scholarly journals Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to α-difluoromethylornithine

1991 ◽  
Vol 278 (3) ◽  
pp. 871-874 ◽  
Author(s):  
J M Hyttinen ◽  
M Halmekytö ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Ornithine Decarboxylase ◽  
Reverse Transcription ◽  
Copy Number ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Dot Blot ◽  
Semiquantitative Assessment ◽  
Myeloma Cells ◽  
Human Myeloma Cells

We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.

scholarly journals Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells

1995 ◽  
Vol 310 (1) ◽  
pp. 299-303
Author(s):  
J Wahlfors ◽  
S Myöhänen ◽  
V P Korhonen ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Enzyme Activity ◽  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Copy Number ◽  
Methylation Status ◽  
Gene Copy Number ◽  
Myeloma Cell ◽  
Gene Copy ◽  
Myeloma Cell Line ◽  
Myeloma Cells

(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Resistance to methotrexate due to gene amplification in a patient with acute leukemia.

1984 ◽  
Vol 2 (1) ◽  
pp. 16-20 ◽  
Author(s):  
M D Carman ◽  
J H Schornagel ◽  
R S Rivest ◽  
S Srimatkandada ◽  
C S Portlock ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Copy Number ◽  
Cdna Probe ◽  
Leukemia Cell ◽  
Gene Copy Number ◽  
Leukemia Cell Line ◽  
Gene Copy ◽  
Acute Myelocytic Leukemia ◽  
Human Leukemia ◽  
Dot Blot

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.

scholarly journals Genome instability drives epistatic adaptation in the human pathogen Leishmania

2021 ◽  
Vol 118 (51) ◽  
pp. e2113744118
Author(s):  
Giovanni Bussotti ◽  
Laura Piel ◽  
Pascale Pescher ◽  
Malgorzata A. Domagalska ◽  
K. Shanmugha Rajan ◽  
...  
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Biomarker Discovery ◽  
Genome Instability ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Snorna Gene ◽  
Ideal System

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

scholarly journals Genome plasticity in Paramecium bursaria revealed by population genomics

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yu-Hsuan Cheng ◽  
Chien-Fu Jeff Liu ◽  
Yen-Hsin Yu ◽  
Yu-Ting Jhou ◽  
Masahiro Fujishima ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Copy Number ◽  
Population Genomics ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Paramecium Bursaria ◽  
Gene Copy ◽  
Genome Plasticity ◽  
Ciliate Species ◽  
Number Variation

Abstract Background Ciliates are an ancient and diverse eukaryotic group found in various environments. A unique feature of ciliates is their nuclear dimorphism, by which two types of nuclei, the diploid germline micronucleus (MIC) and polyploidy somatic macronucleus (MAC), are present in the same cytoplasm and serve different functions. During each sexual cycle, ciliates develop a new macronucleus in which newly fused genomes are extensively rearranged to generate functional minichromosomes. Interestingly, each ciliate species seems to have its way of processing genomes, providing a diversity of resources for studying genome plasticity and its regulation. Here, we sequenced and analyzed the macronuclear genome of different strains of Paramecium bursaria, a highly divergent species of the genus Paramecium which can stably establish endosymbioses with green algae. Results We assembled a high-quality macronuclear genome of P. bursaria and further refined genome annotation by comparing population genomic data. We identified several species-specific expansions in protein families and gene lineages that are potentially associated with endosymbiosis. Moreover, we observed an intensive chromosome breakage pattern that occurred during or shortly after sexual reproduction and contributed to highly variable gene dosage throughout the genome. However, patterns of copy number variation were highly correlated among genetically divergent strains, suggesting that copy number is adjusted by some regulatory mechanisms or natural selection. Further analysis showed that genes with low copy number variation among populations tended to function in basic cellular pathways, whereas highly variable genes were enriched in environmental response pathways. Conclusions We report programmed DNA rearrangements in the P. bursaria macronuclear genome that allow cells to adjust gene copy number globally according to individual gene functions. Our results suggest that large-scale gene copy number variation may represent an ancient mechanism for cells to adapt to different environments.

scholarly journals Histidine Operon Deattenuation in dnaAMutants of Salmonella typhimurium Correlates with a Decrease in the Gene Dosage Ratio between tRNAHis and Histidine Biosynthetic Loci

1999 ◽  
Vol 181 (9) ◽  
pp. 2938-2941 ◽  
Author(s):  
Anne-Beatrice Blanc-Potard ◽  
Nara Figueroa-Bossi ◽  
Lionello Bossi
Keyword(s):  
Salmonella Typhimurium ◽  
Copy Number ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Gene Copy

ABSTRACT Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37°C. This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of thehisR locus, which encodes the tRNAHis. We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close tooriC, and the his operon, far fromoriC. Some of the conclusions drawn here may apply to other operons as well.

Abstract 2351: MYC gene copy number determine MYC expression and sensitivity to a MYC-inhibitor in multiple myeloma cells

2014 ◽  
Author(s):  
Toril Holien ◽  
Kristine Misund ◽  
Glenn Buene ◽  
Oddrun E. Olsen ◽  
Katarzyna A. Baranowska ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Myeloma Cells ◽  
Myc Gene

scholarly journals Determination of Cytochrome P450 2D6 (CYP2D6) Gene Copy Number by Real-Time Quantitative PCR

2005 ◽  
Vol 2005 (3) ◽  
pp. 248-253 ◽  
Author(s):  
Laurent Bodin ◽  
Philippe H. Beaune ◽  
Marie-Anne Loriot
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Copy Number ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Rapid Identification ◽  
Gene Copy ◽  
Cyp2d6 Gene ◽  
Real Time Quantitative Pcr ◽  
Gene Copies

Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number). Using the2−ΔΔCtcalculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies), and 12 samples with duplicated genes. The average ratio ranged from1.02to1.28,1.85to2.21, and2.55to3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

Population differences in antifreeze protein gene copy number and arrangement in winter flounder

Genome ◽  
1991 ◽  
Vol 34 (1) ◽  
pp. 174-177 ◽  
Author(s):  
Pliny H. Hayes ◽  
Peter L. Davies ◽  
Garth L. Fletcher
Keyword(s):  
Multigene Family ◽  
Copy Number ◽  
Tandem Repeats ◽  
Freezing Point ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Gene Organization ◽  
Winter Flounder ◽  
Gene Copy ◽  
Intrapopulation Variation

Many marine fish in polar waters produce antifreeze proteins (AFPs) to depress their serum freezing point to below that of seawater. Winter flounder from the east coast of North America contain multiple AFP gene copies organized both as tandem repeats and as linked but irregularly spaced genes, with the tandemly repeated genes encoding the bulk of the circulating AFPs. We report here on AFP gene organization in winter flounder from nine locations ranging from Long Island, NY to Conception Bay, Nfld. There are clear differences in AFP gene copy number and arrangement between some of the populations. The greatest variation is seen in the size of the tandem component in fish from the warmer, deeper locations. This contrasts to the conservation of organization in the dispersed, β-tubulin multigene family used for comparative purposes. We suggest that variation in AFP gene family size and organization reflects a relaxation of selection in some geographical areas in the postglacial period.Key words: gene dosage, multigene family, tandem repeats, intrapopulation variation, glaciation.

scholarly journals Aneuploidy influences the gene expression profiles in Saccharomyces pastorianus group I and II strains during fermentation.

2021 ◽  
Author(s):  
Roberto de la Cerda ◽  
Karsten Hookamp ◽  
Fiona Roche ◽  
Georgia Thompson ◽  
Soukaina Timouma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Gene Dosage ◽  
Expression Patterns ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Gene Expression Patterns ◽  
Group I ◽  
Saccharomyces Pastorianus ◽  
Group Ii

The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of Group I and II strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.

scholarly journals Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

2000 ◽  
Vol 346 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Michael G. PATON ◽  
S. H. P. Parakrama KARUNARATNE ◽  
Elsa GIAKOUMAKI ◽  
Neil ROBERTS ◽  
Janet HEMINGWAY
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Gene Copy Number ◽  
Absolute Quantification ◽  
Gene Copy ◽  
Common Mechanism ◽  
Control Mechanisms ◽  
Dot Blot ◽  
Culex Mosquitoes ◽  
Pcr Product

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

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