An effective DNA extraction protocol for brown algae

Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular‐based evolutionary studies. Our broad and long‐term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total‐DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total‐ DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques.     

Functional Enhancement of Electrofusion-derived BRIN-BD11 Insulin-secreting Cells After Implantation into Diabetic Mice

Electrofusion-derived BRIN-BD11 cells are glucosesensitive insulin-secreting cells which provide an archetypal bioengineered surrogate β-cell for insulin replacement therapy in diabetes mellitus, 5x10⁶ BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5–16 days (<17mmol/l) and normoglycaemia (<9mmol/l) was achieved by 7–20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10⁻⁹mol/l), cholecystokinin-8 (10⁻⁸ mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05–0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagonlike peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05–0.01). The functional enhancement of electrofusion-derived surrogate β-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist β-cell engineering approaches to therapy for diabetes.     

Replication protein A is sequentially phosphorylated during meiosis

Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.     

ALLOZYME VARIATION IN OLD WORLD RACES OF SORGHUM BICOLOR (POACEAE)

A survey of allozyme variation in cultivated races of sorghum (Sorghum bicolor) was undertaken. Eight plants each of 83 accessions representing the five primary races and five of the intermediate races of sorghum were analyzed for 15 enzyme systems encoded by 27 loci. Low levels of variation were found within accessions, which is typical of self-pollinating species. Little variation was also found among accessions. Compared with other cereals, S. bicolor is depauperate in allozyme variation. We found an average of 1.81 alleles per locus with a mean expected heterozygosity of 0.008 for the accessions and total panmictic heterozygosity of 0.093. Only 9% of the variation present was found within accessions, while 91% was among accessions. Most of the variation present is attributable to differences in geographic origin of the accessions rather than racial differences. Western and eastern Africa have the highest levels of total heterozygosity (0.108 and 0.088, respectively), while southern Africa has the lowest (0.008). Principal component analysis revealed continuous variation among races and geographic regions with the accessions failing to segregate into discrete racial or geographic clusters.     

Large-scale analysis of non-synonymous coding region single nucleotide polymorphisms

MOTIVATION: Single nucleotide polymorphisms (SNPs) are the most common form of genetic variant in humans. SNPs causing amino acid substitutions are of particular interest as candidates for loci affecting susceptibility to complex diseases, such as diabetes and hypertension. To efficiently screen SNPs for disease association, it is important to distinguish neutral variants from deleterious ones. RESULTS: We describe the use of Pfam protein motif models and the HMMER program to predict whether amino acid changes in conserved domains are likely to affect protein function. We find that the magnitude of the change in the HMMER E-value caused by an amino acid substitution is a good predictor of whether it is deleterious. We provide internet-accessible display tools for a genomewide collection of SNPs, including 7391 distinct non-synonymous coding region SNPs in 2683 genes. AVAILABILITY: http://lpgws.nci.nih.gov/cgi-bin/GeneViewer.cgi     

Adult neuron survival strategies--slamming on the brakes

Developing neurons are programmed to die by an apoptotic pathway unless they are rescued by extrinsic growth factors that generate an anti-apoptotic response. By contrast, adult neurons need to survive for the lifetime of the organism, and their premature death can cause irreversible functional deficits. The default apoptotic pathway is shut down when development is complete, and consequently growth factors are no longer required to prevent death. To protect against accidental apoptotic cell death, anti-apoptotic mechanisms are activated in mature neurons in response to stress. Loss or reduced activity of these intrinsic anti-apoptotic 'brakes' might contribute to or accelerate neurodegeneration, whereas their activation might rescue neurons from injury or genetic abnormalities.     

Discovery of induced point mutations in maize genes by TILLING

Background

Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.

Results

We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.

Conclusions

Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.     

A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex(-/-) mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex(-/-) mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex(-/-) AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.     

A test of a potential short cut in the nitrogen cycle: The role of exudation of symbiotically fixed nitrogen from the roots of a N-fixing tree and the effects of increased atmospheric CO2 and temperature

N-fixing trees facilitate the growth of neighboring trees of other species. These neighboring species benefit from the simple presence of the N fixation symbiosis in their surroundings. Because of this phenomenon, it has been hypothesized that a change in atmospheric CO₂ concentration may alter the role of N-fixing trees in their environment. It is thought that the role of N-fixing trees in ecosystems of the future may be more important since they may help sustain growth increases due to increased CO₂ concentration in nitrogen limited forests. We examined: (1) whether symbiotically fixed N was exuded from roots, (2) whether a doubled atmospheric CO₂ concentration would result in increased organic N exudation from roots, and (3) whether increased temperature or N availability affected N exudation from roots. This study analyzed exudation of dissolved organic N from the roots of seedlings of the N-fixing tree Robinia pseudoacacia L. in a full factorial design with 2 CO₂ (35.0 and 70.0 Pa) × 2 temperature (26 or 30 °C during the day) × 2 N fertilizer (0 and 10.0 mM N concentration) levels. Trees with no other source of N except N fixation exuded about 1% to 2% of the fixed N through their roots as dissolved organic N. Increased atmospheric CO₂ concentrations did not, however, increase N exudation rates on a per gram belowground biomass basis. A 4 °C increase in temperature and N fertilization did, however, significantly increase N exudation rates. These results suggest that exudation of dissolved organic N from roots or nodules of N-fixing trees could be a significant, but minor, pathway of transferring N to neighboring plants in a much more rapid and direct way than cycling through death, decomposition and mineralization of plant residues. And, while exudation rates of dissolved organic N from roots were not significantly affected by atmospheric CO₂ concentration, the previously observed CO₂ fertilization effect on N-fixing trees suggests that N exudation from roots could play a significant but minor role in sustaining increases in forest growth, and thus C storage, in a CO₂ enriched atmosphere.