578 N. Peiren et al. Results and discussion Cloning and sequencing of cDNA fragments encoding Api m 6 5′-Rapid ampliﬁcation of cDNA ends (5′-RACE) using gene- speciﬁc primers resulted in an amplicon of approximately 350 bp in size that was cloned in the pCR®4 vector. Trans- formation to chemically competent TOP-10 Escherichia coli yielded a large number of transformants, 10 of which were retained for further analysis. DNA sequences were found to be identical (represented by clones 5.1) and consisted of a putative 5′ untranslated region (UTR) and a coding region for which the predicted protein was identical to that known for Api m 6. 3′-RACE resulted in a fragment of approximately the same size, which was again cloned and sequenced. Here the sequenced clones showed two transcript variants, represented by the inserts of clone 3.3 and clone 3.9. Sub- sequently, we ampliﬁed the complete Api m 6 transcript from a venom gland cDNA preparation using primers designed, respectively, at the extreme 5′- and 3′-ends of clone 5.1 and clone 3.9. Sequencing of the corresponding 528 bp frag- ment revealed two additional minor transcript differences, this time at the 5′-end, 38 and 117 bp upstream of the forward primer, when compared with the insert of clone 5.1. This cDNA sequence was deposited to GenBank (accession number DQ384991), as was a sequence assembly of the inserts of clones 5.1 and 3.9 (accession number DQ384990). Because the latter matched perfectly well the latest honeybee genome assembly (see further), it was named variant 1, whereas DQ384991 became variant 2. Deduced amino acid sequence and protein heterogeneity The deduced amino acid sequences of both transcripts are given in Fig. 1. At the protein level differences were noticed at position 14 (Val against Ile) and at the carboxy terminal region, with two additional residues Leu and Pro. This corresponds with the differences found at the C-terminal ends of the four Api m 6 variants described by Kettner et al. (2001): Api m 6.01 and Api m 6.03 having the same two additional residues, in contrast to the variants Api m 6.02 and Api m 6.04. Further we noticed that the deduced amino acid sequences were 21 residues longer at the N-terminus when compared with the earlier described variants Api m 6.03 and Api m 6.04 (Kettner et al., 2001). However, based on an in silico SigP-NN prediction (Bendtsen et al., 2004), a signal peptide cleavage site was identiﬁed between position 21 and 22, resulting in a mature protein that starts at exactly the same residue. Depending on the cDNA variant that was translated this mature protein will correspond to Api m 6.03 or Api m 6.04. The other two variants described by Kettner et al. (2001) lack the ﬁrst four N-terminal amino acids Phe- Gly-Gly-Phe of the mature protein. We have found no indica- tion that this was the result of a new transcript or alternative splicing variant. Nor could we evidence that the mature Figure 1. Nucleotide and deduced amino acid sequences of Api m 6 transcript variant 1 (A) and 2 (B). The numbers on the left denote the nucleotide numbers.The start and stop codon are underlined. Residues −1 to −21 are from the signal peptide. Residue +1 denotes the ﬁrst amino acid of the mature protein. In (C) the alignment of the deduced amino acid sequences of the two Api m 6 transcript variants is given. proteins of these variants are shortened by enzymatic activity similar to step-wise cleavage of the pro part from promelittin by dipeptidylpeptidase IV (Kreil et al., 1980). Fur- ther we notice that amino acid replacement at position 14 in the deduced amino acid sequence is located in the signal peptide and has no consequences for the mature allergen. Sequence homology and conserved domains Conserved domain search (Marchler-Bauer & Bryant, 2004) of the deduced amino acid sequence from transcript variant © 2006 The Authors Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 577–581