High frequency transformation of Mucor with recombinant plasmid DNA

Heeswijck, R. van; Roncero, M. Isabel G.

doi:10.1007/bf02907500

Cited by 130 publications

(69 citation statements)

References 35 publications

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“…circinelloides R7B was carried out as described elsewhere (van Heeswijck & Roncero, 1984) with the modifications of Wolff et al (2002), as follows. Protoplasts were prepared by enzymic treatment of germlings with a mixture of 125 mg chitosanase-RD (US Biologicals) and 5 U chitinase (from Streptomyces griseus, Sigma) in a total volume of 2 ml.…”

Section: Methodsmentioning

confidence: 99%

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

2007

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Section: Methodsmentioning

confidence: 99%

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

2007

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“…The absence ofa utilisable sexual recombination system for genetic analysis and manipulation (13,31) made it desirable to develop a transformation system and introduce recombinant DNA technology into the study of these organisms. The transformation of M. circinelloides with hybrid plasmid DNA was reported in a previous publication (16). Cloning of a Leu § gene by direct complementation was made possible by the high frequency of transformation and the ability to recover plasmids from the transformant DNA.…”

Section: Introductionmentioning

confidence: 99%

Autonomous replication of plasmids in Mucor transformants

Heeswijck

1986

Carlsberg Res. Commun.

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Evidence is presented for the autonomous replication of recombinant plasmids in Mucor circinelloides Leu* transformants. Plasmids consisting of a unique fragment of Mucor DNA inserted into YRpl7 or pBR322 (i) give a high frequency &transformation (up to 7800 Leu* transformants per lag DNA), (ii) are mitotically unstable, (iii) can be reisolated in an unmodified form from uncut transformant DNA, and (iv) are present as discrete extrachromosomal DNA molecules (as detected by Southern hybridisation).No integration of the transforming DNA can be detected in Mucor transformants containing free plasmid molecules. This excludes the possibility that free plasmids are simply excision products resulting from a reversal of the integration process.Sub-cloning of the recombinant plasmids and analysis of subsequent Leu § transformants show that autonomous replication is independent of the vector sequences and locates a Mucor ars within a 4.4 kb Pstl fragment of the insert DNA.

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“…It is a relatively simple organism with a small genome (1.6 x 107 bp per haploid genome [34]) and an available transformation system (36) which facilitate molecular genetic investigations. M. racemosus is dimorphic: it exhibits filamentous (hyphal) growth in the presence of oxygen, but under anaerobic conditions with a fermentable hexose available, the organism grows as a single-celled, budding yeast (4).…”

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confidence: 99%

Expression of a gene family in the dimorphic fungus Mucor racemosus which exhibits striking similarity to human ras genes.

et al. 1990

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Sporulation, spore germination, and yeast-hypha dimorphism in the ifiamentous fungus Mucor racemosus provide useful model systems to study cell development in eucaryotic cells. Three RAS genes (MRASI, MRAS2, and MRAS3) from M. racemosus have been cloned, and their nucleotide sequences have been determined. The predicted amino acid sequences and the sizes of the three MRAS proteins exhibit a high degree of similarity with other ras proteins, including that encoded by H-ras, which have been implicated in regulation of proliferation and development in eucaryotic cells by mediating signal transduction pathways. The MRAS proteins show conservation of functional domains proposed for ras proteins, including guanine nucleotide interaction domains, an effector domain, a binding epitope for neutralizing antibody Y13-259, and the COOH-terminal CAAX box, which is a site of thiocylation and membrane attachment. Amino acid sequences unique to each MRAS protein occur adjacent to the CAAX box, consistent with the location of the hypervariable region in other ras proteins. Northern (RNA) analysis was used to study expression of the three MRAS genes in relation to cell development. Gene-specific probes for two of these genes, MRAS1 and MRAS3, hybridized to different 1.3-kb mRNA transcripts. The accumulation of these transcripts depended on the developmental stage, and this pattern was different between the two MRAS genes. No transcript for MRAS2 was detected in the developmental stages examined. The unique patterns of MRAS transcript accumulation suggest that individual MRAS genes and proteins may play distinct roles in cell growth or development.The fungus Mucor racemosus is a saprophytic zygomycete that provides a useful system for studying eucaryotic cell differentiation. It is a relatively simple organism with a small genome (1.6 x 107 bp per haploid genome [34]) and an available transformation system (36) which facilitate molecular genetic investigations. M. racemosus is dimorphic: it exhibits filamentous (hyphal) growth in the presence of oxygen, but under anaerobic conditions with a fermentable hexose available, the organism grows as a single-celled, budding yeast (4). The fungus also undergoes morphogenesis during sporulation and germination of two asexual spore types, sporangiospores and arthroconidia, and a sexual spore called a zygospore.Morphogenesis in M. racemosus is correlated with changes in cyclic AMP (cAMP) and phospholipid metabolism (4,10,13,14,(21)(22)(23) large increase in phospholipid metabolism during yeast-tohypha morphogenesis, especially in the turnover of phosphatidylethanolamine and phosphatidylinositol (PI) (13). Cerulenin prevents phospholipid turnover and yeast-to-hypha morphogenesis under conditions in which cell growth continues at rates typical of yeast cells.The implication of cAMP and PI as secondary-messenger molecules in signal transduction pathways involving ras proteins, the ubiquity of ras proteins in eucaryotes, and the putative role of ras in cell differentiation and proliferation (2, 2...

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High frequency transformation of Mucor with recombinant plasmid DNA

Cited by 130 publications

References 35 publications

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

Autonomous replication of plasmids in Mucor transformants

Expression of a gene family in the dimorphic fungus Mucor racemosus which exhibits striking similarity to human ras genes.

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