Cassettes for PCR‐mediated construction of green, yellow, and cyan fluorescent protein fusions in <i>Candida albicans</i>

Gerami-Nejad, Maryam; Berman, Judith; Gale, Cheryl A.

doi:10.1002/yea.738

Cited by 183 publications

(195 citation statements)

References 26 publications

(27 reference statements)

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“…The first copy of DAM1, ASK1, or SPC19 was replaced by HIS1 in the strain SN148. 5Ј Sequences of long primers (see Table S3 in the supplemental material) that were homologous to sequences upstream and downstream of each gene and 3Ј ends that were homologous to the HIS1 gene were used to construct deletion cassettes by PCR using the plasmid HIS1GFP (19) as the template. Each deletion construct carries 90 to 110 bp of upstream and downstream homology regions flanked by the HIS1 gene.…”

Section: Methodsmentioning

confidence: 99%

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Thakur

Sanyal

2011

Eukaryot Cell

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A fungus-specific outer kinetochore complex, the Dam1 complex, is essential in Saccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. Both Candida albicans and Schizosaccharomyces pombe have regional centromeres as opposed to the short-point centromeres of S. cerevisiae. The interaction of one microtubule per kinetochore is established both in S. cerevisiae and C. albicans early during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis in S. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle in S. cerevisiae and C. albicans but only during mitosis in S. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation in C. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochoremicrotubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.The separation of sister chromatids during mitosis is driven by a dynamic interaction between two macromolecular structures: (i) the kinetochore (KT), a complex proteinaceous structure built on the centromere DNA (10, 47), and (ii) the mitotic spindle formed by microtubules (MTs). The process of chromosome segregation has been studied extensively in a wide range of organisms, from simple unicellular yeasts to humans. While the major sequence of events required for chromosome segregation remains the same, a few species-specific differences exist. The microtubule organizing centers (MTOCs) in budding yeasts, called spindle pole bodies (SPBs), are embedded in the nuclear envelope and assemble an intranuclear mitotic spindle (5). Thus, the interaction of spindle MTs with KTs does not require the breakdown of the nuclear envelope during closed mitosis in budding yeasts, and the KT-MT interaction is established early in the cell cycle (12, 23). The fission yeast Saccharomyces pombe also undergoes closed mitosis (23), but SPBs remain outside the nuclear envelope during interphase (27) and spindle MTs are nucleated and interact with KTs only when the duplicated SPBs enter the nuclear membrane following mitotic initiation (14). Metazoan cells, in contrast, undergo open mitosis (22,45) in which the MTs, originating from MTOCs, gain access to KTs only when the nuclear envelope breaks down during mitosis. Finally, the number of MTs that bind to a single chromosome differs in these organisms: in Saccharomyces cerevisiae only one MT binds per KT (52), in S. pombe two or three MTs bind per KT (13), and in metazoans multipl...

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Section: Methodsmentioning

confidence: 99%

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Thakur

Sanyal

2011

Eukaryot Cell

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“…PCR products were digested with XbaI and ligated with the pGEM-T Easy plasmid (Promega) to generate PI78.1. The GFP ORF was amplified by PCR using pGFP-URA3 as the template (a generous gift from Dr Judith Berman, University of Minnesota) (Gerami-Nejad et al, 2001) and the primers Gfcfxbf1 (59-TAACTAGAATGTCTAAAGGTGAAGAATTATTC-39) and Gfcfxbr1 (59-TAATCTAGATTATTTGTACAATTCATCC-39). The PCR product was digested with XbaI and subcloned into the XbaI site of pI78.1 to derive pI82.10 for the TDH3-GFP fusion.…”

Section: Methodsmentioning

confidence: 99%

Dark brown is the more virulent of the switch phenotypes of Candida glabrata

Srikantha

Daniels

et al. 2008

Microbiology

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Candida glabrata undergoes reversible, high-frequency core switching between phenotypes that include dark brown (DB), light brown (LB) and white (Wh). These phenotypes in turn can switch to the irregular wrinkle (IWr) phenotype. Natural isolates, however, express predominantly the DB phenotype, leading to the hypothesis that it has a colonization advantage over the other switch phenotypes. Using the mouse model of systemic infection, results are presented which support this hypothesis. DB has an advantage over other switch phenotypes in colonizing the two major target organs in the mouse model, the spleen and liver. A time-course study reveals that colonization of the major target organs occurs very rapidly (within 2 h) after host injection, and that the DB advantage for spleen and liver colonization is immediate. The DB advantage is maintained during clearing from spleen, liver and kidneys, and during delayed transient brain colonization. These results demonstrate that DB has a colonization advantage over other switch phenotypes, and that the switch phenotype expressed by a colonizing population therefore plays a fundamental role in virulence. It is therefore essential that switching be considered in both in vivo and in vitro studies of C. glabrata virulence.

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“…The HOG1-GFP chimaera was constructed by PCR amplification of the ORF using the primers o-59HHOG1 (59-CCAAAGCTTATGTCTGCAGATGG-AGAATTTACA-39) and o-HOG1fusion2 (59-GGAAAGCTTCCAC-CACCAGCTCCGTTGGCGGAATCCAA-39). After incorporation of this fragment into the pGEM-T vector (Promega), it was excised as a 1138 bp fragment that was subsequently introduced in the large HindIII fragment of the pGFP-URA3 plasmid (Gerami-Nejad et al, 2001). This construction was integrated at the HOG1 locus after digestion with SalI to render the construction linear.…”

Section: Methodsmentioning

confidence: 99%

The Pbs2 MAP kinase kinase is essential for the oxidative-stress response in the fungal pathogen Candida albicans

et al. 2005

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The human fungal pathogen Candida albicans responds to stress by phosphorylation of the Hog1 MAP kinase. PBS2 was cloned and shown to encode the MAP kinase kinase that is involved in this activation, as determined by immunoblot analyses using antibodies that recognize the active form of the target Hog1 protein. Characterization of pbs2 mutants revealed that they were sensitive to both osmotic and oxidative stress and that they, interestingly, displayed differential behaviour from that of hog1 mutants, losing viability when exposed to an oxidative challenge more rapidly than the hog1 strain. Hog1 and Pbs2 were also shown to be involved in the mechanism of adaptation to oxidative stress, as evidenced by the enhanced susceptibility to oxidants of pbs2 and hog1 mutants, compared with the wild-type strain, when cells were previously exposed to a low, sub-lethal concentration of hydrogen peroxide and by the PBS2-dependent diminished activation of Hog1 MAP kinase in the adaptive process. Studies with a chimaeric Hog1-green fluorescent protein fusion revealed that this protein was localized throughout the cell (being excluded from the vacuole), but concentrated in the nucleus in response to NaCl stress, a process that was dependent on the Pbs2 protein. Both Hog1 and Pbs2 also play a role in controlling the phosphorylation state of the other MAP kinases Mkc1 and Cek1, involved respectively in cell-wall integrity and invasive growth. Furthermore, it is demonstrated that PBS2 plays a role in cell-wall biogenesis in this fungal pathogen, as its deletion renders cells with an altered susceptibility to certain cell wall-interfering compounds. INTRODUCTIONEukaryotic cells respond to environmental changes through signal-transduction pathways. MAPK (mitogen-activated protein kinase) routes consist of a three-kinase module: the MAP kinase kinase kinase, the MAP kinase kinase and the MAP kinase, which are activated by sequential phosphorylation in response to different extracellular signals. These pathways are conserved through evolution (Kültz & Burg, 1998) and their functionality is well-documented in some model organisms, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe (Banuett, 1998;Gustin et al., 1998). In budding yeast, six MAPK pathways have been described that perform essential functions in fungal physiology, such as mating, pseudohyphal/invasive growth, cellwall integrity, STE vegetative growth (SVG), spore-wall assembly and adaptation to high osmolarity (HOG) (Gustin et al., 1998;Posas et al., 1998). This last route is activated in response to osmotic and -as described recently -oxidative stress (Haghnazari & Heyer, 2004;Singh, 2000) and mutant cells in some elements of the pathway are sensitive to both kinds of stresses. Osmosensitivity is partially explained by the inability to increase intracellular glycerol under restrictive conditions in order to counteract the extracellular turgor (Albertyn et al., 1994). Under hyperosmotic conditions, hog1 and pbs2 mutant strains display different alterations, suc...

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Cassettes for PCR‐mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans

Cited by 183 publications

References 26 publications

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Dark brown is the more virulent of the switch phenotypes of Candida glabrata

The Pbs2 MAP kinase kinase is essential for the oxidative-stress response in the fungal pathogen Candida albicans

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