Plasmid K1 Research Articles

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis.

The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.

Use of gene shuffles to study the cytoplasmic transcription system operating on Kluyveromyces lactis linear DNA plasmids.

A modified double selection approach for manipulation of the cytoplasmic plasmids k1 and k2 from dairy yeast Kluyveromyces lactis has been exploited to investigate promoter and gene function. Using TRP1-mediated integration of a LEU2 gene fusion, we have shown that expression of the selection marker is strictly dependent on the k2 promoter UCS5. Also, k2ORF6, the gene encoding the RNA polymerase specific for UCS recognition, is functional when shuffled between the plasmids. Once transplaced onto k1 by means of gene shuffling, the hybrid ORF6 complemented an orf6 deletion created on plasmid k2 eventually yielding yeast strains that contained only two recombinant plasmids: a k2 derivative (rk2/6) with a k2orf6::TRP1 gene deletion, and a k1 derivative (rk1/6) carrying the transplaced ORF6 allele along with the LEU2 marker. This interchangeability of both UCS promoter activity and gene function between k2 and k1 supports the concept of an autonomous transcription system that operates on these nonconventional yeast plasmids.

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Kluyveromyces lactis killer plasmid pGKL2: molecular analysis of an essential gene, ORF5.

The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called P k1 containing most of the terminal repeat sequence was isolated and examined for promoter activity. P k1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn 903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative ( lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of P k1.The promoter activity of P k1 fragment was independent of the presence or absence of killer plasmids. It is not known whether P k1 can also function bidirectionally on the natural k1 plasmid. The possible functions of P k1 for killer plasmid gene expression and plasmid replication are discussed.

Characterization and sequence of lupin mitochondrial plasmid-like DNA.

Two minicircular DNAs of 1.2 kb (K1) and 1.4 kb (K2) were found in mitochondria of fertile lupin (Lupinus albus). The plasmid-like DNA, K1, was cloned, labelled and hybridized with mitochondrial DNA from three different species of lupin. We have found no evidence for integrated copies of K1 in any of the mitochondrial genomes probed in this study. No sequence homology between plasmid K1 and K2, and no homology of either with chloroplast DNA, has been detected. The K1 DNA is two-fold more abundant than the K2 DNA and about seven-fold more abundant than a unique segment of the mtDNA. The entire nucleotide sequence of the K1 DNA has been determined. This sequence exhibits a 340 base pair region with highly organized repeats. The sequence of K1 shows no substantial homology with sequence of other mitochondrial plasmids of higher plants.

Genome organization of the killer plasmid pGK12 from Kluyveromyces lactis.

We have determined the entire sequence of the plasmid K2 from Kluyveromyces lactis which is involved in the maintenance of both killer plasmids in the cell. K2 shares many of the characteristics of the smaller killer plasmid K1: high A+T content (74.7%) and very compact genomic organization. K2 contains ten open reading frames. Some of them overlap on different strands and some on the same strand. Northern blotting of K2 transcripts shows that at least eight ORFs are transcribed. Analysis of the predicted aminoacid sequence of ORF2 from K2 reveals homology with the aminoacid sequence of ORF 1 from K1 and with several viral DNA polymerases. The sequence of K2 from Saccaromyces cerevisiae F102-2 was also determined. Only one nucleotide difference was found between the K2 sequence from the two yeasts. This mutation does not change the genome organization of the plasmid and has only minimal effect on the structure of the encoded proteins.

A transcriptional barrier to expression of cloned toxin genes of the linear plasmid k1 of Kluyveromyces lactis: evidence that native k1 has novel promoters.

The killer toxin of Kluyveromyces lactis consists of three polypeptides encoded by the linear plasmid k1. We re-introduced the entire k1 sequence, cloned on a circular replicating plasmid, into K. lactis strains lacking k1, and found that the resulting transformants did not produce toxin. The barrier to expression was found to be transcriptional: the four transcripts of native k1 were absent, and instead shorter, aberrant k1 transcripts were made. We determined the precise initiation sites of the four transcripts of native k1: these had very short untranslated leaders and mapped about 14bp downstream of an "upstream conserved sequence" (UCS). It appears that k1 has novel promoters which are inactive on circular plasmids which replicate in the nucleus. This is consistent with the suggestion that native k1 resides in the cytoplasm.

The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them.

The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium. We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol. wt 99,000), beta- (mol. wt 30,000) and gamma- (mol. wt 27,500) subunits. The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated. The N-terminal amino acid sequence of each subunit has been determined. Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits. The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.

Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis.

In killer strains of the yeast Kluyveromyces lactis, production of a protein toxin which inhibits the growth of sensitive yeast cells is associated with the presence of two linear DNA plasmids, k1 and k2. We have determined the nucleotide sequence of the smaller plasmid k1 (8.9kb) which is thought to carry the structural gene(s) encoding the toxin. The plasmid has a low G + C content (26.8%) and contains four long open reading frames which account for over 95% of the total sequence. The longest open reading frame (1146 amino acids) probably corresponds to a structural gene for the killer toxin. Transcripts from three of the putative genes have been detected in K.lactis by Northern hybridisation.

Killer DNA plasmids of the yeast Kluyveromyces lactis

The killer character of Kluyveromyces lactis is cytoplasmically inherited. Two DNA plasmids k1 and k2 (8.8 and 13.4 kilobase pairs, respectively) are associated with the killer system. Either cytoplasmic or nuclear mutations can lead to a loss of the killer activity. Two cytoplasmic non-killer mutants have been specifically studied. When they were crossed to each other, the killer phenotype was restored. Genetic analysis led us to conclude that each of the two mutants carried a mutation in the k1 plasmid and that the recovery of killer phenotype was due to genetic recombination of the plasmids, and not to complementation.

Multiple actin-related sequences in the Drosophila melanogaster genome

In this paper we describe the isolation and characterization of a 7.2 kb D. melanogaster chromosomal DNA fragment (K1) which contains nucleotide sequences complementary to D. melanogaster actin mRNA. Plasmid K1 was identified using a Dictyostelium actin cDNA plasmid, B1, as a probe. D. melanogaster mRNA selected by hybridization with immobilized K1 DNA was translated in vitro to yield products which co-migrate with the D. melanogaster actins I, II and III in two-dimensional gel electrophoresis and bind to DNAase I agarose. A physical map localizing restriction endonuclease cleavage sites in the K1 DNA fragment and the direction of transcription is presented. The position of the coding region has been localized by hybridization with labeled B1 DNA and with labeled poly(A)-containing D. melanogaster RNA. On the basis of hybridization of labeled subfragments of plasmid K1 to restriction endonuclease-cleaved D. melanogaster embryo DNA, we conclude that the nucleotide sequence of the presumptive coding region is responsible for labeling of a pattern of multiple restriction fragments from embryo DNA. The chromosomal locus from which DNA fragment K1 is derived has been localized by in situ hybridization to two closely linked bands in the region 88F. Related DNA sequences corresponding to putative actin genes have also been mapped cytologically. These results support the hypothesis that the genes for actin in D. melanogaster are members of a closely related family of coding sequences.