YEAST MOLECULAR GENETICS (B)

Homologous recombination is quite a frequent event!

Gene replacement one step

Deleting a yeast gene There are a number of different ways to generate the piece of DNA for yeast transformation, i.e. the marker flanked by fragments with DNA from YFG1 It can be done using restriction enzymes and DNA ligation It can be done by PCR/restriction/ligation; the entire plasmid is amplified by PCR with the exception of the ORF; restriction sites in the PCR primers generate a site where the marker can be cloned in It can be done by PCR without any cloning step; in two separate PCR reactions the flanking regions of YFG1 are amplified and used in a second round as primers to amplify the marker gene; this requires the primers to be designed accordingly (see below) It can also be done with long PCR primers, in which only the marker is amplified and recombination is mediated by the primer sequences; as little as 30bp can be enough to mediate recombination; in such cases the use of a heterologous marker is recommended to make integration in the right place more reliable The latter two approaches do not even require the gene to be cloned!! A gene deletion project hence may take only a couple of days First PCR to amplify the flanking parts of your favourite gene YFG1 URA3 Second PCR to amplify the marker URA3 Final PCR product ready for transformation

Smart gene deletion There are very smart ways to make most out of a gene deletion/disruption approach, depending on the marker cassette used For instance, if the marker cassette contains in addition the lacZ reporter gene a precise fusion can be generated that places the lacZ gene under control of the yeast promoter of YFG1 If such a construct is used for gene deletion in a diploid, it can be used to study the expression of the gene by monitoring b-galactosidase activity in that diploid and after sporulation of the diploid the mutant phenotype can be studied in the haploid progeny In a similar way, a gene can be tagged. For instance, if the casette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection URA3 lacZ YFG1 Diploid cell

Smart gene deletion In a similar way, a gene can be tagged. For instance, if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection and purification For instance, there are now sets of strains available in which each yeast protein has been tagged with GFP or TAP-tag YFG1 URA3 GFP

Smart gene deletion There are some ways to delete a yeast gene without leaving any trace behind, i.e. no marker gene This is very important if one wants to re-use the marker in order to make many deletions in one and the same strain (there are strains with more than 20 deletions!) It is also important for industrial yeast strains; when one wants to engineer those at the end no foreign DNA should be left behind (but for hardliners on genetic engineering the intermediate presence of foreign DNA in a yeast is already ”dangerous”) All these methods use homologous recombination a second time, i.e. to pop-out the integrated DNA again An example for this are the loxP-kanR-loxP cassettes; recombination between the two loxP cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid); recombination just leaves behind a single loxP site

Gene replacement two steps Questa tecnica può essere utilizzata solo con marcatori selezionabili e controselezionabili Facciamo un esempio….

Smart gene deletion A very useful marker to work with is URA3 because one can select for and against its presence Selection for URA3 is of course done on medium lacking uracil Selection against URA3 uses the drug 5-flouro-orotic acid, which is toxic to URA3 cells An example is shown below URA3 plasmid YFG1 genome YFG1 URA3 Integration of the plasmid, which only contains YFG1 flanking regions, creates a duplication; recombination between the blue sequences leads to a pop-out of the entire plasmid plus the YFG1 coding region

From gene disruption to transposon mutagenesis The gene deletion/disruption technique has been taken a step further to be used in random mutagenesis For this a gene library is first constructed as discussed before such that the inserted yeast DNA can be cut out with NotI, an enzyme that only cuts a very few times in the yeast genome Then this library is mutagenised with a transposon in E. coli, where the Tn randomly integrates into the yeast DNA Subsequently, the entire mix of NotI fragments is transformed into yeast where it is expected to replace genes; with about 30,000 yeast clones a more then 90% coverage of the genome is achieved The Tn used is a quite sophisticated example of such a transposon, that can be partially cut out again through the lox-sites. This creates a tag, which allows immunolocalisation of the gene product TR: Tn3 terminal inverted repeats Xa: Factor Xa cleavage recognition site loxR: lox site, target for Cre recombinase lacZ: 5'-truncated lacZ gene encoding b- galactosidase URA3 gene from S. cerevisiae tet: tetracycline resistance gene res: Tn3 site for resolution of transposition intermediate loxP: lox site, target for Cre recombinase 3xHA: Hemagglutinin (HA) triple epitope tag

From gene disruption to transposon mutagenesis The reason why transposon mutagenesis is so powerful lies in the fact that the gene affected by the insertion can be determined very easily For this, the entire genomic DNA of the mutant is isolated and cut with an enzyme that does not cut within the transposon In this way of course many fragments are generated but only one will contain the transposon plus some flanking yeast DNA Ligation generates a circular plasmid that can be transformed into E. coli and further analysed Sequencing using a primer binding to the transposon but directing into the yeast DNA will reveal exactly where the transposon was integrated when the sequence is compared to that of the yeast genome This method works so well that it has been used for a comprehensive genome analysis For instance, we have recently screened 25,000 Tn-mutants for a number of properties and could allocate functions to a number of uncharacterised genes with relevance to stress tolerance Derivative of the transposon with antibiotic markers are very useful tools to mutagenise and study industrial strains

Analisi genetica delle tetradi Consente di attribuire una funzione a geni mutati (analisi fenotipo dell’aploide mutato)

Visualizza le interazioni proteina-proteina Two-hybrid system Visualizza le interazioni proteina-proteina

Getting further: two-hybrid system The yeast two hybrid system is a method to detect the interaction of two proteins in the yeast cell and it can be used to select for an interacting partner of a known protein The original version uses a transcriptional read-out to monitor interaction, nowadays there are also other methods The method is so powerful since it is not restricted to yeast proteins; the interacting partners can origin from any organism; in fact some versions do not use any yeast sequences Basis for the system is the modular nature of transcription activators that consist of exchangeable DNA binding and transcriptional activation domains The gene of interest, the bait, is cloned in fusion with a DNA binding domain, such as that of the E. coli lexA protein The potential binding partner, the target or prey, which may be a library, is cloned in fusion to a transcriptional activation domain, such as that from VP16, a viral protein Only when bait and target interact, a reporter gene whose only promoter is a lexA binding site will be activated lexA site reporter

Application of the yeast two-hybrid system The possible applications to the two-hybrid system are absolutely tremendous The system can be used to detect interaction between two proteins The system can be used to characterise the domains and residues in the two proteins that mediate interaction; this can be done by mutagenesis and the use of a counterselectable reporter, such as URA3 The system can of course be used to find interaction partners The system can be used to find proteins that regulate the interaction between two proteins The system can be used to screen for drugs that inhibit the interaction between two proteins The system is actually used to construct an genome-wide map of protein interactions in yeast; using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible protein intercations etc................

Use of recombinant yeast in industrial biotechnology

Yeast as cell factory for recombinant protein production (RPP)

Looking for unconventional yeasts

Promoters to be used in RPP

Promoters to be used in RPP

RPP in S. cerevisiae Usata nella terapia riperfusiva post-infarto per minimizzare il danno ossidativo! Farmaco salvavita

RPP in S. cerevisiae

RPP in Pichia pastoris

Which fermentation process for your strain?