Biomedical subjects

D K Ann

Publications and source records attributed to D K Ann.

At least 37 records · Page 2Linked to original sources

PubMed1995

Hamster monomorphic arylamine N-acetyltransferase: expression in Escherichia coli and purification.

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC. The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of a novel protein regulated by opioid treatment of NG108-15 cells.

A new cDNA clone, NGD5, has been identified from a subtraction cDNA library constructed with mRNA isolated from control neuroblastoma x glioma NG108-15 cells and cells treated for 48 h with the delta-opioid agonist, D-Ala2, D-Leu5 enkephalin (DADLE). NGD5 mRNA is decreased, in a naloxone-reversible manner, upon long-term treatment of NG108-15 cells with DADLE, indicating that this clone may be related to opioid receptor function. Northern analysis indicates that NGD5 mRNA is expressed in rat brain. Two similar nearly full-length NGD5 clones, NGD5A and NGD5B, were isolated from a lambda gt10 NG108-15 cDNA library and sequenced. The predicted 40-kDa peptide encoded for by the NGD5 cDNA has no significant homology to the recently cloned mu, delta or kappa opioid receptors nor to any other known proteins.

Amino Acid Sequence↗

PubMed1994

Cloning of human and mouse brain cDNAs coding for S1, the second member of the mammalian elongation factor-1 alpha gene family: analysis of a possible evolutionary pathway.

We previously reported the cloning of a rat S1 cDNA whose deduced amino acid sequence shares high similarity (92%) with that of mammalian elongation factor-1 alpha (EF-1 alpha), a protein involved in the binding of aminoacyl-tRNA to the ribosome during peptide synthesis. We report here the isolation of a full-length cDNA from a mouse brain library and a partial-length cDNA from a human hippocampus library which share extensive sequence similarity to rat S1 cDNA. We show that, as with mammalian EF-1 alpha S, the predicted primary amino acid sequences of rat, mouse, and human S1 are almost identical, except for one conservative substitution. These results indicate that mouse and man contain a second member of the EF-1 alpha gene family, the S1 gene. They also suggest that our result obtained in rat may be extrapolated to mouse and man.

Amino Acid Sequence↗

PubMed1994

Control of keratin gene expression by vitamin A in tracheobronchial epithelial cells.

Vitamin A (retinol) treatment induces (and/or enhances) mucous cell differentiation and alters keratin gene expression in cultured airway epithelial cells of human and nonhuman primate origin. We observed that retinol greatly reduced the synthesis of keratins 5, 6, 14, 16, and 17, but slightly enhanced keratins 7, 8, 10, 13, 15, 18, and 19. These changes were also reflected at the mRNA level as demonstrated by cell-free translation and by cDNA cloning of human keratin genes based on differential hybridization. One of these cDNA clones, HT27, isolated from the cDNA library of human tracheobronchial epithelial cells and whose expression in cultured cells was greatly suppressed by retinol, had a nucleotide sequence identical to the C-terminus of keratin 16. The identity of this clone was further confirmed by Western blot analysis using an antibody specific to the 15-amino acid synthetic peptide and the C-terminal sequence. Using this cDNA clone and two known keratin clones, pKA1 (keratins 5 and 6) and pKB2 (keratin 14), we found the levels of these corresponding mRNAs in cultured cells to be reduced 10- to 25-fold after treatment of cells with vitamin A. The inhibition was time- and dose-dependent with respect to retinol and was sensitive to prior treatment with cycloheximide. However, nuclear run-on transcriptional assays revealed no significant reduction of the synthesis of these messages in retinol-treated cultures. Furthermore, no change in the half-life of these mRNAs was observed in cells after the retinol treatment. Based on these results, we conclude that vitamin A indirectly controls the synthesis of these keratins at the post-transcriptional level.

Amino Acid Sequence↗

PubMed1993

The helix-loop-helix proteins (salivary-specific cAMP response element-binding proteins) can modulate cAMP-inducible RP4 gene expression in salivary cells.

Salivary-specific and cAMP-inducible expression of the rat proline-rich protein gene RP4 is dependent on a 28-base pair sequence of a salivary-specific cAMP response element (SCRE) (Lin, H. H., and Ann, D. K. (1992) Gene Expression 2, 365-377). To unravel its trans-acting factor(s), we used double-stranded oligoprobes corresponding to the SCRE to screen a randomly primed lambda gt11 cDNA expression library made from RNA of rat salivary cells. In this report, we describe the cDNA cloning of these helix-loop-helix SCRE-binding proteins (SCBPs) and demonstrate that there are at least three isoforms in salivary cells, namely SCBP alpha, SCBP beta, and SCBP gamma. RNA polymerase chain reaction and sequence analyses further confirmed the existence of these three different SCBP isoforms, which code for putative proteins of 707, 706, and 682 amino acids, respectively. Expression of the cloned SCBP cDNAs in salivary cells stimulates the expression of a cotransfected reporter construct containing multicopies of the SCRE cloned upstream of the thymidine kinase promoter and the chloramphenicol acetyltransferase structural gene. This stimulation is much more pronounced in transfections in which SCBP alpha and SCBP beta are cotransfected than when they are transfected individually. Furthermore, when low concentrations of SCBP alpha and SCBP beta are cotransfected with the SCRE reporter gene, coexpression of the catalytic subunit of protein kinase A is required to efficiently activate the expression of the reporter gene. These results strongly suggest that the observed stimulation of the SCRE is achieved through the coordinated expression of the SCBP alpha, SCBP beta, and protein kinase A activities, perhaps via a direct association of the two SCBPs and their phosphorylation by protein kinase A. We conclude that the isolated SCBP alpha and SCBP beta cDNAs encode transcription activators that participate in the control of the inducible RP4 gene expression in salivary cells.

Amino Acid Sequence↗

PubMed1993

Macaque salivary proline-rich protein: structure, evolution, and expression.

Proline-rich proteins are a family of proteins that exhibit unique features including an unusual high proline content and salivary-specificity. As a major constituent in the salivary secretion of higher primates, proline-rich proteins may have biological roles in oral lubrication and protection. In this article, the genomic structure and regulation by cAMP of one of the macaque salivary proline-rich protein genes, MnP4, is reviewed. The evolution of this multigene family of proteins is also discussed.

Amino Acid Sequence↗

PubMed1993

Isoproterenol/tannin-dependent R15 expression in transgenic mice is mediated by an upstream parotid control region.

Transgenic mice were used to locate the cis-acting DNA elements that are essential for tissue-specific and inducible expression of the rat proline-rich protein gene, R15. Chimeric genes with up to 10 kb of R15 5'-flanking region fused to chloramphenicol acetyltransferase (CAT) or polyomaviral large T-antigen (PyLT) reporter genes were tested. Our results demonstrate that (1) the isoproterenol/tannin-inducible, parotid-specific transgene expression requires an upstream cis-regulatory domain, namely the parotid control region, which extends from -6 to -1.7 kb of the R15 gene; (2) this parotid control region functions with a heterologous promoter and is indispensable for achieving a reproducible chromosomal position-independent transgene expression; (3) deletion of the R15 5'-flanking region up to -1.7 kb results in a pleiotropic effect on the transgene expression, which includes ectopic (nonsalivary) reporter expression and lack of inducibility by either the beta-agonist isoproterenol or dietary tannin stimulation; (4) when the -10 to -6 kb region from the R15 gene is deleted in the construct, the inducible expression in the parotid glands of the transgenic mice decreases by over 30-fold, but position-independent and tissue-specific transgene expression is retained. Moreover, the mechanism of induction by either catecholamine isoproterenol or dietary tannin appears to be through a beta 1-adrenergic receptor-mediated pathway for both normal (non-transgenic) and transgenic animals.

Animals↗

PubMed1992

Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.

Amino Acid Sequence↗

PubMed1992

Characterization of the statin-like S1 and rat elongation factor 1 alpha as two distinctly expressed messages in rat.

Previously, we reported a rat S1 protein that is antigenically related to statin, a nonproliferating cell-specific marker; however, it shares high homology with the known human elongation factor-1 alpha (EF-1 alpha). To differentiate S1 from rat EF-1 alpha and to study their respective regulation for expression, a rat EF-1 alpha cDNA clone was isolated and characterized. The nucleotide and deduced amino acid sequences of this partial rat EF-1 alpha cDNA are compared with that of human and mouse as well as with rat S1. Both their messages were detected in rat brain by EF-1 alpha- or S1-specific probes. However, the mRNA encoding EF-1 alpha is more abundant than that encoding S1. S1 and EF-1 alpha expression were investigated in the parotid and submandibular glands of untreated rats and those treated with isoproterenol, a proliferation-inducing catecholamine. Quantitative solution hybridization demonstrated a dramatic reduction (approximately 68%) in the S1 mRNA following isoproterenol injection in proliferation-responsive parotid glands and a mild reduction (approximately 20%) of S1 steady-state messages in the proliferation-refractile submandibular glands. A slight increase or no changes of EF-1 alpha levels in both parotid and submandibular glands following isoproterenol treatment are also observed. Therefore, the EF-1 alpha and S1 genes are different genes, both expressed and regulated in vivo, but in differential quantitative and qualitative patterns.

Amino Acid Sequence↗

PubMed1992

Reciprocal expression of c-jun, proline-rich protein and amylase genes during rat parotid salivary gland development.

We have investigated the temporal expression and cellular localization of the c-jun proto-oncogene and two major rat parotid gland secretory protein genes, PRP (proline-rich protein) and amylase, during postnatal development. c-jun mRNA steady-state levels increased at days 1, 7 and 14 after birth and decreased to basal levels at 21 days and older. PRP mRNA was first detected at 14 days and abruptly increased to adult levels at day 21. Amylase transcripts were first seen at day 7 and progressively increased to adult levels by 28 days. In situ hybridization demonstrated c-jun mRNA accumulation in the differentiating acinar cells and the ducts. The c-jun mRNA accumulation with time corresponds with the proliferative activity reported to occur in these two cellular populations. PRP transcripts were present exclusively in the well differentiated acinar cells while the accumulation of amylase mRNA corresponded to the progressive commitment of parotid cells to acinar differentiation. Our data suggest that during the postnatal development of the rat parotid gland: (a) c-jun expression associates with parotid gland proliferation and precedes the expression of PRP and amylase genes, and (b) activation of PRP and amylase genes is not concomitant and apparently occurs only in differentiating acinar cells.

Age Factors↗

PubMed1992

Identification of cis- and trans-acting factors regulating the expression of rat salivary-specific RP4 gene.

The molecular basis of tissue-specific and cyclic AMP (cAMP)-inducible gene expression in salivary glands is not well understood. Previously, we cloned a salivary-specific proline-rich protein gene, RP4. To analyze the cis-regulatory element(s) that mediates the regulation of this rat salivary RP4 gene, chimeric pRP4CAT constructs containing up to 1.7 kb of the 5'-flanking region of RP4 fused to a reporter gene were transiently transfected into salivary cells. Deletion studies suggest that a 159 bp (-147/+12) fragment of the RP4 5'-flanking region is sufficient to confer salivary-specific induction by agents that can raise intracellular cAMP concentration. Further delineation of this essential sequence revealed that a segment from -136 to -109 is necessary and sufficient to confer cAMP responsiveness in a salivary-specific manner when linked to a heterologous promoter. However, this 28 bp fragment (-136/-109) does not contain an identical match to the consensus cAMP response element (CRE). DNA mobility shift binding assays establish that a sequence-specific DNA-protein complex is formed between this DNA fragment and nuclear proteins from salivary cells, but not with nuclear proteins from HeLa cells, which contain canonical CRE binding proteins (CREBs). Taken together, these data demonstrate that we have identified a 28 bp cis-regulatory element in the RP4 gene that mediates salivary-specific cAMP-inducible gene expression. We propose that the novel salivary-specific CRE binding protein (SCBP) is a key regulator for salivary cAMP-inducible gene expression.

Animals↗

PubMed1991

Sequence and expression of the MnP4 gene encoding basic proline-rich protein in macaque salivary glands.

We report here the macaque MnP4 cDNA and genomic sequences which encode a basic proline-rich protein (PRP), which is synthesized in macaque parotid gland and submandibular gland. The locations of intron positions and the prototype of the tandem 20-amino-acid repeat motif with the sequence, PPPPGKPQGPPQQGGNKPQG, in MnP4, were compared to those in related genes encoding PRP and glutamic/glutamine-rich proteins (GRP) in humans and rodents. Exceedingly high homology of the first exon and 40-bp region immediately upstream of exon I is observed with other PRP genes of all species studied. In order to identify the regulatory elements involved in control of MnP4 gene expression, a rat submandibular gland-derived cell line (RSMT-A5) was transfected with MnP4-cat constructs that contained the promoter and 5'-flanking regions of the macaque MnP4 gene fused to the bacterial cat gene. Deletion analysis revealed that putative positive and negative regulatory elements reside between nucleotides (nt) -107 and +5, and nt -586 and -108, respectively. As part of this study, the promoter of the macaque MnP4 gene appears to be salivary gland specific. This salivary gland-specific gene expression attests to the complexity of transcriptional regulation in eukaryotes.

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin.

Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in growth-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation factor 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than 20% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse fibroblasts, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions -1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Molecular characterization of rat multigene family encoding proline-rich proteins.

Three members of the rat proline-rich protein multigene family have been characterized. Each of these genes, RP4, RP13, and RP15, contains three exons and they are approximately 4.8, 5.7, and 5.4 kb, respectively. The DAN sequences of RP4 and RP13 are greater than 93% homologous in the 3.1-kb segment extending from the 5'-upstream region (approximately nucleotide -930) to 238 nucleotides after the second exon/intron junction; however, regions further downstream, intron II and exon III, share less than 43% identity. In contrast, exon III from RP15, RP13, and the previously sequenced mouse PRP gene MP2 are more than 73% conserved. These analyses suggest that the duplication of the ancestral genes to RP13 and RP4 occurred prior to the divergence of the rat PRP genes. The results also indicate that in the past 21.5 million years, multiple recombination events have resulted in a very high degree of divergence among intron II and exon III of RP4 and RP13. This divergence is due in part to the insertion of members of the rat long interspersed repeat DNA family at -930 bp upstream from the transcription initiation site and within intron II of RP13. Comparisons of the nucleotide sequences and organization of exon I with the genomic organization of PRP and glutamic acid/glutamine-rich protein genes in this and previous studies reveal striking resemblance among these genes. These observations are consistent with the notion that this super multigene family arose from duplication of progenitor genes via unequal crossing over events. In addition, the results suggest that concerted evolution has occurred within the tandemly repeated motif of exon II.

Amino Acid Sequence↗

PubMed1991

Isoproterenol downregulation of statin-related gene expression in the rat parotid gland.

Statin, a 57 kilodalton (kDa) nuclear protein, is characteristically found in nonproliferating cells in culture as well as nondividing cells of a wide range of highly differentiated tissues. Moreover, cells in culture that are statin positive lose this statin expression when re-entering the cell-cycle traverse. In this work, statin expression was investigated in the parotid gland of untreated rats and those treated with isoproterenol (IPR), a proliferation-inducing catecholamine. Indirect immunofluorescence microscopy revealed specific nuclear staining with anti-statin monoclonal antibody (S-44) in the acinar and ducts cells of the untreated rats but significantly reduced in those induced with isoproterenol. To characterize the protein recognized by S-44, protein extracts from both tissues were immunoblotted and incubated with S-44. The antibody reacted specifically with a 48 kDa protein in the extract of the parotid glands from untreated rats while no reaction was detected in that of the proliferation-induced ones. These observations along with the result that a statin-like (S1) transcript is downregulated by isoproterenol in the parotid glands further support the notion that the disappearance of statin-related expression is associated with the IPR-induced proliferation in the rat parotid glands. The discrepancy between the apparent molecular mass of the protein identified by S-44 in nonproliferating parotid cells and that of statin originally found in fibroblasts, suggests that either a modified form of statin may be present in the parotid gland, or this 48 kDa protein may be a member of the nonproliferative statin-like family.

Animals↗

PubMed1988

Molecular evolution of the mouse proline-rich protein multigene family. Insertion of a long interspersed repeated DNA element.

Proline-rich proteins (PRPs) in the salivary glands of mice, rats, and hamsters are encoded by tissue-specific inducible multigene families. Mouse PRP genes are located on chromosome 8, and transcription is dramatically induced (about 70-fold) by isoproterenol treatment. Clones containing two nonallelic PRP genes (MP2 and M14) were isolated from cosmid and phage libraries of CD-1 mouse genomic DNA. The cloned regions comprise a contiguous block of 77 kilobase pairs of the mouse genome. Restriction mapping established the physical lineage of PRP genes MP2 and M14, and they are tandemly arrayed. The DNA sequence analysis presented in this report suggests that genes M14 and MP2 (Ann, D. K., and Carlson, D. M. (1985) J. Biol. Chem. 260, 15863-15872) arose via a gene duplication of a common ancestor. Two major differences between M14 and MP2 were observed. PRP gene MP2 has 13 tandemly arrayed 42-nucleotide repeats in exon II, whereas M14 has 17 repeats, and PRP gene M14 has an insertion by transposition of a 2-kilobase pair member of the long interspersed repeated DNA (LINE) family (LIMd) into intron I. The evolution of this PRP multigene family has been dominated by intra-exonic amplification of repeating nucleotide units coding for these and other proline-rich repeated peptides and by gene duplication. The LIMd element gives rise to heterogenous EcoRI, BamHI, and HindIII restriction enzyme patterns, and this insertion is also present in BALB/c, C57BL/6J, and DBA/2J mice.

Amino Acid Sequence↗

PubMed1988

Retinol-regulated gene expression in human tracheobronchial epithelial cells. Enhanced expression of elongation factor EF-1 alpha.

Conducting airway epithelial cells requires vitamin A or its synthetic chemicals (retinoids) for their survival and for the expression of normal mucociliary functions. By using molecular cloning, we have shown that one of the effects of retinol on cultured human tracheobronchial epithelial (HTBE) cells is the enhancement (from 2- to 4-fold) of the mRNA encoding the elongation factor EF-1 alpha. Sequence analysis has shown that clone HT7, which was identified by differential hybridization procedures, contained a cDNA insert which encoded a protein closely resembling (81%) elongation factor EF-1 alpha from brine shrimp and completely identical to the published sequence of human elongation factor EF-1 alpha (Brands, H.H.G.M., Maassen, J.A., Van Hemert, F.J., Amons, R., and Moller, W. (1986) Eur. J. Biochem. 155, 167-171). Regions of homology of HT7 to EF-Tu from yeast mitochondria, plant chloroplasts, and Escherichia coli are also evident. A single RNA band at 1700 bases was observed for both untreated and retinol-treated HTBE cells, and for mouse liver and parotid glands when Northern transfer from denaturing agarose gel was probed with a 32P-labeled HT7 insert. An enhanced amino acid incorporation and increased protein content per cell for HTBE cells grown in the presence of retinol were observed. Results presented by these studies indicate that retinol may regulate the transcription of a factor required for translation.

Amino Acid Sequence↗

PubMed1988

The preparation of poly(A)+mRNA from the hagfish slime gland.

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.

Animals↗