The high degree of conservation of the core sequence of the "cockroach-types" of AST and their widespread distribution suggest that they should be considered a ubiquitous family of peptides within the invertebrates, regulating a range of important physiological processes. These functional processes, by either neural or humoral routes of action, include the inhibition of endocrine function, interneuronal functions, neuromodulatory roles, myotropic and myoendocrine roles, and direct action on biosynthetic pathways. The myomodulatory function appears to be conserved through evolutionary time, whereas the JH inhibitory activity appears to be confined to specific orders. This suggests that the myomodulatory role was the more ancestral of these two particular functions. Certainly, further purification and gene cloning as a means to precursor identification and functional analysis will be a prerequisite to understanding the diverse functions of this peptide family.

Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diuloptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-propeptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentiafly related peptides that may also be processed. Southem blot analysis revealed the presence of a single copy of the

Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.

Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA. The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured. With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition. The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence. The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage. The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state. Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence induding an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Allatostatins are a family of insect neuropeptides that inhibit -juvenile hormone binsynthesis by the corpora allata. We have characterized cDNA and gcnomic DNA sequences that specify a preproallatostatin precursor in the oviparous cockroach Peripluriefa an~rricarin. Comparison of this prccursor with that previously described [Donly, B. C., Ding, Q., Tobe, S. S. &L Bcndena, W. G. (1993) Proc,. Nut/ Accrd. Sci.USA 90, 8807-88 1 1 ] for the viviparous cockroach Diploptei-ci pzi~ic'tutii revealed several cc)tnnion fcalures. First, thc precursors are remarkably similar in size and the organization of thc peptides within the precursor is conserved. The separation of the peptides into groups by acidic domains within the precursor has been maintained. The F nmericcinu precursor contains 14 allatostatin-like peptideb thal contain the core C-terminal sequence (Tyr/Phe)-Xaa-Phe-Gly-(l,eu/llc)-NH,, as compared to the I). piirictutu prccursor, which contains 13. Five of the peptides are perfectly conscrvcd between the two species. The rcmiiinder, with one exception, contain amino acid suhstitutions in the N-terminal address porlion of thc pcptide.Several features of expression are also similar between these two species. In both, u singlc copy gene specifies a large allatostatin transcript of 5.0 kb in I? unit>ric:cuici and 9.2 kb in D. putrrldu. I n F! w w r icum, allatostatin transcripts appear to be produced by numerous cells in different IXgiOllS of the brain.Kqywnrds: allatostatin ; Diploptera; Periplanete; juvenile horrnone; inscct.Rcproduction and inctmiorphosis in most insect species arc controlled by quantitative changes in the biosynthesis and secrction of the sesquitcrpenoid juvenile hormone from the corpora allata 11, 21. The rate of juvenile hormone biosynthesis in differcnt insect species appears to he conlrollcd by stimulatory and inhibitory factors (allatotropins and allatostatins, respectively), which for the most part originate in the brain and are transportcd via nerves to the corpora allata [ 2 ] . The presence of inhibitory molecules originating from the brain was first demonstrated using the cockroach as a modcl system. Transection of the braincorpora rtllata connective nervi corporis allati in several cockroach species resulted in stimulation of the rates of juvenile hormone biosynthesis [ 31. Recent experiments in a cockroach system 141 also suggest that ovarian factors may act on the brain to stimulate juvenile hormone biosynthesis by the corpora allata either by activating release of an allatotropic factor or by limiting the releasc of allatostatins.The only allatotropin characterized to date is an u-amidiited 13-ami no-acid neuropeptidc isolated from brain of the moth IS, 161.Cockroaches display several rcproducti ve inodcs that range frnrn oviparity to ovoviviparity to viviparity 117, 1 XI. The more pi-imitive [ 191 oviparous species, I-! n~rtwricwtcc, has ovcrlapping and continuous ovarian cycles that appear to br accompanied by on/off switching of juvenile hornione biosynthesis by cells of the...

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