580 N. Peiren et al. assembly scaffolds 16.18 and 16.19.These genome sections apparently assembled separately because of sequence variation found in the two haplotypes sequenced for the Honeybee Genome Project.This effect is probably widespread in assemblies of this and other draft genome sequences. In fact, this effect is seen further down chromosome 16 (scaf- folds 16.11 and 16.12), where the hypervariable immune effector apidaecin (Casteels et al., 1994) has apparently disrupted the genome assembly. An understanding of allelic variation at speciﬁc genes might help unite unassembled parts of the bee genome. As a corollary, misassembled sections of the bee genome might indicate biologically important variation in genes adjacent to these gaps. Experimental procedures Bee venom glands Honeybees (Apis mellifera carnica) were all taken at the hive entrance of a single colony from the apiary of the Ghent University (Belgium). The venom glands of 320 bees were dissected under anaesthesia by chilling. First, the whole sting apparatus was removed from the abdomen and submerged in RNALater® (Ambion, Austin, TX, USA).Subsequently, the glands were separated from the reservoir and collected all together in 100 µ l fresh solution. Homogenization and mRNA isolation was done using the Micro-FastTrack™ 2.0 Kit (Invitrogen, Carlsbad, CA, USA) following the protocol for fresh and frozen tissue. After spectrophotometric yield determination, mRNA was stored in elution buffer at −80 °C until ready for use. cDNA preparation and primer development Venom gland cDNA was prepared using AMV reverse transcriptase and the oligo dT primer from the cDNA Cycle® Kit (Invitrogen) according to the manufacturer’s instructions. To ﬁnd the corre- sponding genome sequence for primer development, the Api m 6 amino acid sequence (P83563) was BLAST-searched against an early release of the honeybee genome (Amel_1.2) at http:// www.hgsc.bcm.tmc.edu/projects/honeybee. The target domain was located on group 16.4 and extended from position 258118– 258415 (total length of the group: 806 207 bp). Subsequently Api m 6 gene-speciﬁc primers (GSP) for rapid ampliﬁcation of cDNA ends (RACE; see further) were developed using the Primer3 software on the world wide web (Rozen & Skaletsky, 2000). This gave the following result: GSP forward primer (GSP-fw) 5′-TTGGAG- GATTTGGAGGCTTGGAGGA-3′ and GSP reverse primer (GSP-rv) 5′-GCATTTAGATCGCGGAACGCATACCT-3′. Their suitability for further analysis was tested by a simple polymerase chain reaction (PCR) using the venom gland cDNA preparation (see above) as template. The resulting amplicon was sequenced for conﬁrmation. 5′-Rapid ampliﬁcation of cDNA ends (5′-RACE) and 3′-RACE RACE ready cDNA was prepared by following the protocol described in the GeneRacer™ Kit (Invitrogen). Brieﬂy, 100 ng of bee venom gland mRNA was subsequently treated with calf intestinal phosphatase and tobacco acid pyrophosphatase, to be ligated at its 5′-end with the GeneRacer™ RNA oligo. This ligated mRNA was reverse transcribed using the SuperScript™ III RT and the GeneRacer™ oligo dT primer to create RACE-ready ﬁrst-strand cDNA with known priming sites at the 5′- and 3′-ends. Generation of 5′-RACE fragment was done by PCR, using a combination of GeneRacer™ 5′-primer and GSP-rv, whereas the 3′-ampliﬁcation needed a combination of GSP-fw and GeneRacer™ 3′ primer. Both reactions were done in an EppeΛdorf Mastercycler using a touchdown protocol. Cloning and DNA sequencing PCR products were cloned in the pCR®4-TOPO® vector. Individual transformants were picked and analysed for the presence of insert by PCR.The corresponding amplicons were used for sequence analysis. DNA sequencing was performed using a Perkin Elmer ABI Prism 377 (Perkin Elmer, Wellesley, MA, USA) automated DNA sequencer. PCR product was treated with shrimp alkaline phosphatase (1 U/µ l, Amersham E70092Y, Amersham Biosciences, Buckingham, UK) and exonuclease I (20 U/µ l, Epicentre Biotechniologies ×40505k, Madison, WI, USA) for 15 min at 37 °C, followed by 15 min at 80 °C to inactivate the enzymes. This material was then used for cycle sequencing without any further puriﬁcation, using the ABI Prism BigDye V 3.1 Terminator Cycle Sequencing kit.The sequencing con- ditions were 30 s at 96 °C, 15 s at 50 °C and 4 min at 60 °C for 27 cycles. Primers used for sequencing were GSP-fw, GSP-rv or GeneRacer™ 5′-primer. Cycle sequence products were precipi- tated by adding 25 µ l of 95% ethanol and 1 µ l 3 M sodium acetate, pH 4.6 to each cycle sequencing reaction (10 µ l). The samples were placed at −20 °C for 15 min and centrifuged at 14 000 r.p.m. (12 225 g) for 15 min. After precipitation, an additional wash of the pellet was performed with 125 µ l of 70% ethanol and centrifuged at 14 000 r.p.m. (12 225 g) for 5 min. The pellet was dried in a Speedvac concentrator, redissolved in loading buffer and run on a 48 cm 4.25% acrylamide/bisacrylamide (29 : 1) gel. Analysis of sequence data Partial cDNA clones and the complete cDNA sequence generated above were aligned with honeybee genome assembly 4.0 (Amel_4.0–20061003) using BLASTN (Altschul et al., 1997), with- out ﬁlters. Sequences were also compared by BLASTN with unscaffolded contigs. Several thousand base pairs of ﬂanking DNA on either side of Api m 6 (e.g. on Contig5539) was used to conﬁrm that this contig and unassembled Contig14926 were in fact derived from the same genome location, despite showing substan- tial genome sequence variation at and near Api m 6. Unscaffolded Contig14926 was used, via BLASTN, to span the gap between assembled scaffolds 16.18 and 16.19. Sequence variation 2 5′- and 3′ UTR regions of Api m 6 was characterized by alignment of DQ384991 and Contigs14926 and 5539. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. Banks, B.E.C. and Shipolini, R.A. (1986) Chemistry and pharma- cology of honey bee venom. In Venoms of the Hymenoptera (Pick, T. ed.), pp. 329–416. Academic Press, London. Bendtsen, J.D., Nielsen, H., von Heijne, G. and Brunak, S. (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–795. © 2006 The Authors Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 577–581