Biomedical subjects

C C Hu

Publications and source records attributed to C C Hu.

At least 37 records · Page 2Linked to original sources

PubMed1998

Production of infectious RNA transcripts from full-length cDNA clones representing two subgroups of peanut stunt virus strains: mapping satellite RNA support to RNA1.

Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV-ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups.

Amino Acid Sequence↗

PubMed1998

Derived protein and cDNA sequences of hamster amelogenin.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

Amelogenin↗

PubMed1998

Proteolytic activity of opossum tooth extracts.

Amelogenins are the main component of the developing enamel matrix. In placental mammals, amelogenins are rapidly cleaved following their secretion. HPLC fractionation of tooth extracts produces a complex chromatographic profile. The fractions are rich in amelogenin cleavage products that generally retain the amino-terminus of the parent protein but have varying lengths of peptide removed from the original carboxyl-terminus. In contrast, HPLC fractionation of opossum tooth extracts produces a simple profile with a single major chromatographic peak. SDS-and Western blot analyses demonstrated that most of the amelogenin consisted of a prominent protein band that migrated at 28 kDa. Mass spectroscopy confirmed the presence of two uncleaved, alternatively spliced forms of opossum amelogenin, Op202 and Op57, but did not detect major amelogenin cleavage products evident in tooth extracts from placental mammals. Amino acid composition analysis supported the conclusion that uncleaved amelogenin is the major component in the developing enamel matrix. Enzymogram analyses using gelatin, casein and recombinant amelogenin as substrates, comparing porcine, rat and opossum tooth extracts, suggested that fewer proteinases are present in opossum. These results identify potentially significant differences in the proteolytic processing of amelogenins between metatherian and eutherian mammals.

Amelogenin↗

PubMed1998

Purification, characterization, and cloning of enamel matrix serine proteinase 1.

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.

Amelogenesis↗

PubMed1998

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA.

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.

Amino Acid Sequence↗

PubMed1998

Biochemical characterization of recombinant mouse amelogenins: protein quantitation, proton absorption, and relative affinity for enamel crystals.

Four recombinant mouse amelogenins, which varied by the presence or absence of the exon 4 encoded segment as well as the carboxyl-terminus were heterologously expressed and purified from bacteria. The rM193 and rM179 contain the carboxyl-terminus, whereas the rM180 and rM166 do not. The rM193 and rM180 contain the polypeptide segment encoded by exon 4 of the amelogenin gene. A precisely weighed sample of purified rM179 was quantified by Lowry, Bicinchoninic Acid and Bradford assays. It was determined that these protein quantification methods characteristically under or overestimate the amount of amelogenin. The calculated correction factors were: Lowry (x 1.35), BCA (x 1.96), and Bradford (x 0.78). Recombinant mouse amelogenin (rM179) was characterized with respect to its hydrogen ion binding properties. The protein absorbs 11.9 +/- 1.7 protons during a pH change from 8.0 to 5.0, suggesting that amelogenins buffer the enamel fluid in vivo. Crystal binding experiments were performed using rM193, rM180, rM179 and rM166. The carboxyl-terminus enhanced the binding of amelogenin to enamel crystals while the exon 4 encoded segment did not appreciably affect crystal binding.

Absorption↗

PubMed1998

Murine enamelin: cDNA and derived protein sequences.

Enamelin is the largest enamel protein. Recently we reported the characterization of a cDNA clone encoding porcine enamelin. The secreted protein has 1104 amino acids--over 6 times the length of amelogenin (173 amino acids) and almost 3 times the lengths of sheathlin (395 amino acids) and tuftelin (389 amino acids). Immunohistochemistry has shown that uncleaved porcine enamelin concentrates at the growing tips of the enamel crystallites while its cleavage products localize to rod and interrod enamel. Here we report the isolation and characterization of cDNA encoding murine amelogenin and demonstrate the tooth specificity of porcine enamelin. The murine clone is 4154 nucleotides in length and encodes a protein of 1274 amino acids. In the absence of post-translational modifications murine enamelin has an isotope averaged molecular mass of 137 kDa and an isoelectric point of 9.4. Multiple tissue Northern blot analyses detect porcine enamelin mRNA in developing teeth but not in liver, heart, brain, spleen, skeletal muscle and lung. Mouse and porcine enamelin share 61% amino acid identity and 75% DNA sequence identity. Mouse enamelin has 14 tandemly arranged copies of an 11 amino acid segment that is found only once in porcine enamelin.

Amino Acid Sequence↗

PubMed1998

Enamel matrix serine proteinase 1: stage-specific expression and molecular modeling.

Enamel proteins are cleaved by proteinases soon after their secretion by ameloblasts. Intact proteins concentrate in the outer enamel at or near the growing tips of the enamel crystallites while cleavage products accumulate in the deeper enamel. In the transition and early maturation stages there is a dramatic increase in proteolytic activity. This activity, coupled with the diminished secretory and increased reabsorptive functions of ameloblasts, leads to a precipitous fall in the amount of enamel protein in the matrix. Recently we have cloned and characterized an mRNA encoding a tooth-specific serine proteinase designated enamel matrix serine proteinase 1 (EMSP1) [Simmer et al., JDR (1998) 77: 377]. EMSP1 can be detected in the inner enamel during the secretory stage and its activity increases sharply during the transition stage. Stage-specific Northern blot analysis demonstrates this increase is accompanied by a parallel increase in the amount EMSP1 mRNA. A 3-dimensional computer model of EMSP1, based upon the crystal structure of bovine trypsin, has been generated and analyzed. All six disulfide bridges as well as the active site are conserved. Changes in the peptide binding region and the specificity pocket suggest that interaction of the proteinase with protein substrates is altered, potentially causing a shift in substrate specificity. The calcium binding region of trypsin is thoroughly modified suggesting that the calcium independence of EMSP1 activity is due to an inability to bind calcium. The three potential N-linked glycosylation sites, N104, N139 and N184, are in surface accessible positions away from the active site.

Amino Acid Sequence↗

PubMed1997

Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3.

Strains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80%, the corresponding value between strains ER and J was 91%, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic, encoding a putative movement protein (3a protein) and a coat protein (CP). The intercistronic and 5' untranslated region (UTR) sequences of PSV strains, unlike those of cucumber mosaic cucumovirus (CMV) strains, are highly conserved and thus not useful for distinguishing the two subgroups. However, the 3' UTR sequences of PSV strains, like those of CMV strains, can discriminate between the two subgroups since strains within the same subgroup are 95% identical in their 3' UTRs whereas those in different subgroups are only 74-78% identical. PSV-W and PSV-ER RNA4s were determined to be 994 and 1006 nucleotides long, respectively. PSV 3a and CP genes have higher percentage nucleotide sequence identities to those of tomato aspermy cucumovirus than to those of CMV.

Amino Acid Sequence↗

PubMed1997

Cloning, characterization, and heterologous expression of exon-4-containing amelogenin mRNAs.

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s). Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms that are secreted and their biochemical properties. Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing. The largest amelogenin cDNA encoded a 194-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment. Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional splicing variants could contain the exon 4 coding region. Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages. These messages encode amelogenin polypeptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length. The isotope-averaged molecular weights for the deduced, single-phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively. Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4. The exon-4-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI).

Amelogenin↗

PubMed1997

Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of porcine enamelin mRNAs.

Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.

Amino Acid Sequence↗

PubMed1996

Cloning, DNA sequence, and alternative splicing of opossum amelogenin mRNAs.

The enamel layer that covers the surfaces of teeth is thickest and most highly mineralized in mammals. The durability of mammalian enamel may have allowed for selection against the lifelong replacement of teeth that is observed in other vertebrates. Variation in enamel structure among animals is thought to be the result of evolutionary changes in the constituents of the developing enamel matrix. In placental mammals, the principal component of this matrix is amelogenin. We have determined the complete primary structures of two opossum amelogenins through a combination of protein sequencing, cloning, and DNA sequencing. RNA messages were cloned that encode 202- and 57-residue amelogenins, which are presumed to be expressed from the same gene but differ due to alternative splicing of identical pre-mRNAs. Edman degradation of the larger amelogenin ran for 42 cycles and yielded the sequence: IPLPPHPGHPGYINFS YEVLTPLKWYQSMMRQQYPSYGYEPM. The derived 202-residue amelogenin, assuming that serine 16 is phosphorylated, has an isotope-averaged molecular mass of 23,023.75 Daltons and a pI of 6.2. This is the largest amelogenin yet characterized. The increase in length is due to the presence of a 30-residue tandem repeat of QP(I/M) in exon 6 in the same position as a similar, but shorter, repeat expressed from the bovine X-chromosome. The 57-residue amelogenin, which is known from other organisms as the leucine-rich amelogenin protein (LRAP), has an isotope-averaged molecular mass of 6764.75 Daltons and a pI of 5.5. The opossum enamel protein is highly homologous to those previously characterized in eutherians and demonstrates that amelogenins were refined structurally prior to the metatherian/eutherian divergence between 100 and 150 million years ago.

Alternative Splicing↗

PubMed1996

Cloning, cDNA sequence, and alternative splicing of porcine amelogenin mRNAs.

In mammals, the organic matrix of developing enamel is dominated by amelogenins. To investigate the expression of proteins secreted into the developing enamel matrix, we have constructed a porcine enamel organ epithelia-specific cDNA library. The amelogenin fraction of the cDNA library was characterized by the cloning of amelogenin-specific polymerase chain-reaction (PCR) amplification products, 5' and 3' rapid amplification of cDNA ends (RACE), and by helper phage rescue of unamplified clones. Clones were characterized that encode porcine amelogenin isoforms 173, 157, 56, 41, and 40 amino acids in length. The structure of the porcine amelogenin gene differs from that of any of those yet described. There are two homologous but distinct exons 1, 2, and 7. One of the two exon 7s can vary in length depending upon the selection of either of two polyadenylation signal/cleavage sites. As a rule, a given exon 1 always pairs with the same exon 2 but can be associated with either exon 7. Despite significant sequence divergence within these exons, no differences are observed in exons 3, 5, and 6. We interpret these findings as evidence of a single amelogenin gene expressed from two promoters; however, the results do not exclude the existence of a second amelogenin gene. The variability generated through the use of alternate promoters and exon 7s primarily affects the non-coding regions of the message. A given amelogenin isoform expressed from the two promoters displays four amino acid differences within the signal peptide, while the secreted proteins are identical. Similarly, the alternative use of exon 7 does not alter the structure of the protein products. The pattern of RNA splicing of amelogenin pre-mRNAs is different for the transcripts expressed from the two promoters. The 173- and the 56-residue amelogenins can be expressed from either promoter, while the 157-residue amelogenin is generated by only one of the two promoters.

Alternative Splicing↗

PubMed1996

Comparative HPLC, SDS-PAGE, and immunoblot analyses of dental enamel proteins.

The primary structures of amelogenins expressed from different genes vary because of DNA sequence divergence and variations in alternative RNA splicing. The pattern of splicing is unique for each amelogenin gene yet investigated, even when two copies of the gene are expressed in the same cell. Despite the high conservation of amelogenin sequences, diversity in the pattern of RNA splicing leads to significant differences in the number and character of amelogenin isoforms in the developing enamel matrix. Since conservation of molecular structure is an indicator of functional significance, we compared enamel protein preparations from rat, porcine, rabbit, and opossum developing tooth organs. Enamel extracts were fractionated by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were performed with polyclonal antibodies raised against recombinant murine amelogenin and the polypeptide encoded by murine exon 4. The opossum enamel extract produced the simplest chromatogram, suggesting that fewer proteins are secreted into the developing enamel matrix. The predominant opossum amelogenin has an apparent molecular mass of 28 kDa and reacts strongly with the recombinant amelogenin antibody but is not recognized by the murine exon 4 antibody. Opossum amelogenin mRNA was amplified with murine amelogenin primers specific for the amino- and carboxyl-terminal coding regions. The mobility of the amplification products on 4% agarose gels indicates that the leucine-rich amelogenin polypeptide (LRAP) is expressed in the opossum and that the major amelogenin is larger than its homologue in the mouse. We conclude that the alternative splicing of amelogenins pre-dates the metatherian and eutherian divergence over 100 million years ago.

Alternative Splicing↗

PubMed1995

The conserved, hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels.

Although the Mr values of the coat proteins (CPs) of several cucumoviruses have been calculated from their deduced amino acid sequences to be approximately 24,000, the experimentally determined M(r) values using the Laemmli SDS-PAGE system were 30,000-31,000. Examination of the amino acid composition revealed that these CPs are neither highly acidic nor highly basic. Post-translational glycosylation or phosphorylation were also ruled out as contributing factors to the observed anomalous electrophoretic mobility because the products of in vitro translation of cucumovirus RNA 4 and in vivo bacterial expression of the cloned CP gene co-migrated with authentic cucumovirus CPs. Comparison of the hydropathy profiles of the CPs revealed the presence in each of a strikingly similar, highly hydrophilic N-terminal domain of 30-32 amino acid residues that contains a cluster of basic amino acids, mainly arginine. Selective chemical cleavage at tryptophan residues in the CPs of cucumoviruses, known to contain single tryptophan residues, yielded two peptides; an N-terminal peptide that contained the conserved hydrophilic domain and a C-terminal peptide. SDS-PAGE analysis showed that the N-terminal, but not the C-terminal, peptide exhibited the anomalous electrophoretic mobility.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of a mouse amelogenin expressed in Escherichia coli.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Amelogenin↗

PubMed1994

Alternative splicing of the mouse amelogenin primary RNA transcript.

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5' ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5' untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.

Alternative Splicing↗