|Zhu, Yu Cheng|
Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2003
Publication Date: 3/15/2004
Citation: ARAKANE, Y., HOGENKAMP, D., ZHU, Y., KRAMER, K.J., SPECHT, C.A., BEEMAN, R.W., KANOST, M.R., MUTHUKRISHNAN, S. 2004. CHARACTERIZATION OF TWO CHITIN SYNTHASE GENES OF THE RED FLOUR BEETLE, TRIBOLIUM CASTANEUM, AND ALTERNATE EXON USAGE IN ONE OF THE GENES DURING DEVELOPMENT. Journal Of Insect Biochemistry And Molecular Biology 34: 291-304. Interpretive Summary: Synthesis of chitin is a critical and precisely regulated step during insect molting, but very little is known about the enzymes that control this process, chitin synthases. Any perturbation or disruption of the delicate process of re-growing a new cuticle will lead to insect death. We cloned and characterized two chitin synthase genes from the red flour beetle in order to better understand how insects choreograph the shedding of the old skin and the re-growing of the new. Studies such as these will lead to better understanding of insect growth and development and better strategies for disrupting the associated genes for pest control.
Technical Abstract: Two chitin synthase (CHS) genes of the red flour beetle, Tribolium castaneum, were sequenced and their expression patterns during development analyzed. Amino acid sequences within the CHS catalytic domain were highly similar to CHS genes from other insects, nematodes and fungi. The DNA sequences of both genes, TcCHS1 and TcCHS2, were determined by amplification of overlapping PCR fragments from two BAC DNAs. The TcCHS genes were mapped to different linkage groups in Tribolium. Each ORF was identified and full-length cDNAs were amplified, cloned and sequenced. The longest ORFs found in the cDNAs were 4,674 bp in TcCHS1 and 4,392 bp in TcCHS2, which code for transmembrane proteins of 1,558 and 1,464 amino acids, respectively. The organizations of the two TcCHS genes are different. The most important difference between the two TcCHS genes is the presence of alternate exons encoding 59 amino acids in TcCHS1, but absent in TcCHS2. Alternate exons encoding the 59-amino acid long region were also conserved in CHS genes from several other insect species including Manduca sexta (tobacco hornworm), Drosophila melanogaster (fruit fly), and Anopheles gambiae (malaria mosquito). Analysis of the expression of the two TcCHS genes using RNA prepared from several different developmental stages of Tribolium revealed differences in their patterns of expression. TcCHS1 is expressed predominantly in the embryonic and pupal stages, whereas TcCHS2 is prevalent in the late larval and adult stages. Alternate exon 8b of TcCHS1 is utilized over a much narrower range of development than exon 8a. We propose that the isoforms of the TcCHS1 enzyme are used predominantly for the formation of chitin in embryonic and pupal cuticles, whereas TcCHS2 is utilized primarily for the synthesis of peritrophic matrix-associated chitin in the midgut.