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Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti
Author(s) -
Lowenberger C. A.,
Smartt C. T.,
Bulet P.,
Ferdig M. T.,
Severson D. W.,
Hoffmann J. A.,
Christensen B. M.
Publication year - 1999
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1046/j.1365-2583.1999.810107.x
Subject(s) - biology , intron , genomic dna , signal peptide , gene isoform , gene , microbiology and biotechnology , complementary dna , defensin , peptide sequence , genetics , amino acid , coding region , exon
Aedesaegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 μM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter–and intra–isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty–eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty–six amino acids, and the mature protein domain of forty amino acids, Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF 168.