Uninduced Levels Research Articles

Regulation of LPS-mediated induction of C/EBPδ gene expression in livers of young and aged mice

The C/EBP family of transcription factors plays a major role in the regulation of families of stress response genes, in particular, the acute phase response genes. We have examined expression of the C/EBPδ gene during the bacterial lipopolysaccharide mediated induction of the acute phase response in livers of young (4 months) and aged (24–28 months) male C57Bl/6 mice by Northern, Western, and Southwestern analyses. C/EBP δ mRNA is present at a low constitutive level, is induced by lipopolysaccharide, and reaches the same induced level in young and aged mice. Aged mice, however, show a higher constitutive, uninduced mRNA pool level and a delay in recovery to uninduced levels after lipopolysaccharide treatment. C/EBP δ mRNA is observable 30 min after lipopolysaccharide in total RNA, cytoplasmic and polysomal fractions. Specific full length 28-kDa nascent peptides are detectable in polysomes 90 min after lipopolysaccharide. mRNA and nascent peptides cosediment with large polysomes and C/EBP δ mRNA is shifted to larger polysomes in lipopolysaccharide treated aged mice, consistent with an increased rate of initiation. Specific DNA-binding activity of C/EBP δ protein in nuclear extracts was examined by electromobility shift and antibody supershift assay. The levels of C/EBP δ binding-activity, are consistent with the changes in mRNA levels in young lipopolysaccharide treated livers. These studies support our hypothesis that aged mice exhibit a state of chronic inflammation or stress in the absence of a stressor.

Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth.

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mM salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein.

The dnaK operon of Streptomyces coelicolor contains four genes (5'-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G+C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor--one controlling the dnaK operon and another controlling the groE genes.

Distribution patterns of HSP 90 protein in rice

Rice seedlings exposed to high and low temperatures, salinity and water stress accumulate 87 and 85 kDa stress-associated-proteins (SAP), collectively referred to as SAP 90. In the present study, we provide evidence that SAP 87 of rice is a member of the eukaryotic stress 90 HSP 90 family. Further, high uninduced levels of rice SAP HSP 90 exist in lemma, palea and culm tissues. Dry seeds of rice, wheat, maize and sorghum also contain high uninduced amounts of this protein. Seed-derived callus tissue show high constitutive levels of this protein as well. When seed and callus tissues were subjected to heat shock, the levels of this protein declined. Tissue print-immunoblot analysis using anti SAP 90 antibodies revealed higher accumulation of SAP HSP 90 in the vascular bundles/procambial cells and in the outermost cell layers of various leaves as well as in the meristematic cells of the stem apex. In seeds, this protein was most abundant in the seed coat and whole embryo.

Transcriptional regulation of delta-aminolevulinic acid dehydratase synthesis by oxygen in Bradyrhizobium japonicum and evidence for developmental control of the hemB gene.

An increased demand for cytochromes is associated with symbiotic development and microaerobic metabolism in the bacterium Bradyrhizobium japonicum, and evidence suggests that hemB, rather than hemA, is the first essential bacterial heme synthesis gene in symbiosis with soybean. Steady-state levels of mRNA and protein encoded by hemB were strongly and rapidly induced by O2 deprivation as determined by RNase protection and immunoblot analyses, but hemH message was not induced. Oxygen limitation resulted in a greater-than-10-fold increase in the rate of hemB mRNA synthesis as determined by transcriptional runoff experiments, whereas hemH transcription was unaffected by the O2 status. Thus, hemB is a regulated gene in B. japonicum and is transcriptionally controlled by O2. Unlike the expression in parent strain I110, hemB expression was not affected by O2 in the fixJ strain 7360, and O2-limited cultures of the mutant contained quantities of hemB mRNA and protein that were comparable to uninduced levels found in aerobic cells. In addition, spectroscopic analysis of cell extracts showed that increases in b- and c-type cytochromes and the disappearance of cytochrome aa3 in response to microaerobic growth in wild-type cells were not observed in the fixJ mutant. FixJ is a key transcriptional regulator that mediates O2-dependent differentiation in rhizobia, and therefore hemB expression is under developmental control. Furthermore, the data suggest a global control of cytochrome expression and heme biosynthesis in response to the cellular O2 status.

Characterization of two nuclear proteins that interact with cytochrome P-450 1A2 mRNA. Regulation of RNA binding and possible role in the expression of the Cyp1a2 gene.

Regulation of the expression of the cytochrome P-450 la2 gene (cyp1a2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Some reports have proposed involvement of post-transcriptional mechanisms [Adesnik, M. & Atchison, M. (1986) Crit. Rev. Biochem. 19, 247-305; Silver, G. & Krauter, K. S. (1990) Mol. Cell. Biol. 10, 6765-6768]. Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYP1A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3' untranslated region of CYP1A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYP1A2 mRNA. Treatment of mice with an inducer of CYP1A2, 3-methylcholanthrene, increases the binding of the 46-kDa protein and decreases the binding of the 37-kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYP1A2 mRNA by 3-methylcholanthrene. Phosphorylation of the 46-kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46-kDa but not the 37-kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre-mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3' untranslated region of CYP1A2, it is possible that the 37-kDa protein has a role in the maintenance of uninduced levels of CYP1A2 mRNA, while the 46-kDa protein could be important in the maturation of elevated levels of CYP1A2 pre-mRNA, during induction.

Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor.

SoxR protein of Escherichia coli governs a global response against superoxide-generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance. A redox signal activates SoxR post-translationally to trigger transcription of a second regulatory gene, soxS. Activated and non-activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription. SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near-consensus -10 and -35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp). We have constructed and analyzed site-specific deletions that alter the promoter element spacing. Reducing the spacer length to 16-18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat. More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment. Gel mobility-shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase. Thus, activated SoxR remodels the unusual configuration of the wild-type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the -10 and the -35 elements.

Leishmania major:Promastigotes Induce Expression of a Subset of Chemokine Genes in Murine Macrophages

Recent studies suggest thatLeishmania majorpromastigotes infect cultured macrophages in a stealthy fashion, activating little or no host gene expression and often interfering with the host's ability to respond to further stimulation. Here we examined macrophage transcription at early times following infection, when virulent parasites must execute steps required for survival. Stationary-phase promastigotes induced rapid and transient expression of transcripts of the chemokines JE (human MCAF/MCP-1) and KC (human GRO) in bone marrow-derived macrophages from BALB/c mice. JE and KC expression rose four- to sixfold shortly after infection and returned to uninduced levels by 4–24 hr. In contrast, chemokines MIP-1α, C10, and RANTES were not induced, nor were TGF-β, IL-10, IL-12, or i-NOS. Chemokine induction did not occur following ingestion of latex beads, implicating a parasite-specific stimulus. Elevated expression of a subset of chemokines is the earliest known transcriptional response of macrophages toL. majorinfection and potentially may provide a signal for the initiation of downstream immunological responses which occurin vivo,such as cytokine induction and chemotaxis of monocytes and macrophages. Thus,Leishmaniahas a remarkable ability to take an active role in either inducing or preventing the expression of distinct sets of host genes during macrophage invasion and successful intracellular parasitism.

Functional characterization of p53 molecules expressed in human squamous cell carcinomas of the head and neck

Mutation of the p53 tumor suppressor gene has been demonstrated in a large proportion of human head and neck squamous cell carcinomas (HNSCCs) and has been assumed to play a role in the pathogenesis of these tumors, although no formal evidence of functional aberration has been demonstrated. In this study, we isolated cDNA clones encoding the entire p53 coding region from six human HNSCC cell lines that showed aberrant patterns of p53 expression in the parental cells, analyzed their nucleotide sequences, and characterized their function in vivo. cDNAs cloned from four cell lines harbored alterations within the p53 coding sequence (one missense mutation, one missense mutation plus in-frame deletion, one splice donor mutation, and a 1-nt insertion). HN30 cells, which contained wild-type p53 nucleotide sequences, showed a high constitutive level of protein expression. HN26 cells contained wild-type coding sequences but did not express the 53-kDa protein, although the mRNA was transcribed and a molecule of increased molecular mass (70 kDa) was observed by western blotting. Functional studies revealed that none of the four proteins encoded by mutant cDNAs were able to transactivate expression of a reporter plasmid containing a wild-type p53 consensus binding site when cotransfected into p53-null cells, whereas molecules encoded by wild-type p53 cDNAs increased reporter gene expression about a hundredfold over uninduced levels. Co-expression of each mutant cDNA with wild-type p53 cDNA and a wild-type p53-responsive reporter gene demonstrated that each of the proteins encoded by mutant cDNAs harbored some degree of inhibitory activity that varied depending on the mutation present. Thus, aberrant p53 function as a result of mutation or altered expression characterizes oral squamous cell carcinomas. The inhibitory activity of these molecules may be a mechanism for deregulation of the function of co-expressed wild-type p53 that may be of importance during the early stages of tumor development.

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.

Strategies for maximizing metallothionein promoter regulated recombinant protein production in mammalian cell cultures

A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn(+2) or Cd(+2)) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd(+2) dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd(+2) although cell-associated Cd(+2) levels remain high. Further addition of Cd(+2) to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd(+2) is sequestered into a form which does not stimulate the MT promoter. Cd(+2) dosing regimes which maintained cell associated Cd(+2) concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd(+2) loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line.

Expression of the 1,25-dihydroxyvitamin D3-24-hydroxylase gene in rat intestine: response to calcium, vitamin D3 and calcitriol administration in vivo.

The 25(OH)D3/1,25(OH)2D3 24-hydroxylase (24-hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca2+ concentrations. However, the participation of Ca2+ in the expression of the 24-hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca2+ and the D3 and/or 1,25(OH)2D3 status on the 1,25(OH)2D3-mediated inducibility of the 24-hydroxylase gene in rat duodenum. Hypocalcemic D-depleted rats were supplemented with calcium alone to normalize serum Ca2+ without normalizing the D3 status or were acutely or chronically supplemented with D3 or 1,25(OH)2D3. Messenger RNA for the 24-hydroxylase was undetectable in the intestine of hypocalcemic D-depleted rats, and short- or long-term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)2D3 administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca2+, and the D3 or 1,25(OH)2D3 status. Moreover, 24-hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)2D3, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time-course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)2D3 supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down-regulated the responsiveness in the continuous presence of 1,25(OH)2D3. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)2D3 on the in vivo induction of the 24-hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)2D3 concentrations, the 24-hydroxylase efficiently controls the amount of 1,25(OH)2D3 in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone.

Regulation of the Human Cellular Glutathione Peroxidase Gene During In Vitro Myeloid and Monocytic Differentiation

AbstractWe have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL-60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro–induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.

Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet

Ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylase activities, and contributions of the P450 cytochromes CYP1A1, CYP2B1 and 2, CYP2C6 and CYP2C12 to these metabolic activities, were evaluated in hepatic microsomes from ad libitum and calorie restricted female Fischer 344 rats across an age continuum from 1 to 26 months. The presence of CYP1A in microsome preparations was confirmed by western blot analysis. Uninduced levels of EROD and PROD peak in very young animals, decline to about 3 months of age, and do not exhibit an additional substantive decline after about 3 months of age. Monoclonal antibody (MAb) 1-7-1 (anti-CYP1A) strongly inhibited EROD activity in all microsome preparations, with the highest levels of inhibition in microsomes from 1- and 3-month-old AL animals. PROD activity in 1-month uninduced animals was apparently not attributable solely to CYP2B1 and 2, since it was inhibited by about 30% in both 1- and 26-month-old AL rats by an MAb specific for CYP2C12. However, in CR rats, CYP2C12 did not contribute to PROD activity. Approximately 40% of PROD activity in old AL rats and 20% of PROD activity in old CR rats was inhibited by an MAb specific for CYP2C6. These data indicate that long-term calorie restriction modulates either the expression or post-translational modification patterns of constitutive P450 isozymes in rats as they age, with P450 patterns in calorie restricted rats more closely resembling those found in young animals.

A thin layer chromatographic technique for detecting inducers of Agrobacterium virulence genes in corn, wheat and rye

A method is described for detecting plant metabolites capable of inducing the virulence genes of Agrobacterium tumefaciens. The method uses A. tumefaciens containing a plasmid with an inducible virulence gene fused to a galactosidase gene (virE::lacZ). Thin layer chromatography plates are overlayed with agar containing the indicator bacterium and a chromogenic galactoside (X-gal). Virulence gene inducing plant metabolites induce galactosidase which releases an aglycone readily oxidized by air to a blue pigmented zone at the Rf of the inducer. The method has been used to demonstrate the presence of virulence gene inducers in corn, wheat and rye. The uninduced background level of galactosidase also permits detection of bacterial growth inhibitors after a longer incubation period.

Effect of Dietary Alpha-Linolenic, Eicosapentaenoic and Docosahexaenoic Acids on Liver Microsomal Mixed-Function Oxidases in Rats

This study was undertaken to compare the effects of three major dietary n-3 fatty acids, i.e. alpha-linolenic acid (C18:3, n-3; LN), eicosapentaenoic acid (C20:5, n-3; EPA) and docosahexaenoic acid (C22:6, n-3; DHA), on liver microsomal mixed-function oxidase activities with and without phenobarbital (PB) induction. Male Sprague-Dawley rats were fed a 5% lipid diet containing either LN, EPA or DHA as major constituent fatty acid (about 40%) of dietary lipids for 15 days. The fatty acid compositions of liver microsomal lipids of each n-3 fatty acid group were characteristically different, particularly in the n-3 fatty acid compositions, but were nearly the same with and without PB treatment. Dietary LN, EPA and DHA did not affect the levels of PB-inducible cytochrome P-450 species nor the activities of aminopyrine N-demethylase and aniline hydroxylase. However, the PB-uninduced constitutive levels of cytochromes P-450 increased in the following order with a slight but significant difference between LN and DHA groups: LN group < EPA group < DHA group. Both the PB-uninduced activities of aminopyrine N-demethylase and aniline hydroxylase changed in a very similar manner to the constitutive levels of cytochromes P-450. These results suggest that the uninduced constitutive level of certain cytochrome P-450 species is modulated differentially by dietary LN, EPA and DHA, whereas dietary LN, EPA and DHA do not affect the PB-inducible levels of cytochrome P-450 species.

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis.

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

Mouse β-Globin DNA-Binding Protein B1 is Identical to a Proto-Oncogene, the Transcription Factor Spi-1/PU.1, and is Restricted in Expression to Hematopoietic Cells and the Testis

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

The Escherichia coli cAMP receptor protein (CRP) represses the Rhizobium meliloti dctA promoter in a cAMP-dependent fashion.

The expression of the Rhizobium meliloti C4-dicarboxylic acid permease gene (dctA) is controlled by the sensor DctB and the transcriptional regulator, DctD. The R. meliloti Dct system has been reconstituted in Escherichia coli. Expression of the dctA promoter is DctBD dependent and is induced in the presence of C4-dicarboxylic acids (dCA). Other carbon sources also influence dctA expression. We demonstrate that the cAMP receptor protein (CRP) has a repressive effect on the dctA promoter. A mutated CRP molecule (CRP-H159L), unable to activate catabolic promoters (but still proficient in DNA binding), gives similar results. This suggests that the CRP-cAMP complex represses the dctA promoter activity by direct interaction with the DNA. Direct binding of the CRP-cAMP complex to the dctA promoter was confirmed in vitro by gel mobility-shift assays. Sequence analysis of the dctA promoter indicates that the most likely binding sites for CRP are the two confirmed DctD-binding sites. It is proposed that the CRP-cAMP complex competes with DctD for occupancy of these sites. Since in the presence of CRP-cAMP complex the uninduced levels of dctA expression are reduced, whereas induced levels are largely unaffected, such competition appears to be an essential regulatory feature of dctA expression.

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.