The influx of serum albumin to enamel matrix in rat incisors after trauma.

The most frequent results of trauma to tooth germs are enamel hypoplasia and enamel hypocalcification. These differing results may be due to the stage of amelogenesis at which trauma occurs. The cellular and biomolecullar events involved in the genesis of these defects are poorly understood. We hypothesized that one factor involved is the possibility that relatively high levels of serum albumin enter the enamel matrix through the damaged enamel organ, and impair mineralization of the matrix. The present study was undertaken to immunohistochemically and autoradiographically localize serum albumin in the enamel organs of rat incisors after trauma was inflicted to the mandibular incisor region of 4-day-old rats. Hemorrhage was seen surrounding the enamel organ and between the detached secretory-stage ameloblasts. One day after trauma, the most intense immunohistochemical (IHC) staining for albumin was localized in the outer layer of the enamel matrix adjacent to the detached secretory-stage ameloblasts. Albumin was also detected autoradiographically in the secretory-stage ameloblasts layer and enamel matrix. These findings indicate that serum albumin can leak between the detached ameloblasts and penetrate the enamel matrix after trauma. Leaked albumin was still present in the matrix during the maturation stage. Leaked albumin in the developing enamel could inhibit crystal growth and result in hypocalcification.

[Effect of experimentally induced trauma to the incisors in rats]. / Experimentálisan létrehozott trauma hatása patkánymetszókön.

The purpose of this study was to produce developmental lesions of enamel in rats by a quick and simple method and to examine the enamel hypoplasia caused by trauma. 4-day-old and 3-week-old Wistar rats were used. Trauma was produced by needle pricking and hitting on the mandibula. After one week incisors were removed together with jaws and prepared for microradiography, X-ray diffraction, light microscope and transmission electron microscope examination. Microradiographic findings in the needle pricking group revealed hypocalcified and hypoplastic enamel. The incidence was 73.7%. In the hitting group of 4-day-old rats the incidence was lower, 70.0%. Microradiographic findings in this group were diffuse hypoplasia in the surface enamel. Hitting of 3-week-old rats produced no enamel hypoplasia. Crystallinity of hypoplastic enamel was reduced in both a-axis and c-axis direction. Histological findings in 4-day-old rats revealed disarranged and detached ameloblasts at location trauma. These results indicate that a needle pricking method involving secretory stage ameloblasts of 4-day-old rat incisors can induce a considerable amount of enamel hypoplasia.

Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes.

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3--4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses.

Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.

The immunohistochemical localization of Bax and Bcl-2 and their relation to apoptosis during amelogenesis in developing rat molars.

Bax and Bcl-2 are members of a family of intracellular, membrane-associated proteins that regulate programmed cell death. It has been suggested that, when Bax predominates, programmed cell death is accelerated and the apoptosis inhibitory activity of Bcl-2 is suppressed. The present study was undertaken to immunohistochemically (IHC) localize Bax and Bcl-2 in the cells of the enamel organ during amelogenesis in rat molars. Also, apoptotic cells were detected by TUNEL staining. The IHC intense localization of Bcl-2 and light staining for Bax in the pre-ameloblasts suggest that apoptosis is inhibited in the proliferating pre-ameloblasts. This is consistent with an absence of TUNEL staining for apoptosis in these cells. However, in the late secretory and transition ameloblasts, and adjacent stratum intermedium, evidence of apoptosis of the ameloblasts was observed. Bax and Bcl-2 were co-localized in the proximal ends of late secretory, transition and early maturation-stage ameloblasts, but immunoreactivity for Bax markedly increased in the proximal ends of late secretory and transition ameloblasts, while the Bcl-2 staining appeared to be lighter. This suggests that Bax antagonized Bcl-2 function, limiting the ability of Bcl-2 to prolong cell survival. In the early maturation stage, Bax staining faded while the immunoreactivity for Bcl-2 increased. Evidence of distinct apoptosis was reduced in the early maturation stage ameloblasts. When related to the occurrence of apoptosis during amelogenesis, the relative intensity of expression of Bax and Bcl-2 changed in a pattern consistent with that observed in other cell lines. This indicates that these proteins play essential roles in the process of amelogenesis, as predicted by their proposed mechanisms of action in the control of apoptosis.

Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer.

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.

Activation of blood coagulation factor IX by gingipains R, arginine-specific cysteine proteinases from Porphyromonas gingivalis.

The effect of two arginine-specific cysteine proteinases (gingipains R) from Porphyromonas gingivalis, an aetiological factor of adult periodontitis, on the activation of human factor IX was investigated in the presence of ethylene glycol, an activity enhancer of activated factor IX (factor IXa), with the use of a fluorogenic oligopeptide substrate. Each gingipain R rapidly activated factor IX but the 95 kDa proteinase complex (HRgpA) that contains both haemagglutinin/adhesion and catalytic domains was 2.4-fold more efficient than the single-chain 50 kDa gingipain R (RgpB), which has only a catalytic domain. SDS/PAGE and N-terminal sequence analysis of factor IX digestion fragments indicated that, like all endogenous activators, gingipains R also produce factor IXabeta via an IXa intermediate. Significantly, phospholipids augmented the activation of factor IX by HRgpA but not by RgpB in a Ca(2+)-dependent manner. In the presence of both cofactors the kinetic efficiency of HRgpA to activate factor IX (k(cat)/K(m)=1.9x10(6) M(-1).s(-1)) was 8.5-fold higher than that of RgpB (k(cat)/K(m)=2.3x10(5) M(-1).s(-1)) and double that of the factor VIIa-tissue factor complex, but 8-fold lower than that for factor XIa. A comparison of the relative activation rates of factor IX, factor X and prothrombin directly in plasma by HRgpA suggests a significant contribution for factor IX conversion in blood coagulation induced by gingipains R. Taken together, gingipains R are the first-reported activators of factor IX of bacterial origin. By this effect they could be involved in the production of thrombin as well as the subsequent generation of prostaglandins and interleukin 1, all of which have been found to be associated with the development and progression of periodontitis.

In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time.

PURPOSE: Recombinant human serum albumin (rHSA), secreted by a Pichia pastoris expression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. METHODS: Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. RESULTS: Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. CONCLUSION: The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411.

Primary structure of guinea pig plasma prekallikrein.

A full length guinea pig plasma prekallikrein (PK) cDNA was cloned from a liver cDNA library. The nucleotide sequence with 2242 bp was analyzed and the amino acid sequence with 618 residues was deduced. Kallikrein was purified from guinea pig plasma and cleavage site in the activation was determined. The amino acid sequence around the cleavage site -368Ile-Asp-Ala-Arg-Ile-Val-Gly-375Gly- differed from that of the human PK -368Thr-Ser-Thr-Arg-Ile-Val-Gly-375Gly-. Protease substrates containing penta-peptides which mimicked the sequence of the cleavage sites from P3 to P2' of guinea pig Hageman factor (HF) and PK were synthesized, and kinetic analyses of the hydrolysis by guinea pig activated HF (HFa) and kallikrein were carried out. The combination between HFa and the PK mimicking peptide provided the best kinetics. These results in part explain why the cascade activation of PK by HFa is predominant in the guinea pig system.

Determination of the cross-linked residues in homo-dimerization of S19 ribosomal protein concomitant with exhibition of monocyte chemotactic activity.

When S19 ribosomal protein molecules are intermolecularly cross-linked by a transglutaminase-catalyzed reaction, the monocyte chemotactic activity is newly expressed. Heparin, at a concentration of 1 U/ml, greatly augmented the cross-linking reaction. This augmentation was due to binding affinity of S19 ribosomal protein to heparin. The major heparin-binding region of S19 ribosomal proteins was identified to Lys23-Lys-Ser-Gly-Lys-Leu-Lys29, using region-directed mutant proteins. The amino acid residues of S19 ribosomal protein used for the intermolecular cross-linkage were then determined by the peptide map analysis with amino acid sequencing and by the site-directed mutagenesis; Gln137 and Lys122 were used in the intermolecular cross-linkage.

Organization of the chemokine gene cluster on human chromosome 17q11.2 containing the genes for CC chemokine MPIF-1, HCC-2, HCC-1, LEC, and RANTES.

To understand the organization of the human CC chemokine gene cluster on chromosome 17q11.2, we determined the nucleotide sequence of a region 181 kb long containing five CC chemokine genes, MPIF-1 (SCYA23), HCC-2 (SCYA15), HCC-1 (SCYA14), LEC (SCYA16), and RANTES (SCYA5), by the random shot-gun method. The four CC chemokine genes, MPIF-1, HCC-2, HCC-1, and LEC, are clustered within a region 40 kb long, whereas the RANTES gene is located approximately 10 kb apart from the four chemokine gene minicluster. These chemokine genes are arranged in the same orientation, and their sizes are relatively long, 3.1 (HCC-1)-8.8 kb (RANTES) when compared with other CC chemokine genes, such as MIP-1alpha/LD78alpha (SCYA3) (1.9 kb) and MCP-1 (SCYA2) (1.5 kb). In contrast to most other human CC chemokine genes that consist of three exons, the MPIF-1 and HCC-2 genes, separated by 12 kb, have four exons. When the nucleotide sequences of the MPIF-1 and HCC-2 genes are compared, they are well conserved, including introns and flanking sequences, except for the middle region of the long first intron, indicating that they have been generated recently in evolutionary terms by duplication. In addition to the CC chemokine genes, more than 30 exons are identified in the sequenced region by similarity search against expressed sequence tags (ESTs) and also by the gene prediction program GenScan. This indicates that the chemokine cluster sequenced in this study is a gene-rich region in the human genome.

The immunohistochemical localization of the interferon-gamma and granulocyte colony-stimulating factor receptors during early amelogenesis in rat molars.

Previous studies, in which the known janus kinase and signal transducer and activator of transcription (STAT) isoforms were immunohistochemically mapped in developing rat molars, implicated a sizeable list of cytokine superfamily receptor (CSR)/signal-transduction pathway (STP) linkages in the cells of the enamel organ involved in the events leading directly to early amelogenesis. Various combinations of upregulated janus kinases and STATs are known to be linked to single or small groups of CSRs. On the basis of the previous observations it was hypothesized that the interferon-gamma receptor (IFNgamma r) and the granulocyte colony-stimulating factor receptor (G-CSF receptor) would be localized in specific sites in the cells of the enamel organ during early amelogenesis. To verify this, whole-head, freeze-dried sections were here obtained at the level of the mandibular first and second molar from newborn and 5-day-old rats. These sections were not demineralized or fixed, reducing the possibility of false-negative results. Antibodies to the IFNgamma r and the G-CSF receptor were localized using a modification of the avidin-biotin complex method. In the newborn rats, IFNgamma r was localized in the preameloblasts in the cervical loop, the proximal and distal ends of presecretory ameloblasts, the outer enamel epithelium, the dental lamina, and in bone. In 5-day-old rats, it was confined to the proximal ends of the presecretory and secretory ameloblasts. The G-CSF receptor was observed in the molars of newborn rats in the preameloblasts, the proximal and distal ends of the presecretory ameloblasts, outer enamel epithelium, and in bone. In 5-day-old rats, G-CSF receptor was localized in the preameloblasts, the proximal ends of presecretory and secretory ameloblasts, the stellate reticulum, the outer enamel epithelium, and in bone. These findings indicate that the IFNgamma r and the G-CSF receptor, and their downstream STP linkages, are upregulated in the cells of the enamel organ and may be involved in the events leading directly to early enamel formation.

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8.

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.

Reversed-phase ion-pair chromatographic analysis of tetracycline antibiotics. Application to discolored teeth.

A high-performance liquid chromatographic method with diode array detection was developed to simultaneously separate tetracycline antibiotics and applied to the analysis of discolored teeth. By a reversed-phase ion-pair chromatographic system using pentanesulfonate as a counter ion, minocycline, oxytetracycline, tetracycline and demeclocycline were eluted in this order, and they showed base-line separation within 9 min. When using oxytetracycline as an internal standard, the quantitative ranges were between 2.5 ng/ml and 7.5 microg/ml. Powdered dentine (10 mg) and enamel (40 mg) prepared from discolored primary teeth were sonicated in 0.25 ml of 10 mM HCl containing oxytetracycline (0.75 microg/ml) and 50 mM EDTA-2Na, thereafter the supernatants were chromatographed. Eluates from both discolored tooth samples were identified as minocycline based on diode array spectra of their peaks, while minocycline was not detected in any samples from nondiscolored normal teeth, indicating that discoloration of the tested teeth was due to minocycline incorporated into dentine and enamel. Replicate quantitative analyses of the identical tooth substances showed that intra- and inter-assay C.V.s were 2.63 and 4.95% for dentine, and 5.42 and 10.88% for enamel. Application of the developed method to nine discolored teeth revealed that the incorporated minocycline ranged from 20.13 to 84.62 ng/mg of dentine and 0.89 to 7.87 ng/mg of enamel.

The ornithine transcarbamylase (OTC) gene: mutations in 50 Japanese families with OTC deficiency.

Mutations in the OTC gene in 50 Japanese families with OTC deficiency were reviewed in relation to the phenotype of the patients and predicted structure of the mutant enzyme. Similar to other X-linked diseases, mutant alleles in OTC deficiency are highly heterogeneous. Mutations observed in male patients with neonatal onset of the disease included base insertion/deletion, exon skipping, and nonsense and missense mutations in exon 4, 5, 6, or 7. OTC activity was essentially undetectable in this group of patients. These mutations possibly resulted in unstable mRNA or truncated protein, or involved the active site or core domain of the enzyme leading to structural changes. In male patients with late onset, abnormalities observed were missense mutations in exons 2, 4, 8, 9, and 10, and missense mutations plus donor site errors involving exons 4, 5, and 6. OTC activity in these patients was 8.1 +/- 6.3% of the control and most mutations occurred on the surface of the protein. In female patients, age at onset ranged from 19 months to 7 years, depending on residual OTC activities (4.5 to 33% of the control). Most mutations in this group were similar to those seen in male patients with neonatal onset, i.e., nonsense and missense mutations in exons 5 and 6, and exon skipping, leading to null enzyme activity. These collective data can serve for genetic counseling and monitoring in prenatal care.

Activation of blood coagulation factor X by arginine-specific cysteine proteinases (gingipain-Rs) from Porphyromonas gingivalis.

The effect of two arginine-specific cysteine proteinases (gingipain Rs) from Porphyromonas gingivalis, a causative bacterium of adult periodontitis, on human blood coagulation was investigated. Activated partial thromboplastin time and prothrombin time were shortened by these proteinases, with a 95-kDa gingipain R containing adhesin domains being 5-fold more efficient in comparison to a 50-kDa gingipain R containing the catalytic domain alone. The 50-kDa enzyme reduced each coagulation time in several plasmas deficient in various coagulation factors, while it was ineffective in factor X-deficient plasma unless reconstituted with this protein. Each proteinase activated factor X in a dose- and time-dependent manner, with Michaelis constants (Km) being found to be lower than the normal plasma factor X concentration, strongly suggesting that factor X activation by gingipain Rs, especially the 95-kDa form which is strongly activated by phospholipids, could occur in plasma. This is the first report of factor X activation by bacterial proteinases and indicates that the gingipain Rs could be responsible for the production of thrombin and, indirectly, with the generation of prostaglandins, interleukin-1, etc., which have been found to be associated with the development of periodontitis induced by P. gingivalis infections. Furthermore, the data support the hypothesis that induction of blood coagulation by bacterial proteinases may be a causative agent in the pathogenesis of disseminated intravascular coagulation in sepsis.

Human glutamate pyruvate transaminase (GPT): localization to 8q24.3, cDNA and genomic sequences, and polymorphic sites.

Two frequent protein variants of glutamate pyruvate transminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a "half-YAC" from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2,7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the results of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of an NlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this world-wide polymorphism.