA and universal tryptophan codon UGG. That of the other is 5'-C-C-A-3', which may base-pair exclusively with UGG. These two tRNA genes are expressed in the cell. All these findings suggest strongly that, in M. capricolum, UGA codes for tryptophan using the opal tRNAUCA but not tRNACCA.RESULTS AND DISCUSSION UGA Codons in M. capricolum S3 and L16 Genes. As reported in a previous paper (1), we isolated the recombinant plasmid pMCB1088 containing a 9-kilobase-pair fragment of M. capricolum DNA. The fragment contains the genes for at least nine ribosomal proteins-S3, S5, S8, S14, S17, L5, L6, L16, and L18-as deduced from its encoded protein sequences being highly homologous with the corresponding E. coli ribosomal protein sequences (refs. 1 and 2; unpublished results). Fig. 1 shows the complete nucleotide sequence of a 629-base-pair (bp) HindIII fragment which is a part of the insert of pMCB1088 (see refs. 1 and 2). The DNA corresponds to the 3' half of the S3 gene and about 90% of the L16 gene from the 5' terminus. When the M. capricolum sequences are aligned with the E. coli protein sequences (3, 4) ( Fig. 1), four UGA (opal) codons are found within the reading frames. The possibility that these UGA codons are termination signals can be excluded by their occurrence in the regions having extensive sequence homologies with the E. coli proteins. More importantly, three out of the four UGA codons appear at the positions corresponding to tryptophan in the E. coli proteins. This suggests that UGA is a sense codon, probably for tryptophan, in M. capricolum. No UGG codon for tryptop...

Prothoracicotropic hormone (PTTH), a brain secretory polypeptide of insects, stimulates the prothoracic glands to produce and release ecdysone, the steroid essential to insect development. The complementary DNAs encoding PTTH of the silkmoth Bombyx mori were cloned and characterized, and the complete amino acid sequence was deduced. The data indicated that PTTH is first synthesized as a 224-amino acid polypeptide precursor containing three proteolytic cleavage signals. The carboxyl-terminal component (109 amino acids) that follows the last cleavage signal represents one PTTH subunit. Two PTTH subunits are linked together by disulfide bonds, before or after cleavage from prepro-PTTH, to form a homodimeric PTTH. When introduced into Escherichia coli cells, the complementary DNA directed the expression of an active substance that was functionally indistinguishable from natural PTTH. In situ hybridization showed the localization of the prepro-PTTH mRNA to two dorsolateral neurosecretory cells of the Bombyx brain.

Silk gland is a larval specific tissue of lepidopteran insects and begins to degenerate shortly before pupation. Programmed cell death (PCD) of the anterior silk gland of Bombyx mori last instar larvae was studied in vivo and in vitro, focusing on the effects of 20- hydroxyecdysone (20E). The glands began to exhibit signs of PCD in vivo 2 days after gut purge and completed PCD by 48 h. In vitro, 20E prematurely induced PCD, and its completion took 144 h (6 days). An oligo-nucleosomal ladder pattern was observed in DNA extracted at the end of PCD. Caspase 3 inhibitor inhibited attainment of full PCD, but it did not block chromatin condensation as revealed by acridine orange staining. alpha-Amanitin inhibited the PCD induced by 20E in vitro if added to the culture in the first 8 h. Similarly, cycloheximide and emetine completely blocked PCD when applied in the first 18 h of culture with 20E. These results indicate that 20E-stimulated transcription and protein synthesis for PCD are completed in 8 h and 18 h, respectively. Nevertheless, withdrawal of 20E from the medium at different times showed that 20E must be present in vitro for 42 h to elicit full PCD. Current results indicate that the effects of 20E on the progression of PCD are mediated by two distinct processes - one through nuclear hormone receptors, and the other independent from de novo gene expression.

Many insects exhibit stereotypic instinctive behavior [1-3], but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates [4, 5]. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. Here, we identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, we mapped comprehensive activity patterns of the silkmoth brain in response to female sex pheromones. We found that Hr38 can also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, we constructed a neural activity map of the fly brain that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies.

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ABSTRACT-Bombyxin is a 5 kDa secretory brain peptide that belongs to the insulin family. Bombyxin of the silkmoth Bombyx mori can induce adult development when injected into brain-removed dormant pupae of the saturniid moth Samia cynthia ricini by activating the prothoracic glands to synthesize and release ecdysone. Bombyx bombyxin has been shown to lower the concentration of the major haemolymph sugar, trehalose, and to elevate the trehalase activity in the midgut and muscles in Bombyx, but the doses required to be effective are higher than the amounts in the feeding larvae. The exact physiological function of bombyxin in Bombyx itself is therefore still obscure, but its insulin-like structure suggests it has important roles. Bombyxin comprises a mixture of highly heterogeneous molecular forms whose amino acid sequences have 40% identity with human insulin. The Bombyx bombyxin gene encodes a precursor consisting of the signal peptide, B chain, C peptide, and A chain, in that order from the N terminus. So far, 32 bombyxin genes have been identified in Bombyx, and they are classified into 7 families, A to G, according to their sequence similarity. The bombyxin genes have no introns and cluster in unique distribution patterns. The gene arrangement in the cluster has been classified into three categories: gene pairs, gene triplets, and single genes. Nucleotide sequence analysis indicates that equal and unequal crossings-over and duplications may have generated these unique distribution patterns. The Bombyx bombyxin genes are expressed predominantly in the brain and at low levels in a number of other tissues. Genes of all 7 families are expressed in four pairs of the medial neurosecretory cells of the brain. Detailed examination indicated that only a limited number of genes in the A, B and C family members are expressed and that their expression shows a gene-arrangement-dependent pattern.

Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20-hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR-A and usp-2 , but not EcR-B1 or usp-1 , may be components of the ecdysone receptor complex. Up-regulation of E75A, BHR3 , and three BR-C isoforms, but not E75B , appeared to be associated with the induction of PCD. β β β β FTZ-F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.