Structure and regulation of expression of the lysozyme gene from the tobacco hornworm, Manduca sexta

Amy Beth Mulnix, Purdue University

Abstract

A cDNA library was constructed from poly A+ RNA isolated from the fat body of induced M. sexta larvae. Screening of the library with lysozyme-specific nucleotide probes identified a cDNA encoding lysozyme. The nucleotide sequence of the cDNA was determined. The cDNA was 840 bp in length, encoding a putative leader sequence and the secreted protein. A non-translated region 425 bp in length was present at the 3 terminus. The lysozyme cDNA was used to investigate the expression of the lysozyme gene. The fat body contained the highest steady-state levels of lysozyme RNA in the naive insect. All tissues examined had accumulated lysozyme RNA 24 hr after injection with peptidoglycan fragments. The kinetics of induction of lysozyme RNA were further examined in both the total and poly A+ RNA pools isolated from fat body. Increases in the relative abundances of lysozyme RNA were evident 2 hr after treatment with peptidoglycan, peaked at $\sim$12 hr after treatment and remained elevated for at least an additional 36 hr. These results are consistent with the hypothesis that expression of the lysozyme gene following treatment with peptidoglycan fragments is regulated at the level of transcription. A genomic fragment containing the lysozyme gene was isolated and the nucleotide sequence of the gene determined. The coding sequence for lysozyme is contained on 3 exons. The transcription start site was determined to be 38 nucleotides upstream of the translation start site. The copy number of the gene could not be determined, however the data suggest that the gene is present at a low frequency. Allelic variation of the locus containing the gene was identified in our laboratory population of insects.

Degree

Ph.D.

Advisors

Dunn, Purdue University.

Subject Area

Entomology|Molecular biology

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