In vivo site-directed mutagenesis using oligonucleotides.

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination.

Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination.

A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae.

A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-microm ([cir+]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-microm ([cir0]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir+] and [cir0] strains.

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae.

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.

Molecular engineering with the FRT sequence of the yeast 2 microm plasmid: [cir0] segregant enrichment by counterselection for 2 microm site-specific recombination.

Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.

The CDC6 gene is required for centromeric, episomal, and 2-microns plasmid stability in the yeast Saccharomyces cerevisiae.

Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested. A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure. A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids.