Search results for "Erich"
showing 10 items of 805 documents
Functional Characterization of a Guanylyl Cyclase-activating Protein from Vertebrate Rods
1996
The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Act…
Midbiotics: conjugative plasmids for genetic engineering of natural gut flora.
2019
ABSTRACT The possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of …
Estimating the extent of horizontal gene transfer in metagenomic sequences
2008
Abstract Background Although the extent of horizontal gene transfer (HGT) in complete genomes has been widely studied, its influence in the evolution of natural communities of prokaryotes remains unknown. The availability of metagenomic sequences allows us to address the study of global patterns of prokaryotic evolution in samples from natural communities. However, the methods that have been commonly used for the study of HGT are not suitable for metagenomic samples. Therefore it is important to develop new methods or to adapt existing ones to be used with metagenomic sequences. Results We have created two different methods that are suitable for the study of HGT in metagenomic samples. The …
Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II.
2008
AbstractThree isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, an…
Transcription in bacteriophage f1-infected Escherichia coli: RNA synthesized on DNA of deletion mutant PII shows the existence of a two-site terminat…
1984
Two different transcripts are synthesized on the DNA of deletion mutant PII of bacteriophage f1 in E. coli cells infected with this miniphage. Both RNA species appear to be primary transcripts and differ by about 100 nucleotides at their 3'OH end. Mapping of these molecules on the miniphage genome suggests that a two-site terminator is active at the end of the I region of transcription of bacteriophage f1.
Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli
2016
Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in d…
A Gene-Specific Requirement for FACT during Transcription Is Related to the Chromatin Organization of the Transcribed Region
2006
The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced …
Why are the genomes of endosymbiotic bacteria so stable?
2003
The comparative analysis of three strains of the endosymbiotic bacterium Buchnera aphidicola has revealed high genome stability associated with an almost complete absence of chromosomal rearrangements and horizontal gene transfer events during the past 150 million years. The loss of genes involved in DNA uptake and recombination in the initial stages of endosymbiosis probably underlies this stability. Gene loss, which was extensive during the initial steps of Buchnera evolution, has continued in the different Buchnera lineages since their divergence.
Homemade Site Directed Mutagenesis of Whole Plasmids
2009
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensi…
Genome size reduction through multiple events of gene disintegration in Buchnera APS
2001
The evolution of the endosymbiont Buchnera during its adaptation to intracellular life involved a massive reduction in its genome. By comparing the orthologous genes of Buchnera, Escherichia coli and Vibrio cholerae, we show that the minimal genome size of Buchnera arose from multiple events of gene disintegration dispersed over the whole genome. The elimination of the genes was a continuous process that began with gene inactivation and progressed until the DNA corresponding to the pseudogenes were completely deleted.