Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-16
2003-05-13
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S004000, C435S006120, C435S029000, C435S252300, C435S254210, C435S254220, C435S471000, C435S483000, C435S320100, C536S023100
Reexamination Certificate
active
06562595
ABSTRACT:
1. INTRODUCTION
This invention relates to the discovery of nourseothricin (NST) drug sensitivity in the pathogenic yeast,
Candida albicans
and in
Saccharomyces cerevisiae
. In particular, the present invention relates to a cognate drug resistance marker system for use in gene transformation and disruption experimentation. Specifically, the present invention provides a modified nourseothricin/streptothricin resistance gene, SAT, for expression in
C. albicans
. The present invention also provides a cell, nucleic acid molecule, and vector comprising the modified SAT1 nucleic acid sequence. The present invention further provides a SAT expression module for gene knock-outs.
2. BACKGROUND OF THE INVENTION
Opportunistic fungi are a rapidly emerging class of microbial pathogens causing systemic fungal infection or “mycosis” in patients immunocompromised either by illness (e.g., AIDS) or standard medical treatment (e.g., organ transplants, chemotherapy, radiation therapy). Candida spp. rank as the predominant genus of such fungal pathogens. In recent years, rapid and reliable diagnosis of fungal infection has advanced primarily through the application of molecular biological techniques. Understanding the pathogenesis of this organism, from which novel treatment strategies will develop, is also dependent on improved techniques in molecular genetics.
The recent commitment by the Stanford Sequence Center to sequence the entire
C. albicans
yeast genome will accelerate our understanding in both the biology and eventual treatment of candidiasis. The DNA sequence resulting from this enterprise however offers only a prediction towards potential pathogenesis pathway(s) and antifungal targets. Maximum information gained from this effort requires experimentation. The ability to study the role of any particular gene, both by abolishing its function through gene disruption experiments, as well as overproducing its gene product through transformation experiments, directly tests the predictions made by bioinformatic analysis. As
C. albicans
is an imperfect fungus which lacks a sexual cycle and is fixed in the diploid state, gene disruption experiments are more cumbersome, requiring replacement of both alleles of the target gene before an examination of its null phenotype be determined. To this end, improved DNA methodologies are required for experimentation in
C. albicans.
Currently, auxotrophic markers are employed to select for precise genetic alterations in
C. albicans
. Auxotrophic markers are recessive mutations, usually in biosynthetic genes, which can be complemented by either supplementing the yeast strain with the desired requirement (e.g., uridine) or by transformation of the wild type gene. A number of non-reverting, auxotrophic mutations, to which the complementing wild type gene has been cloned, are available for genetic manipulations in
C. albicans
(Pla et al., 1996 Yeast 12:1677-1702). CAI4, the standard
C. albicans
strain employed by researchers, contains a single auxotrophic marker—a homozygous null mutation in the CaURA3 gene. The utility of this strain stems largely from a “URA-blaster” gene disruption procedure developed for
C. albicans
by Fonzi and Irwin (1993 Genetics 134:717-728) which utilizes a CaURA3 gene flanked by direct repeats of the
Salmonella typhimurium
HisG gene. This Ura-blaster cassette is used to replace part of the target gene in vitro. The resulting disruption cassette is then transformed into CAI4, whereby through homologous recombination, Ura+ transformants harboring a heterozygous mutation for the target gene are selected. Counterselection on 5-fluoroorotic acid (5-FOA), relying on intrachromosomal recombination between HisG repeats, excises the CaURA3 gene, leaving a single copy of the HisG sequence within the target gene, and allowing reuse of the auxotrophic marker-based disruption cassette for disruption of the target gene's second allele.
Despite a reliance on auxotrophic markers to select for successful DNA transformation or gene disruption, this dependency comes with significant limitations. Firstly, analysis is restricted to the genetic background to which the auxotrophic mutation has been introduced and the complementing gene available. This severely restricts genetic analyses of clinical isolates which lack auxotrophic markers. Alternatively, a specially constructed strain containing the appropriate auxotrophic marker must first be constructed, a procedure which is both time consuming and problematic. A second common problem associated with auxotrophic markers is the limited number of stable mutations constructed in a particular strain background. As outlined above, CAI4, the most widespread
C. albicans
strain used for genetic manipulation, maintains only a single auxotophic marker. Although, the URA3 marker can be reused in gene disruption experiments, this process has significant drawbacks, and more sophisticated manipulations (for example, the selection and stable maintenance of a second gene) are difficult. Auxotrophic mutations also potentially affect physiological processes such as pathogenicity, rendering the strain inappropriate for virulence studies (Pla et al., 1996, Yeast 12:1677-1702). Therefore, a strain maintaining multiple auxotrophic mutations must be complemented for each mutation in order to perform virulence studies, and even under such conditions, issues of haplo-insufficiency add further complexity to the utility of such a multiply-marked
C. albicans
strain. In theory, the Ura-Blaster method overcomes this issue of limited auxotrophic markers for multiple gene disruptions by the ability to reuse the Ura3 marker. In practice however, additional problems develop, most notably the introduction of extragenic mutations which accumulate through successive counterselections on 5-FOA; which itself is a mutagenic compound. Repeated use of the procedure, for example in the construction of a double homozygote strain, may add multiple extragenic mutations; any of which can potentially contribute to phenotype(s) unlinked to either of the disrupted loci and consequently complicate interpretation of the result. Another problem common to auxotrophic mutations is the altered growth rate they impart, in addition to their potential for contributing a further variable into phenotypic analyses. For example, despite the addition of supplementary Uridine to hyphal-inducing media, CAI4 neither forms as extensive hyphae, nor switches from the budding form to hyphal form as rapidly as its Ura3+ parent strain, SC5314.
Historically auxotrophic markers have contributed tremendously to basic research of the bakers' yeast,
Saccharomyces cerevisiae
. However, a clear trend towards the use of a dominant drug selectable marker has developed, principally by an international consortium of researchers participating in the
S. cerevisiae
genome knock out project. To this end, a single dominant selectable marker has been constructed, comprising the
E. coli
-derived kanamycin resistance gene, Kan
R
, flanked by
Ashbya gossypii
TEF3 promoter and terminator regulatory sequence (Wach et al., 1994, Yeast 10:1793-1808; Jimenez and Davies, 1980, Nature 287:869-871). This KanMX module is expressed in
S. cerevisiae
and confers resistance to the Kanamycin-related aminoglycoside, geneticin, allowing selection for the desired strain when plated in the presence of the drug after transformation. The use of this KanMX module in place of auxotrophic markers solves many of the above discussed problems associated with their use. Genetic manipulations employing this dominant selectable marker can now be carried out directly in any
S. cerevisiae
strain. Studies comparing Kan
r
-marked versus wild type strains incubated together in a chemostat reveal no detectable difference in growth rate associated with the maintenance of the KanMX module. Moreover, no indirect effects on physiological, developmental, or morphological processes are detected. Because the KanMX disruption module is completely heterologous, the efficiency of proper inte
Bussey Howard
Davison John
Roemer Terry
Guzo David
Leffers, Jr. Gerald G.
McGill University
Pennie & Edmonds LLP
LandOfFree
Dominant selectable marker for gene transformation and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Dominant selectable marker for gene transformation and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Dominant selectable marker for gene transformation and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3068946