With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis1 with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis2. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.

Targeted screening for induced mutations

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1357&context=usdaarsfacpub

History, current status and collaborative research projects for Bemisia tabaci

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

High-fidelity mRNA amplification for gene profiling.

It is paradoxical that exotic species invade and displace native species that are well adapted to local environments. Yet, even those exotics that eventually become abundant and widespread, often do so only after having failed to establish following multiple earlier introductions. The first pattern, while not generally discussed in this context, is usually explained by exotic species pre-adaptations for human-altered environments and by a release from enemies. It can be understood further by examining the superior quality of colonists from large species-rich regions and the historical contingency of evolution. The second pattern is generally explained by invoking demographic and environmental stochasticity; how? ever, it can be understood further by examining the role of environmental variation over space and by metapopulation dynamics. These pro? cesses provide a context in which these patterns of invasion are not paradoxical, but instead, expected.

https://www.jstor.org/stable/info/2665407

The paradox of invasion

Most successful existing vaccines rely on neutralizing antibodies, which may not require specific anatomical localization of B cells. However, efficacious vaccines that rely on T cells for protection have been difficult to develop, as robust systemic memory T-cell responses do not necessarily correlate with host protection. In peripheral sites, tissue-resident memory T cells provide superior protection compared to circulating memory T cells. Here we describe a simple and non-inflammatory vaccine strategy that enables the establishment of a protective memory T-cell pool within peripheral tissue. The female genital tract, which is a portal of entry for sexually transmitted infections, is an immunologically restrictive tissue that prevents entry of activated T cells in the absence of inflammation or infection. To overcome this obstacle, we developed a vaccine strategy that we term 'prime and pull' to establish local tissue-resident memory T cells at a site of potential viral exposure. This approach relies on two steps: conventional parenteral vaccination to elicit systemic T-cell responses (prime), followed by recruitment of activated T cells by means of topical chemokine application to the restrictive genital tract (pull), where such T cells establish a long-term niche and mediate protective immunity. In mice, prime and pull protocol reduces the spread of infectious herpes simplex virus 2 into the sensory neurons and prevents development of clinical disease. These results reveal a promising vaccination strategy against herpes simplex virus 2, and potentially against other sexually transmitted infections such as human immunodeficiency virus.

/pdf/a-vaccine-strategy-that-protects-against-genital-herpes-by-2c8j1mhx72.pdf

A vaccine strategy that protects against genital herpes by establishing local memory T cells

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1x105 to 4.3x106 conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30°C, compared to 15 and 20°C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).

/pdf/selection-of-beauveria-bassiana-and-metarhizium-anisopliae-235i18gdio.pdf

Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

http://www.scielo.br/pdf/bjm/v34n2/arq05.pdf

Genetic variability of brazilian Alternaria spp. isolates as revealed by RAPD analysis

The solitary egg parasitoids (Hymenoptera: Scelionidae) of Pentatomidae species complex are potential candidates for soybean (Glycine max L.) integrated pest management programs in Brazil. The correct identification of species and strains of these parasitoids is necessary to implement the use of biological control. Polymerase chain reaction (PCR) was used to generate random amplification of polymorphic DNA (RAPD) from male individuals of Telenomus podisi Ashmead and Trissolcus spp. collected in a soybean field in Brasilia, Brazil in 1996. The analysis of 18 primers used to screen Trissolcus spp. individuals revealed different patterns related to the species analyzed. These results indicate the potential of RAPD markers to differentiate T. teretis Johnson, T. urichi Crawford and T. basalis (Wollaston). The 13 selected primers used to analyze T. podisi individuals produced 103 clear polymorphic scorable markers. Different genotypes were observed within this population analyzed, although the cluster analysis applied to the RAPD data showed high homogeneity (similarity >78.2%) among individuals.

/pdf/rapd-analysis-revealing-polymorphism-in-egg-parasitoids-of-3l0xu3gn9b.pdf

RAPD analysis revealing polymorphism in egg parasitoids of soybean stink bugs (Hemiptera: Pentatomidae)

foram selecionados para analise 388 caracteres binariosgerados por 31 primers. A analise fenetica destes dados revelou uma altahomogeneidade (similaridade ≥ 79,5%) entre os isolados, principalmente entreos brasileiros (similaridade ≥ 98,3%), os quais foram obtidos de regioesgeograficamente proximas e do mesmo hospedeiro,

/pdf/polymorphism-of-the-grasshopper-schistocerca-pallens-4j3qyi4lez.pdf

Polymorphism of the Grasshopper Schistocerca pallens (Thunberg) (Orthoptera: Acrididae) and its Natural Pathogen Metarhizium flavoviride Gams & Rozsypal (Hyphomycetes), Revealed by RAPD Analysis

Salah M. Aljanabi

Papers

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques

Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

Genetic variability of brazilian Alternaria spp. isolates as revealed by RAPD analysis

RAPD analysis revealing polymorphism in egg parasitoids of soybean stink bugs (Hemiptera: Pentatomidae)

Polymorphism of the Grasshopper Schistocerca pallens (Thunberg) (Orthoptera: Acrididae) and its Natural Pathogen Metarhizium flavoviride Gams & Rozsypal (Hyphomycetes), Revealed by RAPD Analysis