Table 1.

PCR products of the nucleotide binding domain of the partial hsc70 and hsp70 genes in the diplopod Tachypodoiulus niger

Accession no.	Sequence similarity	Degenerate primers	Template	PCR product (bp)
AM502906	hsc70	Fw: 5′ATYAAYGARCCIACKGCIGCIGCWATTGCITATGG3′ Rev: 5′GAYGARGCWGTTGCITAYGGIGCWGCIGTICARGC3′	mRNA	543
AM502907			gDNA	701
AM502908	hsp70		gDNA	549
AM502909	grp78		gDNA	540

Animals were collected from a beech forest stand, kept at 16 °C at a 12 h/12 h light/dark photoperiod in transparent plastic boxes and fed with partially decayed leaves of the Common Hazel (Corylus avellana). Heat shock was carried out in a heating cabinet for 1 h at 30 °C followed by 15 min at 40 °C. Total genomic DNA (gDNA) was isolated after removal of the alimentary tract using a modified method of Zhang and Hewitt (Zhang and Hewitt 1998). The total RNA was isolated using an invertebrate RNA isolation kit (Peqlab, Erlangen, Germany). A reverse transcription was carried out with SuperScript® II RT and oligo(dT)_12–18-primer (Invitrogen Life Technologies, Darmstadt, Germany). Degenerate primers and PCR programs (3 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at 56 °C and 1.5 min at 72 °C followed by a final 10 min at 72 °C) were adopted from Köhler et al. (Köhler et al. 1998) and slightly modified when necessary. PCR products were purified by agarose gel electrophoresis and subsequently cloned into the pCR4-TOPO-vector (Invitrogen, Groningen, The Netherlands). The plasmid DNA was isolated from positive clones with the E.Z.N.A. plasmid miniprep kit II (Peqlab). A custom sequencing was carried out in both directions and confirmed twice by Seqlab, Sequencing Laboratories (Göttingen, Germany). The BLAST searches (Altschul et al. 1990) were used for sequence identification. Alignments of the hsp70 sequences were carried out using ALIGN program (Smith and Waterman 1981)