The human tuftelin gene: cloning and characterization.
Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5' RACE and 3' RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.
Mao
Z
Z
Dental Research Unit, Department of Oral Biology, Institute of Dental Sciences, Hebrew University, Hadassah Faculty of Dental Medicine, Jerusalem, Israel.
Shay
B
B
Hekmati
M
M
Fermon
E
E
Taylor
A
A
Dafni
L
L
Heikinheimo
K
K
Lustmann
J
J
Fisher
L W
LW
Young
M F
MF
Deutsch
D
D
D024506:5' Flanking Region / Q000235:genetics; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003746:Dental Enamel Proteins / Q000235:genetics*; D005091:Exons; D005796:Genes / Q000235:genetics*; D006801:Humans; D007438:Introns; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014083:Tooth Germ / Q000378:metabolism; D024363:Transcription Initiation Site
D018076:DNA, Complementary; D003746:Dental Enamel Proteins; C038456:tuftelin; D004247:DNA