Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Depurination of Brome mosaic virus RNA3 inhibits its packaging into virus particles

Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication.

The effects of O6-methylguanine on the reactions involved in initiation of DNA replication were investigated by measuring the interactions of SV40 T antigen with oligonucleotides substituted with the methylated base. O6-Methylguanine residues were positioned in either binding site I or binding site II of the SV40 origin of replication. Binding of purified T antigen, measured by both nitrocellulose filter binding and delayed oligonucleotide migration, was unaffected by the presence of seven methylated bases in binding site II. Single substitutions within binding site I were sufficient to inhibit T-antigen binding, and the extent of inhibition was dependent on the position of O6-methylguanine in the DNA sequence. Unwinding by T antigen was analyzed by measuring displacement of a single-stranded oligonucleotide from similarly substituted, partially duplex substrates. The presence of three O6-methylguanine residues in binding site I facilitated the helicase activity of T antigen. In contrast, single O6-methylguanine bases inhibited unwinding. A correlation was observed between the position of the methylated base and the inhibition of both binding and unwinding by T antigen.     

清醒猫摄食过程中的胃电活动

本文报告慢性清醒猫正常摄食过程中胃电的变化,以及脑室注射心得安和动物麻醉对胃电的影响。结果显示,空腹饥饿状态下,猫胃电图上出现具有特征性的高振幅的“饥饿波”;摄食时胃电慢波抑制,可见较小快波;进食后半小时左右,胃电开始出现每分钟4—5次的振幅逐渐增大的正弦形慢波,多数慢波负载有快波。脑室注射心得安,可使饱猫胃电慢波抑制期出现饥饿波。动物在饥饿时麻醉,胃电慢波幅度显著降低,饥饿波完全不出现,苏醒后逐渐恢复。     

Methods for peptide identification by spectral comparison

Background  

Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies.     

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction.     

Proteolytic fragments of Alzheimer's disease-associated presenilin 1 are present in synaptic organelles and growth cone membranes of rat brain

Previous studies have demonstrated the molecular linkage of three causative genes for early-onset Alzheimer's disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the approximately 20-kDa C-terminal and approximately 30-kDa N-terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N- and C-terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.