Contrary to prior indications, the glycosidic sweeteners stevioside and rebaudioside A are degraded to the diterpenoid aglycone steviol by rat intestinal microflora in vitro. Additional studies with steviol-17-[14C] show almost total absorption from the rat lower bowel following intracecal administration.
We have isolated a cDNA clone encoding a precursor form of the diuretic hormone from the tobacco hornworm Manduca sexta (Mas-DH). Translation of the cDNA revealed a 138-amino acid precursor consisting of the Mas-DH amino acid sequence bounded by dibasic amino acid processing sites, a putative signal sequence, and additional peptide sequence on either side of the Mas-DH coding sequence. The region of the precursor upstream of the mature Mas-DH sequence shows limited (28%) homology to the cryptic region of the ovine corticotropin-releasing factor precursor. The Mas-DH RNA is 1.5-1.6 kilobases long; it is present in both the heads and bodies of adult and larval insects. In prewandering fifth stadium larvae, Mas-DH mRNA is expressed in brain, nerve cord, gut, and Malpighian tubules, but not in the fat body. There is a single genomic copy of the Mas-DH gene; the message is multiply spliced.Manduca sexta diuretic hormone (Mas-DH) is an amidated 41-amino acid peptide that was isolated from pharate adult tobacco hornworm heads on the basis of induction of fluid loss in beheaded adult cabbageworms (1). This peptide shows homology to a family of vertebrate neuropeptides, including sauvagine, corticotropin-releasing factor, and urotensin I (1). Since the isolation of Mas-DH, other peptides of related structure, which function in regulating fluid balance in insects, have been isolated from Manduca (2) and from two orthopteran genera: Locusta (3, 4) and Acheta (5). Mas-DH itself has been shown to induce water loss in both lepidopteran (1, 6) and orthopteran (7) species. The structural and biological properties of these related insect diuretic hormones have been reviewed recently (8).Due to the importance of Mas-DH and to the possibility of improving the utility for insect control of recombinant baculoviruses expressing this insect hormone (9), we have determined a precursor sequence for Mas-DH after isolation of a cDNA clone for the peptide.t In addition to the coding sequence for the mature diuretic hormone and a signal sequence, the precursor peptide encodes additional peptide sequences on either side of the coding region and is bounded by processing sites similar to processing sites extensively characterized in mammalian systems (10). Mas-DH is expressed in both heads and bodies of pharate adult and prewandering fifth stadium larvae; in these larval bodies, Mas-DH mRNA is observed in nerve cord, gut, and Malpighian tubules. The gene for the hormone appears to be present as a single copy in the M. sexta genome; multiple splicing events are required for mRNA assembly.
MATERIALS AND METHODSDNA Synthesis and Sequencing. Degenerate oligonucleotides were purchased from Clontech. Nondegenerate oligonucleotides were synthesized by using an Applied Biosystems model 380A DNA synthesizer. Plasmid DNA was sequenced using the chain-termination method (11) and the Sequenase kit (United States Biochemical).Reverse Transcriptase-PCR. Two sets of degenerate oligonucleotides were used to reverse transcribe RNA for later amplificati...
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