Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

Antibodies to tubulin were prepared in rabbits by immunization with reduced-carboxymethylated calf-brain tubulin. In immunodiffusion tests the antibodies showed full cross reactivity with the immunogen as well as with native calf-brain tubulin. The same antibodies showed cross reactivity with a factor in extract of cotton (Gossypium hirsutum L.) cotyledons but there was no full immunological identity between calf-brain tubulin and this factor. A solid-phase radioimmunoassay for quantitative estimation of this plant tubulin-like factor was developed. It measured the binding of antibodies to immobilized calf-native tubulin. Competition between the unknown soluble tubulin-like factor, and immobilized tubulin was assayed at serum dilution of 1:50. Extraction conditions which preserved the antigenic properties of the tubulin-like factor from cotton cotyledons were defined. The radioimmunoassay measured quantities of the tubulin-like factor in the range of 0.1–10 g-equivalents of calf-brain tubulin. Immediately after homogenization of the tissue only 25% of the total amount of tubulin-like activity was present in soluble form, while most of it remained in the insoluble fraction. Apparent maximal solubilization was achieved spontaneously 10 h after homogenization or by treatment with guanidine hydrochloride. These results indicate that in this material, tubulin is not released immediately by homogenization but remains assembled in microtubules and-or in a bound or sequestered form.Abbreviations NRS normal rabbit serum - RCM reduced-carboxymethylated     

Site-specific integration of an F'' lac pro factor in the region of the replication origin (oriC) of E. coli

Summary An episome, F 128, which carries approximately 8x10⁴ base pairs of chromosomal DNA homologous to the lac pro region of the E. coli chromosome, has been found to integrate into the oriC region of the chromosome in a site specific reaction. While the event appears to be recA-dependent, no homology between the episome and this region of the chromosome was detected. The Hfr strains formed result from the integration of intact F 128 molecules. The structure of the Hfr strains generated has been determined and their transfer properties analyzed.     

基于蛋白质二级结构热稳定性的正交方法

对蛋白质热稳定性的研究是解析蛋白高级结构,开发蛋白功能及新药物研发过程中的一个重要环节,是对其结构分析的一个重要关切点.观测蛋白质的圆二色光谱随温度程序变化而改变是研究其热稳定性的常用手段,传统的实验方法为选用某一单波长作为测试点,通过连续升温测试蛋白在单波长下的圆二色变温曲线,然后拟合出Tm值,此方法所得的信息有限,...     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Age‐related memory vulnerability to interfering stimuli is caused by gradual loss of MAPK‐dependent protection in Drosophila

Age‐related memory impairment (AMI) is a common phenomenon across species. Vulnerability to interfering stimuli has been proposed to be an important cause of AMI. However, the molecular mechanisms underlying this vulnerability‐related AMI remain unknown. Here we show that learning‐activated MAPK signals are gradually lost with age, leading to vulnerability‐related AMI in Drosophila. Young flies (2‐ or 3‐day‐old) exhibited a significant increase in phosphorylated MAPK levels within 15 min after learning, whereas aged flies (25‐day‐old) did not. Compared to 3‐day‐old flies, significant 1 h memory impairments were observed in 15‐, 20‐, and 30‐day‐old flies, but not in 10‐day‐old flies. However, with post‐learning interfering stimuli such as cooling or electric stimuli, 10‐day‐old flies had worse memory performance at 1 h than 3‐day‐old flies, showing a premature AMI phenomenon. Increasing learning‐activated MAPK signals through acute transgene expression in mushroom body (MB) neurons restored physiological trace of 1 h memory in a pair of MB output neurons in aged flies. Decreasing such signals in young flies mimicked the impairment of 1 h memory trace in aged flies. Restoring learning‐activated MAPK signals in MB neurons in aged flies significantly suppressed AMI even with interfering stimuli. Thus, our data suggest that age‐related loss of learning‐activated neuronal MAPK signals causes memory vulnerability to interfering stimuli, thereby leading to AMI.     

Using all Gene Families Vastly Expands Data Available for Phylogenomic Inference

Traditionally, single-copy orthologs have been the gold standard in phylogenomics. Most phylogenomic studies identify putative single-copy orthologs using clustering approaches and retain families with a single sequence per species. This limits the amount of data available by excluding larger families. Recent advances have suggested several ways to include data from larger families. For instance, tree-based decomposition methods facilitate the extraction of orthologs from large families. Additionally, several methods for species tree inference are robust to the inclusion of paralogs and could use all of the data from larger families. Here, we explore the effects of using all families for phylogenetic inference by examining relationships among 26 primate species in detail and by analyzing five additional data sets. We compare single-copy families, orthologs extracted using tree-based decomposition approaches, and all families with all data. We explore several species tree inference methods, finding that identical trees are returned across nearly all subsets of the data and methods for primates. The relationships among Platyrrhini remain contentious; however, the species tree inference method matters more than the subset of data used. Using data from larger gene families drastically increases the number of genes available and leads to consistent estimates of branch lengths, nodal certainty and concordance, and inferences of introgression in primates. For the other data sets, topological inferences are consistent whether single-copy families or orthologs extracted using decomposition approaches are analyzed. Using larger gene families is a promising approach to include more data in phylogenomics without sacrificing accuracy, at least when high-quality genomes are available.     

Protoplast fusion in Bacillus species produces frequent,unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.