ABSTRACT
STUDY QUESTION: Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells? SUMMARY ANSWER: Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation. WHAT IS KNOWN ALREADY: In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo. STUDY DESIGN, SIZE, DURATION: We isolated testicular cells enriched for interstitial cells from frozen-thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34-/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination. MAIN RESULTS AND THE ROLE OF CHANCE: From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34-/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties. WIDER IMPLICATIONS OF THE FINDINGS: A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.
Subject(s)
Cell Differentiation/genetics , Leydig Cells/metabolism , Multipotent Stem Cells/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Spermatogenesis/genetics , Aged , Animals , Cell Culture Techniques/methods , Cells, Cultured , Culture Media , Heterografts , Humans , Male , Mice , Mice, Knockout , Middle Aged , Prostatic Neoplasms/pathology , Receptors, LH/genetics , Testosterone/bloodABSTRACT
Chronic periaortitis is thought to result from an autoallergic reaction to oxidized low-density lipoprotein (OxLDL). No data exist on lipid profile and atherosclerotic biomarkers. We investigated circulating levels of OxLDL and of anti-OxLDL (aOxLDL) antibodies in patients with chronic periaortitis using the cross-sectional case-control study on 20 patients with chronic periaortitis. Patients were compared to 20 age- and sex-matched controls. aOxLDL antibodies were measured by ELISA and expressed as mean optical density values at 450 nm from duplicate measurements (OD(450)). aOxLDL antibody titers (median [interquartile range]) did not differ significantly between patients and controls (aOxLDL-IgM: 0.70 [0.24-1.08] vs. 0.54 [0.25-0.73] OD(450); aOxLDL-IgG: 0.59 [0.38-0.75] vs. 0.41[0.33-0.63]OD(450)). Female patients had higher aOxLDL-IgM levels than male patients (1.02 [0.46-1.38] vs. 0.29 [0.22-0.84] OD(450); P = 0.05). aOxLDL-IgM titers were lower in patients with cardiovascular disease (CVD) than in patients without CVD (0.22 [0.16-0.37] vs. 0.92 [0.70-1.30] OD(450); P = 0.003) and correlated positively with HDL-cholesterol (r = 0.47, 95% CI 0.02-0.69; P = 0.03) and inversely with diastolic blood pressure (r = -0.46, 95% CI -0.75 to -0.01; P = 0.03) and OxLDL/apoB ratio (r = -0.41, 95% CI -0.73 to 0.04; P = 0.06). No differences or associations were found between aOxLDL-IgG titers and other variables between or within patients and/or controls. In patients OxLDL levels correlated with smoking pack-years (r = 0.58, 95% CI 0.17-0.81; P = 0.007). Data suggest a differing innate immune response to OxLDL in patients with chronic periaortitis compared to controls. Whether this response is causally related to chronic periaortitis development remains to be clarified.
Subject(s)
Autoantibodies/blood , Lipids/blood , Lipoproteins, LDL/blood , Retroperitoneal Fibrosis/blood , Aged , Antihypertensive Agents/immunology , Antihypertensive Agents/therapeutic use , Apolipoproteins B/blood , Apolipoproteins B/immunology , Atherosclerosis/blood , Atherosclerosis/immunology , Autoantibodies/immunology , Case-Control Studies , Cross-Sectional Studies , Female , Humans , Hypertension/drug therapy , Hypertension/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Lipoproteins, LDL/immunology , Male , Middle Aged , Retroperitoneal Fibrosis/epidemiology , Retroperitoneal Fibrosis/immunology , Smoking/blood , Smoking/epidemiology , Smoking/immunology , Treatment OutcomeABSTRACT
An expanding body of research indicates that exposure to contaminants may impact marine mammal health, thus possibly contributing to population declines. The harbor seal population of the San Francisco Bay (SFB), California, has suffered habitat loss and degradation, including decades of environmental contamination. To explore the possibility of contaminant-induced health alterations in this population, blood levels of polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (DDE), and polybrominated diphenyl ethers (PBDEs) were quantified in free-ranging seals; relationships between contaminant exposure and several key hematological parameters were examined; and PCB levels in the present study were compared with levels determined in SFB seals a decade earlier. PCB residues in harbor seal blood decreased during the past decade, but remained at levels great enough that adverse reproductive and immunological effects might be expected. Main results included a positive association between leukocyte counts and PBDEs, PCBs, and DDE in seals, and an inverse relationship between red blood cell count and PBDEs. Although not necessarily pathologic, these responses may serve as sentinel indications of contaminant-induced alterations in harbor seals of SFB, which, in individuals with relatively high contaminant burdens, might include increased rates of infection and anemia.
Subject(s)
Dichlorodiphenyl Dichloroethylene/blood , Insecticides/blood , Phoca/blood , Polybrominated Biphenyls/blood , Polychlorinated Biphenyls/blood , Water Pollutants, Chemical/blood , Animals , Female , Gas Chromatography-Mass Spectrometry , Male , San FranciscoABSTRACT
In neurons, neuropeptides and other synaptic components are transported down the axon to the synapse in vesicles using molecular motors of the kinesin family. In the synapse, these neuropeptides are found in dense core vesicles (DCVs), and, following calcium-mediated exocytosis, they interact with receptors on the target cell. We have developed a rapid, large-scale technique for purifying peptide-containing DCVs from specific nuclei in the central nervous system. By using differential velocity gradient and equilibrium gradient centrifugation, neuropeptide-containing DCVs can be separated by size and density from optic nerve (ON) and its termini, the lateral geniculate nuclei and the superior colliculi. Isolated DCVs contain neuropeptides (substance P and brain-derived neurotrophic factor), synaptic vesicle (SV) membrane proteins (SV2, synaptotagmins, synaptophysin, Rab3 and synaptobrevin), SV-associated proteins (alpha-synuclein), secretory markers for DCVs previously isolated (secretogranin II), and beta-amyloid precursor protein. By using electron microscopic techniques, DCV were also visualized and shown to be immunoreactive for neuropeptides, neurotrophins, and SV membrane proteins. Because of the interesting group of physiological and potentially pathophysiological proteins associated with these vesicles; this isolation procedure, applicable to other CNS nuclei, should represent an important research tool.
Subject(s)
Geniculate Bodies/chemistry , Neuropeptides/isolation & purification , Optic Nerve/chemistry , Secretory Vesicles/chemistry , Substance P/isolation & purification , Superior Colliculi/chemistry , Amyloid beta-Protein Precursor/isolation & purification , Animals , Brain-Derived Neurotrophic Factor/isolation & purification , Chromogranins , Membrane Glycoproteins/isolation & purification , Nerve Tissue Proteins/isolation & purification , Proteins/isolation & purification , Rabbits , Synucleins , alpha-SynucleinABSTRACT
Insulin is capable of regulating cellular and metabolic processes as well as gene expression. In recent years, enthusiasm has surfaced for using insulin-mimetics to study the mechanism of action of insulin. Vanadate and selenate are two compounds that have been found to mimic the action of insulin on control of blood glucose levels in vivo. Vanadate has also been shown to regulate the expression of several enzymes both in vivo and in vitro, however, studies concerning selenate's ability to regulate expression have not been reported. In this study we show that administration of vanadate or selenate to streptozotocin-induced diabetic rats not only normalizes blood glucose levels similarly to insulin but also positively affects the expression of two key metabolic enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS). Both G6PDH and FAS activity are significantly decreased in diabetic animals compared to non-diabetic control. Treatment of the diabetic animals with either insulin, vanadate or selenate restored both activities to about 80-90% of control. All treatment conditions exhibited activities significantly higher than those determined for the diabetic group but did not differ significantly from each other. Increases in G6PDH or FAS activity are due to increases in mRNA level. Increase in both G6PDH and FAS mRNA was comparable to the observed increase in activity suggesting that regulation of expression by the mimetics occurs pretranslationally.
Subject(s)
Fatty Acids/biosynthesis , Glucosephosphate Dehydrogenase/drug effects , Hypoglycemic Agents/pharmacology , Selenium Compounds/pharmacology , Vanadates/pharmacology , Animals , Blood Glucose , Diabetes Mellitus, Experimental , Fatty Acids/genetics , Gene Expression , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Insulin/metabolism , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Selenic Acid , Weight GainABSTRACT
Ethanol has been shown to induce the activity of glucose-6-phosphate dehydrogenase (G6PDH). To clarify the mechanism behind this induction, we examined the role of acetaldehyde (AA), the first product of ethanol metabolism. In primary adult rat hepatocytes maintained in chemically defined medium, we examined the effect of AA on G6PDH activity, mRNA levels and lipid synthesis. We observe a 40% increase in G6PDH activity and a similar increase in mRNA levels, following exposure to 100 microM AA. The increase in activity was found to be maximal at 24 h while mRNA levels increased over controls as early as 3 h. The induction in G6PDH by AA was found to occur at lower concentrations and earlier time points than those reported using ethanol. The role of insulin, a known inducer of G6PDH activity was studied alone and in combination with AA on both G6PDH activity and mRNA levels as well as lipid biosynthesis. Insulin (300 ng/ml) was found to increase G6PDH activity, mRNA levels and [14C]-acetate incorporation into lipid. It was also shown to have an additive effect with AA on G6PDH activity, suggesting their actions are mediated via different mechanistic pathways. No change in [14C]-acetate incorporation into lipid, however, was observed with acetaldehyde alone.
Subject(s)
Acetaldehyde/pharmacology , Glucosephosphate Dehydrogenase/biosynthesis , Lipids/biosynthesis , Liver/drug effects , RNA, Messenger/metabolism , Animals , Blotting, Northern , Cells, Cultured , Drug Synergism , Glucosephosphate Dehydrogenase/genetics , Insulin/pharmacology , Liver/cytology , Liver/enzymology , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Venous central oximetry (ScO2) in high-risk pediatric surgical patients (myopathies, congenital diaphragmatic hernia) is an useful guide to treatment. ScO2 was monitored in 15 patients (4 neonates) during and after thoracic interventions or interventions involving manipulation of the liver. Oximetrix ScO2 is not more invasive than a catheter of common stiffness, but the size 4F remains large for neonates. The physiological or pathological signification of its variation has to be deduced from clinical evaluation. Interpretation may be easier during anesthesia and in the absence of sepsis. ScO2 can be used either as a sensitive monitoring with usually an early response, to evaluate judicious treatment, or as an indirect way to evaluate cardiac output if the other factors of the Fick relation can be estimated or measured.
Subject(s)
Cardiomyopathies/surgery , Hernias, Diaphragmatic, Congenital , Monitoring, Intraoperative , Oxygen/blood , Resuscitation , Adolescent , Adult , Child , Child, Preschool , Hernia, Diaphragmatic/surgery , Humans , Infant, Newborn , VeinsABSTRACT
Histidine-rich glycoprotein (HRG) is a monomeric plasma glycoprotein involved in the modulation of coagulation and fibrinolysis. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HRG-specific cDNA probe, the HRG gene was assigned to chromosome 3. One hybrid that was known to contain only a segment of chromosome 3 also reacted positively with the HRG probe. Hybridization analysis with a set of chromosome 3-specific probes showed that the segment of chromosome 3 present in this hybrid is missing the region pter-p14, which indicates that HRG is not located in this region. No restriction fragment length polymorphisms were detected for HRG with 10 commonly used restriction enzymes.
Subject(s)
Chromosomes, Human, Pair 3 , Glycoproteins/genetics , Proteins/genetics , Animals , Blotting, Southern , Chromosome Mapping , Genes , Humans , Hybrid CellsABSTRACT
Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u-PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI-1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.
Subject(s)
Endothelium, Vascular/cytology , Fibrinolytic Agents/metabolism , Plasminogen Activators/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Urokinase-Type Plasminogen Activator/metabolism , Biological Factors/pharmacology , Cells, Cultured , Cytokines , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fibrinolysin/pharmacology , Gene Expression Regulation/drug effects , Humans , Plasminogen Activators/genetics , Plasminogen Activators/physiology , Plasminogen Inactivators/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/physiologyABSTRACT
The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The monokine tumor necrosis factor (human recombinant TNF) increased the production of PAI by cultured human endothelial cells from umbilical vein (twofold) and from foreskin microvessles (four to eight fold). This was demonstrated by titration of endothelial cell-conditioned medium with t-PA, by reverse fibrin autography, and by immunoprecipitation of [35S]PAI-1 by anti-PAI-1 IgG. TNF also induced a marked increase of PAI-1 messenger RNA (mRNA) in the cells. The stimulation of PAI activity by TNF was seen at 4 U/mL and reached a maximum at 500 U/mL. Human recombinant lymphotoxin and interleukin-1 (alpha and beta) also stimulated the production of PAI activity, while interleukin-6 was ineffective. Separate additions of TNF or interleukin-1 (IL-1) at optimal concentrations (500 U/mL and 5 U/mL, respectively) resulted in a comparable stimulation of PAI production by endothelial cells. The simultaneous addition of both mediators resulted in an additive effect. The effect of TNF could not be prevented by the addition of polymyxin B or by anti-IL-1 antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion by endothelial cells. Two hours after a bolus injection of 250,000 U/kg TNF into rats, a fivefold increase in circulating PAI levels was found. In the next ten hours, the levels returned to normal. Blood platelets do not significantly contribute to the increase in circulating PAI, because the number of platelets did not change after TNF injection and the amount of PAI in blood platelets is not sufficient for several hours during an increase in PAI activity. The acute phase reactants, fibrinogen and alpha 2-antiplasmin in rat plasma, were altered little if any two to 24 hours after injection of 250,000 U/kg TNF. In vitro, TNF did not change PAI production by human and rat hepatocytes in primary monolayer culture. Therefore, it is most likely that vascular endothelial cells contribute to the increased amount of circulating PAI induced by TNF in vivo. This increase in PAI activity might decrease fibrinolysis.
Subject(s)
Endothelium, Vascular/metabolism , Glycoproteins/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Animals , Blotting, Northern , Cells, Cultured , Gene Expression Regulation/drug effects , Glycoproteins/genetics , In Vitro Techniques , Lymphotoxin-alpha/pharmacology , Male , Plasminogen Inactivators , RNA, Messenger/genetics , Rats , Rats, Inbred Strains , Tissue Plasminogen Activator/biosynthesisABSTRACT
The plasminogen activator inhibitor (PAI-1) from endothelial cells is a potentially important regulator of plasminogen activator activity. Cultured human endothelial cells increase their PAI-1 production upon stimulation with LPS and TNF, agents that are known to cause an increase in PAI-1 levels in vivo. We isolated a PAI-1 cDNA probe, and by RNA hybridization analysis studied the regulation of PAI-1 mRNA synthesis in human endothelial artery cells. Freshly isolated endothelial cells do not contain detectable amounts of PAI-1 mRNA, but after adherence and incubation for 18 h in growth medium produce considerable amounts of PAI-1 activity and contain PAI-1 mRNA levels comparable to those found in subcultured cells. When subcultured endothelial cells are incubated for 6 h with LPS or TNF, both species of PAI-1 mRNA increase 10 to 20 fold, while PAI-1 activity in the growth medium increases only 1.5 to 2 fold. Stimulation of endothelial cells in the presence of cycloheximide (CHX) results in superinduction of mainly the 3.0 kb PAI-1 mRNA. The 3' end of this mRNA contains a 60 bp AT-rich sequence, that resembles 3' sequences present in a number of other genes superinducible with CHX.
Subject(s)
Endothelium, Vascular/cytology , Gene Expression Regulation , Glycoproteins/genetics , RNA, Messenger/genetics , Base Sequence , Blotting, Northern , Cells, Cultured , Cloning, Molecular , Culture Media , Cycloheximide/pharmacology , DNA Probes , Electrophoresis, Agar Gel , Humans , Lipopolysaccharides/pharmacology , Nucleic Acid Hybridization , Plasminogen Inactivators , RNA/isolation & purification , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.
Subject(s)
Genes , Glycoproteins/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cosmids , Exons , Female , Humans , Introns , Molecular Sequence Data , Placenta/metabolism , Plasminogen Activators/antagonists & inhibitors , Plasminogen Inactivators , PregnancyABSTRACT
A region located upstream of the uvrB promoters P1 and P2 was found to cause high plasmid loss when cloned in multicopy vectors. Two sequence elements responsible for this phenomenon were identified by mapping of spontaneous mutations that restore plasmid maintenance: a sequence known to have in vitro promoter activity and a partially overlapping sequence that shows extensive homology to recognition sites for the DnaA protein. Accordingly alterations in the level of DnaA protein in vivo were found to affect the extent of plasmid loss. A possible role for interaction of the DnaA protein with the region of interest is discussed in relation to regulation of uvrB expression.
Subject(s)
Bacterial Proteins/genetics , DNA Repair , Escherichia coli/genetics , Genes, Bacterial , Genes, Regulator , Base Sequence , Cloning, Molecular , DNA Replication , Gene Expression Regulation , Genetic Linkage , Promoter Regions, GeneticABSTRACT
The transcriptional activity of the tandem promoters of the Escherichia coli uvrB gene was measured in vivo. Both promoters are shown to be inducible by UV irradiation. P1, the most proximal promoter, is responsible for the main part of transcription both in uninduced and induced cells. Plasmids have been constructed carrying small deletions in the lexA binding site that overlaps with P2, the distal promoter. These deletions result in constitutive transcription from P1. This indicates that the DNA region which contains P2 functions mainly as a target site for regulation of P1 transcription in vivo.