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Gene Profiling in Human Periodontal Ligament Fibroblasts by Subtractive Hybridization
T. Yamamoto,
F. Myokai,
F. Nishimura,
T. Ohira,
N. Shiomi,
K. Yamashiro,
H. Arai,
Y. Murayama and
S. Takashiba*
Department of Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;
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Figure 1. Unique gene expression in HPFs and HGFs. (A) Osteocalcin mRNA expression in HPFs and HGFs as revealed by PCR analysis. The arrowhead at the right indicates the position of the amplicon. Complementary DNA (10 ng) from each cell was amplified by PCR with human osteocalcin primers (5'-ATGAGAGCCCTCACACTCCTC-3' and 5'-CAGGGGATCCGGGTAGGGGAC-3'). The amplification was performed for 40 cycles, and the annealing temperature was 55°C. M: 100-bp ladder. (B) Messenger RNA expression of PDL-29 in HPFs and HGFs. Messenger RNA from HPFs and HGFs was hybridized with PDL-29 cDNA probe. For an internal control, the mRNAs were also hybridized with human β-actin probe. The donor is #1 in Fig. 1C . (C) Densitometric analysis of signal intensity. Relative signal intensities for PDL-29 mRNA from three different donors are shown after normalization against the signal for β-actin mRNA.
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Figure 2. Structure of the PDL-29 gene. (A) PDL-29, PET112 cDNA sequences, and their chromosomal localization. Nucleotides 1-1984 of PDL-29 and 28-2011 of PET112 completely match, whereas only PDL-29 contains the insertion sequence just before the poly (A)-tail. Nucleotides 1-28 of PET112 and 1-161 of PDL-29 are localized to exon 1, and nucleotides 1530-2180 of PDL-29 are localized to exon 15. Filled area in PDL-29: insertion sequence (197 bp) just before the poly (A)-tail. Filled area in PET112: 5' region of PET112 different from that of PDL-29. Full-length PDL-29 (2180 bases) was submitted to GenBank (Nov. 1, 1998) and has been assigned accession no. AB019410.
(B) Insertion sequence in the 3'-UTR of PDL-29. Nucleotides 1984-2180 of PDL-29 and the poly (A)-tail are displayed. Horizontal bars indicate putative cis-acting elements of known molecules, and similarities are indicated in percentages. GH, bovine growth hormone; TCR Vβ2.2, human T-cell receptor Vβ2.2; OC, rat osteocalcin; ML-MuMTV, mouse mammarytumor-virus long terminal repeat; GPT, mouse N-acetylglucosamine-l-phosphate transferase; LYZ, chicken lysozyme; and PAI-2, human plasminogen activator inhibitor type-2.
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Figure 3. Expression of PDL-29 mRNA in rat periodontium. In situ hybridization on periodontal tissue sections from Wistar rat was performed with an anti-sense probe from human PDL-29 cDNA (A,B,C). Dense signals are observed in GFs and PFs. When a sense riboprobe is used, no signal is observed in the tissue (D,E). Neighboring sections were stained with hematoxylin and eosin (F,G). Gingival tissue (B,D,F) and periodontal ligament (C,E,G) correspond to the areas in panel A. Bar equals 300 µm. to, tooth; gt, gingival connective tissue; ab, alveolar bone; pl, periodontal ligament.
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Journal of Dental Research, Vol. 82, No. 8,
641-645 (2003)
DOI: 10.1177/154405910308200814
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