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The chromosome copy number of the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Spaans SK, van der Oost J, Kengen SW - Extremophiles (2015)

Bottom Line: While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined.In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase.An apparent correlation between the presence of histones and polyploidy in Archaea is observed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Wageningen University, PO Box 8033, 6700 EJ, Wageningen, The Netherlands, sebastiaan.spaans@wur.nl.

ABSTRACT
The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed.

No MeSH data available.


Chromosome copy number quantification by “agarose gel method”. a Agarose gel containing cell lysate samples of three independent cultures, in duplicate, flanked by a 1-kb GeneRuler DNA ladder. The white line indicates non-adjacent lanes of the same gel. The DNA concentration and chromosome copy number of each sample was quantified by comparing the fluorescence intensity of the gel bands to that of the left and right ladder. b The number of chromosome copies per cell. This was calculated by dividing the chromosome copy number of the lysate by the original cell density of each replicate
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Fig2: Chromosome copy number quantification by “agarose gel method”. a Agarose gel containing cell lysate samples of three independent cultures, in duplicate, flanked by a 1-kb GeneRuler DNA ladder. The white line indicates non-adjacent lanes of the same gel. The DNA concentration and chromosome copy number of each sample was quantified by comparing the fluorescence intensity of the gel bands to that of the left and right ladder. b The number of chromosome copies per cell. This was calculated by dividing the chromosome copy number of the lysate by the original cell density of each replicate

Mentions: To make growth curves, five independent cultures of T. kodakarensis were grown on pyruvate, and their average OD600 values and their variances were used to construct Fig. 1. The results show that growth is highly reproducible, which is a prerequisite for reliable chromosome copy number quantification methods. Before an exact chromosome copy number quantification was performed, however, a preliminary in-gel quantification was applied first to determine the probability of polyploidy in T. kodakarensis, and hence if a more exact quantification method was worth applying. This was done by preparing cell lysate samples, in duplicate, of three independent cultures having an OD600 of ~0.76. Corresponding cell densities were determined and aliquots of the 6 samples obtained were loaded on an agarose gel flanked by a known amount of a 1-kb GeneRuler DNA ladder (Thermo Scientific) as shown in Fig. 2a. The DNA concentration and chromosome copy number of each sample was quantified by measuring the fluorescence intensity of the corresponding gel bands and comparing it to the ladder containing known concentrations of DNA. Together with the original cell density of each sample, the number of chromosome copies per cell was calculated (supplementary file). The results are shown in Fig. 2b. These results clearly indicate polyploidy in T. kodakarensis, suggesting an average chromosome copy number of 17.8 ± 3.8. It was therefore decided to determine the chromosome copy number by a more exact quantification method as well.Fig. 2


The chromosome copy number of the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Spaans SK, van der Oost J, Kengen SW - Extremophiles (2015)

Chromosome copy number quantification by “agarose gel method”. a Agarose gel containing cell lysate samples of three independent cultures, in duplicate, flanked by a 1-kb GeneRuler DNA ladder. The white line indicates non-adjacent lanes of the same gel. The DNA concentration and chromosome copy number of each sample was quantified by comparing the fluorescence intensity of the gel bands to that of the left and right ladder. b The number of chromosome copies per cell. This was calculated by dividing the chromosome copy number of the lysate by the original cell density of each replicate
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4502288&req=5

Fig2: Chromosome copy number quantification by “agarose gel method”. a Agarose gel containing cell lysate samples of three independent cultures, in duplicate, flanked by a 1-kb GeneRuler DNA ladder. The white line indicates non-adjacent lanes of the same gel. The DNA concentration and chromosome copy number of each sample was quantified by comparing the fluorescence intensity of the gel bands to that of the left and right ladder. b The number of chromosome copies per cell. This was calculated by dividing the chromosome copy number of the lysate by the original cell density of each replicate
Mentions: To make growth curves, five independent cultures of T. kodakarensis were grown on pyruvate, and their average OD600 values and their variances were used to construct Fig. 1. The results show that growth is highly reproducible, which is a prerequisite for reliable chromosome copy number quantification methods. Before an exact chromosome copy number quantification was performed, however, a preliminary in-gel quantification was applied first to determine the probability of polyploidy in T. kodakarensis, and hence if a more exact quantification method was worth applying. This was done by preparing cell lysate samples, in duplicate, of three independent cultures having an OD600 of ~0.76. Corresponding cell densities were determined and aliquots of the 6 samples obtained were loaded on an agarose gel flanked by a known amount of a 1-kb GeneRuler DNA ladder (Thermo Scientific) as shown in Fig. 2a. The DNA concentration and chromosome copy number of each sample was quantified by measuring the fluorescence intensity of the corresponding gel bands and comparing it to the ladder containing known concentrations of DNA. Together with the original cell density of each sample, the number of chromosome copies per cell was calculated (supplementary file). The results are shown in Fig. 2b. These results clearly indicate polyploidy in T. kodakarensis, suggesting an average chromosome copy number of 17.8 ± 3.8. It was therefore decided to determine the chromosome copy number by a more exact quantification method as well.Fig. 2

Bottom Line: While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined.In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase.An apparent correlation between the presence of histones and polyploidy in Archaea is observed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Wageningen University, PO Box 8033, 6700 EJ, Wageningen, The Netherlands, sebastiaan.spaans@wur.nl.

ABSTRACT
The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed.

No MeSH data available.