RESUMEN
An acetylated sugar, sucrose octaacetate (SOA), tastes bitter to humans and has an aversive taste to at least some mice and other animals. In mice, taste aversion to SOA depends on allelic variation of a single locus, Soa. Three Soa alleles determine 'taster' (Soa(a)), 'nontaster' (Soa(b)), and 'demitaster' (Soa(c)) phenotypes of taste sensitivity to SOA. Although Soa has been mapped to distal Chromosome (Chr) 6, the limits of the Soa region have not been defined. In this study, mice from congenic strains SW.B6-Soa(b), B6.SW-Soa(a), and C3.SW-Soa(a/c) and from an outbred CFW strain were genotyped with polymorphic markers on Chr 6. In the congenic strains, the limits of introgressed donor fragments were determined. In the outbred mice, linkage disequilibrium and haplotype analyses were conducted. Positions of the markers were further resolved by using radiation hybrid mapping. The results show that the Soa locus is contained in an approximately 1-cM (3.3-4.9 Mb) region including the Prp locus.
Asunto(s)
Cromosomas , Ratones Endogámicos/genética , Sacarosa/análogos & derivados , Sacarosa/metabolismo , Gusto/genética , Animales , Animales no Consanguíneos , Mapeo Cromosómico/métodos , Cricetinae , Femenino , Genotipo , Desequilibrio de Ligamiento , Masculino , Ratones , Ratones Endogámicos C3H/genética , Ratones Endogámicos C57BL , Fenotipo , Umbral Gustativo/fisiologíaRESUMEN
Getting pure populations of taste buds suitable for molecular analysis has hampered the characterization of genes specifically expressed in taste cells. To solve this problem, we prepared specific cDNA libraries from small numbers of taste cells and surrounding epithelium isolated by laser capture microdissection (LCM) and report the discovery of a rhesus monkey novel gene (rmSTG) expressed specifically in taste cells, as found by differential screening of the cDNA libraries and RNA in situ hybridization. RNA in situ hybridization shows the preferential expression of this gene in taste buds from circumvallate, foliate, and fungiform papillae of the tongue. RT-PCR and Northern analysis of RNA from different non-taste organs showed no expression, pointing to a very specialized function of the protein in taste cells. Analysis of extended cDNAs and genomic DNA showed two exons and one intron. Northern analysis of circumvallate papillae showed a transcript of 1.3 kb as established in the gene model. BLAST search analysis showed that the human homolog is localized in the recently completely sequenced HLA class I region of Chromosome 6p21 and is sublocalized to the main susceptibility region for psoriasis vulgaris. The predicted gene encodes a protein of 314 amino acids with an N-terminal signal peptide and cleavage site, suggesting a membrane-bound or secreted protein with an extracellular role in taste cell physiology. The monkey, human, and mouse STG proteins contain potential O-glycosylation sites and tandem repeats inside a region showing approximately 50% similarity with prion proteins.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Primates , Papilas Gustativas/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , Exones , Expresión Génica , Biblioteca de Genes , Glicosilación , Humanos , Hibridación in Situ , Intrones , Rayos Láser , Macaca mulatta , Ratones , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
The Soa bitter-sensitivity and Prp salivary-protein loci map to distal mouse chromosome six. No recombination has been found between sucrose octaacetate (SOA)-avoidance phenotype and PRP haplotype in any mouse population. Soa and Prp, therefore, are either very near each other or identical. To assess the latter possibility, two type-A, proline-rich protein genes (MP2 and M14), situated approximately 30 kb apart at the Prp locus, were separately transferred from an SOA-taster inbred strain (SWR) to an SOA-nontaster inbred strain (FVB). Five MP2-transgenic mice and seven M14-transgenic mice were insensitive to 1 mM SOA in two-bottle tests, thus retaining the nontaster FVB phenotype. Each transgenic mouse was mated to control FVB mice. Their transgene-positive F1 and F2 offspring also were insensitive. Transgene expression varied among the founder lines, but SWR-like expression levels, higher than background FVB expression levels, were found in submandibular gland tissue of adult transgenic mice from two MP2 lines and one M14 line. F3 mice from one of these MP2 lines were mated to F2 mice from the M14 line. Nine offspring inherited both transgenes. All nine were insensitive to 1 mM SOA. These findings indicated that expression of mRNAs for both type-A Prp genes alone or together did not enhance SOA taste sensitivity in nontaster mice.
Asunto(s)
Conducta Animal/fisiología , Péptidos/genética , Sacarosa/análogos & derivados , Gusto/fisiología , Animales , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Dominios Proteicos Ricos en Prolina , TransgenesAsunto(s)
Saliva/fisiología , Cromosomas Humanos Par 12/genética , Complemento C3/genética , Glicoproteínas/genética , Hemólisis/fisiología , Humanos , Péptidos/genética , Fosfoproteínas/genética , Polimorfismo Genético/genética , Dominios Proteicos Ricos en Prolina , Proteínas/genética , Proteínas y Péptidos Salivales/genética , Esferocitosis Hereditaria/genética , Gusto/genética , Gusto/fisiologíaRESUMEN
The human salivary proline-rich proteins (PRPs) are coded by six closely linked genes on chromosome 12p13.2. Two of the PRP genes, PRH1 and PRH2, code for acidic PRPs that are functionally important in binding to teeth surfaces, in maintaining a supersaturated concentration of salivary calcium, and in promoting adherence of potentially pathogenic oral bacteria to the teeth surfaces. A frequent acidic PRP electrophoretic variant is found in Afro-Americans and was presumed to be located in the 44 amino acid carboxy-terminal portion of the full length protein (150 amino acids). This region (and especially the carboxy-terminal dipeptide) may be crucial in promoting bacterial adherence to teeth surfaces. We here report, by DNA sequence analysis, a C-->A(2957) transversion that causes a Q147K change in the PR1' protein. The mutation is located two residues from the carboxy-terminal dipeptide and thus might influence its bacterial adherence property. We also report a PCR-based assay for the C-->A(2957) mutation and show its applicability in an Afro-American population (n=61) where the allele frequency (as determined from phenotyping of saliva proteins and PCR DNA analysis) was found to be notably high (0.16).
Asunto(s)
Bacteriemia/genética , Población Negra/genética , Mutación/genética , Péptidos/genética , Proteínas y Péptidos Salivales/genética , Cromosomas Humanos Par 12/genética , Humanos , Prolina , Dominios Proteicos Ricos en Prolina , Proteínas Salivales Ricas en ProlinaRESUMEN
The ability of two mouse PRP gene promoters to direct the expression of the bacterial lacZ reporter gene was tested in transgenic mice. Transgenes A1-lacZ and C1-lacZ consisted of 8.2 kb A1 and 7.8 kb C1 PRP promoters respectively fused to the lacZ coding sequence. A1 and C1 are two A-type PRP genes isolated from the inbred SWR mice, which show the same gene structure and similar sequence to the closely related MP2 and M14 PRP genes previously cloned from outbred CD-1 mice. We here show that both A1-lacZ and C1-lacZ transgenes have very similar expression patterns: (1) they expressed the lacZ gene in all 14 established transgenic lines under normal (non-stimulated) conditions; (2) the expression was restricted to the granular convoluted tubular cells of the submandibular glands; (3) the expression was developmentally regulated beginning at sexual maturation and lasting to at least 1.5 years of age; and (4) expression in some lines was probably influenced by sex hormones, since higher expression was found in males than in females. A1-lacZ and C1-lacZ are the first transgenes derived from the PRP/GRP (glutamine/glutamic acid-rich protein) gene superfamily to be expressed in the granular convoluted tubular cells (with known endocrine functions), rather than in the acinar cells (with mainly exocrine functions) of the submandibular glands.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Operón Lac , Prolina/metabolismo , Regiones Promotoras Genéticas , Glándula Submandibular/metabolismo , Animales , Células Cultivadas , Clonación Molecular , Femenino , Hormonas Esteroides Gonadales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factores Sexuales , Glándula Submandibular/citología , Transgenes , beta-Galactosidasa/metabolismoRESUMEN
Histatins are small molecular weight salivary proteins that are important in the non-immune host defense system. Two frequent cis-linked coding-change mutations were previously described in exon 5 of the HIS2 gene of Blacks. The polymorphic mutant allele was termed HIS2(2) and the wild-type allele HIS2(1). We here describe two new non-coding change polymorphisms of the HIS2 gene: a deletion in intron 5 (7183-7198 del) and a C-->T mutation in exon 5 [C-->T (7104)] that characterize two new HIS2 alleles, HIS2(3) and HIS2(4) respectively. Both mutations occur on a HIS2(1) background. The HIS2(3) allele occurred only in Afro-Americans, but not in 67 Japanese, 51 Chinese and 50 Whites. Among 66 random DNA samples from Afro-Americans, frequencies of HIS2(1), HIS2(2), HIS2(3) and HIS2(4) were 0.67, 0.22, 0.05 and 0.07 respectively, with a heterozygosity of 0.45. The frequencies of the HIS2(4) allele in 50 Whites and 50 Chinese were 0.06, and 0.1 respectively. In a comparison of 60 matched saliva and DNA samples from the Afro-American population, the DNA-based mutation analysis reliably identified salivary histatin phenotypes. The salivary histatin polymorphism (inferred from PCR analysis) was used to test a biologically plausible hypothesis, that the mutant histatin phenotype (coded by the HIS2(2) allele) confers relative resistance to severe and fatal malaria. In a study of 185 Black Tanzanian subjects, there were no significant differences in HIS2(2) allelic frequencies between the various test groups: for 86 cerebral malaria subjects, 54 uncomplicated malaria subjects, and 45 combined asymptomatic parasitemia and health controls, HIS2(2) frequencies were 0.16, 0.17 and 0.17 respectively. Thus, there was no support for the hypothesis in this population.
Asunto(s)
Malaria Cerebral/genética , Polimorfismo Genético , Proteínas/genética , Proteínas y Péptidos Salivales/genética , Población Negra/genética , Niño , Preescolar , Frecuencia de los Genes/genética , Genotipo , Humanos , Inmunidad Innata/genética , Lactante , Malaria Cerebral/inmunología , Mutación , Fenotipo , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , TanzaníaRESUMEN
Six closely linked PRP (proline-rich protein) genes code for many salivary PRPs that show frequent length and null variants. From determined protein sequences and DNA sequence analysis of variant alleles, we here report the coding and molecular basis for Con (concanavalin A-binding) and Po (parotid "o") protein polymorphisms. The Con1 glycoprotein is encoded in exon 3 of a PRB2 allele (PRB2L CON1+) with a potential N-linked glycosylation site. Because of a probable gene conversion encompassing > or = 684 bp of DNA, the "PRB2-like" Con2 glycoprotein is encoded in exon 3 of a PRB1 allele (PRB1M CON2+) with a potential glycosylation site. The PmF protein is also encoded in the PRB1M CON2+ allele, thus explaining the previously reported association between Con2 and PmF proteins. A PRB2L CON1 allele contains a single nt missense change [TCT(Ser)-->CCT (Pro)] that abolishes the potential N-linked glycosylation site (NKS-->NKP) in the Con1 protein, and this explains the Con- type. The Po protein and a glycoprotein (II-1) are encoded in the PRB4 gene, and both proteins are absent in the presence of a mutation in the PRB4M PO- allele that contains a single nt change (G--C) at the +1 invariant position of the intron 3 5'donor splice site. The genetically determined absence of the II-1 glycoprotein leads to altered in vitro binding of Streptococcus sanguis 10556 to salivary proteins, which suggests a biological consequence for null mutations of the PRB4 gene.
Asunto(s)
Variación Genética , Hominidae/genética , Péptidos/genética , Polimorfismo Genético , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Concanavalina A/metabolismo , Cartilla de ADN , Exones , Femenino , Glicosilación , Humanos , Masculino , Datos de Secuencia Molecular , Núcleo Familiar , Linaje , Biosíntesis de Péptidos , Reacción en Cadena de la Polimerasa , Dominios Proteicos Ricos en Prolina , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/biosíntesis , Proteínas y Péptidos Salivales/genética , Homología de Secuencia de AminoácidoRESUMEN
The decoded amino acid sequence of a salivary protein variant, histatin 3-2 (formerly termed Pb c), that is found primarily and in high frequency in Black populations was determined by genomic PCR and direct sequencing of the HIS2(2) allele. Two different mutations that cause coding changes were found in exon 5. The first mutation is a single nucleotide (T-->A) substitution that causes a TAT (Tyr)-->TAA (Stop) change at residue 28. This premature stop mutation results in a 27 amino acid histatin 3-2 protein, which is 5 amino acids smaller than the common histatin 3-1 allelic protein (a product of the HIS2(1) allele). The second mutation, a single nucleotide (G-->A) substitution (located only 19 nucleotides upstream of the first mutation) causes a CGA (Arg)-->CAA (Gln) change at residue 22, which eliminates a proteolytic cleavage site. These two mutations explain the differences in electrophoretic patterns of HIS2(1) versus HIS2(2) coded histatin peptides and may have functional significance. Each mutation alters a different DNA restriction site, and this provides a DNA-based test for the mutations. This test should greatly simplify population and family studies of this protein polymorphism, since the saliva-based test is considerably more problematic. Elucidation here of the derived protein sequence of the variant histatin 3-2 protein may also facilitate functional studies.
Asunto(s)
Mutación Puntual , Proteínas/genética , Proteínas y Péptidos Salivales/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Población Negra/genética , Cartilla de ADN/genética , Enzimas de Restricción del ADN , Variación Genética , Histatinas , Humanos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Six closely linked PRP (proline-rich protein) genes code for salivary PRPs that show frequent length and null polymorphisms. We report assignment of Ps proteins to the PRB1 gene, the derived primary structures of Ps 1 and Ps 2 proteins, and the molecular basis for some null alleles among PRB1-coded PRPs (Ps, PmF, PmS, and Pe). The derived primary structures of Ps 1 and Ps 2 proteins were determined by sequencing exon 3 of the different-length PRB1M (medium) and PRB1L (large) copies from subject C.J. with the Ps 1-2 phenotype. The PRB1L copy (coding for Ps 2) contained three additional tandem repeats within the Ps coding region, and the different-length Ps 1 and Ps 2 proteins can be explained on this basis. The molecular basis for the Ps 0 and the Pe- phenotypes was determined in another individual (M.V.O., a PRB2/1 fusion-gene heterozygote) with a single PRB1L copy. A premature stop mutation (CGA [Arg]-->TGA [stop]) occurred at residue 61 in the Ps-coding region. The identical mutation was found in the PRB1L and PRB1/2S (small) copies of a second individual (E.A.) with reduced Pe protein and the Ps 0 phenotype. This individual is a PRB1/2 fusion-gene heterozygote (Azen et al. 1992) with probably three mutated PRB1 copies (PRB1L-PRB1L-PRB1/2S). DNA sequences of the postulated crossover region of the PRB1/2S fusion-gene copy supported the postulated crossover. The PmF- and PmS- phenotypes in the three subjects were due to both the stop mutation and the lack of suitable proteolytic cleavage sites in the PRB1-coded precursor proteins.
Asunto(s)
Péptidos/genética , Polimorfismo Genético , Proteínas y Péptidos Salivales/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Exones , Humanos , Datos de Secuencia Molecular , Fenotipo , Dominios Proteicos Ricos en Prolina , Homología de Secuencia de Ácido NucleicoRESUMEN
Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.
Asunto(s)
Evolución Biológica , Familia de Multigenes , Fosfopéptidos/genética , Proteínas/genética , Proteínas y Péptidos Salivales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Exones , Biblioteca Genómica , Histatinas , Humanos , Intrones , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
We present the nucleotide sequences of four members of the six-member human salivary proline-rich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3' end of IVS2 and the 3' end of exon 3.
Asunto(s)
Evolución Biológica , Familia de Multigenes , Péptidos/genética , Proteínas y Péptidos Salivales/genética , Alelos , Secuencia de Bases , Exones , Conversión Génica , Reordenamiento Génico , Humanos , Intrones , Datos de Secuencia Molecular , Prolina , Dominios Proteicos Ricos en Prolina , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de SecuenciaRESUMEN
Human salivary PRPs are determined by six closely linked genes on chromosome 12p13.2. The many PRPs show complex electrophoretic patterns that differ between individuals and reflect numerous genetic polymorphisms. Frequent length and null polymorphisms are common among PRPs. Common themes emerge as a background for these PRP polymorphisms. First, posttranslational proteolysis occurs with double-banded patterns among acidic PRPs and the generation of numerous basic PRPs derived from precursor proteins. Specific mutations may interfere with proteolysis, preventing generation of double-banded acidic PRPs (as with the Pa protein) or of small basic PRPs from precursor proteins (as with Pm proteins). Second, single cysteine substitutions in PRPs (Pa from PRH1 and G1 8 from PRB3) may lead to disulfide bonded homodimers as well as heterodimers with salivary peroxidase. Third, frequent homologous and unequal crossing-over within the PRP gene cluster leads to frequent protein size-variants (intragenic events as with the G1 protein variants) and the generation of the PRB2/1 fusion gene (intergenic event) with deletion of the PRB1 coding region and absence of multiple PRB1 coded proteins (Ps, Pm, Pe) in PRB2/1 homozygotes. Fourth, null mutations may also be produced (as with PsO and G1 0) by single nucleotide changes.
Asunto(s)
Polimorfismo Genético , Proteínas y Péptidos Salivales/genética , Alelos , Secuencia de Aminoácidos , Humanos , Péptidos/genética , Prolina/genética , Dominios Proteicos Ricos en Prolina , Proteínas Salivales Ricas en ProlinaRESUMEN
Strain distribution patterns among recombinant inbred strains suggested that a locus influencing taste sensitivity to sucrose octaacetate was on chromosome 6. A location for Soa was established by linkage analysis of behavioral and electrophoretic data from outbred and congenic strains and from test-cross progeny. Haplotyping of 41 outbred CFW-Cr animals with a cDNA probe showed perfect cosegregation of Soa and Prp, a gene for salivary proline-rich proteins. Five of twelve B6.SW-Soaa strains were found to retain Ldr-1, lactate dehydrogenase regulator-1, on chromosome 6 as an allelic passenger from the SWR/J donor strain (source of the Soaa Taster allele). Centimorgan distance was estimated using the ABP/Le linkage-testing strain (non-Taster, Soab) and the SWR/J strain (Taster, Soaa) in a testcross breeding system. The data are consistent with a position for the Soa locus on mouse chromosome 6, 62 cM from the centromere.
Asunto(s)
Mapeo Cromosómico , Ratones Endogámicos/genética , Recombinación Genética/genética , Sacarosa/análogos & derivados , Gusto/genética , Animales , Cruzamientos Genéticos , Femenino , Ligamiento Genético/genética , Marcadores Genéticos/genética , Masculino , Ratones , Modelos Genéticos , Fenotipo , Umbral Gustativo/fisiologíaRESUMEN
The PRB2/1 fusion gene is produced by homologous and unequal crossing-over between PRB1 and PRB2 genes that code for basic salivary proline-rich proteins (PRPs). To determine the molecular basis for the PRB2/1 fusion gene, the DNA sequence was determined for the PRB2/1 gene and was compared with those of the PRB1 and PRB2 genes. From these comparisons, the crossing-over is postulated to occur in a 743-bp region of identity, with only 1-bp mismatch between the PRB1 and PRB2 genes, in the third intron outside the coding region of the two genes. This region of virtual complete identity is the largest found between any of the six closely linked PRB genes and may facilitate recombination. Since the coding region of PRB1 is completely absent from the PRB2/1 gene, salivas from two white PRB2/1 homozygotes were studied to determine which polymorphic PRPs were missing from the salivas. Polymorphic PRPs Pe, PmF, PmS, and Ps were found to be missing from the salivas. However, a white individual lacking the same salivary PRPs is a PRB2/1 heterozygote with one PRB1 allele. The explanation for the missing salivary proteins in this individual is unknown. The PRB2/1 gene is relatively frequent in several populations of unrelated individuals, including American blacks (n = 41), American Utah whites (n = 76), and mainland Chinese (n = 131), with gene frequencies of .22, .06, and .09, respectively. Evidence for the occurrence of PRB1/2 heterozygotes is also presented.
Asunto(s)
Intercambio Genético/genética , Péptidos/genética , Proteínas y Péptidos Salivales/genética , Secuencia de Bases , Southern Blotting , Deleción Cromosómica , Tamización de Portadores Genéticos , Homocigoto , Humanos , Datos de Secuencia Molecular , Dominios Proteicos Ricos en Prolina , Mapeo RestrictivoRESUMEN
To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions conditioned to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.
Asunto(s)
Glándulas Salivales/citología , Amilasas/genética , Amilasas/metabolismo , Animales , Northern Blotting , Calcio/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Toxina del Cólera/farmacología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Hidrocortisona/farmacología , Insulina/farmacología , Macaca , Glándula Parótida/citología , Glándula Parótida/efectos de los fármacos , Glándula Parótida/enzimología , Péptidos/genética , Péptidos/metabolismo , Dominios Proteicos Ricos en Prolina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/enzimología , Glándula Submandibular/citología , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/enzimología , Factores de TiempoRESUMEN
A search was made for expression of genes for proline-rich proteins (PRPs) and other salivary-type proteins, including statherin and histatins, in taste-bud tissues of mice and primates because of previous genetic findings in mice (Azen et al., 1986) that Prp and taste genes for certain bitter substances are either the same or closely linked. Taste-bud tissues and other tissues were tested for specific mRNAs with labeled DNA probes by Northern blotting and in situ hybridization. It was found that PRP mRNAs were present in von Ebner's glands of mice and macaques, and that there was a much greater degree of PRP mRNA induction in mouse parotid (16-fold) than in von Ebner's gland (two-fold) after in vivo isoproterenol stimulation. This difference may be due, in part, to differences in autonomic nerve innervation. Statherin and histatin mRNAs were found in macaque taste-bud tissues containing von Ebner's gland, and statherin protein was found in human von Ebner's gland by immunohistochemistry. The finding of PRP gene expression in von Ebner's gland, whose secretions have been suggested to play a role in taste stimulation, adds further support to a possible function of PRPs in bitter tasting. The possible functions of statherin and histatins in von Ebner's gland secretions may be related to statherin's regulation of salivary calcium and histatins' antibacterial and antifungal properties.
Asunto(s)
Péptidos/genética , Proteínas/genética , ARN Mensajero/análisis , Glándulas Salivales/química , Proteínas y Péptidos Salivales/genética , Papilas Gustativas/química , Lengua/química , Animales , Northern Blotting , Sondas de ADN , Humanos , Inmunohistoquímica , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Péptidos/análisis , Dominios Proteicos Ricos en Prolina , Proteínas/análisis , Proteínas y Péptidos Salivales/análisis , Gusto/genética , Gusto/fisiología , Lengua/anatomía & histologíaRESUMEN
From electrophoretic analysis, we identified in the saliva of an Ashkenazi Jew a disulfide-bonded major glycoprotein variant (Gl 8) that is a product of the proline-rich protein (PRP) locus PRB3. A previous study of this variant protein misidentified it as Pa 2 and as a product of a different PRP locus. The other PRB3 allele in this individual is an apparent null. To identify the mutations, we sequenced the tandemly repetitious exon 3 (the major protein-coding portions) of both alleles. A CGT----TGT (Arg----Cys) mutation was found in one allele (PRB3Scys), which accounts for the disulfide-bonded and peroxidase-modifying properties of Gl 8. A single nucleotide insertion was found in the other allele (PRB3Mnull) that leads to a frameshift with a premature termination codon that causes an apparent lack of gene expression. Null alleles are frequent at PRP loci coding for basic and glycosylated PRPs, and the mechanism described might explain other null phenotypes among PRPs. From nucleotide comparisons, a model of intragenic unequal crossing-over is proposed to explain, in part, the generation of the PRB3Mnull allele. The Gl 8 protein variant is found in Ashkenazi Jews (gene frequency around .008) but not in the general white, black, or Japanese populations. It is interesting that products of different PRP genes, Gl 8 from PRB3 and Pa 1 from PRH1, are both disulfide bonded and probably modify salivary peroxidase (part of an important intraoral antibacterial system) through formation of disulfide-bonded heterodimers.
Asunto(s)
Expresión Génica , Péptidos/genética , Proteínas y Péptidos Salivales/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , Disulfuros , Electroforesis en Gel de Poliacrilamida , Exones , Mutación del Sistema de Lectura , Humanos , Judíos/genética , Masculino , Datos de Secuencia Molecular , Peroxidasa/metabolismo , Fenotipo , Prolina/metabolismo , Dominios Proteicos Ricos en Prolina , Saliva/enzimología , Proteínas Salivales Ricas en ProlinaRESUMEN
Human statherin (STT) is a low-Mr (43 amino acids) acidic phosphoprotein secreted mainly by salivary glands. It acts as an inhibitor of precipitation of Ca.phosphate salts in the oral cavity. DNA (12.2 kb) was isolated from human genomic phage lambda libraries as a series of overlapping clones, and the nucleotide sequence of the STT-encoding gene (STT) was determined. The transcribed region spans 6.5 kb and contains six exons and five introns. Upstream DNA (1.6 kb) was also sequenced and a number of possible regulatory elements were identified. The exon-intron boundaries of the STT gene roughly coincide with the protein-coding regions of the mRNA and with the functional domains of STT. This pattern of organization has been seen in a variety of eukaryotic genes and is consistent with the domain theory of gene evolution.