Re: [Tracker] pdf indexing problem

[Björn Johansson <bjorn_johansson bio uminho pt>]

Hi,
I use this pdf as experiment:

http://dl.dropbox.com/u/1263722/goldstein_1999.pdf

It is a journal article and I search for the 
authors last name "mccusker", which tracker-needle does not find.

at the end of this email is the output from doing 

/usr/libexec/tracker-extract -v 3 -f goldstein_1999.pdf 

the name "mccusker" can be found under "plainTextContent",

I saw there is a new version 0.10.1 depending on a new version of libpoppler. Perhaps this would solve the problem?

cheers,
bjorn

On Wed, Mar 2, 2011 at 14:17, Aleksander Morgado <aleksander lanedo com> wrote:

>
> The text I want to search for is inside the pdf. tracker-extract spits
> out all the text from the pdf and I can find my search term in this
> text. Tracker-needle finds nothing though.

Extraction works properly then.

What's the exact term you're looking for?

It may be filtered out if the term is a number (although it can be
configured not to do so), or if the term is a common word, or if the
term is less than 3 characters (IIRC)

--
Aleksander

-- 
______O_________oO________oO______o_______oO__ 
BjÃrn Johansson
Assistant Professor
Departament of Biology
University of Minho
Campus de Gualtar
4710-057 Braga
PORTUGAL
http://www.bio.uminho.pt
http://sites.google.com/site/bjornhome
Work (direct) +351-253 601517
Private mob. +351-967 147 704
Dept of Biology (secretariate) +351-253 60 4310
Dept of Biology (fax) +351-253 678980


/usr/libexec/tracker-extract -v 3 -f goldstein_1999.pdf 
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<--- [1|0] tracker_extract_get_metadata_by_cmdline(uri:'file:///home/bjorn/Dropbox/Public/goldstein_1999.pdf', mime:(null))
---- [1|0]ÂÂ Guessing mime type as 'application/pdf' for uri:'file:///home/bjorn/Dropbox/Public/goldstein_1999.pdf'
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---- [1|0]ÂÂ Found 4 metadata items
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---- [1|0]Â a nfo:PaginatedTextDocument ;
ÂÂÂ Ânfo:pageCount 13 ;
ÂÂÂ Ânie:plainTextContent "ïïïïï\nYeast 15, 1541â1553 (1999)\nThree New Dominant Drug Resistance Cassettes for\nGene Disruption in Saccharomyces cerevisiae\nALAN L. GOLDSTEIN1 AND JOHN H. MïCUSKER1*\n1\nDepartment of Microbiology, 3020 Duke University Medical Center, Durham, NC 27710, U.S.A.\nDisruption-deletion cassettes are powerful tools used to study gene function in many organisms, including\nSaccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance\ncassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three\nnew dominant drug resistance cassettes by replacing the kanamycin resistance (kanr) open reading frame from the\nkanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring\nresistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25\n(natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are\ncloned into pFA6, and so are in all other respects identical to pFA6âkanMX3 and pFA6âkanMX4. Most tools and\ntechniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These\nnew heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not aïect\ngrowth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae\nstrains with multiple mutations within a single strain. Copyright  1999 John Wiley & Sons, Ltd.\nïïï ïïïïï â PCR-targeting; pFA plasmids; dominant drug resistance marker; kanMX; yeast genome analysis;\ngene deletion; functional analysis; hygromycin B; nourseothricin; bialaphos; phosphinothricin\nINTRODUCTION\nDominant drug resistance markers are commonly\nused genetic tools in fungi other than Saccharomy-\nces cerevisiae (Austin et al., 1990; Avalos et al.,\n1989; Chakraborty et al., 1991). In place of domi-\nnant drug resistance markers, the genetic manipu-\nlation of S. cerevisiae has almost exclusively\ninvolved auxotrophic mutations. However, the\nextensive use of auxotrophic mutations is unfor-\ntunate because it limits analysis to auxotrophic\nstrains and because auxotrophies are frequently\n*Correspondence to: John H. McCusker, PhD, Box 3020, Duke\nUniversity Medical Center, Dept. of Microbiology, Durham,\nNC 27710, U.S.A. Tel: (919) 681-6744; fax: (919) 684-8735;\ne-mail: mccusker abacus mc duke edu\nContract/grant sponsor: North Carolina Biotechnology Center,\nU.S.A.; Contract/grant number: #9605-ARG-0044.\nContract/grant sponsor: Duke University Medical Center,\nU.S.A.\nCCC 0749â503X/99/141541â13$17.50\nCopyright  1999 John Wiley & Sons, Ltd.\ndeleterious, (e.g. see Atkinson et al., 1980; Baganz\net al., 1997; Schroeder and Breitenbach, 1981).\nSome dominant drug resistance markers have\nbeen developed for use in S. cerevisiae (reviewed\nin Van den Berg and Steensma, 1997). However,\nof these dominant drug resistance markers,\nonly the G418 resistance conferring kanMX\ncassettes (Wach et al., 1994) have achieved wide\nuse in the S. cerevisiae community, including its\nuse for whole genome analysis of S. cerevisiae\n(http://sequence-www.stanford.edu/group/yeastâ\ndeletionâproject/deletions3.html). The widespread\nuse of the kanMX cassettes is due to their design\nfeatures: (a) strong G418 resistance phenotype\nwhen integrated into the genome in single copy;\n(b) ability to be PCR ampliïed for constructing\nsite-directed deletionâdisruption mutations; and\n(c) complete lack of homology to the yeast\ngenome.\nReceived 9 November 1998\nAccepted 12 June 1999\n 1542\nA. L. GOLDSTEIN AND J. H. MïCUSKER\nTable 1. S. cerevisiae strains.\nStrain Genotype Reference\nYJM145 HO/HO gal2 McCusker et al., 1994\nYJM237 MATa/MAT ho/ho LYS2/lys2-101 McCusker et al., 1994\nÂÂÂÂÂÂ LYS5/lys5-101 gal2/gal2 \nÂÂÂÂÂÂ MAT ho::hisG ura3 \nÂÂÂÂÂÂ Derived from YJM237 ho/ho::kanMX4 \nÂÂÂÂÂÂ Derived from YAG44 ho/ho::hphMX4 \nÂÂÂÂÂÂ Derived from YAG44 ho/ho::natMX4 \nÂÂÂÂÂÂ Derived from YAG44 ho/ho::patMX4 \nÂÂÂÂÂÂ Derived from YJM237 ho/ho::patMX4 \nÂÂÂÂÂÂ MAT \nÂÂÂÂÂÂ MAT ura3-1 \nYJM799\nYAG44\nYAG48\nYAG51\nYAG62\nYAG77\nS1\nS19\nWe have expanded the MX drug marker arma-\nmentarium by creating three new dominant drug\nresistance markers, each with a unique resistance\nphenotype. Because these new drug markers are\nbased on the kanMX cassettes, they have the\nvirtues of the kanMX cassettes. These novel domi-\nnant drug resistance markers allow strains to be\nuniquely marked in competition experiments to\nlook for subtle phenotypic diïerences and are also\nparticularly well-suited for standard genetic uses,\nsuch as the construction of multiple mutations\nin one strain and creating diïerently marked\nmutations to look for synthetic phenotypes.\nMETHODS\nStrains and media\nEscherichia coli host strains used were XL1-Blue\nor DH5 . S. cerevisiae strains are listed in Table 1.\nYeast extract peptone dextrose (YPD), synthetic\ndextrose (SD) and yeast extract peptone ethanolâ\nglycerol (YPEG) were prepared as previously\ndescribed (Rose et al., 1989). Yeast transformants\ncontaining the G418 (kanMX), hygromycin B\n(hphMX) or nourseothricin (natMX) resistance\ncassettes were selected on YPD supplemented with\n200 g/ml geneticin (Life Technologies), 300 g/ml\nhygromycin B (Boehringer-Mannheim Co. or\nCalbiochem-Novabiochem Co.), or 100 g/ml\nclonNAT (Hans-Knoll Institute fur Naturstoï-\nÂ\nÂ\nForschung, Jena, Germany), respectively. Yeast\ntransformants containing the phosphinothricin\nresistance (patMX) cassette were selected on\nSDP (1Â7 g/l Difco Yeast Nitrogen Base with-\nout (NH4)2SO4 and without amino acids, 1 g/l\nCopyright  1999 John Wiley & Sons, Ltd.\nThis\nThis\nThis\nThis\nThis\nThis\nThis\nThis\nstudy\nstudy\nstudy\nstudy\nstudy\nstudy\nstudy\nstudy\nï-proline, and 20 g/l dextrose) and either 200 g/ml\nbialaphos (Shinyo Sangyo Co., Ltd, Tokyo,\nJapan) or 600â800 g/ml glufosinate (Aldrich).\nAll antibiotics were ïlter-sterilized and added\nto autoclaved medium. Solid media contained\n2% agar.\nConstruction of dominant drug resistance cassettes\nNew dominant drug resistance cassettes based\nupon the kanMX3 and kanMX4 cassettes were\nconstructed by double fusion PCR (Amberg et al.,\n1995; Dillon and Rosen, 1990; Ho et al., 1989) in\nthree steps (Figure 1). First, three sequences were\nampliïed separately: (a) the TEF gene promoter\n(P-TEF) and adjacent multicloning sequence\n(MCS) from pFA6; (b) a dominant drug resistance\nORF; and (c) the transcriptional terminator from\nthe TEF gene (T-TEF), including adjacent MCS\nfrom pFA6. The primers used in each reaction\ncreated PCR products with overlapping termini.\nSecond, the P-TEF and dominant drug resistance\nORF PCR products were joined in the ïrst PCR\nfusion reaction. Finally, the P-TEF-dominant drug\nresistance ORF fused PCR product was combined\nwith the T-TEF PCR product in the second fusion\nreaction to create the ïnished cassette.\nEach 50 l reaction contained 10 mï KCl,\n10 mï (NH4)2SO4, 20 mï TrisâHCl (pH 8Â8),\n2 mï MgSO4, 0Â1% Triton X-100, 0Â2 mï dNTPs,\n0Â5 ï sense primer, 0Â5 ï antisense primer, 10â\n20 ng DNA template and 2 U VentR DNA\nPolymerase (NEB). Because of their high GC\ncontent, PCR ampliïcation of the nourseothricin\nN-acetyltransferase (nat1) and phosphinothricin\nN-acetyltransferase (pat) ORFs contained 5%\nYeast 15, 1541â1553 (1999)\n 1543\nTHREE NEW DOMINANT DRUG RESISTANCE CASSETTES\nFigure 1. Construction of new dominant drug-resistance MX cassettes. (A)\nSchematic diagram of strategy for PCR construction of MX cassettes. Each\ncassette consists of three components, the dominant drug resistance marker\n(DDRM) open reading-frame, ïanked by upstream (P-TEF) and downstream\n(T-TEF) regulatory sequences. In the primary reaction each component is\nPCR-ampliïed separately using primers to create overlapping termini. The\ndark lines outside the boxes represent the primers used for each reaction.\nStraight lines are completely homologous to the target DNA, while bent lines\nrepresent primers that create overlapping termini. In successive PCR reactions\n(ïrst and second fusions), the individual components are fused together by\nvirtue of their overlapping termini. (B) The letters above each primer indicate\nwhich oligonucleotide was used for constructing each cassette (sequence in\nTable 2).\nDMSO. Hot start ampliïcation was initiated with\na 1 min 94 C denaturation, followed by 30 ampli-\nïcation cycles (94 C for 1 min, 55 C for 1 min,\n72 C for 3 min), and terminated with a 20 min\n72 C extension. PCR products from each reaction\nwere gel-puriïed and approximately 10 ng of each\nDNA was used in subsequent ampliïcations.\nThe plasmid pFA6âkanMX3 was the DNA\nsource for amplifying P-TEF and T-TEF ïanked\nby direct repeats, while pFA6âkanMX4 was the\nDNA source for amplifying P-TEF and T-TEF\nwithout ïanking repeats (Wach et al., 1994). Plas-\nmids pLG90 (Gritz and Davies, 1983), pHN15\n(unpublished; nat1 Accession No. X73149), and\npSVB26-pat (derivative of pES6.1; Strauch et al.,\n1988) were used as sources for the hygromycin B\nphosphotransferase (hph), nat1 and pat ORFs,\nCopyright  1999 John Wiley & Sons, Ltd.\nrespectively. The primers used for each reaction\nare listed in Figure 1B and their sequences listed in\nTable 2.\nConstruction of plasmids containing dominant drug\nresistance cassettes\nEach dominant drug resistance cassette was sub-\ncloned into the pFA6 vector backbone by ligation\ninto the BamHI and SpeI sites of pFA6a (Wach\net al., 1994). The resulting plasmids are listed in\nTable 3.\nThe hphMX4 and natMX4 dominant drug\nresistance cassettes were also subcloned into\npAG22, a derivative of the yeastâE. coli shuttle\nvector pRS316 (Sikorski and Hieter, 1989).\npRS316 was digested with KpnI and XbaI to\nYeast 15, 1541â1553 (1999)\n 1544\nTable 2.\nA. L. GOLDSTEIN AND J. H. MïCUSKER\nOligonucleotides.\nPrimer Sequence 5 to 3 Comments\nPR29 Universal cassette construction\nPR30 natMX cassette construction\nPR31\nPR32\nPR33\nPR34\nPR35\nPR36\nPR37\nPR38\nPR43\nPR44\nPR45\nPR46\nPR78\nPR79\nPH7\nPH8\nJM37\nJM7\nJM8\nCACATACGATTTAGGTGACAC\nGGTAAGCCGTGTCGTCAAGAGTGGTACCCATGGTT\nGTTTATGTTC\nGCTCTACATGAGCATGCCCTGCCCCTAATCAGTAC\nTGACAATAAAAAG\nAATACGACTCACTATAGGGAG\nACCACTCTTGACGACACGGCTTACC\nGGGGCAGGGCATGCTCATGTAGAGC\nCGTCGCGGTGAGTTCAGGCTTTTTACCCATGGTT\nGTTTATGTTC\nCAGCACTCGTCCGAGGGCAAAGGAATAATCAGTA\nCTGACAATAAAAAG\nAAAAAGCCTGAACTCACCGCGACG\nTTCCTTTGCCCTCGGACGAGTGCTG\nCTCAACTGGTCTCCTCTCCGGAGAACCCATGGTT\nGTTTATGTTC\nCCAGTTAGGCCAGTTACCCAGATCTAATCAGTACTG\nACAATAAAAAG\nTCTCCGGAGAGGAGACCAGTTGAG\nTTAGATCTGGGTAACTGGCCTAACTGG\nCCTTGACAGTCTTGACGTGC\nCGCACTTAACTTCGCATCTG\nCGCAAGTCCTGTTTCTATGC\nCTACGTTGCCTCCATCG\nCCTCGACATCATCTGCCC\nCCTCATAAGCAGCAATCAATTCCATCTATACTTTA\nAAGCATAGGCCACTAGTGGATCTG\nCTTTTATTACATACAACTTTTTAAACTAATATACA\nCATTTCAGCTGAAGCTTCGTACGC\nremove most of the multi-cloning site, blunt-ended\nwith Klenow/T4 DNA polymerase, and self-ligated\nto create pAG22. The cassettes were subcloned\ninto the single NotI site of pAG22 and the orien-\ntation of each cassette was determined by digestion\nwith ScaI. The resulting plasmids are listed in\nTable 3.\nTargeted disruption and cassette replacement\nTargeted gene disruptions with dominant drug-\nresistance cassettes were PCR-ampliïed in 50 l\nreactions containing 50 mï KCl, 20 mï TrisâHCl\n(pH 8Â4), 1Â5 mï MgCl2, 0Â05% Tween 20, 0Â2 mï\ndNTPs, 100 g/ml acetylated bovine serum albu-\nmin, 0.5 ï sense primer, 0.5 ï antisense primer,\n10 ng DNA template and 5 U Taq DNA Polym-\nerase (GibcoBRL). Because of the high GC con-\ntent of the pat and nat1 ORFs, PCR ampliïcation\nCopyright  1999 John Wiley & Sons, Ltd.\nnatMX cassette construction\nUniversal cassette construction\nnatMX cassette construction\nnatMX cassette construction\nhphMX cassette construction\nhphMX cassette construction\nhphMX cassette construction\nhphMX cassette construction\npatMX cassette construction\npatMX cassette construction\npatMX cassette construction\npatMX cassette construction\nHomologous to TEF promoter\nHomologous to TEF terminator\nVerify homologous integration at ho locus\nVerify homologous integration at ho locus\nVerify homologous integration at ho locus\nho targeting primer\nho targeting primer\nreactions of the natMX and patMX cassettes were\nsupplemented with 5% DMSO. Ampliïcation of\nthe cassettes was initiated with a 1 min 94 C de-\nnaturation, followed by 30 ampliïcation cycles: (a)\n94 C for 1 min, 55 C for 1 min, 72 C for 3 min for\nplasmid-borne MX4 cassettes; or (b) 94 C for 30 s,\n55 C for 10 s, 72 C for 3 min for plasmid-borne\nMX3 cassettes. Reactions were terminated with a\n20 min 72 C extension. The short annealing time\nused for amplifying the MX3 cassettes eliminates\nmost of the unwanted PCR products often\nobserved when amplifying the direct repeat\ncontaining MX3 cassettes (Goldstein et al., 1999).\nThe same reaction conditions as used for tar-\ngeted gene disruptions were used for exchanging\ndominant selectable markers by targeted gene\nreplacement. Ampliïcation of the selectable\nmarkers and ïanking TEF regulatory sequences\nwas initiated with a 3 min 94 C denaturation,\nYeast 15, 1541â1553 (1999)\n 1545\nTHREE NEW DOMINANT DRUG RESISTANCE CASSETTES\nTable 3.\nPlasmids.\nName\nMX4 cassettes in pFA6\npAG25\npAG29\npAG32\nMX3 cassettes in pFA6\npAG35\npAG31\npAG34\nMX4 cassettes in pAG22\n(derived from pRS316)\npAG36\npAG26\nOther plasmids\npFA6kanMX4\npFA6kanMX3\npFA6a\npHN15\nPRS316\npAG22\npLG90\npSVB26-pat\nComments Reference\nnatMX4 This study\npatMX4 This study\nhphMX4 This study\nnatMX3 This study\npatMX3 This study\nhphMX3 This study\nnatMX4 CEN URA3 This study\nhphMX4 CEN URA3 This study\nSource of NAT1 ORF\nCEN URA3\nDerived from pRS316;\nmodiïed MCS\nSource of HPH ORF\nSource of PAT ORF\nfollowed by six ampliïcation cycles: 94 C for 20 s,\n55 C for 30 s, 72 C for 1 min. A second round of\n25 ampliïcation cycles was 94 C for 20 s, 72 C for\n1Â5 min. Reactions were terminated with a 10 min\n72 C extension.\nBetween 500 ng and 2 g of PCR product were\nused to transform S. cerevisiae strains using the\nlithium acetate method (Geitz et al., 1995). Before\nplating transformants onto selective media, the\ncells were grown for 2â4 h in YPD at 30 C on a\nrotator, to allow for _expression_ of the transformed\ndrug resistance marker. Cultures transformed with\nthe pat cassettes were pelleted and resuspended in\nsterile water before plating onto selective media.\nHomologous integration of dominant drug resist-\nance cassettes was veriïed by colony PCR\n(Niedenthal et al., 1996). In instances where colony\nPCR did not yield a product, genomic DNA was\nisolated (Ausubel et al., 1995) and PCR-ampliïed\nusing the same conditions as colony PCR.\nNeutrality of dominant drug marked cassettes\nTo determine whether the dominant drug-\nresistance cassettes were phenotypically neutral,\neach of the dominant drug markers was integrated,\nCopyright  1999 John Wiley & Sons, Ltd.\nWach et al., 1994\nWach et al., 1994\nWach et al., 1994\nKrugel, unpublished\nÂ\nSikorski and Hieter, 1989\nThis study\nGritz and Davies, 1983\nWohlleben, unpublished\nseparately, into one copy of the ho locus of\nYJM237 (McCusker et al., 1994), a diploid, pro-\ntotrophic S288c background strain. Each drug-\nresistant strain was streaked for single colonies on\nYPEG plates to select against petites. A single\ncolony from each strain was inoculated into 2 ml\nof YPD and grown overnight at 30 C on a rotator.\nA 1/50 dilution of each culture was grown to 1Â0\nOD600 in YPD at 30 C. Equal amounts of each\ndrug-resistant strain were mixed and approxi-\nmately 1 103 colony forming units (cfu) of the\nmixture were inoculated into 1 l YPD and grown\nat 30 C to a cell density of approximately\n2 108 cells/ml. Approximately 1 103 cfu of the\nmixed culture were passaged successively to 2 more\nliters of YPD. The initial cell densities (1 cell/ml)\nand ïnal cell densities of each 1 l culture were\nchosen to maximize the amount of time spent in\nexponential phase and thereby accentuate any\npossible eïect of the drug resistance markers on\ngrowth. At each time point (the initial inoculation\nof the mixed cell culture into YPD and when each\n1 l culture reached approximately 2 108 cfu),\nbetween 1â4 102 cfu were plated onto three\nYPD plates and grown at 30 C until colonies\nYeast 15, 1541â1553 (1999)\n 1546\nappeared on the plates. These plates were then\nreplica-plated to medium containing one of\neach of the antibiotics and the resistant colonies on\neach plate were counted. The total number of\npopulation doublings for the mixed culture was\ncalculated from the initial and ïnal cell densities\nof each 1 l culture ([ïnal cell conc.]/[initial cell\nconc.]Y2n, where nYnumber of population\ndoublings).\nPlasmid requests\nSend plasmid requests to John McCusker (fax:\n919) 684-8735, or e-mail: mccusker abacus \nmc.duke.edu).\nRESULTS\nAntibiotics and media conditions for selecting\ndominant drug-resistant S. cerevisiae\ntransformants\nWe have developed three new heterologous\ndominant drug-resistance markers for use in con-\nstructing targeted disruptionâdeletion mutations\nin S. cerevisiae. These markers confer resistance\nto the antibiotics nourseothricin, bialaphos/\nphosphinothricin, and hygromycin B. As described\nbelow, resistance genes to these antibiotics have\nbeen engineered into the MX disruptionâdeletion\ncassettes.\nInitially we determined the range of antibiotic\nconcentrations to use for selecting each new domi-\nnant drug resistance cassette by ïrst making both\nYPD and SD plates with a range of antibiotic\nconcentrations. Next, we streaked these plates\nwith unmarked haploid (YJM799) and diploid\n(YJM145) S. cerevisiae strains to ïnd which media\nand drug concentrations inhibited their growth.\nFurther reïnements of selection conditions were\ncarried out as we constructed the cassettes and\ntransformed them into S. cerevisiae.\nThe aminoglycoside nourseothricin is a complex\nof streptothricin sulphates C and D from Strepto-\nmyces noursei. Resistance to nourseothricin is\nconferred by the nat1 gene from S. noursei,\nwhich encodes nourseothricin N-acetyltransferase\n(Krugel et al., 1993). Nourseothricin is commer-\ncially available as clonNAT (Hans-Knoll Institute\nÂ\nfur Naturstoï-Forschung, Jena, Germany; fax:\nÂ\n+49 3641 65 66 00). S. cerevisiae strains containing\na natMX cassette can be selected for on YPD\nplates containing 100 g/ml clonNAT.\nCopyright  1999 John Wiley & Sons, Ltd.\nA. L. GOLDSTEIN AND J. H. MïCUSKER\nHygromycin B is an aminoglycoside produced\nby Streptomyces hygroscopicus (Pettinger et al.,\n1953). The Hph resistance gene from Klebsiella\npneumoniae encodes hygromycin B phosphotrans-\nferase (Gritz and Davies, 1983). S. cerevisiae\nstrains containing a hphMX cassette can be\nselected for on YPD plates containing 300 g/ml\nhygromycin B.\nBialaphos, which is produced by Streptomyces\nhygroscopicus and S. viridochromogenes, is a pep-\ntide consisting of two alanine residues and the\nglutamate analogue phosphinothricin (Thompson\nand Seto, 1995). Phosphinothricin inhibits gluta-\nmine synthase (Lea et al., 1984). The pat resistance\ngene from S. viridochromogenes Tu94 encodes phos-\nÂ\nphinothricin N-acetyltransferase (Strauch et al.,\n1988; Wohlleben et al., 1988). Bialaphos is com-\nmercially available in a puriïed form (Shinyo\nSangyo Co., Ltd, Tokyo, Japan; fax 81-3-5449-\n3598), while ïï-phosphinothricin is commercially\navailable as glufosinate (Aldrich). Because bial-\naphos is a peptide and phosphinothricin an amino\nacid analogue, synthetic minimal medium, which\ndoes not contain peptides or amino acids that\nwould compete for uptake, is used to select trans-\nformants. Since nitrogen source aïects peptide and\namino acid transport in S. cerevisiae (Cooper,\n1982), we evaluated the sensitivity of yeast to\nbialaphos and phosphinothricin using ammonium\nsulphate (5 g/l), proline (1 or 4 g/l) or leucine\n(4Â5 g/l). We found 1 g/l of proline as the nitrogen\nsource gives the best balance between sensitivity to\nthe antibiotics and speed of growth (data not\nshown). S. cerevisiae strains containing a patMX\ncassette can be selected on SDP plates contain-\ning either 200 g/ml bialaphos or 600â800 g/ml\nglufosinate.\nIn our hands, bialaphos-containing antibiotic\nplates are fungistatic and have a dependable shelf\nlife of only about 1 month when stored at 4 C.\nWhen selecting S. cerevisiae cells transformed\nwith a kanMX cassette on 200 g/ml G418, there is\nfrequently an appreciable background of small\ncolonies which presumably arise from abortive\ntransformation events. Using the plate conditions\ndescribed above, the background level of trans-\nformed colonies selected on hygromycin B or\nbialaphos/glufosinate plates is similar to, or less\nthan, the background on G418 plates. For select-\ning hphMX transformants, higher concentrations\nof hygromycin B can reduce the background.\nTransformation plates containing 100 g/ml\nclonNAT have virtually no background, which,\nYeast 15, 1541â1553 (1999)\n 1547\nTHREE NEW DOMINANT DRUG RESISTANCE CASSETTES\nFigure 2. Dominant drug resistance MX plasmids. (A) Multicloning site and restriction sites of\ndominant drug resistant marker (DDRM) MX3 and MX4 plasmids. The dashed line shows that\nthe MX4 cassette, lacking the direct repeats, was originally cloned into the PmeI site (Wach et al.,\n1994), leaving the site unusable. The solid line shows that the MX3 cassette was cloned into the\nBglII and PmeI sites, leaving both functional (Wach et al., 1994). The name of each plasmid is\nlisted to the right of the schematic representation of each open reading frame. (B) MCS and\norientation of cassettes in E. coliâS. cerevisiae shuttle plasmid. Each of the MX4 cassettes was\ncloned into the single NotI site of pAG22. The orientation of each cassette, determined by ScaI\ndigestion, is shown by the direction of the arrow in the DDRM open reading frame. The name of\neach plasmid is listed to the right of the ORFs.\ncombined with its low cost, makes use of this\nantibiotic very attractive.\nCharacteristics of cassettes and plasmids\nAs shown in Figure 1 and described in Materials\nand Methods, the new dominant drug resistance\ncassettes were assembled by double-fusion PCR.\nThe construction of the deletionâdisruption cas-\nsettes is based upon the widely used kanMX3 and\nkanMX4 cassettes, containing the kanamycin\nresistance gene from the E. coli transposon Tn903,\nwhich confers G418 resistance to S. cerevisiae\n(Wach et al., 1994). The new disruptionâdeletion\ncassettes described here replace the kanr ORF with\neither the pat, nat1 or hph antibiotic resistance\nopen reading frames. In all other respects these\ncassettes are identical to kanMX3 and kanMX4.\nBecause of this characteristic, most reagents and\nprotocols used with the kanMX cassettes can be\nused with the new cassettes. In particular, the same\nprimers can be used to amplify all of the cassettes\nfor site-speciïc integration.\nThe cassettes are bounded by the pFA6 multi-\ncloning site (MCS), as shown in Figure 2. During\ndouble-fusion PCR construction of the new cas-\nsettes, a glycine codon was engineered as the\nCopyright  1999 John Wiley & Sons, Ltd.\nsecond codon for each antibiotic resistance ORF.\nThe ïrst G of the glycine codon maintains the 5\nterminal NcoI site found in the kanMX cassettes.\nIn addition to these features, the promoter and\nterminator sequences of the MX3 cassettes are\nïanked by 466 bp direct repeats, which facilitate\nloss of the cassette by homologous recombination.\nDuring construction of the patMX cassettes the\nnative GTG start codon of the pat ORF\nwas changed to ATG by primer-mediated PCR\nmutation (oligo PR43).\nEach of the new MX4 and MX3 based deletionâ\ndisruption cassettes were subcloned into pFA6 so\nthat the MCS in these plasmids remains identical\nto the MCS of pFA6âkanMX4 and pFA6â\nkanMX3 (see Figure 2 and Table 3). In addition,\nthe hphMX4 and natMX4 cassettes were also\nsubcloned into pAG22, a derivative of the yeastâE.\ncoli shuttle vector pRS316 (CEN URA3) (Sikorski\nand Hieter, 1989). To create pAG22, the DNA\nbetween the KpnI and XbaI restriction sites of the\npRS316 MCS was removed. The MX4 cassettes\nwere digested with NotI and cloned into the single\nNotI site of pAG22 (see Figure 2 and Table 3). The\norientation of the inserts was determined by ScaI\ndigestion (data not shown).\nYeast 15, 1541â1553 (1999)\n 1548\nFidelity of homologous integration\nTo test the ïdelity of homologous integration\nof the new MX4 dominant drug markers, each\ncassette was targeted for de novo homologous\nintegration at the ho locus. The kan, hph, nat\nand patMX4 cassettes were PCR ampliïed with\nprimers JM7 and JM8 and transformed into\nstrain YJM237, selecting for the appropriate anti-\nbiotic resistance phenotype. Genomic DNA was\nextracted from antibiotic-resistant colonies and\nanalysed by PCR for homologous integration.\nPrimers JM37 (within the TEF terminator) and\nPH7 (500 nt upstream of ho) amplify an approxi-\nmately 600 nt PCR product when a MX4 cassette\nis integrated at the ho locus. The following ratios\n(homologously integrated:colonies tested) were\nobtained: G418, 5:6; hygromycin B, 6:6; nour-\nseothricin, 6:6; bialaphos, 3:4. These ratios of\nhomologous integration are similar to those\nobtained with other MX cassettes (Wach et al.,\n1994; Shoemaker et al., 1996; Goldstein et al.,\n1999).\nUnique resistance phenotypes of cassettes\nAn isogenic series of diploid strains, each het-\nerozygous at the ho locus for one of the cassettes,\nwas constructed to assess cross-resistance of the\ndominant drug resistance cassettes. To create the\nisogenic set of strains, the kanMX4 cassette was\nïrst PCR-ampliïed with primers JM7 and JM8 to\ntarget the cassette to the ho locus, and transformed\ninto the diploid YJM237 to create G418 resistant\nstrain YAG44. The remaining strains were\ncreated by cassette exchange. Each cassette was\nampliïed using primers PR78 and PR79, which are\nhomologous to the TEF promoter and terminator\nregions, respectively. The cassette PCR products\nwere then individually transformed into YAG44.\nCassette exchange was veriïed by PCR analysis\nusing primers PH7 and PH8. These primers\nïank the ho locus so that amplifying genomic\nDNA with them allows the cassettes to be\ndiïerentiated by size.\nApproximately 5 102 cells of strains YJM237\n(ho/ho), YAG44 (ho/ho::kanMX4), YAG48 (ho/\nho:hphMX4), YAG51 (ho/ho::natMX4), and\nYAG62 (ho/ho::patMX4) were plated separately\non each antibiotic plate (see Materials and\nMethods for plate formulations) and incubated at\n30 C. After a 1 week incubation, each type of\nantibiotic plate only allowed growth of S. cerevi-\nsiae strains containing resistance cassettes to that\nCopyright  1999 John Wiley & Sons, Ltd.\nA. L. GOLDSTEIN AND J. H. MïCUSKER\nantibiotic (data not shown). These data demon-\nstrate that each dominant-drug-marked MX\ncassette confers a unique resistance phenotype.\nNeutrality of dominant drug resistance markers\nTo assess the neutrality of the new dominant\ndrug resistance markers, the pat, nat, hph and\nkanMX4 cassettes were integrated into one\ncopy of the ho locus of the prototrophic S288c\ndiploid strain YJM237 (McCusker et al., 1994).\nIt has been previously demonstrated that the\nkanMX cassette integrated at the ho locus in a\ndiploid S288c strain is neutral for growth under\na variety of conditions (Baganz et al., 1997).\nThe growth of strains YAG44 (ho/ho::kanMX4),\nYAG48 (ho/ho::hphMX4), YAG51 (ho/ho::nat\nMX4), and YAG77 (ho/ho::patMX4) were com-\npared in competition experiments. As a control to\ndemonstrate that growth diïerences could be\ndetected under these experimental conditions, the\nprototrophic S288c background strain S1 and its\nspontaneous ura3 derivative S19 were compared in\na separate competition experiment.\nAs described in Materials and Methods, the\nexponentially growing drug-resistant strains were\npooled, and the pool was grown under non-\nselective conditions for approximately 84 popu-\nlation doublings. Triplicate samples were removed\nat the beginning of the experiment and approxi-\nmately every 27â28 population doublings there-\nafter, and the fraction of each strain determined by\nreplica-plating to selective media. As shown in\nFigure 3A, the percentage of the ura3 strain S19\nfell from 40% of the mixed culture at initial\ninoculation to just 25% of the culture after\napproximately 84 generations, demonstrating that\ngrowth diïerences can be observed under these\nexperimental conditions.\nFigures 3B and 3C show competition exper-\niments between strains containing the dominant\ndrug resistance cassettes. The percentage of each\ndrug-resistant strain within the mixed cultures\nremained remarkably stable, changing no more\nthan 3Â0% after approximately 84 population\ndoublings. Although more precise comparisons\nof growth under non-selective conditions can be\nobtained in a chemostat culture (Baganz et al.,\n1997), within the limitations of serial transfer\nbetween batch cultures the pat, nat and hphMX\ncassettes are phenotypically neutral.\nIn addition to growth rate, each of the MX4\nmarked strains was assayed for its ability to\nYeast 15, 1541â1553 (1999)\n 1549\nTHREE NEW DOMINANT DRUG RESISTANCE CASSETTES\nFigure 3. In vitro neutrality of dominant drug resistance MX cassettes in\nS. cerevisiae. For competition experiments, the individual strains were pooled\nand grown for approximately 84 population doublings under non-selective con-\nditions (YPD, 30 C). At the beginning of the experiment, and approximately\nevery 28 population doublings thereafter, three aliquots of cells were removed\nfrom the culture, diluted, and plated in onto non-selective plates. After colonies\nformed, each plate was replica-plated onto selective plates to determine the\nphenotype of each colony. From this data we reconstructed the composition of\nthe culture and determined the percentage of each strain within the culture.\nEach data point on the graphs is the average of three measurements. (A)\nControl to show that depletion of a strain is detectable under the experimental\nconditions using unmarked wild-type strain S1 and ura3 strain S19. (B and C)\nGrowth comparison of strains containing dominant drug-resistance markers\nhomologously integrated into the ho locus: natMX4 (YAG51), hphMX4\n(YAG48), patMX4 (YAG77) and kanMX4 (YAG44). The kanMX4 cassette\nwas previously shown to have little eïect on growth (Baganz et al., 1997).\nsporulate. Each strain sporulated as eïciently as\nthe parental YJM237 strain (data not shown).\nDISCUSSION\nThe long tradition of manipulating S. cerevisiae\nwith auxotrophic mutations started with the pro-\ntotrophic selection of diploids and complement-\nation testing (Pomper and Burkholder, 1949).\nComplementation of S. cerevisiae auxotrophic\nmutations has since been widely used to select for\ntransformation by plasmids and for gene disrup-\ntions. Clearly, the yeast community has been well\nserved by auxotrophic mutations.\nGiven the wide use and long history of auxo-\ntrophic mutations, why do yeast geneticists need\nheterologous dominant drug resistance markers?\nUnfortunately, unlike heterologous dominant drug\nresistance markers, auxotrophic mutations have\nCopyright  1999 John Wiley & Sons, Ltd.\nlimitations as selectable markers. One obvious\nlimitation is that one must use a strain with one or\nmore auxotrophic mutations, which restricts the\nanalysis of prototrophic non-laboratory strains\n(McCusker et al., 1994; Mortimer et al., 1994).\nAnother limitation is that gene conversion of\nchromosomal auxotrophic mutations by the trans-\nforming DNA can be a signiïcant problem\nwhen introducing site-speciïc deletionâdisruption\nmutations into the genome (Langle-Rouault and\nJacobs, 1995).\nIn addition to these limitations, auxotrophic\nmutations also have inherent problems in that they\nfrequently have undesirable, and sometimes unpre-\ndictable, phenotypes. An example of an unex-\npected phenotype is the osmotic sensitivity of aro7\nmutants (Ball et al., 1986). Auxotrophic mutations\ncan also have synthetic phenotypes in combination\nwith other mutations, such as the synthetic\nYeast 15, 1541â1553 (1999)\n 1550\nlethality of (a) arginine auxotrophic and can1\nmutations (Whelan et al., 1979) and (b) tryp-\ntophan and ergosterol auxotrophic mutations\n(Gaber et al., 1989).\nAuxotrophy also interferes with the two inten-\nsively studied nitrogen-starvation-regulated cellu-\nlar diïerentiation processes in S. cerevisiae,\nsporulation and formation of pseudohyphae. By\ndeïnition, auxotrophs require supplementation of\ndeïned growth medium with nitrogen-containing\ncompounds, such as amino acids and bases. There-\nfore, it is not surprising that some auxotrophic\nmutations and/or their required nutritional\nsupplements aïect sporulation eïciency (e.g. see\nAtkinson et al., 1980; Freese et al., 1984;\nSchroeder and Breitenbach, 1981; Varma et al.,\n1985). In addition, auxotrophy interferes with\nformation of pseudohyphae; that is, pseudohyphal\nformation is best observed with prototrophic\nstrains in minimal medium with no added\nnutritional supplements (J. Heitman, personal\ncommunication).\nFinally, many auxotrophic mutations have\ndemonstrable growth defects even in rich medium\n(e.g. see Smith et al., 1995, 1996). The growth\ndefects of auxotrophic mutations make their use in\ndetecting subtle growth phenotypes, whether in\nsmall competition experiments or in whole genome\nanalysis, questionable at bestâa point speciïcally\nmade by Baganz et al. (1997). In summary, con-\nsiderable caution is required in using auxotrophic\nmutations, and there are powerful arguments\nagainst the use of auxotrophic mutations in some\ncircumstances.\nOne solution to the inherent problems and limi-\ntations of auxotrophic mutations are dominant\ndrug resistance markers. Such markers are exten-\nsively used in bacterial genetics and in fungi other\nthan S. cerevisiae. One of the advantages of hetero-\nlogous, dominant drug-resistance markers is that\nthey can be used in any strain and, in particular,\nwould facilitate the analysis of prototrophic non-\nlaboratory S. cerevisiae strains (McCusker et al.,\n1994; Mortimer et al., 1994).\nRecently, a heterologous, dominant G418 resist-\nance marker has been developed for use in S.\ncerevisiae (Wach et al., 1994). The G418 cassette\ncan be used in virtually any strain, including\nprototrophs, and has been shown to be phenotypi-\ncally neutral (Baganz et al., 1997). Perhaps the\nmost important feature of the MX cassettes is that\nthey are completely heterologous to the S. cerevi-\nsiae genome. Since heterologous markers do not\nCopyright  1999 John Wiley & Sons, Ltd.\nA. L. GOLDSTEIN AND J. H. MïCUSKER\nhomologously recombine with genomic DNA,\ndirected site-speciïc integration eïciencies are\nhigher than cassettes containing selectable S. cer-\nevisiae genes. This is of particular concern when\ntransforming with PCR products containing short\nregions of gene homology.\nAlthough this limitation of homologous inte-\ngration cassettes can be reduced by the creation of\nspecialized designer strains carrying deletions of\ncommonly used S. cerevisiae auxotrophic markers\n(Brachmann et al., 1998; Sikorski and Hieter,\n1989), it would be useful to have additional\nheterologous, dominant resistance markers. Such\nmarkers would facilitate the construction of\nmultiple mutations in one strain and would make\nit easier to perform competition experiments to\ndetect subtle phenotypes. To expand the choices\nof heterologous markers, we have created three\nnew dominant drug-resistance markers by replac-\ning the kanr ORF of the kanMX cassettes with\nresistance genes to the antibiotics hygromycin B,\nnourseothricin and bialaphos/phosphinothricin.\nLike G418, hygromycin B and nourseothricin\nare aminoglycosides that inhibit translation.\nBialaphos, a tripepetide that contains the\nglutamate analogue phosphinothricin, inhibits\nglutamine synthase. Unlike the aminoglycosides,\nbialaphos is most eïective in deïned media,\nenabling the simultaneous selection for drug\nresistance and prototrophies.\nOne of the major motivations for creating these\nnew dominant drug resistance markers was to\nfacilitate the construction of multiply marked\nstrains. Since G418, hygromycin B and nour-\nseothricin are all aminoglycosides, we determined\nwhether there was any cross-resistance between\nthese antibiotics and their resistance genes. Even\nafter a week-long incubation at 30 C, we saw no\nsign of cross-resistance, consequently multiply\nmarked strains can easily be selected for on plates\ncontaining two or more antibiotics.\nThe lack of cross-resistance between the antibi-\notic resistance markers also means that it is quite\neasy to exchange MX markers within a strain.\nSince all of the MX markers contain the same\npromoter and terminator regions, an MX cassette\ncan be replaced with a diïerent cassette simply by\nusing PCR primers complementary to the ïanking\nregulatory regions. Since the regulatory regions\nare approximately 200 bp, this exchange is quite\neïcient. The isogenic strains constructed for the\nneutrality experiments (discussed below) were\nconstructed in this way by ïrst creating an\nYeast 15, 1541â1553 (1999)\n 1551\nTHREE NEW DOMINANT DRUG RESISTANCE CASSETTES\nho/ho::kanMX4 deletion strain de novo and then\nreplacing the kanMX4 cassette in this strain with\nthe other dominant drug resistance markers.\nAlthough the direct-repeat-containing MX3 cas-\nsettes were designed to facilitate marker exchange,\nwe have found that marker loss in MX3 containing\nstrains is ineïcient. Having a positive selection for\nmarker exchange would make marker loss much\neasier. To this end, we constructed fusions between\nthe Candida albicans GAL1 ORF and both the kanr\nand nat1 ORFs. Work by Platt had shown that\nconstitutive _expression_ of GAL1 conferred toxicity\nto 2-deoxygalactose in S. cerevisiae cells, therefore\nmarker loss of a kanr-GAL1 or nat1âGAL1 fusion\nshould confer 2-deoxygalactose resistance (Platt,\n1984). Unfortunately, strains with integrated\ncopies of either fusion construct were not sensitive\nto 2-deoxygalactose. Although gal1 deletion\nstrains containing either GAL1 fusion construct\ncould grow on YP plates with galactose as the sole\ncarbon source, their growth was much slower than\nwild-type cells. These results suggest that galac-\ntokinase activity in the fusion construct containing\nstrain is not suïcient to confer 2-deoxygalactose\nsensitivity.\nThe functional analysis of a gene or family of\ngenes often includes deleting the gene(s) and\nassessing the eïect of its loss on growth under a\nvariety of conditions. In many cases, mutants have\nsubtle phenotypes which can only be discerned in a\ncompetition experiment (e.g. see Smith et al., 1995,\n1996). One way to determine subtle phenotypic\ndiïerences in small-scale competition experiments\nis to mark each competing strain with a diïerent\ngenetic marker, such as diïerent dominant drug\nresistance markers. However, it is imperative for\nsuch quantitative analysis that the dominant drug\nresistance marker(s) be phenotypically neutral.\nSince it had been previously demonstrated that\nthe ho locus is phenotypically neutral for growth in\ndiploid strains (Hammond et al., 1994; Yocum,\n1983; Baganz et al., 1997), we chose that location\nfor integrating the dominant drug resistance cas-\nsettes. The ïrst competition was a control exper-\niment using two isogenic strains, one wild-type and\none an auxotroph, to demonstrate that growth\ndiïerences could be detected under our experimen-\ntal conditions. Since his3 mutations were either\ndeleterious or beneïcial to growth, depending\nupon culture conditions (Baganz et al., 1997), and\nleu2 mutations neutral (Palmero et al., 1997), we\ntested the eïect of uracil auxotrophy on growth.\nWe found that after approximately 84 population\nCopyright  1999 John Wiley & Sons, Ltd.\ndoublings in rich medium, a ura3 mutant strain\nwas depleted by 15% compared to its wild-type\nparent. By contrast, isogenic diploid strains diïer-\ning only by the dominant drug resistance marker\nat the ho locus were phenotypically neutral for\ngrowth with changes of no more than 3Â0%\nobserved. Therefore, the new dominant drug\nresistance markers can be used to detect subtle\ngrowth diïerences between strains.\nThere are a variety of uses for these new domi-\nnant drug resistance cassettes, including standard\ngenetic uses such as creating multiple mutations\nin one strain or creating diïerently marked mu-\ntations to look for synthetic phenotypes. As\ndescribed in this report, because the markers are\nphenotypically neutral, they are ideally suited to\ndetect subtle growth diïerences in competition\nexperiments. Perhaps most importantly, these het-\nerologous cassettes, along with the kanr cassette,\nare ideal for marking and creating deletionâ\ndisruptions in prototrophic strains. Non-\nlaboratory strains will become increasingly\nimportant as the use of S. cerevisiae continues to\nexpand as a model system in ïelds such as\nwhole genome analysis, population dynamics,\nfungal pathogenicity and quantitative genetics\n(McCusker et al., 1994).\nACKNOWLEDGEMENTS\nThis work was supported by funds from the North\nCarolina Biotechnology Center (#9605-ARG-\n0044) and Duke University Medical Center. We\nthank Drs Hans Kruegel and Wolfgang Wohlleben\nÂ\nfor plasmids. 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ÂÂÂ Ânfo:tableOfContents "Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae bookmark1 " .

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