Genomic and transcriptional analysis of Api m 6 579 Figure 2. Alignment of two in silico spliced honeybee genome sequences with different cDNA fragments of the bee venom allergen Api m 6. (A) Api m 6 was present in mapped scaffold 16.18 from genome assembly 4.0 (positions marked above the bar, along with coordinates for the corresponding Contig5339). Api m 6 is characterized by two introns (depicted by black triangles; splicing sites given on top) and this haplotype of the genome assembly showed a perfect sequence-level match with cloned 5′- (clone 5.1) and 3′-RACE fragments (clone 3.9). The predicted protein from this sequence is identical to the described Api m 6 variant 1. (B) A second transcript variant was found by cDNA sequencing (full cDNA). This transcript has a nearly identical match to unmapped scaffold GroupUn.6097 (Contig14926), the only difference being a TT-insertion mutation (depicted by a white triangle) in the 5′-UTR. The 3′-end of transcript variant 2 corresponds also to another cloned 3′-RACE fragment (clone 3.3). Fragment lengths (in bp) are given below the bar and in boxes. 1 revealed a trypsin inhibitor like cysteine rich (TIL) domain from residue 37–91 (score: 37.3; E-value: 5e-04). This family contains trypsin inhibitors as well as a domain found in many extracellular proteins. The domain typically contains 10 cysteine residues that form ﬁve disulphide bonds in the combination 1–7, 2–6, 3–5, 4 –10 and 8–9. The assumption that Api m 6 represents a trypsin inhibitor was already made by Banks & Shipolini (1986), although the protein was at that time hardly characterized and certainly not yet named as such. In fact, they described two peptides H1 (17 residues) and H3 (35 residues), which later were found to correspond with the N-terminal ends of Kettner’s isoforms of Api m 6, differing from each other only in that one lacks the ﬁrst four residues. The amino acid compositions were similar to a protease inhibitor that was earlier puriﬁed from bee venom by Shkenderov (1973). Although no proteolytic activity could be assigned to the peptides H1 and H3, it appears now that Api m 6 has a molecular weight quite near that of Shkenderov’s protease inhibitor, i.e. 9000 Da. Noncoding sequence variation and the genome assembly A BLASTN search (Altschul et al., 1997) of the complete cDNA against honeybee genome assembly 4.0 (Amel_4.0– 20061003) revealed two hits with very high scores/E-values: scaffold 16.18 (Contig5539), score: 486; E-value: e-136 and unmapped scaffold GroupUn.6096 (Contig14926), score: 371; E-value: e-101. DNA sequence comparison demonstrated a 100% match between Contig5539 (between position 37212 and 36406, introns excluded) and an assembly of the RACE fragments from clone 5.1 and clone 3.9 (Api m 6 variant 1, Fig. 2A). On the other hand, Contig14926 (between position 16221 and 17048, introns excluded) was identical to the cod- ing region of the full size Api m 6 transcript and the insert of clone 3.3 (Api m 6 variant 2, Fig. 2B). However, the UTR of the full size cDNA shows an indel of two consecutive thymines, found upstream from position 41 in DQ384991. There are also eight indels (at four locations) and one G to A transition in the 3′ UTR of variant 1 when aligned with var- iant 2. These results demonstrate the existence of several different transcript variants of the bee venom allergen Api m 6, originating from genome-level variation at a single locus. Api m 6 shows a substantial amount of sequence varia- tion (nearly all haplotypes sequenced to date differ in at least one place) as well as sections of repetitive simple sequences (monobasic A or T runs, as well as an AT dinu- cleotide repeat; Fig. 1). It is conceivable that this variation was the root cause for misassembly at this section of the draft honeybee genome sequence. First, it is evident that assembly scaffolds 16.18 and GroupUn.6095 are homolo- gous, and that GroupUn.6095 spans the gap between © 2006 The Authors Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 577–581