Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families. These families are created based on experimentally characterized proteins and are populated by sequences from public databases with significant similarity. Protein biochemical information is continuously curated based on the available literature and structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification (i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties. This resource has been available for over 10 years to the scientific community, contributing to information dissemination and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/.

The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics

The problems of determination and elaboration of developmental patterns are more accessible in the case of visible patterns such as anthocyanin pigmentation patterns. Anthocyanin pigments offer the added advantage that they are completely dispensible in plant growth and development, which means that manipulation of anthocyanin pigmentation patterns generally has no deleterious or pleiotropic effects on the plant. Furthermore, many anthocyanin genes are known to act cell autonomously, so patterns of expression can visualized on a cell by cell basis. Coen et al. (1988) have discussed the basic principles behind flower color patterns with emphasis on Antirrhinum majus. Here I will focus on flower color patterns in Petunia hybrida and explain how the use of transgenes to manipulate patterns provides a tool with the potential to improve our understanding of how patterns are determined and elaborated.

Elicitation of Organized Pigmentation Patterns by a Chalcone Synthase Transgene

A specific tetracycline-induced, low-molecular-weight RNA encoded by the inverted repeat of Tn10 (IS10).

Plasmodesmata are intercellular organelles in plants that allow the passage of molecules between plant cells. Movement through plasmodesmata may allow transcription factors expressed in one cell to move into adjacent cells, thereby regulating gene expression non-cell autonomously. The two animations illustrate (i) movement of a protein through an individual plasmodesma and (ii) an experiment to detect the movement of the transcription factor through plasmodesmata from the L1 layer of a plant meristem into the L2 and L3 layers. These two animations would be useful in teaching plant biology or plant development or a cell biology class discussing mechanisms of intercellular transport.

Movement of Macromolecules in Plant Cells Through Plasmodesmata

Forty years ago, R. Alexander Brink1 first proposed that in addition to their genetic function, chromosomes possess a paragenetic function. By paragenetics, he was referring to chromosomally based mechanisms in epigenetics—the catchall term we now use for heritable changes in gene expression associated with mechanisms superimposed upon an unchanged primary DNA sequence. One example of epigenetic regulation with significant impact on biotechnology is transgene silencing. In many animal and plant systems cytosine methylation is a reversible covalent modification often associated with transgene silencing2. However, epigenetic gene regulation occurs in organisms without DNA methylation3, indicating that methylationindependent mechanisms exist for heritably perpetuating altered transcriptional states. A recent article in Nature describes a very interesting gene, MOM (Morpheus molecule, for the Greek god of dreams), which is required to maintain transcriptional silencing of a heavily methylated transgene array in Arabidopsis4. When this gene is disrupted, the transgene array becomes transcriptionally active, yet the extensive cytosine methylation associated with the array remains. This work, together with previous studies on another mutant in Arabidopsis (antisense MET1), suggests that extensive DNA methylation is neither required nor sufficient for transgene silencing (summarized in Fig. 1). Transgenes or endogenous genes, organized in direct or inverted repeats, often become transcriptionally silenced2. In organisms with DNA methylation mechanisms, the inactivation often correlates with extensive DNA methylation relative to the transcriptionally active state. In addition to cis-inactivation, some sequences can transcriptionally inactivate homologous sequences in allelic or nonallelic positions, and this inactivation is heritable in the absence of the inducing transgene. These trans-inactivation events are very similar to cases of endogenous gene silencing referred to as paramutation5. Mutations in MOM could provide an important tool for genetic engineering, as silent transgenes are activated in the absence of pleiotropic developmental abnormalities. MOM efficiently reactivates two different transcriptionally silent transgenes, suggesting that the MOM protein maintains transcriptional silencing in Arabidopsis4. If mutations in the MOM ortholog in crop species behave similarly, introducing transgenes into mutant lines could mitigate the problem of transgene silencing. Efficient resilencing of the transgene in heterozygous MOM/mom plants4 will require that mom be homozygous in marketable crop varieties. The MOM gene is the first known molecular component essential for transcriptional silencing of transgenes that does not affect DNA methylation levels. All other mutants that relieve transcriptional silencing also have reduced levels of DNA methylation within the transgene locus6,7. The putative protein sequences of the two silencing mutants that have been cloned provide a link with chromatin remodeling. The predicted MOM gene product is a 2001-amino acid novel nuclear protein that has a region of similarity to the C-terminal portion of the putative helicase region of SWI2/SNF2 DNAdependent ATPases. Another gene in Arabidopsis that is required for transgene silencing7, DDM1, is also a member of the SWI2/SNF2 family of DNA-dependent ATPases8. DDM1 is most similar to the subset of putative helicases involved in chromatin remodeling8, and it shares sequence similarity throughout a larger region of SWI2/SNF2 relative to MOM4,8. DDM1 was originally identified by a screen for mutants that exhibited reduced methylation in the highly repeated centromeric and ribosomal sequences9. Multiple alleles of ddm1 were subsequently isolated as mutants that activate a transcriptionally silent transgene7. In ddm1 mutants, transgene activation correlated with decreased methylation7. Whereas MOM plants have normal developmental phenotypes after nine generations of inbreeding4, plants homozygous for ddm1 for several generations frequently show developmental abnormalities that when outcrossed segregate as new mutations independent of ddm110. As many of these mutations are semidominant and unstable, they are likely to represent epimutations that result from the inappropriate regulation of particular genes. Similarly, reduced expression of the major DNA methyltransferase in Arabidopsis, MET1, by means of antisense, also produces plants with abnormal development11. When antisense MET1 plants were tested for relief of transgene silencing, DNA methylation of the transgene was significantly reduced, but the transgene remained silent (see Fig. 1)7. The observation that DNA methylation is neither sufficient (mom) nor required (antisense MET1) for the maintenance of transgene silencing raises the issue of what aspects of chromatin are mediating silencing. These mutants are an excellent resource for addressing this issue. Silencing Morpheus awakens transgenes

Silencing Morpheus awakens transgenes.

In flowering plants, transformation with a transgene whose coding sequence is homologous to an endogenous plant gene, and whose expression is driven in the sense orientation by a strong promoter, often produces the opposite outcome of that which was intended. Instead of over-expression of the gene product, transcripts from both the endogenous gene and the transgene are found to be “cosuppressed,” resulting in a null phenotype. Nuclear run-on experiments have shown that the cosuppressed state is posttranscriptional; experiments that alter transgene dosage suggest that the induction of cosuppression is threshold-dependent; and cytoplasmic RNA viruses that are homologous to a strongly expressed nuclear gene are subject to cosuppression, suggesting that it occurs in the cytoplasm (de Carvalho et al. 1992de Carvalho et al. 1995; Lindbo et al. 1993; Smith et al. 1994; Mueller et al. 1995; Goodwin et al. 1996). A preferred interpretation of these observations, presented in detail by Smith et al. (1994), is that the production of nuclear transcripts to levels exceeding some threshold concentration somehow activates a sequence-specific turnover process that acts in the cytoplasm. This phenomenon is referred to variously as “sense suppression,” “sense cosuppression,” or simply “cosuppression.” It can be distinguished from promoter-homology-dependent suppression, a gene silencing phenomenon that occurs at the transcriptional level, requires promoter homology, and is thought to be a consequence of ectopic pairing (for review, see Matzke and Matzke 1995; see also Vaucheret et al.; Meyer; both this volume). Sense cosuppression also can be distinguished from suppression caused by antisense transgenes (Jorgensen et al. 1996).

Richard A. Jorgensen

Papers

Elicitation of Organized Pigmentation Patterns by a Chalcone Synthase Transgene

A specific tetracycline-induced, low-molecular-weight RNA encoded by the inverted repeat of Tn10 (IS10).

Movement of Macromolecules in Plant Cells Through Plasmodesmata

Silencing Morpheus awakens transgenes.

Sense Cosuppression of Flower Color Genes: Metastable Morphology-based Phenotypes and the Prepattern-threshold Hypothesis