TY - JOUR
T1 - Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast
AU - Tagwerker, Christian
AU - Dupont, Christopher L.
AU - Karas, Bogumil J.
AU - Ma, Li
AU - Chuang, Ray Yuan
AU - Benders, Gwynedd A.
AU - Ramon, Adi
AU - Novotny, Mark
AU - Montague, Michael G.
AU - Venepally, Pratap
AU - Brami, Daniel
AU - Schwartz, Ariel
AU - Andrews-Pfannkoch, Cynthia
AU - Gibson, Daniel G.
AU - Glass, John I.
AU - Smith, Hamilton O.
AU - Venter, J. Craig
AU - Hutchison, Clyde A.
N1 - Funding Information:
Synthetic Genomics Inc. (SGI); all authors were supported by SGI (in part); National Science and Engineering Research Council of Canada (NSERC, Postdoctoral Fellowship) and SGI [to B.J.K.]. Funding for open access charge: JCVI.
PY - 2012/11
Y1 - 2012/11
N2 - Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8′, similar to that of Saccharomyces cerevisiae (38′), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.
AB - Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8′, similar to that of Saccharomyces cerevisiae (38′), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.
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U2 - 10.1093/nar/gks823
DO - 10.1093/nar/gks823
M3 - Article
C2 - 22941652
AN - SCOPUS:84868275346
SN - 0305-1048
VL - 40
SP - 10375
EP - 10383
JO - Nucleic acids research
JF - Nucleic acids research
IS - 20
ER -