A pentadecanucleotide sequence, TTTCAACAAATAAGT, contiguous with the 5′‐end of Saccharomyces cerevisiae tRNA‐Leu3 coding sequence acts as a positive modulator of transcription in a homologous in vitro system. To determine whether modulation also takes place in vivo, the amber suppressor forms of tRNA‐Leu3 genes with different 5′‐flanking sequences were generated by site‐specific mutagenesis and cloned into YCp19, a yeast vector maintained at 1‐2 copies per cell. These plasmids were transformed into S. cerevisiae strains marked with amber mutations lys2‐801, met8‐1, and tyr7‐1. The ability of the tRNA‐Leu3 amber suppressor genes (tDNA‐Leu3A) to suppress functionally lys2‐801 and tyr7‐1 mutations in the yeast host strain correlated well with template activities measured in vitro. We conclude that the plasmid‐borne tRNA gene acts as an effective suppressor from the plasmid and the conserved pentadecanucleotide sequence modulated the expression of yeast tRNA‐Leu3 in vivo as well as in vitro. This regulatory sequence if found associated with genes coding for a number of tRNAs which are abundant in yeast. We postulate that this sequence represents a mechanism by which production of specific tRNAs can be enhanced to match demand created by codon use preferences.

A 3778-bp DNA sequence of the insecticidal protoxin gene coding sequence and flanking regions from Bacillus thuringiensis subspecies berliner 1715 has been determined. The protoxin is composed of 1155 amino acids, deduced from the nucleotide sequence, and has a calculated molecular mass of 130,615 daltons. To determine the DNA portion that encodes toxicity, sequential deletions were constructed from the 3' end of the coding region using nuclease Bal-31. Using these mutants in an insect bioassay, we found that an amino-terminal 612-amino-acid peptide is toxic, whereas, a 603-amino-acid peptide is not toxic to insects. Ninety percent of the amino acid residues were homologous to the protoxins from closely related subspecies kurstaki HD-1-Dipel and sotto. The differences occurred both in the amino-terminal half, or toxic portion, and in the carboxy-terminal half. These differences were clustered in several regions. From comparative analysis of subspecies berliner and kurstaki, we propose a model whereby the protoxin molecule is divided into distinct structural and functional domains. These domains may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.

Bacillus thuringiensis subspecies kurstaki (Btk) and subspecies berliner (Btb) both produce lepidopteran-specific larvicidal protoxins with different activities against the same insect species. Toxic activity resides in the amino-terminal half of both protoxins, whereas the carboxy-terminal half of the molecules is not required for toxicity. The protoxins are 90% homologous, with a major cluster of differences in the amino-terminal half, and a 26 consecutive amino-acid insertion within the carboxy-terminal half of the Btk protoxin. Protoxin chimeras composed of the amino-terminal half of one subspecies and the carboxy-terminal half of the other were generated. Wild-type and chimeric protoxins were compared in bioassays against tobacco hornworm larvae. The amino-terminal half, the toxin itself, dictates specific larvicidal activity.

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