Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach

Abstract

A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination.

Live Cell Imaging in Fission Yeast

“…cDNAs encoding FP tags can easily be fused to the 3 ′ terminus of a gene at its genomic locus in less than a week via polymerase chain reaction techniques (Wach et al 1997;Bähler et al 1998;Janke et al 2004), whereas recombinase-mediated cassette exchange (RMCE) and marker switch approaches that take marginally longer support tagging at the amino terminus (MacIver et al 2003;Watson et al 2008;Tang et al 2011). The aim is to support stable expression at wild-type levels, because the single integrated FP-tagged allele remains under the control of the endogenous promoter at the endogenous chromosomal location.…”

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

“…Marker reconstitution mutagenesis was used to screen for mutants as described by Tang et al (2011). First, a fusion PCR fragment was amplified using four primers (GGCGGA GATATC GTTTTC TTGCTC TGTTTT C, GGTACC ACCAGC TGAAGA TGATAC AACTCT AC, CATCTT CAGCTG GTGGTA CCACTA TGTATC, and GCTATA CGATAT CCAGAT CTACCC AAAGTT CCTCAT GAG, as p1, p2, p3, and p4, respectively), digested with BglII and EcoRV, and then subcloned into the NruI-BglII site of p208H5cdU4 + , generating p208-act1.…”

“…To isolate an allelic series of actin mutants in fission yeast, we performed a screen using marker reconstitution mutagenesis (Tang et al 2011), a novel and highly efficient reverse genetics approach. As shown in Figure 1A, a mutagenic PCR fragment of act1-his5c (the whole act1 + gene and the C-terminal part of the his5 + gene) was transformed into a strain with an integrated his5cD fragment (a fragment of his5 + with a 50-bp C-terminal part truncation) adjacent to the endogenous act1 + locus.…”

“…In fission yeast, actin is present in different cytoskeletal structures, such as patches containing branched actin filaments, cables, and the cytokinetic ring composed of linear and unbranched actin filaments (Marks et al 1986;Machesky and Gould 1999;Feierbach and Chang 2001;Noguchi and Mabuchi 2001;Arai and Mabuchi 2002;Nakano et al 2002;Kovar et al 2003). These features combined with the ease of genetic manipulation and the availability of mutants affecting 40 proteins involved in cytokinesis provide an opportunity to dissect actin function in all aspects of cell physiology, particularly in cytokinesis.In this study, we utilized a highly efficient approach known as marker reconstitution mutagenesis (Tang et al 2011) to generate an allelic series of actin mutants. We have identified 39 mutant alleles with amino acid changes distributed throughout the protein and, in most cases, defective in distinct cellular processes.…”

“…In this study, we utilized a highly efficient approach known as marker reconstitution mutagenesis (Tang et al 2011) to generate an allelic series of actin mutants. We have identified 39 mutant alleles with amino acid changes distributed throughout the protein and, in most cases, defective in distinct cellular processes.…”