Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.     

格网尺度下典型旅游城市生态服务价值估算和时空分异特征——以三亚为例

以典型旅游城市三亚市为案例地，利用2006-2018年4期Landsat遥感影像数据，借助ENVI、ArcGIS平台定量识别土地利用演变特征，在1km×1km格网尺度下估算旅游地生态系统服务价值，并结合空间探索性数据分析揭示生态系统服务价值时空分异特征及其与旅游地发展的时空耦合关系。结果表明：（1）2006-2018年间，三亚市生态系统服务价值总量呈逐年下降趋势，由6.73×10⁹元降至5.76×10⁹元，累计减少9.78×10⁸元；（2）空间格局上，三亚市呈"南低北高"空间分异格局，2006-2018年增值区域连片分布于崖州区、天涯区、吉阳区南部区域，且呈逐年减少趋势，减值区域集聚于天涯区东北部、海棠区；（3）空间集聚上，生态系统服务价值截面各年份均呈显著空间正相关且相关性先降后增。高高集聚区位于天涯区北部区域，低低集聚区分布于沿海、海湾地区；（4）旅游发展与生态系统服务价值时空演化特征关联性较强。三亚市天涯区北部林地生态环境良好，生态系统服务价值略有下降但绝对数值稳定，是生态系统服务价值主要来源；旅游发展较为迅速的三亚湾、崖州湾以及海棠湾，相对增值区域较多，但绝对生态系统服务价值损失显著，严重滞后于其他区域。     

Germination,reproduction and interference in the amphicarpic annual Emex spinosa (L.) Campd

Summary A comparison was made of the dimorphic subterranean and aerial achenes of the annual Emex spinosa (L.) Campd. Subterranean achenes were less dormant, had a higher percentage of viability, germinated at a faster rate, were less temperature-dependent in germination and produced larger seedlings than aerial achenes. Subterranean achenes germinated, equally well in light or dark but aerial achenes had a strong light requirement. There was a linear relationship between numbers of weight of aerial achenes and plant size in the field. More resources were allocated to reproduction in plants restricted in size because of environmental conditions. Subterranean achene production was less affected than aerial by density, nitrogen levels, varying field conditions and interference between plants grown, from subterranean and aerial achenes. In mixtures of such plants, those from subterranean achenes had larger leaf area and stems and more aerial achenes. These differences were not apparent in monocultures by the time of harvest. Seed dimorphism is more likely in short-lived fugitive species and the different roles associated with dimorphism (persistence in situ versus dispersal in E. spinosa) are seen as an ecological advantage in such species.     

The physical mapping of bacteriophage T5 transfer tRNAs.

Transfer RNAs, isolated from Escherichia coli F cells infected with T5 bacteriophage, were charged with radioactive amino acids and used in RNA-DNA hybridization studies to detect and locate T5 tRNA cistrons in the T5 DNA chromosome. Hybridization of 14 3H-aminoacyl-tRNA species, including purified T5 [35S]Met-tRNAm and [35S]Met-tRNAf, to the separated strands of T5+ DNA indicates that most, if not all, of the T5 tRNAs are transcribed from the continuous heavy strand of T5 DNA. Heteroduplex mapping of eight mutant T5 DNA deletions has enabled us to locate and determine the size of these deleted segments. By correlating this information with the presence and absence of specific tDNA sequences in these mutants, as determined by tRNA-DNA hybridization, we were able to define the physical limits of four tDNA-containing loci along the T5 DNA molecule. A physical map for 15 tRNA species examined indicates that the structural genes for these tRNAs are clustered within a segment length of T5 DNA that represents approximately 11.2% of the total wild type T5 DNA. The existence of the deletion mutants indicates that T5 tRNAs are dispensable for T5 replication under the growth conditions and for the host employed.