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PubMed2000

A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells.

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.

Animals↗
PubMed2002

Epithelial inflammation response induced by Shigella flexneri depends on mucin gene expression.

The protective effects of different mucin gene profiles on gut protection were assessed by the evaluation of TNFalpha production by intestinal epithelial cells infected by Shigella flexneri. Three HT-29 cell lines were used: HT29-G(-) (enterocyte-like cells, secreting no mucins), HT29-FU (highly expressing MUC2 and MUC4) and HT29-MTX (highly expressing MUC3 and MUC5AC). These cells were infected either by an invasive (M90T) or the control isogenic (BS176) strains of S. flexneri, and TNFalpha mRNA production was quantified by competitive PCR. In the HT29-G(-) cells, M90T induced an increased production of TNFalpha mRNA compared to BS176, giving a TNFalpha ratio of 5.6 +/- 3.3. In contrast, similar levels of TNFalpha mRNA were detected in HT29-FU and HT29-MTX cells stimulated with either M90T or BS176, giving ratios of 1.4 +/- 1.3 and 1.0 +/- 0.1, respectively. The results suggest that mucin genes have abilities to protect epithelial cells against S. flexneri. Furthermore, the difference in the TNFalpha ratio between the HT29-FU and HT29-MTX cells suggests distinct protective effects for these two mucin-secreting epithelial cells.

Cells, Cultured↗
PubMed2001

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

Adenosine Triphosphate↗
PubMed2026

Frataxin deficiency drives cardiac dysfunction and transcriptional dysregulation in Friedreich ataxia iPSC model.

Friedreich ataxia (FRDA) is a progressive neuromuscular degenerative disorder caused by GAA repeat expansions in the FXN gene, leading to frataxin deficiency and multisystem pathology. Cardiomyopathy is the leading cause of mortality in individuals with FRDA. To investigate the cellular and molecular mechanisms underlying FRDA-associated cardiac dysfunction, we employed induced pluripotent stem cell (iPSC) lines derived from three individuals with FRDA, each paired with an isogenic control line generated through CRISPR/Cas9-mediated excision of the pathogenic GAA repeat expansion. Correction of the mutation restored FXN expression to levels comparable to healthy donor iPSCs, and all lines differentiated efficiently into cardiomyocytes. Functional analysis revealed significant contractile abnormalities in FRDA cardiomyocytes and multicellular cardiac microtissues, including prolonged contraction and relaxation times and faster beating rates, consistent with clinical observations of cardiac contractile dysfunction. FRDA cardiomyocytes also exhibited pathological features such as increased cell size, irregular calcium transients, elevated mitochondrial reactive oxygen species levels, increased mitochondrial fission and increased cell death. These phenotypes were exacerbated by pathological levels of iron supplementation in culture media, highlighting the heightened sensitivity of frataxin-deficient cardiomyocytes to iron-induced metabolic stress. RNA sequencing revealed a distinct transcriptional profile associated with frataxin deficiency. MEG3 and PCDHGA10 were consistently dysregulated across all three FRDA-iPSC lines and may represent early molecular markers of FRDA cardiomyopathy. Functional interrogation of these candidates demonstrated that targeted silencing of MEG3 or PCDHGA10 in FRDA cardiomyocytes significantly reduced disease‑associated cell death without affecting FXN expression. Notably, PCDHGA10 silencing also normalized elevated mitochondrial reactive oxygen species, whereas MEG3 silencing did not, highlighting gene‑specific contributions to FRDA cardiomyocyte survival. Collectively, these findings identify MEG3 and PCDHGA10 as functionally relevant regulators of FRDA cardiomyocyte pathology.

Friedreich Ataxia↗
PubMed2001

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes.

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Amino Acid Sequence↗
PubMed2003

Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2.

A hallmark of highly malignant human gliomas is their infiltration of the brain. We analyzed a large number of primary human glioma biopsies and found high levels of expression of an angiogenic regulator, angiopoietin-2 (Ang2), in the invasive areas, but not in the central regions, of those tumors. In the invasive regions where Ang2 was overexpressed, increased levels of matrix metalloprotease-2 (MMP-2) were also apparent. Consonant with these features, intracranial xenografts of glioma cells engineered to express Ang2 were highly invasive into adjacent brain parenchyma compared with isogenic control tumors. In regions of the Ang2-expressing tumors that were actively invading the brain, high levels of expression of MMP-2 and increased angiogenesis were also evident. A link between these two features was apparent, because stable expression of Ang2 by U87MG cells or treatment of several glioma cell lines with recombinant Ang2 in vitro caused activation of MMP-2 and acquisition of increased invasiveness. Conversely, MMP inhibitors suppressed Ang2-stimulated activation of MMP-2 and Ang2-induced cell invasion. These results suggest that Ang2 plays a critical role in inducing tumor cell infiltration, and that this invasive phenotype is caused by activation of MMP-2.

Angiogenesis Inducing Agents↗
PubMed2000

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Base Sequence↗
PubMed2005

The tumor suppressor PTEN is necessary for human Sprouty 2-mediated inhibition of cell proliferation.

Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.

Adaptor Proteins, Signal Transducing↗
PubMed2026

DENND3-p.R534S disrupts dyadic microdomain architecture to drive potentially pro-arrhythmic calcium and electrophysiologic instability.

AIMS: Inherited ventricular arrhythmias (VAs) frequently occur in the absence of pathogenic variants in canonical ion channel genes, suggesting alternative mechanisms of electrical instability. DENND3 is a guanine nucleotide exchange factor that regulates Rab GTPase-mediated trafficking, but its role in cardiac excitation-contraction coupling and membrane microdomain organization remains undefined. METHODS AND RESULTS: We studied induced pluripotent stem cell-derived cardiomyocytes generated from a CRISPR/CAS9-engineered ultra-rare DENND3-p.R534S variant-inserted line (previously identified in an idiopathic ventricular fibrillation pedigree) and matched isogenic controls. Multielectrode array recordings, live-cell calcium imaging, super-resolution imaging using expansion microscopy, and biochemical analyses were used to assess electrical activity, calcium handling, membrane architecture, and calcium release unit organization. Potentially therapeutic studies were performed using genetic and pharmacologic inhibition of Rab11b. DENND3-p.R534S induced pluripotent stem cell-derived cardiomyocytes exhibited multicellular electrical instability characterized by increased beat-to-beat variability, arrhythmic activity, conduction slowing, and prolonged excitation-contraction delay. These abnormalities were accompanied by heterogeneous and dyssynchronous calcium cycling despite preserved expression of major calcium-handling proteins. Super-resolution imaging revealed disruption of BIN1-dependent membrane architecture and nanoscale uncoupling of Cav1.2 and RyR2. Inhibition of Rab11b restored BIN1 organization, re-established dyadic coupling, normalized calcium cycling, and improved electrical stability. CONCLUSION: These findings support a model in which altered trafficking balance contributes to disruption of membrane microdomain organization, leading to dyadic uncoupling, calcium instability, and electrical dysfunction. Modulation of the Rab11b-mediated trafficking pathway restored structural and functional abnormalities, supporting the trafficking-associated pathway as a potential therapeutic target in DENND3-associated ventricular arrhythmia.

Myocytes, Cardiac↗
PubMed1998

Evaluation of transgenic event 176 "Bt" corn in broiler chickens.

A 38-d feeding study evaluated whether standard broiler diets prepared with transgenic Event 176-derived "Bt" corn (maize) grain had any adverse effects on male or female broiler chickens as compared to diets prepared with nontransgenic (isogenic) control corn grain. No statistically significant differences in survival or BW were observed between birds reared on mash or pelleted diets prepared with transgenic corn and similar diets prepared using control corn. Broilers raised on diets prepared from the transgenic corn exhibited significantly better feed conversion ratios and improved yield of the Pectoralis minor breast muscle. Although it is not clear whether this enhanced performance was attributable to the transgenic corn per se, or due to possible slight differences in overall composition of the formulated diets, it was clear that the transgenic corn had no deleterious effects in this study.

Animal Feed↗
PubMed2006

Assessment of Populus wood chemistry following the introduction of a Bt toxin gene.

Unintended changes in plant physiology, anatomy and metabolism as a result of genetic engineering are a concern as more transgenic plants are commercially deployed in the ecosystem. We compared the cell wall chemical composition of three Populus lines (Populus trichocarpa Torr. & A. Gray x Populus trichocarpa Bartr. ex Marsh., Populus trichocarpa x Populus nigra L. and Populus deltoides x Populus nigra) genetically modified to express the Cry3A or Cry3B2 protein of Bacillus thuringiensis (Bt) with the cell wall chemistry of non-transformed isogenic control lines. Three genetically modified clones, each represented by 10 independent transgenic lines, were analyzed by pyrolysis molecular beam mass spectrometry, gas chromatography/mass spectrometry and traditional wet chemical analytical methods to assess changes in cell wall composition. Based on the outcome of these techniques, there were no comprehensive differences in chemical composition between the transgenic and control lines for any of the studied clones.

Bacillus thuringiensis↗
PubMed2004

Chronic rejection: insights from a novel immunosuppressive-free model of kidney transplantation.

The use of immunosuppressive drugs in models of chronic rejection may limit their usefulness for mechanistic studies. We have developed a new minor histocompatibility-mismatched rat kidney transplant model without the need for immunosuppression. Kidneys from LEW (RT1(l)) donors were transplanted to congenic WF.1L (RT1(l)) recipients and compared with the reversed strain combination and isogenic controls. Urinary protein excretion was measured serially in all recipients; kidneys were harvested 90, 120, and 180 d after transplantation for morphologic analysis and cytokine gene expression. In vitro lymphocytic reactivity and cytokine analysis of mixed lymphocyte reaction (MLR) culture supernatants by ELISA was also carried out. LEW into WF.1L kidney grafts developed proteinuria starting 120 d after transplantation and were associated with morphologic changes of focal segmental glomerulosclerosis together with interstitial cell infiltrates, upregulated gene expression of IL-1beta, IL-2, and TNF-alpha/-beta, as well as IL-2, IFN-gamma, and TNF-alpha production by lymphocytes in MLR culture supernatants. WF.1L kidneys transplanted into LEW recipients did not develop chronic rejection and had upregulation of Th2 cytokines, both within the allograft and in MLR supernatant of recipient lymphocytes cultured with WF.1L cells. Furthermore, these lymphocytes produced both Th1 and Th2 cytokines when cultured with WF cells, unlike lymphocytes from the LEW isografts, which produced Th1 cytokines when challenged with WF cells. These studies show that indirect allorecognition can cause strain-dependent chronic rejection associated with Th1-like cytokine production, whereas production of Th2 cytokines is associated with protection from the development of chronic rejection.

Animals↗
PubMed2006

Antibody and complement mediated injury in transplants following sensitization by allogeneic blood transfusion.

BACKGROUND: Many patients on the waiting list for transplants are sensitized from previous blood transfusions, pregnancy, or transplants. We investigated the role of complement in acute and chronic pathology in hearts transplanted to sensitized rats. METHODS: Blood was transfused from allogeneic PVG.R8 rats or control isogeneic PVG.1U rats to C6-sufficient and -deficient PVG.1U rats. Three weeks later hearts were transplanted from PVG.R8 donors and low-dose cyclosporin A was initiated. RESULTS: Allogeneic but not isogeneic blood transfusion elicited strong immunoglobulin (Ig) M, IgG1 and IgG2b alloantibody responses. Sensitization caused accelerated acute rejection of cardiac allografts by C6-sufficient recipients (4 days). In contrast, allografts functioned over 40 days in all C6-deficient recipients, but sensitization caused increased interstitial fibrosis and chronic vasculopathy. Circulating alloantibodies were associated with deposits of C4d on the vascular endothelium together with pericapillary accumulation of neutrophils and macrophages in the grafts. In contrast, T cells accumulated in periarterial lymphatics that did not have C4d deposits. CONCLUSIONS: Presensitization by allogeneic blood transfusion causes accelerated acute graft rejection in the presence of the complete complement cascade. In the absence of C6, macrophages colocalized with deposits of C4d and T cells accumulated in the periarterial lymphatics.

Animals↗
PubMed1992

Isolation and characterization of Escherichia coli mutants affected in aerobic respiration: the cloning and nucleotide sequence of ubiG. Identification of an S-adenosylmethionine-binding motif in protein, RNA, and small-molecule methyltransferases.

We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on non-fermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7 kb portion of gyrA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26.5 kDa. This gene (ubiG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42-44% identity to fungal acetyl-CoA synthetases.

Aerobiosis↗
PubMed1993

Mutants of Escherichia coli affected in respiration: the cloning and nucleotide sequence of ubiA, encoding the membrane-bound p-hydroxybenzoate:octaprenyltransferase.

A mutant of Escherichia coli has been isolated that is unable to grow aerobically on non-fermentable substrates, but able to grow anaerobically on glycerol with alternative electron acceptors such as fumarate. Nitrate as electron acceptor supports anaerobic growth on glycerol, but not on succinate or lactate. Oxygen consumption rates by cell-free extracts with succinate, lactate or glycerol 3-phosphate as substrates were low relative to activities in an isogenic control strain but were restored in vitro by adding ubiquinone-1. Transformation of the mutant with a cloned 2.6 kb ClaI-PvuII fragment of chromosomal DNA restored cellular quinone levels and growth on succinate. The plasmid also complemented a previously isolated ubiA mutant for aerobic growth on non-fermentable substrates. The nucleotide sequence of the cloned fragment revealed a fragment of plsB (91.7 min on the E. coli chromosome map) and three open reading frames (ORFs), one of which (ORF3) encodes a protein with a predicted molecular mass of 32511 Da. The hydrophobicity profile of the ORF3 protein is characteristic of a membrane protein with five hydrophobic regions and is very similar to that of the Saccharomyces cerevisiae COQ2 gene product (p-hydroxybenzoate:polyprenyltransferase, required for the second step of ubiquinone biosynthesis) and to the product of the E. coli cyoE gene. Complementation of ubi mutants with various deletion derivatives of the cloned DNA fragment confirms that ORF3 is ubiA. ORF3 is closely linked to ubiC (ORF2), which encodes chorismate lyase.

Alkyl and Aryl Transferases↗
PubMed2026

Functional validation of the Plasmodium falciparum K13 C580Y mutation in recently collected Ethiopian isolates.

Recent genomic investigation in Ethiopia identified the first detection of the Plasmodium falciparum Kelch13 (K13) C580Y substitution in the Horn of Africa. To assess its functional impact, we introduced C580Y into two recently collected Ethiopian clinical isolates using CRISPR-Cas9 genome editing. Ring-stage survival assays showed significantly elevated in vitro dihydroartemisinin survival in edited parasites relative to isogenic controls, demonstrating that C580Y confers artemisinin tolerance in contemporary Ethiopian parasite genetic backgrounds.

CRISPR-Cas9↗
PubMed2002

Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes.

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a hemB mutant of S. aureus 8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of clfA and fnb genes by the hemB mutant compared to its isogenic parent. The influence of the hemB mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the hemB mutant, either by complementing with intact hemB or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the hemB mutation occurred independently from agr, a major regulatory locus of virulence factors in S. aureus. Both agr-positive and agr-lacking hemB mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins.

Adhesins, Bacterial↗
PubMed1975

Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli.

A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.

Chromosome Mapping↗