View larger version (52K):
[in a new window]
|
Figure 1. Histology of pulp, detection of cDNAs, and reverse Northern analysis. (A) Histology of pulp tissues. The wounded and healthy teeth were fixed with PBS containing 4% paraformaldehyde, demineralized with 10% EDTA for 4 wks, and embedded in paraffin. Hematoxylin and eosin staining was performed on serial sections 7 µm thick. The pulps 1 wk after cavity preparation (a,b) and healthy pulp (c,d) are shown. Pulpal cells underneath the cavity are indicated by the arrow, and neither reparative dentin formation nor apparent disruption of the odontoblast layer is observed (a,b). Bar equals 300 µm.
(B) Procedure of subtractive hybridization. The target and driver single-stranded (ss) cDNAs bound to the beads were synthesized from the total RNA (100 ng) isolated from the wounded and healthy pulp tissues. The target complementary sscDNA (c-sscDNA) was synthesized from the target sscDNA-beads with an EcoRI-dT primer (5'-GGCGAATTCTGCAGTTTTTTTTTTTTTT-3'). After auto-subtraction, the target c-sscDNA was subtracted twice from the driver sscDNA beads. The subtracted c-sscDNA was amplified by polymerase chain-reaction (PCR) with use of the EcoRI-dT primer, and was displayed on a 3% agarose gel. The procedure is described in detail in the Appendix.
(C) Display of amplified cDNAs. The wounded pulp cDNA subtracted from the healthy pulp cDNA was used as WIN cDNA, while the healthy pulp cDNA subtracted from the wounded pulp cDNA was used as WSP cDNA. The subtracted cDNA was amplified, and the product underwent gel electrophoresis. Lanes: 1, wound-inductive (WIN) cDNA; 2, wound-suppressive (WSP) cDNA; M, 100-bp DNA ladder.
(D) Messenger RNA expression of genes in pulp tissues. The cDNAs (WIN-2, 6, 10, 11, 13, WSP-1, 2, 6, 7, GAPDH) underwent electrophoresis on an agarose gel (Ag). They were then transferred to a membrane, and hybridized with the probe from wounded pulp tissue (Wo) and healthy pulp tissue (He). Relative signal intensity (each cDNA/GAPDH) is shown in the upper panel. *WIN-11 was used as a probe for screening of the cDNA library. Two independent hybridizations were performed, and a typical result is shown.
(E) WIN-11 mRNA in adult rat tissues. WIN-11 mRNA was detected in the tissues shown by an arrow in the upper panel, while ß-actin mRNA was detected in the tissues shown by an arrow in the lower panel. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. Three independent hybridizations were performed, and a typical result is shown.
|
, common region in rFIP-2A and rFIP-2B; 
, region of rFIP-2B different from that of rFIP-2A. 
, cDNA containing the specific region for rFIP-2A or rFIP-2B was cloned and used as a template for riboprobes of in situ hybridization. The procedure is described in detail in the Appendix. (B) Structure of rFIP-2s. Deduced amino acid sequences of rFIP-2s and hFIP-2 are shown. The signal peptide domain is boxed in green, the putative leucine-zipper domains are boxed in black, and the zinc-finger domain is boxed in blue. The putative bZIP motif is boxed in red. *Same amino acid as the sequence above; –, missing amino acid. (C) Expression of rFIP-2 mRNA. Complementary DNA was amplified by PCR with the primers as described below. The PCR products for rFIP-2A, B, and ß-actin underwent electrophoresis on a gel. The specific primers were designed: rFIP-2A sense, 5'-TGCCCAGCCAGCCTCCTACC-3'; rFIP-2B sense, 5'-ATCTCTGTGGCCGGACCTGTTACC-3'; and rFIP-2A and rFIP-2B antisense, 5'-CCACTTCGATTCCCACACTC-3'. For an internal control, specific primers for rat ß-actin were designed: sense, 5'-TTGTAACCAACTGGGACGATATGG-3'; and antisense, 5'-GATCTTGATCTTCATGGTGCTAGG-3'. Two independent PCRs were performed, and typical results are shown. Lane M, 100-bp DNA ladder. Relative signal intensity (each mRNA from/ß-actin mRNA) is shown in the lower panel. 