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P M Reddy

Publications and source records attributed to P M Reddy.

At least 19 recordsLinked to original sources
PubMed2001

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies.

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.

Acetophenones↗
PubMed2001

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens.

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.

Colony Count, Microbial↗
PubMed2001

Effect of ATP sensitive potassium channel modifiers on antinociceptive effect of metoclopramide.

Metoclopramide, a prokinetic drug, has been documented to produce antinociceptive response in animal models through opioid pathways. Morphine has been shown to act through ATP sensitive potassium channels (KATP) to produce antinociceptive response. However, such a possibility has not been examined for metoclopramide. The present study investigated this using pharmacological tools. Acetic acid induced abdominal constriction assay procedure was utilized to assess antinociception. The results confirmed that metoclopramide has antinociceptive response. Glibenclamide, a KATP channel blocker, pretreatment antagonized this response. Where as, in minoxidil pretreated animals, metoclopramide elicited an enhanced antinociceptive response. Glibenclamide and minoxidil, which are known KATP channel blocker and opener respectively, interfered with metoclopramide antinociception. These finding are suggestive of a role for KATP channels in metoclopramide antinociception in mice.

Adenosine Triphosphate↗
PubMed2000

NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase.

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

Acetophenones↗
PubMed2000

Pharmaco-economic analysis of psychotropic drugs.

OBJECTIVES: To analyse cost and adverse reactions of psychotropic drugs for their cost-effective use. METHODS: Four hundred and sixty nine psychotropic formulations from CIMS, June 1998 were evaluated for (a) extent of variation in retail price for same strength and dosage form, (b) role of number of companies manufacturing the same formulation and (c) companies pricing their product at price less than average of maximum and minimum price in relation to number of products marketed by them. The side effects of antipsychotic and antidepressant drugs were graded for their severity and cumulative side effects score. Side effect index and cost index were calculated on relative basis and their product was used as cost benefit index. RESULTS: Fifty per cent of psychotropic drugs had less than 100% price variation with highest of 2049% for risperidone 4 mg tablets. A direct relationship existed between the drug cost and price variation wherever the variation crossed 200%. Similar trend was noticed between the minimum price variations and the number of companies marketing the product. There was no appreciable relationship between number of products marketed and pricing by the manufacturer. Cumulative side effect score was lowest (10) for trifluoperazine and pimozide and highest (15) for risperidone amongst antipsychotic drugs, whereas amongst antidepressants fluoxetine had lowest (1.75) and amitryptyline had highest (28.5) cumulative side effect score. CONCLUSION: One has to be more careful while selecting a brand of a drug when price variation is more (200-2049%). Trifluoperazine (1.0) and fluoxetine (1.7) were found to be most economical with better cost benefit index compared to thioridazine (494.2) and clomipramine (113.0) in their respective groups. Thus our analysis provides basic information regarding cost effective therapy with psychotropic drugs.

Cost-Benefit Analysis↗
PubMed1999

Widespread occurrence of the homologues of the early nodulin (ENOD) genes in Oryza species and related grasses.

Eighty accessions representing 23 species from the genus Oryza were examined for the presence of homologues of early nodulin (ENOD) genes. Southern analyses indicated a widespread distribution of homologues of ENOD genes across all the genomes of rice as well as other monocots. The degree of cross-hybridization of the legume ENOD genes with sequences in the genomes of various species, as revealed by hybridization differentials measured in terms of signal intensities, however, suggests that the homologues of ENOD genes are conserved to varied extents in different Oryza species. The presence of homologues of ENOD genes in a wide variety of plant species denotes that the biological functions of early nodulins may be diverse, and not restricted to nodule organogenesis alone. The fact that ENOD gene homologues exist widely both in dicots and monocots provides evidence that these homologues have arisen from a common ancestral plant.

DNA, Plant↗
PubMed1999

Rice ENOD40: isolation and expression analysis in rice and transgenic soybean root nodules.

The early nodulin ENOD40 has been proposed as playing a pivotal role in the organogenesis of legume root nodules. We have isolated the ENOD40 gene homologues ObENOD40 and OsENOD40 from the wild and cultivated rice genotypes Oryza brachyantha and Oryza sativa, respectively. Rice ENOD40s contain a sequence at the 5' end (region I) for encoding an oligopeptide that is highly conserved in all legume ENOD40s. Furthermore, at the 3' end (region II), the nucleotide sequence of rice ENOD40s exhibited a considerable homology to the corresponding region in legume ENOD40s. Among various organs of the rice plant, expression of OsENOD40 was detected only in stems. In situ hybridization studies revealed that, within the stem, transcription of OsENOD40 is confined to parenchyma cells surrounding the protoxylem during the early stages of development of lateral vascular bundles that conjoin an emerging leaf. Expression pattern of OsENOD40 promoter-GUS fusion in nodules developed on transgenic hairy roots of soybean was also found to be restricted to peripheral cells of nodule vascular bundles, thus evidencing that rice ENOD40 promoter activity is essentially the same as that of soybean ENOD40. Taken together, these results strongly suggest that OsENOD40 and legume ENOD40s share common, if not identical, functions in differentiation and/or function of vascular bundles.

Amino Acid Sequence↗
PubMed1998

Isolation, analysis and expression of homologues of the soybean early nodulin gene GmENOD93 (GmN93) from rice.

Rice (Oryza sativa var. Nipponbare) possesses two different homologues of the soybean early nodulin gene GmENOD93 (GmN93). Analysis of the cDNA clones of rice homologues showed that OsENOD93a has an open reading frame (ORF) with a coding sequence homology of 58.2% to GmENOD93, whereas the ORF of OsENOD93b has displayed a homology of 42.3%. OsENOD93a and OsENOD93b genes are differentially expressed in different parts of the rice plant, as well as in cultured cells induced or non-induced with chitin oligomer. In intact rice tissues, OsENOD93b was most abundantly expressed in roots and at much lower levels in etiolated and green leaves, whereas the expression of OsENOD93a was very low in roots and etiolated leaves, and was not detected in green leaves. The level of OsENOD93a expression was enhanced markedly in suspension-cultured cells, whereas that of OsENOD93b did not increase. The application of chitin oligomer, an elicitor which induces a defence response in plants, did not significantly alter the expression of both these homologues in suspension cultures.

Amino Acid Sequence↗
PubMed1996

Chloride transport in primary cultures of rabbit colonocytes at different stages of development.

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway.

Animals↗
PubMed1996

Isolation and characterization of nucleolin gene as one of the vitamin A-responsive genes in airway epithelium by a palindromic primer-based mRNA differential display method.

A palindromic primer-based mRNA differential display method has been used to isolate various vitamin A-responsive genes from primary cultures of monkey tracheobronchial epithelial cells. This method, as compared with the original mRNA differential display (mDD) method described by Liang and Pardee, used only one arbitrarily designed primer instead of two in the polymerase chain reaction. The single-primer mDD method has several advantages over the two-primer mDD system, especially in the reamplification and the selection of 5'-end cDNA clone. To verify the usefulness of this approach, one of these differential display bands, M34, was initially chosen for further amplification and cloning. The clone derived from the M34 band has a DNA sequence with > 90% homology to the human nucleolin gene. Furthermore, DNA sequencing confirms that both 5' and 3' ends of the insert of M34 contain the invertly repetitive nucleotide sequence that was used to direct this cloning. Nucleolin is a multifunctional phosphoprotein that plays an important role in ribosome biogenesis and mRNA stability. Northern blot analysis demonstrated that in addition to the elevation by vitamin A, the level of nucleolin message is significantly higher in fetal than in adult tracheobronchial epithelial cultures. Furthermore, in situ hybridization demonstrated that the amount of nucleolin message is significantly higher in both basal and ciliated cell types than in mucous and intermediary cell types. These results support the feasibility that the single-primer mDD technique can be used to isolate vitamin A-responsive genes with a palindromic nature.

Animals↗
PubMed1996

Altered growth and attachment of rabbit crypt colonocytes isolated from different developmental stages.

The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function. Colonic epithelia from newborn, weanling, and adult animals were separated from the muscle and subjected to enzymatic digestion. A mixture of 0.05% Pronase, 0.015% collagenase IV, and 0.023% DTT was determined to be optimal for the isolation of newborn and weanling colonocytes. This solution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth. Colonocyte viability of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a approximately 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes revealed only a approximately 10% increase. The colonocytes also showed an age-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen IV; newborn and weanling colonocytes show > 80% attachment, whereas adult colonocytes showed only a 45% attachment. The efficacy of attachment to Matrigel compared with that on plastic also differed with age, representing 9.3-, 5.5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively. Newborn and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and protein content compared with those grown on plastic. There was no difference in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.

Age Factors↗
PubMed1995

Cypermethrin-induced in vivo alterations in the carbohydrate metabolism of freshwater fish, Labeo rohita.

Aspects of carbohydrate metabolism under in vivo conditions were analyzed in functionally different tissues of the freshwater fish, Labeo rohita, exposed to a lethal (LC50/96 hr = 5.24 micrograms liter-1) and sublethal concentration (0.52 micrograms liter-1) of cypermethrin for 4 days. All exposed fish exhibited a hyperglycemic condition. An increase in tissue lactate with a decrease in pyruvate, total carbohydrates, and glycogen contents was noted. Activity of lactate dehydrogenase was elevated, indicating a shift toward anaerobiosis. TCA cycle enzymes, namely succinate dehydrogenase and malate dehydrogenase, were inhibited. In most cases changes were more pronounced during a lethal exposure compared to sublethal exposure period. The data indicate that the fish has adopted a compensatory mechanism to derive energy during pyrethroid toxicosis.

Anaerobiosis↗
PubMed1995

Glutathione S-transferases in tracheobronchial epithelium.

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.

Animals↗
PubMed1994

Genomic footprinting and sequencing of human beta-globin locus. Tissue specificity and cell line artifact.

In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.

Adult↗
PubMed1994

Protein metabolism in brain and muscle tissues of Mus booduga following repeated oral benzenehexachloride feeding.

Protein metabolism was studied in the brain and muscle tissues of mice, Mus booduga after administering orally 50 mg/kg body weight of benzenehexachloride (BHC) daily for 1, 5 and 15 days. Both tissues exhibited considerable decline in all the protein fractions such as total, soluble and structural proteins. This corroborates with the increased levels of free amino acids (FAA) and protease. To fortify these alterations, elevation in the activities of aspartate aminotransferase (AAT), alanine aminotransferase (AlAT) and glutamate dehydrogenase (GDH) were noticed. The two nitrogenous end products namely, ammonia and urea levels were also increased. These results clearly demonstrate the impairment of protein metabolism due to sublethal BHC toxicity.

Administration, Oral↗
PubMed1993

Erythroid differentiation of mouse erythroleukemia cells results in reorganization of protein-DNA complexes in the mouse beta maj globin promoter but not its distal enhancer.

Dimethyl sulfoxide (DMSO) induction of mouse erythroleukemia (MEL) cells represents a well-defined in vitro system of terminal erythroid differentiation. We have studied the molecular mechanisms of transcriptional activation of the mouse beta maj globin gene during MEL cell differentiation by analyzing nuclear factor-DNA interactions in vivo at the gene's upstream promoter and a distal enhancer, 5'HS-2. Genomic footprinting data indicate that three motifs, CAC, NF-E2/AP1, and GATA-1, of the 5'HS-2 enhancer are bound with nuclear factors in MEL cells both prior to and after DMSO induction. No obvious conformational change of these nuclear factor-DNA complexes could be detected upon terminal differentiation of MEL cells. On the other hand, DMSO induction of MEL cells leads to the formation of specific nuclear factor-DNA complexes at several transcriptional regulatory elements of the mouse beta maj globin upstream promoter. Our genomic footprinting data have interesting implications with respect to the molecular mechanisms of transcriptional regulation and chromatin change of the mouse beta maj globin gene during erythroid differentiation.

Animals↗
PubMed1993

Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Base Sequence↗