Multiple Antibiotic Resistance Plasmids Allow Scalable , PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

Plasmid-mediated antibiotic resistance is commonly used as selection criterion for genetically engineered bacteria and fungi due to its effi ciency and the availability of the respective drugs from commercial sources (1,2). For instance, many functional and genetic studies of yeast and other organisms require convenient plasmid templates for PCR-mediated gene disruptions, deletions (3), the recently developed bridge-induced translocation (BIT) system (4,5) and for many other purposes.


Introduction
Plasmid-mediated antibiotic resistance is commonly used as selection criterion for genetically engineered bacteria and fungi due to its effi ciency and the availability of the respective drugs from commercial sources (1,2).For instance, many functional and genetic studies of yeast and other organisms require convenient plasmid templates for PCR-mediated gene disruptions, deletions (3), the recently developed bridge-induced translocation (BIT) system (4,5) and for many other purposes.
Combining two or three resistance genes simultaneously on the same DNA vector can facilitate selection and eliminate false positives (spontaneous acquisition of resistance to three diff erent drugs is rather improbable, excluding resistance phenotypes caused by the elevated effl ux of drugs from the cell) (2,6).Moreover, a strict selection in combination with stable maintenance will be very advantageous when using shutt le plasmids as shuttle vectors for cloning purposes in yeast.For this reason, we decided to develop a plasmid that is highly selective for both expression purposes as well as for the preparation of functional analysis constructs, allowing a substantial decrease in unwanted background resistance.This has been achieved by combining the coding sequences for antibiotic resistance to three widely available drugs: ampicillin, kanamycin (geneticin in yeast) and hygromycin B, in a specially engineered order.The result was the construction of two plasmids (pMM and pMM-CEN) conferring simultaneous resistance to these drugs, while lending themselves to be easily manipulated and utilized as vectors.As an experimental application of these new vectors, the pMM plasmid was employed as PCR template in the BIT process (5).BIT utilizes homologous recombina-tion to physically bridge two loci of choice within the yeast genome, with a double-stranded DNA amplicon (cassett e) amplifi ed from a pMM template, in which a positively selectable marker is fl anked by short regions of homology (typically 65 bp) to the two loci of choice.

Plasmid construction
All described constructs were built on the pFA6a--kanMX4 (accession number AJ002680; European Molecular Biology Laboratory, EMBL) plasmid platform (10).Firstly, the hygromycin B resistance gene (hphMX4) was amplifi ed from plasmid pAG32 using PCR (11).The PCR product was designed in such a way that it contained a BamHI restriction site at the 5' end, the Hyg R gene, and the KpnI-BglII site at the 3' end of the amplicon.Primers used for construction, verifi cation and testing are reported in Table 1.
All PCR reactions were carried out using Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA, USA) following the manufacturer's instructions.The amplifi ed fragments were purifi ed aft erwards using the Qiagen PCR product purifi cation kit (Qiagen GmbH, Hilden, Germany).Plasmid pFA6a-kanMX4 was linearized using Bam-HI-BglII (New England Biolabs, Ipswich, MA, USA) and hphMX4, a hygromycin B resistance gene ending with appropriate restriction sites, was ligated into the resulting gap using standard protocols (7).The DNA fragment was designed in such a way that it restored functional BamHI and BglII sites aft er ligation.The DNA construct was used to transform genetic recombination-defi cient Sure2 and XL1-Blue E. coli strains to ensure the lack of recombination events between neighbouring DNA translational elongation factor (TEF) elements, and the strains were plated on plates containing either or both ampicillin and hygromycin B (in the case of strain Sure2), or on plates with one or more of the ampicillin, kanamycin and hygromycin B (for strain XL1-Blue).Aft er one day of growth, plasmids were isolated from one of the two strains using the Promega mini-prep kit (Promega, Madison, WI, USA) and used to transform the other E. coli strain.The DNA construct was verifi ed by PCR with primers specifi c for the kanamycin resistance, the hphMX4 and the combination of the two genes by restriction fragment length polymorphism (RFLP, using AatII, BglII, BamHI-BglII and MluI restriction enzymes) and fi nally validated by transforming E. coli DH5α and selecting on LB agar plates containing kanamycin, hygromycin B and ampicillin (7).The result ing plasmid was named pMM and it contained genetic determinants conferring simultaneous resistance to ampicillin, kanamycin (geneticin for yeast) and hygromycin B. Between the kanamycin and hphMX4 resistance genes there is a preferential cloning site that allows the use of both KpnI and BglII restriction enzymes.These sites were utilized when constructing pMM-CEN.Building upon the pMM cloning platform, a full length ARS1--CEN4 DNA fragment with compatible ends derived by PCR from plasmid pYAC3 (12) was inserted between KpnI and BglII sites.The ARS1-CEN4 fragment was obtained using ARS-CEN4 primer set (Table 1) and ligated using a standard ligation protocol.Because only one pair of convenient restriction sites was used for cloning of the ARS--CEN4 region, we decided to add a polylinker with the unique sites PacI, XmaI, KasI and insert it into the KpnI site.The resulting construct was verifi ed by PCR using primers specifi c for kanMX4, hphMX4, ARS-CEN4 and combinations of these genes.Additionally, restriction analysis was performed using the same enzymes as for pMM.Final validation was obtained by transformation of bacteria and yeast followed by selection on media containing ampicillin, kanamycin, hygromycin B in diff erent combinations (for bacteria) and G418 and hygromycin B for yeast.

Transforming Saccharomyces cerevisiae
Saccharomyces cerevisiae San1 cells were transformed with the standard procedure described by Burke et al. (9) with the slight modifi cation of allowing cells to grow longer (1.5 h in YPD) under non-selective conditions before they were plated on selective medium to give them a better chance to express all resistance genes.Bacterial transformation was performed according to commonly known procedures (7).

Antibiotic resistance test
Bacterial colonies were picked and grown overnight in LB both supplemented with 100 μg/mL of ampicillin.The following day, 5 μL of every overnight culture were inoculated into the same medium.Cultures were grown 6 h at 37 °C and then 5 mL of fresh selective medium containing one or a combination of two or three antibiotics were inoculated with 10 μL of these cultures and grown overnight.The following day, the absorbance was measured at 660 nm using a GeneQuant™ pro spectrophotometer (GE Healthcare, Litt le Chalfont, UK).
LB plates supplemented with increasing concentrations of the antibiotics were used to assess the minimum inhibitory concentration (MIC) of the drugs.pMM-containing E. coli DH5α strains were examined for susceptibility to ampicillin, kanamycin and hygromycin B. The strains were transformed with appropriate plasmids for the experiments: pFA6a-kanMX4 to determine resistance to kanamycin, pUC18 to ampicillin and pAG32 to hygromycin B. Wild type strain was used as a control.Final cell counts were adjusted to 10 3 , 10 4 , 10 5 and 10 6 CFU per mL of fresh culture per plate.Aft er 24 h of incubation at 37 °C, microbial growth was checked to determine the MIC value of the various antibiotics.

Plasmid copy number determination
Plasmid copy number was determined using an approach adapted from a previous report (13).First, an internal standard plasmid was constructed using the pGEM ® -T Easy vector system (Promega).The internal standard plasmid contained both, one gene characteristic of the plasmid to be titrated (bla gene) and one specifi c for the bacterial chromosome (dxs).The relative primer sets are specifi ed in Table 1.A PCR mixture containing Kapa HiFi polymerase (Kapa Biosystems) and two sets of primers was amplifi ed twice at the limiting number of 20 and 21 cycles using a Thermo Hybaid Sprint PCR thermal cycler (Ashford, Middlesex, UK).For the internal standard plasmid, the PCR reaction yielded two bands, which were subsequently subjected to densitometric analysis using a Pharmacia-LKB (Uppsala, Sweden) laser scanner densitometer.The intensity of each band was measured three times, and the average value was used to calculate the intensity ratio, which was normalized to 1.For each measurement, two independent references were used (1 and 2) and as an internal control, the copy number of reference 2 was determined using the values of reference 1 and vice versa.When PCR amplifi cation was carried out on genomic DNA of cells containing a plasmid, the reaction also yielded two bands: a plasmid-borne bla fragment and a chromosomal dxs.Calculating their ratio and comparing it to the normalized value of the internal standard, the relative plasmid copy number per cell was calculated.For each sample, references 1 and 2 were used, and the average value was accepted.In total, these measurements were repeated 12 times, every time using two internal standards and those from samples 1 to 3.

Bacterial transformation effi ciency
Both of the constructed plasmids, pMM and pMM--CEN, were used to transform E. coli DH5α strain and the transformants were selected on ampicillin, kanamycin, hygromycin B, ampicillin-kanamycin, ampicillin-hygromycin B, kanamycin-hygromycin B and ampicillin-kanamycin-hygromycin B plates (100 μg/mL of each antibiotic).Aft er incubation, number of CFU was determined using a colony counter (Gallenkamp Co, Ltd, London, UK).The transformation effi ciency was calculated as the number of transformants per μg of DNA.

Bridge-induced translocations
Bridge-induced translocation (BIT) is a system that allows to produce chromosomal translocations between any two genomic loci of choice on diff erent chromosomes by means of a PCR-derived DNA bridge called translocation cassett e.This is a fragment of DNA where a resistance gene is fl anked by two short regions (usually around 64 bp), each homologous to its chromosomal target.pMM and pMM-CEN off er the possibility, as DNA templates, to amplify cassett es with kanamycin, hygromycin and kanamycin-hygromycin on the same amplicon.In addition, pMM-CEN allows the amplifi cation of a DNA cassett e harbouring the CEN4 and an ARS between the kanamycin and hphMX4 resistance genes, thus allowing to test its capacity of bridging two chromosomal portions that do not carry any centromere, a would-be novelty for the BIT system.The pMM plasmid was used as a template for amplifi cation of the BIT translocation cassett e aimed to bridge ADH1 locus localized on chromosome XV and SUC2 locus on chromosome IX in S. cerevisiae as described previously (5).Translocation cassett es were produced using Kapa HiFi polymerase (Kapa Biosystems), which contained the two genes conferring resistance to kanamycin and hygromycin B and were used to transform S. cerevisiae San1 strain.Transformants were initially tested on YPD plates containing G418 and hygromycin B. Recombination of the DNA cassett e ends into each involved chromosome was tested by colony PCR using the mixture of three primers (Table 1), two localized on the genomic sequences and one localized on the translocation cassett e.In the case of chromosome translocation, a product of chromosomal primer and cassett e primer was visible (two bands).Detected translocants were examined by CHEF (contour-clamped homogeneous electric fi eld) analysis as described by Tosato et al. (5) and subsequently by Southern blott ing with the digoxigenin-labelled probes (DIG probe synthesis kit, Roche, Basel, Switzerland) for kanamycin and hphMX4 resistance genes to verify the presence of an aberrant chromosome.Primers used for probe synthesis are shown in Table 1.
These plasmid constructs contain four important gene tic elements placed on the same DNA molecule: a dominant resistance gene for ampicillin (bla), kanamycin (kanMX4 in bacteria) and geneticin (G418 in yeast), hygromycin (hphMX4) (11), and an ARS-CEN4 DNA module (only for pMM-CEN) providing stable maintenance in the yeast cells.The kanMX4 is a DNA segment containing the open reading frame of aminoglycoside phosphotransferase from E. coli transposon Tn903 and the TEF1 (translation elongation factor) promoter and terminator derived from the fi lamentous fungus Ashbya gossypii, providing transcriptional and translational control (10,14).Expression of kanMX4 renders bacteria resistant to kanamycin and yeast cells resistant to geneticin (G418) (15,16).The presence of the hphMX4 gene confers resistance to the antibiotic hygromycin B (11) and the coding region of this Fig. 1. pMM vector map with unique restriction sites.This construct contains functional cassett es conferring resistance to hygromycin (Hyg R ), kanamycin (Kan R ), and ampicillin (Amp R ).Both, kanamycin and hygromycin are under control of the translation elongation factor (TEF) promoter and terminator.Numbers in brachets represent the position of the restriction sites gene is also under the transcriptional and translational control of the TEF1 promoter and terminator.The correct presence of these elements was confi rmed by colony PCRs, as explained in Materials and Methods.For construction of pMM-CEN, the full-length ARS-CEN4 region from the pYAC3 plasmid was inserted between the KpnI and BglII sites.The lack of convenient restriction sites between the markers on pMM-CEN prompted us to add a polylinker fragment in the KpnI site carrying PacI, XmaI and KasI sites.PacI leaves 3' overhang, whereas Xmal and KasI leave 5' overhang, successfully preventing self-ligation.Additionally, they work in the same buff ers and do not exhibit star activity, which makes working with them very simple.These cloning sites can be utilized in several diff erent ways, allowing construction of two-marker cassett es separated by any DNA sequence or two-marker cassett e with a centromeric sequence.Suggested primers for cassett e amplifi cation are shown in Table 1.
These primers were practically tested on the pMM--CEN template and their PCR products showed the correct size (not shown).

Antibiotic resistance tests
Resistance tests indicate that both yeast and bacterial cells were rendered resistant to kanamycin and hygromycin B by the introduction of the pMM plasmid.These two drugs belong to the same group of aminoglycosidic antibiotics and have a similar mode of action (17)(18)(19)(20)(21), which helps to reduce the incidence of spontaneous resistance and lowers the percentage of false positives.Additionally, the plasmid confers resistance to ampicillin thanks to the presence of the b-lactamase gene, thus providing additional means for selection in bacteria (22,23).When bacterial cells were transformed with pMM, they exhibited re-sistance to each of the drugs separately, as well as to all combinations of these agents (ampicillin-kanamycin, ampicillin-hygromycin B, kanamycin-hygromycin B and ampicillin-kanamycin-hygromycin B), rendering this system quite fl exible for the selection of multiple resistances.The same was observed in the case of yeast transformation, where the presence of pMM renders cells able to grow on geneticin-and/or hygromycin B-containing plates (200 μg/mL of G418 and 300 μg/mL of hygromycin B, respectively).Transformation with the second plasmid construct, pMM-CEN, confi rmed these results.However, pMM-containing yeast colonies were, as expected, smaller than pMM-CEN, due to the lack of stable maintenance and transmission of the plasmid within the population.We tested the range of plasmid-mediated resistance on each of the drugs separately and in combinations with each other and determined transformation effi ciency.
The minimum inhibitory concentration (MIC) was tested with E. coli DH5α strains transformed with diff erent plasmids by their ability to produce visible growth on a series of agar plates that contain increasing concentrations of three antibiotics.The concentration of ampicillin on LB agar plates was between 50 and 100 μg/mL, of kanamycin was between 100 and 5000 μg/mL, and of hygromycin B between 100 and 5000 μg/mL.
As shown in Fig. 3, the range of plasmid-mediated resistance to each of the drugs was tested separately and compared with parental plasmid resistance (see Materials and Methods).In the case of ampicillin, all tested bacterial clones were surprisingly resistant up to 10 000 μg/mL.When tested on kanamycin, cells containing pFA6a-kan-MX4 and pMM exhibited resistance up to 1000 μg/mL and no fl uctuations of the values compared to the parental plasmid were observed.A diff erent situation was noticed in the hygromycin B resistance test, in which pMM--containing cells grew in a medium with 2000 μg/mL of the drug, while the parental plasmid pAG32 carrying the resistance gene phpMX4 exhibited only slight growth in 1000 μg/mL of hygromycin B medium.One possible explanation of this phenomenon could be that the phpMX4 promoter on pMM plasmid is subject to a greater rate of activation than when located on pAG32.

Bacterial transformation effi ciency and selection of diff erent markers
Since bacteria harbouring one of our newly constructed plasmids should exhibit resistance to the three markers separately, as well as in mixed confi gurations, we transformed bacterial cells with pMM and pMM-CEN and selected on ampicillin, kanamycin, hygromycin B and the combination of ampicillin-kanamycin, ampicillin--hygromycin B, kanamycin-hygromycin B and ampicillin--kanamycin-hygromycin B plates.Each transformation was repeated 3 times using the same aliquot of pMM and pMM-CEN miniprep.To visualize the infl uence of single, double and triple selection on the number of transformants, transformation effi ciencies were calculated of both plasmids when growing on media containing diff erent combinations of the drugs.The results (Table 2) showed that the highest transformation effi ciency of both plasmids was observed when single selection was applied.These values dropped as expected when two drugs were Fig. 2. pMM-CEN vector map with unique restriction sites.This vector can be used as a shutt le vector due to the presence of CEN4 and ARS elements providing stable maintenance in the buddying yeast.Analogously to the previous vector, this construct confers resistance to kanamycin (Kan R ) and hygromycin (Hyg R ) thanks to the cassett es present within its structure.Moreover, for selection in the bacterial hosts, ampicillin might be used due to the presence of the Amp R gene.TEF=translation elongation factor.Numbers in brachets represent the position of the restriction sites used, with the most toxic being ampicillin-kanamycin.When three markers were combined, the transformation effi ciency was reduced fi ve-(pMM) and tenfold (pMM--CEN).Triple selection allowed only triple-resistant cells to divide and potentially eliminate all background of false transformants.
On the other hand, since both Kan R and Hyg R resistance genes share the same TEF1 promoter/terminator elements, recombination between their copies cannot be excluded, although a rec¯ strain was used.In this case, recombination between two direct DNA repeats represented by the TEF1 promoter sequences on plasmid pMM would lead to the excision of the hphMX4 resistance gene, while recombination between the two TEF1 terminators would lead to excision and loss of the Kan R marker.Therefore, what could have been interpreted as background, i.e. the loss of a resistance phenotype, could very well be due to the loss of a portion of the plasmid carrying that resi-stance gene, which underwent recombination, rather than a classical background due to spontaneous mutation.

Plasmid copy number
The observed high levels of antibiotic resistance could suggest that plasmids exist in high copy numbers in bacterial cells (in yeast cells, pMM-CEN number is limited to one or two due to the presence of centromeric sequences), while the control plasmids, pAG32 and pFA6a--kanMX4 showed approx.15-20 copies per cell (data not shown) and pUC18 is reported to reach a very high copy number of more than 500 (24,25).To our surprise, aft er combining data from 12 independent experiments, we observed that copy number is rather moderate and oscillates around 10 copies per cell (10.77±1.57).Moreover, these plasmids can be stably maintained for almost twenty generations in bacteria.Amp=ampicillin, Kan=kanamycin, Hyg=hygromycin, SEM=standard error of the mean (N=3) Fig. 3. Determination of minimum inibitory concentration (MIC) values with pMM-containing cells on each of the diff erent concentrations of drugs.E. coli DH5μ strains were used for the experiments with the appropriate plasmids: pFA6a-kanMX4 for kanamycin, pUC18 for ampicillin and pAG32 for hygromycin B resistance.The spots represent growth of diff erent dilutions of a cell culture starting with approx.10 3 cells/mL from left to right.Amp=ampicillin, Hyg=hygromycin, Kan=kanamycin, wt=wild type strains

The pMM plasmid as a useful template for the production of BIT DNA cassett es
As an experimental application of these new vectors, the pMM plasmid was employed for the amplifi cation of DNA cassett es used for bridge-induced chromosome translocation (BIT) between the two yeast loci ADH1 on chromosome XV and SUC2 on chromosome IX (Fig. 4).We successfully produced a Kan R -Hyg R DNA cassett e by PCR on the pMM template, utilizing the primers described in Table 1.This DNA cassett e was then used to transform yeast to induce translocations between chromosomes IX and XV.
Aft er colony PCR screen, we were able to fi nd translocants resistant to kanamycin and hygromycin B simultaneously.The stability of pMM-derived cassett e was confi rmed by a CHEF gel when compared to the wild type San1 strain (not shown).Moreover, Southern hybridization revealed strong signal when kanamycin-or hygromycin B-specifi c probe was used and this is a strong evidence that translocation cassett e gets integrated into selected loci and is not lost during cell division.Verifi cation experiments are summarized in Fig. 5.This demonstrated a successful application of this new vector and the relative in vivo stability of the two markers in a recombination profi cient yeast strain.
The added value of this vector with respect to a single-marker one is that, at the onset of selection aft er transformation, one can immediately apply a single (resistance to kanamycin or hygromycin B) or double (resistance to both) selection, thereby imposing a much fl exible and restrictive selective pressure on the cells that have uptaken the transforming DNA plasmid.According to our previous experience with BIT, this vector signifi cantly improved the selection effi ciency, as it was possible to select on much higher doses of one antibiotic, or on two antibiotics, thereby reducing the false-positive background by an estimated 35 or 55 %, respectively.When we performed BIT with a DNA cassett e amplifi ed on the pMM template, we measured the rate of excision between TEF regions by counting cells that lost resistance to one or the other drug, due to direct repeat recombination.The ratio was rather low, as out of 800 yeast colonies originally resistant to both drugs, 62 (7.75 %) exhibited excision of the Kan R marker, and 30 (3.75 %) of the Hyg R .
Recombination between TEF elements on pMM-CEN should also lead to the exclusion of the centromere CEN and ARS elements, thus this plasmid would be quickly Fig. 4. At the top of the fi gure, the PCR amplifi cation of the cassett e carrying the Kan R -Hyg R genes using the pMM template is shown.The primers used, named F (forward) and R (reverse; sequences in Table 1), are drawn in the fi gure as thick black arrows.At the bott om, the scheme of the translocation between chromosomes XV and IX is represented.The primers used to check the correct integration and their relative position are indicated by thin arrows with sequence numbers.The two break lines on chromosome XIV indicate that the drawing of the chromosomes is not proportional S. cerevisiae is a very important organism employed during various genetic analyses in which gene disruption, deletion, tagging, or cloning are the main part of the whole experimental assay.Gene cloning in yeast requires convenient shutt le plasmids (integrative, centromeric or episomal) like the commonly used vectors of the pRS series (26) and their further modifi cations (26)(27)(28).In addition, genome manipulations such as gene deletions or replacements are usually based on drug resistance markers used for gene disruption or tagging (29,30) and require convenient PCR templates for amplifi cation of deletion cassett es.The majority of the vectors present on the market off er the possibility of selection by the means of auxotrophic markers.However, this is not always convenient, as frequently the strain is marker-incompatible (which requires additional manipulation of the genome and generation of designer strains (31,32)).Moreover, in particular approaches, the experimental set up might require growth in rich medium, which renders auxotrophic selection impossible.Transformation-associated background can be another condition rendering some experiments very difficult.A similar system, where the URA3 marker was replaced by the kanMX4 module has been constructed, but until now no double antibiotic selection marker has been available (33).In this work, we observed simultaneous resistance up to two (in the case of yeast) and three (in bacteria) antibiotic agents.Another interesting and important result is that reported in Fig. 3, where the growth tests demonstrated a superior resistance of bacteria to a particular antibiotic when all three, ampicillin, kanamycin and hygromycin B resistance genes were present and expressed on the same plasmid carrier.Although the nature of the metabolic interaction leading to this phenomenon is not clear, its role in the understanding of spontaneous acquisition of multiple antibiotic resistance must be considered.This result warrants an analogous investigation in yeast to verify the possible extension of the phenomenon to eukaryotic model cell system as well.

Conclusions
Our two newly produced plasmid constructs, pMM and pMM-CEN, carry features making them very handy and valuable tools for all modern molecular biology tasks, especially in functional analyses, recombinant DNA work and cloning experiments.The newly constructed pMM and pMM-CEN can be used for these approaches and possess the advantage of permitt ing the amplifi cation of one, two or three antibiotics on the same DNA cassett e, over commonly used vectors.Moreover, pMM-CEN allows the inclusion of a centromere DNA sequence able to stabilize the newly formed transforming chromosome lacking its own centromere DNA sequence.These plasmids can be used completely independently from the auxotrophic markers in the host strain, allowing their utilization in natural yeast isolates where strains were heavily manipulated and no auxotrophic selection marker is available.
Our initial goal was to build a multi-marker plasmid system, believing that this could be used readily as it is, while further applications have proven its additional value as a convenient platform for further genomic modifi cations.The experimental results indicated that clones carrying these constructs could be subjected to very stringent selective conditions like the triple selection that can be successfully used facilitating the limitation of an unwanted background.In order to validate the pMM plasmid ability to be used as a PCR template for genome manipulation, we used it as a template for amplifying a chromosome translocation DNA cassett e used subsequently in the BIT procedure.As expected, the cassett e successfully induced translocation between the ADH1 and SUC2 loci.The resulting clones carried the correct translocated chromosome and exhibited resistance to kanamycin and hygromycin B drugs separately and in combination.The use of double selection lowered the background, probably by allowing only the most fi t clones to survive.Another issue when working with yeast shutt le plasmids is the lack of convenient restriction sites for cloning.To expand more the versatility of pMM-CEN, a polylinker containing unique restriction sites was added.In addition to the ability of this plasmid to be stably maintained and transmitt ed through generations, the presence of these cloning sites will greatly facilitate the work with this vector.

Table 1 .
Primers used for plasmid construction, copy number determination and the translocation cassett e production.Relevant restriction sites in primer sequences are writt en in italic and underlined Plasmid construction and verifi

Fig. 5 .
Fig. 5. a) Colony PCR verifi cation of translocants obtained with Kan R -Hyg R cassett e: lane 1A: ADH1 band obtained on San1 strain.Lane 1B: PCR product obtained from San1 strain carrying the translocated chromosome.The upper band reveals integration of the cassett e into chromosome XV, while the lower band is the ADH1 wild type (wt) band.Analogously, lanes 2A and 2B contain colony PCR products on the wt and translocant strain for the second translocation point (SUC2 on chromosome IX); b) CHEF gel electrophoresis of translocation involving chromosomes XV and IX.Lane 1: wt San1 strain, lane 2: San1 translocant; c) and d) Southern hybridization with DIG probe specifi c for kanamycin and hygromycin respectively, providing direct evidence of translocation cassett e integration into the buddying yeast genome

AknowledgementsR.
Arnak was a PhD student supported by an ICGEB pre-doctoral fellowship; B. Altun was a PhD student supported by the Turkish Government and by the TRIL (Training and Research in Italian Laboratories) program.

Table 2 .
Transformation effi ciency values of plasmids pMM and pMM-CEN determined for diff erent combinations of antibiotics