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E A Azen

Publications and source records attributed to E A Azen.

At least 19 recordsLinked to original sources
PubMed2001

High-resolution genetic mapping of the sucrose octaacetate taste aversion (Soa) locus on mouse Chromosome 6.

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.

Animals↗
PubMed2001

A new gene (rmSTG) specific for taste buds is found by laser capture microdissection.

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.

Amino Acid Sequence↗
PubMed2000

Sucrose octaacetate avoidance in nontaster mice is not enhanced by two type-A Prp transgenes from taster mice.

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.

Animals↗
PubMed1999

Spit is "it".

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Chromosomes, Human, Pair 12↗
PubMed1998

A frequent mutation in the acidic proline-rich protein gene, PRH2, causing a Q147K change closely adjacent to the bacterial binding domain of the cognate salivary PRP (Pr1') in Afro-Americans. Mutations in brief no. 154. Online.

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).

Bacteremia↗
PubMed1997

An expanded histatin gene polymorphism and test of a possible disease resistant phenotype.

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.

Black People↗
PubMed1997

Proline-rich-protein promoters direct LacZ expression to the granular convoluted tubular cells of the submandibular gland in adult transgenic mice.

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.

Animals↗
PubMed1996

PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, II-1, and Po proline-rich proteins.

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.

Alleles↗
PubMed1994

Two coding change mutations in the HIS2(2) allele characterize the salivary histatin 3-2 protein variant.

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.

Alleles↗
PubMed1993

The structure and evolution of the human salivary proline-rich protein gene family.

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.

Alleles↗
PubMed1993

Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family.

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.

Amino Acid Sequence↗
PubMed1993

Genetics of salivary protein polymorphisms.

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.

Alleles↗
PubMed1993

PRBI gene variants coding for length and null polymorphisms among human salivary Ps, PmF, PmS, and Pe proline-rich proteins (PRPs).

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.

Alleles↗
PubMed1992

Chromosome mapping of Soa, a gene influencing gustatory sensitivity to sucrose octaacetate in mice.

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.

Animals↗
PubMed1992

PRB2/1 fusion gene: a product of unequal and homologous crossing-over between proline-rich protein (PRP) genes PRB1 and PRB2.

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.

Base Sequence↗
PubMed1991

Serial cultivation of epithelial cells from human and macaque salivary glands.

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.

Amylases↗
PubMed1990

Structure and sequence determination of the gene encoding human salivary statherin.

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.

Amino Acid Sequence↗