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

Let's admit an unfortunate truth: agricultural research in most of the world is based on old models that no longer serve us well in the twenty-first century as arable land diminishes, populations increase, and climate is changing. A dramatically new vision is needed in order to remake agricultural research, perhaps radically, to address the challenges of the world we find ourselves in today. Here is an attempt to begin to develop such a vision.


(1)

We must learn to value foundational (basic) and applied research equally. Here are three ways to work toward this goal:


Develop the “borderlands” between the Foundational and Applied Research Domains. Recognize and support their unique cultures, while evolving a new culture that bridges the artificial boundary between them.


Develop the borderlands between extramural and intramural research within and between government agencies. Value both domains and foster a new culture of cooperation, common mission, and mutual benefits.


Develop the borderlands between research and extension. Again, foster a new culture of cooperation, common mission, and mutual benefits.




(2)

We need much stronger Foundational Research than currently exists – across the agricultural sciences and in diverse, mission-critical areas that must be defined, prioritized, and supported. Some of these mission-critical areas:


Ecosystems, populations, and communities (agricultural and natural).


Interorganismal interactions (pathogens, pests, pollinators, mutualisms, etc.).


Intraorganismal research: to develop knowledge and understanding leading to predictive modeling of growth, development, physiology, and responses to the environment.


Environmental and earth sciences, including hydrology, soil science, and remote imaging.


Computer and information sciences, mathematical modeling, and cyberinfrastructure in service to, and as a foundation for twenty-first century agricultural sciences: we must learn to harness the evolving Metaweb in service to research, extension, and education in order to foster development of a new agriculture based on intelligent systems, which are part and parcel of the Metaweb. As described by Nova Spivak (http://www.novaspivack.com/science/new-version-of-my-metaweb-graph-the-future-of-the-net), the Metaweb is continuously evolving from the semantic web and social software, bringing together knowledge, and people to produce understanding.




(3)

Applied Science must be reorganized around major concepts, goals, and national and international imperatives, especially:


long-term sustainability: production, water, ecosystems, soil quality, farm communities (long-term sustainability may be defined as: “meeting our needs without compromising the ability of future generations to meet their needs”).


security: energy security, food security, climate security.


health, welfare, and community: nutrition, food quality, robust communities, economic vitality.




(4)

To repeat and extend the first point above (because it is so important), success requires that we foster development of new cultures and new communities that are coincident with needs, not tradition. The needs for new communities are many and varied. It will be important that new communities must arise out of the borderlands between each of the following pairs of communities that are now largely separate:


basic and applied research


research and extension


agriculture and agroforestry


agriculture and ecology


agriculture and the food industry


the food industry and consumers.





To achieve this vision will require us to let go of habits and preconceived notions in order to work toward understanding the diverse cultures and communities that comprise research, extension, and industry and to find new ways of bringing them together productively and creatively in order to create synergies and achieve efficiencies. The specific ways for achieving this vision will be many and varied, and will require the creative efforts of scientists, engineers, funders, industry participants, and policymakers everywhere.

Where should we start? Three principal areas need to be dramatically reorganized: the ways that we fund research, the ways that organize our research institutions, and the ways that we define success and evaluate progress toward the goals above.

Funding priorities need to be dramatically refocused in order to draw researchers out of comfortable isolation into the borderlands where new and challenging interactions between cultures will enable innovative changes, new ways of approaching problems, and disruptive technologies that are needed in order to create a truly sustainable agriculture in sync with sustainable ecosystems and sustainable societies. Probably, the way that funding agencies are organized and funded will need to change in order to make it possible to reorient funding toward new priorities.

Similarly, agricultural research institutions need to be reorganized to bring people together across disciplinary boundaries to populate the borderlands, merge cultures productively, and evolve new cultures and develop new types of institutions. Nothing less than reinventing the nature of agricultural research organizations will suffice, and this reinvention needs to be completed within the next decade if it is to have the needed impact on the necessary timeframe given the increasing demands on agricultural lands, the increasing human population, and changes in climate and health of the ecosystems and the biosphere as a whole.

Finally, of course, none of this can happen unless we also rethink how we evaluate people, programs, and institutions. Expectations and evaluations must be clearly aligned with our new research priorities and the evolving nature of our research organizations. Doing things the way we have always done them is unacceptable if we are to live sustainably for an indefinite time in the only biosphere we have.

/pdf/a-vision-for-21st-century-agricultural-research-18rv00x2dh.pdf

A Vision for 21st Century Agricultural Research

Tn5 is a transposable genetic element which encodes resistance to aminoglycoside antibiotics such as kanamycin and neomycin. This resistance results from the synthesis of the enzyme neomycin phosphotransferase type II (NPTII, also named aminoglycoside 3′-phos-photransferase-II) (Berg et al., 1978). Its structure is in general similar to other transposons with two inverted repeats 1534 bp long (Auerswald and Schaller, 1980) flanking a unique central region approximately 2700 bp long (Berg et al., 1975; Jorgensen et al., 1979).

The Structure of Tn5

Transitive RNAi is a posttranscriptional mechanism of gene silencing that is based on the phenomenon of “transitivity.” This term refers to the spreading of silencing outside of the initial target sequence and is associated with transgene-induced posttranscriptional gene silencing (PTGS). Transitive RNAi is triggered by placing an inverted repeat sequence immediately 3′ of the sense transgene that is to be targeted. Placement of the inverted repeat in this region is thought to increase the efficiency by which RDR6 initiates copying of the transgene to generate double-stranded RNA (dsRNA). In a proof-of-concept approach, we showed that select subsets of genes can be manipulated with transitive RNAi in a high-throughput forward mutagenesis approach (Plant J 61:873–882, 2010). Laser microdissection of Arabidopsis mesophyll cells and en masse cloning of the resulting cDNA libraries into transitive RNAi vectors demonstrated that approximately 15% of genes in the pilot study could generate visible phenotypes, resulting in photosynthetic defects. The capacity for transitive RNAi to silence multiple members of gene family members demonstrated the utility of this approach for forward mutagenesis of redundant gene functions. Targeted silencing of a focused population of gene transcripts by transitive RNAi provides an efficient and complementary approach to procedures that target the entire genome. The ability of RNAi to target closely related genes holds promise for its use in forward mutagenesis of polyploid plants, which exhibit high levels of genetic redundancy. Advantages of transitive RNAi as a forward genetic approach, as well as potential drawbacks to this method, are discussed.

Mutagenesis by Transitive RNAi

The modification of horticultural plant phenotypes by direct gene transfer

Beginning May 2, 2005, the National Institutes of Health (NIH) requests that authors whose research was supported in whole or in part by NIH deposit their peer-reviewed, accepted manuscripts in PubMed Central, NIH's central repository. The NIH intends the manuscript repository to (1) provide an alternative means through which NIH grantees can partially fulfill progress reporting requirements and (2) be part of a central resource through which the public may view research funded by U.S. taxpayers. The full NIH policy can be viewed at http://grants1.nih.gov/grants/guide/notice-files/NOT-OD-05-022.html.

/pdf/aspb-s-response-to-the-nih-s-public-access-policy-5ay7tyvni4.pdf

Richard A. Jorgensen

Papers

A Vision for 21st Century Agricultural Research

The Structure of Tn5

Mutagenesis by Transitive RNAi

The modification of horticultural plant phenotypes by direct gene transfer

ASPB's Response to the NIH's Public Access Policy