Linear DNA Plasmids and Killer System of Kluyveromyces lactis

Gunge, Norio; Tokunaga, Masao

doi:10.1007/978-3-662-07426-8_11

Cited by 7 publications

(9 citation statements)

References 134 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Eukaryotic mRNAs generally contain an m 7 G cap structure at their 5' ends, which is a prerequisite for their stability and efficient translation initiation (72)(73)(74). Even though the pGKL plasmids encode their own putative capping enzyme (K2Orf3p), we found that few pGKL1/2 genes give rise to the frequently 5'-capped transcripts and that most of the transcripts contain a short leader of non-template adenosine nucleotides (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) at their 5' ends that could not have been continuously transcribed from the plasmids (Figs. 2E, 3A and C, 7B;…”

Section: Poly(a) Leadermentioning

confidence: 80%

“…A PCR cassette consisting of a modified T7 promoter (130) and the first 579 nt (including 32 nt of 5' UTR) of the K. lactis ACT gene coding sequence was transcribed using T7 RNA polymerase. The resulting uncapped RNA molecules (1) were capped using a vaccinia virus capping enzyme in the absence (2) or presence of SAM (not shown). The RNAs were incubated with either Rai1 (3) or hDcp2 (not shown).…”

Section: Figurementioning

confidence: 99%

“…Linear plasmids have been found in the cytoplasm of yeast species from nine genera, including Kluyveromyces, Debaryomyces, Saccharomyces, Saccharomycopsis, Wingea, and Pichia (1,2). The overall genetic organization of all yeast cytoplasmic linear plasmids is identical with that of the most studied plasmids, pGKL1 (also termed K1) and pGKL2 (also termed K2), from the yeast Kluyveromyces lactis, where their presence is associated with the killer phenotype (3).…”

Section: Introductionmentioning

confidence: 99%

“…All ORFs are likely expressed independently. Each gene is preceded by an upstream conserved sequence (UCS) motif, which is located approximately −30 nucleotides from the putative start codon for pGKL1 UCSs (AT A / C TGA) or up to −100 nucleotides in the case of pGKL2 UCSs (ATNTGA) (for review see (2,18)). Sequences located between an AUG initiation codon and its respective UCS (inclusive) act as promoters in sensu lato (25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Vopálenský

Sýkora

Mašek

et al. 2018

Preprint

View full text Add to dashboard Cite

Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids from Kluyveromyces lactis as a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5' and 3' RACE analysis of all the pGKL1,2 mRNAs and found them not 3' polyadenylated and containing mostly uncapped 5' poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5' capping and/or 5' polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence that K2ORF3 encodes an mRNA cap guanine-N 7 -methyltransferase and that 5' capped pGKL1,2 transcripts contain N 7 -methylated caps. Translation of pGKL1,2 transcripts is enhanced in lsm1Δ and pab1Δ strains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5' mRNA capping, degree of 5' non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses. doi: bioRxiv preprint 4 synthesized in a template-independent manner. We show that plasmid promoters directly determine the formation of the mRNA 5' end leaders and their capping, pointing to a possible role of the plasmid RNA polymerase in this unusual phenomenon. We also provide the first evidence that MTase is an essential part of the K2Orf3p enzyme and that capped pGKL mRNAs are methylated at the N 7 position of the terminal guanosine moiety. Using in vitro and in vivo approaches, we demonstrate that translation of plasmid-specific mRNAs is independent of major cellular cap-binding proteins, translation initiation factor 4E (eIF4E), and the Pab1 and Lsm1 proteins that were speculated to be possible translational enhancers and/or stabilizers of cellular and viral mRNAs bearing 5' poly(A) stretches (38,39). Our results thus suggest the existence of a novel translation initiation mechanism. Materials and Methods General Strategy for the Modification of pGKL Plasmids Using Homologous Recombination In VivoK. lactis IFO1267 was transformed with a PCR-generated fragment consisting of 5' and 3' ends homologous to the part of the pGKL plasmid to be modified, a non-homologous part that introduces mutation/s into a plasmid-specific protein coding sequence, and a gene encoding a resistance marker (typically against G418 or hygromycin B) whose transcription is driven by the UCR of K1ORF1 or K1ORF2. This type of construct was prepared by fusion PCR; for the organization of such a construct, see Fig. 2A. After transformation, yeast cells were plated onto selective agar media, and their DNA content was analyzed for the presence of the modified pGKL plasmid using agarose electrophoresis. Both modified and wild-type t...

show abstract

Section: Poly(a) Leadermentioning

confidence: 80%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Vopálenský

Sýkora

Mašek

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…The most thoroughly characterized system consists of the nonautonomous killer plasmid pGKL1 (8.9 kb) and the associated autonomous pGKL2 (13.4 kb) from K. lactis Gunge & Tokunaga, 2004;, which have been functionally transferred to Saccharomyces cerevisiae . pGKL1 carries four ORFs of known functions, whereas the larger element pGKL2 has 11 ORFs (Larsen & Meinhardt, 2000), with functions attributed only to ORF2-7 and 10 on the basis of sequence alignments; in some cases experimental evidence also exists.…”

Section: Introductionmentioning

confidence: 99%

Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points

Jeske

Tiggemann

Meinhardt

2006

FEMS Microbiology Letters

View full text Add to dashboard Cite

A pair of linear plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb), resides in the cytoplasm of Kluyveromyces lactis killer strains. The smaller element, actually conferring the killer phenotype, strictly depends on the larger autonomous pGKL2. Here, we have examined the previously uncharacterized pGKL2 open reading frame (ORF)9 (1.34 kb). Northern analysis of a killer plasmid carrying Saccharomyces cerevisiae strain applying an ORF9-specific probe revealed a single transcript closely matching the size of the ORF9 coding region. Multiple transcriptional start points, determined by primer extension analysis, are located 16 nt downstream of a conserved sequence element regarded as the cytoplasmic promoter. In vivo disruption of pGKL2/ORF9 using the cytoplasmically expressible marker-gene LEU2* resulted in the establishment of a three-plasmid system composed of the native cytoplasmic elements pGKL1/2 and a hybrid of the latter, which only remained stable under selective conditions. The native pGKL2, however, did not segregate during prolonged subcultivations, proving an essential function of ORF9 for plasmid maintenance.

show abstract

Linear Protein-Primed Replicating Plasmids in Eukaryotic Microbes

Klassen

Meinhardt

Microbiology Monographs

View full text Add to dashboard Cite

Linear DNA Plasmids and Killer System of Kluyveromyces lactis

Cited by 7 publications

References 134 publications

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points

Linear Protein-Primed Replicating Plasmids in Eukaryotic Microbes

Contact Info

Product

Resources

About