Fig. 4.

hsp60 is the nbl gene. (A) Genetic and physical map of nbl. Linkage map (Top), transcripts (Middle), and physical map (Bottom) places nbl between z54324 and z7144. Numbers in parentheses show recombination events from 2,302 meioses between nbl phenotype and linked genetic markers. (B) DNA sequence analysis from nbl and WT strains revealed a unique thymidine to adenosine mutation in nbl, causing valine-324 to glutamic acid missense mutation in Hsp60 (red arrows). (C) Multiple species alignment of the hsp60 region containing the V324E mutation. Note complete conservation of valine-324 among species, including E. coli. (D) Ribbon drawing structure of one subunit in the cis GroEL (Hsp60) ring. Figures were generated by using molmol (32) and coordinates from Protein Data Bank file 1AON (33). The location of the mutation site is shown in red space-filling form in the apical domain (light blue). Intermediate (green) and equatorial domains (blue) are also shown. (E) Northern blot analysis of hsp60 expression in regenerating caudal fin. hsp60 levels are up-regulated during blastema formation at both 25°C and 33°C. β-actin expression shown as a control. (F) Whole-mount in situ hybridization of hsp60 during regeneration. Fish were incubated at 25°C to ensure expression is not induced by heat-shock treatment. hsp60 expression is first induced beneath the wound epidermis, where mesenchymal tissue disorganization occurs at 24 hpa. At 48 hpa, hsp60 is expressed in the newly formed blastema. At 120 hpa, hsp60 expression is up-regulated in the distal blastema where msxb is expressed.