Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

The molecular biology of barophilic bacteria

Many microorganisms from the deep-sea display high-pressure-adapted — also described as barophilic or piezophilic — growth characteristics. Phylogenetic studies have revealed that a large proportion of the barophilic bacteria currently in culture collections belong to a distinct subgroup of the genus Shewanella, referred to as the “barophile branch.“ Many of the basic properties of barophiles that enable their survival at extremes of pressure remain to be elucidated. However, several genes whose expression is regulated by pressure, or which appear to be critical to baroadaptation, have been uncovered. One such operon, whose presence appears to be restricted to the “barophile branch,” has been identifed in DNA samples obtained from sediments recovered in the deepest ocean trench. In the case of another set of pressure-regulated genes, regulatory elements required for pressure signaling have been uncovered. The nature and regulation of these genes is discussed. Received: February 19, 1997 / Accepted: March 3, 1997     

Seasonal variation in rates of methane production from peat of various botanical origins: effects of temperature and substrate quality

The methane produced in peat soils can vary over the growing season due to variations in the supply of available substrate, the activity of the microbial community or changes in temperature. Our aim was to study how these factors regulate the methane production over the season from five different peat types of different botanical origin. Peat samples were collected on seven occasions between June and September. After each sampling, the peat soils were incubated at five different temperatures (7, 10, 15, 20 and 25 degrees C) without added substrate, or at 20 degrees C with added substrate (glucose, or H(2)/CO(2), or starch). Rates of methane production averaged over the season differed significantly (P<0.05, R(2)=0.76) among the five peat types, the minerotrophic lawn producing the highest rates, and the hummock peat producing the lowest. The seasonal average Q(10) values for each plant community varied between 4.6 and 9.2, the highest value being associated with the ombrotrophic lawn and the lowest value with the mud-bottom plant community. For the unamended peat samples, the rates of methane production from each plant community varied significantly (P<0.05) over the season. This implies that the quality of organic matter, in combination with changes in temperature, explains the seasonal variation in methane production. However, addition of saturating amounts of glucose, H(2)/CO(2) or starch at 20 degrees C significantly reduced the seasonal variation (P<0.05) in methane production in peat from the minerotrophic lawn, wet carpet and mud-bottom plant communities. This suggests that substrate supply (e.g. root exudates) for the micro-organisms also varied over the season at these sites. Seasonal variation in methane production rates was apparent in peat from the hummock and ombrotrophic lawn plant communities even after addition of substrates, suggesting that the active biomass of the anaerobic microbial populations at these sites was regulated by other factors than the ones studied.     

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

Disparate distributions of chemolithotrophs containing form IA or IC large subunit genes for ribulose-1,5-bisphosphate carboxylase/oxygenase in intertidal marine and littoral lake sediments

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.     

Generation of virus‐resistant potato plants by RNA genome targeting

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.     

低强度微波辐射对人精子非热生物效应的研究

选用频率２４５０ＭＨｚ分别在３、５、７、９ｍＷ／ｃｍ２的功率密度,使用新型宽带横电磁传输室（ＢＴＥＭＣＥＬＬ）辐射人精子１小时,我们发现只有５ｍＷ／ｃｍ２的强度对人射子的活动度、存活率、畸形率又穿卵率有显著的影响,这表明在５ｍＷ／ｃｍ２附近存在明显的功率密度窗效应。同时还发现,７ｍＷ／ｃｍ２组的精子的染色体出现畸变。最后对实验结果进行了讨论。     

The role of protected areas in land use/land cover change and the carbon cycle in the conterminous United States

Protected areas (PAs) cover about 22% of the conterminous United States. Understanding their role on historical land use and land cover change (LULCC) and on the carbon cycle is essential to provide guidance for environmental policies. In this study, we compiled historical LULCC and PAs data to explore these interactions within the terrestrial ecosystem model (TEM). We found that intensive LULCC occurred in the conterminous United States from 1700 to 2005. More than 3 million km² of forest, grassland and shrublands were converted into agricultural lands, which caused 10,607 Tg C release from land ecosystems to atmosphere. PAs had experienced little LULCC as they were generally established in the 20th century after most of the agricultural expansion had occurred. PAs initially acted as a carbon source due to land use legacies, but their accumulated carbon budget switched to a carbon sink in the 1960s, sequestering an estimated 1,642 Tg C over 1700–2005, or 13.4% of carbon losses in non‐PAs. We also find that PAs maintain larger carbon stocks and continue sequestering carbon in recent years (2001–2005), but at a lower rate due to increased heterotrophic respiration as well as lower productivity associated to aging ecosystems. It is essential to continue efforts to maintain resilient, biodiverse ecosystems and avoid large‐scale disturbances that would release large amounts of carbon in PAs.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.