scholarly journals Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium

2020 ◽  
Vol 21 (6) ◽  
pp. 2028
Author(s):  
Christelle Durrant ◽  
Jana I. Fuehring ◽  
Alexandra Willemetz ◽  
Dominique Chrétien ◽  
Giusy Sala ◽  
...  
Keyword(s):  
Carbon Source ◽  
Cell Line ◽  
Growth Medium ◽  
Sole Carbon Source ◽  
Chinese Hamster ◽  
Lung Fibroblasts ◽  
Wild Type ◽  
Galactose Metabolism ◽  
Partial Restoration ◽  
Fold Reduction

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

scholarly journals Mutants affecting histidine utilization inAspergillus nidulans

1975 ◽  
Vol 25 (2) ◽  
pp. 119-135 ◽  
Author(s):  
Meryl Polkinghorne ◽  
M. J. Hynes
Keyword(s):  
Carbon Source ◽  
Nitrogen Source ◽  
Sole Carbon Source ◽  
Nitrogen Sources ◽  
Limiting Factor ◽  
Sole Nitrogen Source ◽  
Wild Type ◽  
Exogenous Substrate ◽  
Growth Properties ◽  
Type Strains

SUMMARYWild-type strains ofAspergillus nidulansgrow poorly onL-histidine as a sole nitrogen source. The synthesis of the enzyme histidase (EC. 4.3.1.3) appears to be a limiting factor in the growth of the wild type, as strains carrying the mutantareA102 allele have elevated histidase levels and grow strongly on histidine as a sole nitrogen source.L-Histidine is an extremely weak sole carbon source for all strains.Ammonium repression has an important role in the regulation of histidase synthesis and the relief of ammonium repression is dependent on the availability of a good carbon source. The level of histidase synthesis does not respond to the addition of exogenous substrate.Mutants carrying lesions in thesarA orsarB loci (suppressor ofareA102) have been isolated. The growth properties of these mutants on histidine as a sole nitrogen source correlate with the levels of histidase synthesized. Mutation at thesarA andsarB loci also reduces the utilization of a number of other nitrogen sources. The data suggest that these two genes may code for regulatory products involved in nitrogen catabolism. No histidase structural gene mutants were identified and possible explanations of this are discussed.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line

Genome ◽  
2003 ◽  
Vol 46 (4) ◽  
pp. 707-715 ◽  
Author(s):  
K Neumann ◽  
K M Al-Batayneh ◽  
M J Kuiper ◽  
J Parsons-Sheldrake ◽  
M G Tyshenko ◽  
...  
Keyword(s):  
Cell Line ◽  
Point Mutation ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Single Point Mutation ◽  
Parental Line ◽  
Wild Type ◽  
Drosophila Cell

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were ~1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing ~400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.Key words: dihydrofolate reductase, methotrexate, drug resistance, point mutation.

scholarly journals Inositol Catabolism, a Key Pathway in Sinorhizobium meliloti for Competitive Host Nodulation

2010 ◽  
Vol 76 (24) ◽  
pp. 7972-7980 ◽  
Author(s):  
Petra R. A. Kohler ◽  
Jasmine Y. Zheng ◽  
Elke Schoffers ◽  
Silvia Rossbach
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Mutational Analysis ◽  
Nodule Occupancy ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Catabolic Pathway ◽  
Inositol Dehydrogenase

ABSTRACT The nitrogen-fixing symbiont of alfalfa, Sinorhizobium meliloti, is able to use myo-inositol as the sole carbon source. Putative inositol catabolism genes (iolA and iolRCDEB) have been identified in the S. meliloti genome based on their similarities with the Bacillus subtilis iol genes. In this study, functional mutational analysis revealed that the iolA and iolCDEB genes are required for growth not only with the myo-isomer but also for growth with scyllo- and d-chiro-inositol as the sole carbon source. An additional, hypothetical dehydrogenase of the IdhA/MocA/GFO family encoded by the smc01163 gene was found to be essential for growth with scyllo-inositol, whereas the idhA-encoded myo-inositol dehydrogenase was responsible for the oxidation of d-chiro-inositol. The putative regulatory iolR gene, located upstream of iolCDEB, encodes a repressor of the iol genes, negatively regulating the activity of the myo- and the scyllo-inositol dehydrogenases. Mutants with insertions in the iolA, smc01163, and individual iolRCDE genes could not compete against the wild type in a nodule occupancy assay on alfalfa plants. Thus, a functional inositol catabolic pathway and its proper regulation are important nutritional or signaling factors in the S. meliloti-alfalfa symbiosis.

Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells

1988 ◽  
Vol 8 (3) ◽  
pp. 1137-1146
Author(s):  
C A Weber ◽  
E P Salazar ◽  
S A Stewart ◽  
L H Thompson
Keyword(s):  
Cell Line ◽  
Nucleotide Excision Repair ◽  
Human Gene ◽  
Excision Repair ◽  
Hybrid Cell ◽  
Repetitive Sequences ◽  
Chinese Hamster ◽  
Wild Type ◽  
Wild Type Level ◽  
Nucleotide Excision

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

Cometabolic oxidation of phenanthrene to phenanthrenetrans-9,10-dihydrodiol byMycobacteriumstrain S1 growing on anthracene in the presence of phenanthrene

1999 ◽  
Vol 45 (5) ◽  
pp. 369-376 ◽  
Author(s):  
Saowanit Tongpim ◽  
Michael A Pickard
Keyword(s):  
Carbon Source ◽  
Growth Medium ◽  
Aromatic Compounds ◽  
Sole Carbon Source ◽  
Polycyclic Aromatic Compounds ◽  
P450 Monooxygenase ◽  
Original Source ◽  
Dead End ◽  
Polycyclic Aromatic ◽  
Rhodococcus Strain

Mycobacterium strain S1, originally described as Rhodococcus strain S1 by chemotaxonomic criteria, was isolated by growth on anthracene, and is unable to use any of nine other polycyclic aromatic compounds as carbon source. Metabolism of phenanthrene during growth on anthracene as sole carbon source results in the accumulation of traces of a dihydrodiol metabolite in the growth medium, which, by comparison with authentic standards, has been tentatively identified as phenanthrene trans-9,10-dihydrodiol. Anthracene metabolites were ruled out on the basis of comparisons with authentic anthracene dihydrodiols from Pseudomonas fluorescens D1 and chemically synthesized anthrols. The original source of phenanthrene for dihydrodiol production was phenanthrene present as a <1% contaminant in the anthracene used as carbon source. However, addition of further phenanthrene to the anthracene growth medium increased the level of phenanthrene trans-9,10-dihydrodiol formed. Mycobacterium strain S1 also produced phenanthrene trans-9,10-dihydrodiol when grown in a glucose-salts medium in the presence of phenanthrene. This dihydrodiol is a dead-end metabolite, and neither it nor its parent hydrocarbon are able to support the growth of Mycobacterium strain S1. Studies with metyrapone and ancimidol, which did not inhibit growth on anthracene but did inhibit formation of phenanthrene trans-9,10-dihydrodiol, suggest it is likely the product of a cytochrome P450 monooxygenase-like activity.Key words: phenanthrene trans-9,10-dihydrodiol, Mycobacterium.

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line

1984 ◽  
Vol 4 (7) ◽  
pp. 1320-1325
Author(s):  
P C Ghosh ◽  
R B Wellner ◽  
H C Wu
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Lag Time ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Osmotic Lysis ◽  
Ricin A ◽  
Pinocytic Vesicles

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.

scholarly journals Genetically Modified Strains of Ralstonia eutropha H16 with β-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents

2012 ◽  
Vol 78 (15) ◽  
pp. 5375-5383 ◽  
Author(s):  
Nicole Lindenkamp ◽  
Elena Volodina ◽  
Alexander Steinbüchel
Keyword(s):  
Fatty Acids ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Production Capacity ◽  
Wild Type ◽  
Content Type ◽  
Storage Behavior ◽  
Copolymer Poly

ABSTRACTβ-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate,Ralstonia eutrophaH16 produces the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]. InR. eutropha, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373–5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (phaA,bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, andpcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-typeR. eutrophaand the mutants. In contrast, during the growth on oleate of a multiple mutant lackingphaA,bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16ΔphaAand all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.

Rhizosphere competence of benomyl-tolerant mutants of Trichoderma spp.

1988 ◽  
Vol 34 (5) ◽  
pp. 694-696 ◽  
Author(s):  
Jaleed S. Ahmad ◽  
Ralph Baker
Keyword(s):  
Growth Rate ◽  
Carbon Source ◽  
Trichoderma Harzianum ◽  
Sole Carbon Source ◽  
Linear Growth ◽  
Dry Weight ◽  
Linear Growth Rate ◽  
Wild Type ◽  
Trichoderma Spp ◽  
Rhizosphere Competence

Two strains of Trichoderma harzianum and one each of T. koningii, T. polysporum, and T. viride were mutated for tolerance to the fungicide benomyl. Rhizosphere competence index of several mutants of each strain and species was determined by the rhizosphere competence assay. Most of the mutants and not their wild type parents were rhizosphere competent. When the strains and species were grown in Czapek–Dox broth for 6 days with cellulose as sole carbon source, the mutants produced significantly higher dry weight than their parent wild types. Neither the mutants nor the wild types produced biomass in glucose comparable to that in cellulose. Evidence indicates that Trichoderma spp. were induced by mutation to increase their linear growth rate and to become rhizosphere competent. Tolerance to benomyl does not seem to be a necessary attribute of rhizosphere competence.

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