细胞技术

The cytoskeleton provides the basic architectural organization and shape of the eukaryotic cell, and plays a key role in segregation of the genetic material. A method to visualize the actin and microtubule cytoskeleton in the fungus Ustilago maydis by indirect immunofluorescence is de ...

Critical cellular processes such as DNA replication, DNA damage repair, and transcription are mediated and regulated by DNA-binding proteins. Many efforts have been invested therefore in developing methods that monitor the dynamics of protein-DNA association. As older techniqu ...

As ubiquitous metabolic and signaling intermediaries, kinases regulate innumerable aspects of fungal growth and development. At its simplest, the enzymatic function of a kinase is to transfer a phosphate from a donor molecule (such as adenosine triphosphate) to an acceptor molecule, ...

Biochemical approaches are powerful tools for investigating mechanisms of biological processes. Here, we describe several biochemical approaches that have been successfully in our laboratory to study the filamentous fungus Neurospora crassa. These approaches include pr ...

Digital image analysis has been used to distinguish and quantify leaf color changes arising from a variety of factors. Its use to assess the percentage of leaf area with color differences caused by plant disease symptoms, such as necrosis, chlorosis, or sporulation, can provide a rigorous and qu ...

In host-pathogen interactions, identification of pathogen genes expressed during plant infection poses a challenge, even though these genes may be strongly induced by signals from the host. Here, we describe the application of a PCR-based differential screening method to plant-fun ...

The Real-Time quantitative PCR (qPCR) method has become central for the quantification of gene expression as well as other applications. The major advantages of qPCR are the utilization of small amount of template, high sensitivity and the ability to detect products during the reaction. Af ...

The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. Multiple displacement amplification (MD ...

For an increasing number of microorganisms of scientific and industrial interest, the genome sequences have become available, which in turn has enabled genome-wide microarray studies. Global level transcriptomic analysis has flooded the research community with gene expressi ...

Laser microdissection (LM) has become an important tool for isolating individual cells or cell types from suitably prepared tissue samples. The technique can be used to isolate both fungal and host plant cells after pathogen infection for molecular studies. Sample preparation is a cruci ...

Expression profiling of fungal genes in the arbuscular mycorrhiza (AM) symbiosis has been based on studies of RNA extracted from fungal tissue or mycorrhizal roots, giving only a general picture of overall transcript levels in the targeted tissues. Information about the spatial distri ...

The availability of a large number of gene-disrupted mutants (either from natural mutants’ collections or from knockout projects) is a great advantage for functional analysis studies. However, disfunction of many fungal genes, involved in key developmental processes, leads to dram ...

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of gen ...

Genome sequences of many filamentous fungi are now available and additional genomes are currently being sequenced. One of the next strategic goals is to generate collections of tagged genes in order to establish a link between the several thousands of predicted genes and their function. Tra ...

The availability of complete genome sequences for a number of biologically important fungi has become an important resource for fungal research communities. However, the functions of many open reading frames (ORFs) identified through annotation of whole genome sequences have yet to ...

Genetic transformation is a key technology in modern fungal research. Most commonly, protoplasts are transformed using the polyethylene glycol-mediated transformation protocols. Because protoplasts are generated by treatment of mycelia with a crude enzyme preparation, t ...

The protoplast method for the transformation of filamentous fungi is described in detail, as is the Restriction Enzyme-Mediated Integration (REMI) procedure for introducing tagged mutations into the fungal genome. A split marker method for generating PCR fragments for targeted in ...

Polyamine oxidation is the main catabolic process of polyamines. This process is crucial because not only it participates in the regulation of the endogenous titers of polyamines but also it generates hydrogen peroxide, which can act as a signaling molecule. The recent identification of p ...

Spermine oxidase (SMO), the most recently characterized polyamine metabolic enzyme, catalyzes the direct back-conversion of spermine to spermidine in an FAD-dependent reaction that also yields the byproducts hydrogen peroxide (H2O2) and 3-aminopropanal. These metabolite ...

This chapter describes a protocol to analyze polyamines without any derivatization steps utilizing LC-MS/MS. Polyamines are separated by reversed phase LC prior MS analysis using heptafluorobutyric acid as MS compatible volatile ion-pairing agent, and selective and sensitive ...