California Sea Grant College Program

These analyses have revealed a family of chymotrypsin-like protease genes in Haliotis rufescens that is composed of at least two subfamilies of genes whose expression undergoes a developmental switch. Restriction, Southern and PCR analyses showed that four independent lambda genomic clones have been isolated, and that all contain multiple chymotrypsin gene sequences. These genes were present on three genomic clones in a clustered arrangement and on one clone in tandem. Sequence analyses of two of these genes revealed them to have six exons and five introns in normal orientation. Further, properly positioned canonical "TATA" boxes, polyadenylation sites and normal GT/AG splice sites at the intron-exon junctions suggested these sequences represented normal genes and not pseudogenes. Comparison of the two sequences revealed a high percentage nucleotide identity. Sequence similarities extended to the upstream, downstream and intron sequences. Consistent with this, PCR analyses of genomic DNA spanning the introns revealed product sizes that were predicted from the sequences. Sequence analyses of two chymotrypsin-like protease cDNAs from a Haliotis larval eDNA library allowed their comparison to the coding regions of the two genomic sequences and the adult intestinal eDNA sequence. Both nucleic acid and predicted amino acid sequence comparisons revealed these sequences as all highly similar. However, these comparisons also revealed two chymotrypsin subfamilies, designated herein as HrChymo I and II. Diagnostic restriction analyses of a developmental time series of RT-PCR products revealed a gene switch from expression of the HrChymo I (larval/juvenile) genes to HrChymo II (adult) genes, commencing in post-metamorphic animals at approximately 4 mm in size. In this assay, HrChymo II exon 1 sequences appeared before HrChymo II exon 4 sequences, suggesting a complex switching mechanism between HrChymo I and II sequences.