Klenow! That would polymerise them, given an oligomer to start with and some single-stranded DNA for a template. Klenow was the polymerase that I had planned to use anyhow. How clever. I would use it twice for two purposes. First I would denature my sample, separated into four tubes, add the primers I would later use in the main event, bring to 37 degrees and wait. The polymerase should polymerise all the nucleotides.
We report the synthesis of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2, and evaluate their retention in an E. coli SSO. Correction: Note that in the Degradation of Nucleotide Triphosphates section of the Methods, cells were propagated in media containing 200 µM nucleotide triphosphate, not 200 mM.
We report the first total synthesis of a lipoglycopeptide member of the arylomycin family of natural products, and we demonstrate that it effectively penetrates Gram-negative bacteria and is limited by the same resistance mechanism as other members of the arylomycin class of antibiotics (i.e. mutation in the target protein signal peptidase (SPase)). Unlike the other arylomycins, the lipoglycopeptides are glycosylated, and the structural data reveal that this sugar is most solvent exposed when in complex with SPase.
We characterized the susceptibility of a panel of S. aureus strains to two arylomycin derivatives and observed a wide range of susceptibilities. We found that resistant strains were sensitized by co-treatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. Transcriptional profiling using growth inhibitory concentrations of arylomycin revealed further insight into how this pathogen copes with secretion stress.
HBO was synthesized and incorporated into DNA oligonucleotides and each oligonucleotide was hybridized to a complementary oligonucleotide containing an abasic site at the position opposite HBO. Analysis of steady state emission spectra of the oligonucleotides indicates that the DNA environment selectively stabilizes the proton-transfer product tautomer of HBO, suggesting that flanking bases may be strongly coupled electronically.
Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM. At the end of the section we present a method for scanning EM (SEM) of worms. In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images ().
We report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the post-amplification site-specific labeling of DNA.
We report crystal structures of KlenTaq DNA polymerase at different stages of replicating dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.
To generate residue-specific information on the equilibrium folding of cyt c, we have semisynthesized the protein with specifically deuterated residues. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities of the protein C-D bonds reveal that cyt c undergoes a conformational change at the protein-based heme ligand, Met80, which is then followed by a more global unfolding at 2.3 M GdnHCL.
We report the synthesis and characterization of unnatural base pairs bearing propynyl groups, and their use to site-specifically label amplified DNA via Click chemistry. We demonstrate the attachment of a small molecule biotin tag, and for the first time a protein (SH3), to a large, PCR amplified fragment of DNA, as well as the arraying of two proteins on the same duplex.
The muscles used for locomotion in reside in the body wall. In the adult, there are 95 spindle shaped cells divided among four quadrants just underlying a basement membrane, hypodermis and cuticle. In each quadrant, the cells are arranged in interlocking pairs. In these muscle cells, myofilaments form a lattice that is restricted to a narrow zone of ~1.5 microns, just underlying the basement membrane and hypodermis. By polarized light microscopy, obvious striations are seen; bright (“birefringent”) A-bands alternate with dark I-bands; each I-band contains a row of dense bodies, which are the analogs of Z-discs of vertebrate striated muscle (). Because the striations lie at a slightly oblique angle with respect to the long axis of the worm, this muscle is called “obliquely striated”. Polarized light is also useful for evaluating the second largest set of muscles, those in the pharynx. Below we present a protocol for observing muscle using polarized light.
Plants provide a unique source of diverse secondary metabolites with potentially important biological activities, with one of the most promising classes being the indole alkaloids. One such indole alkaloid natural product is psychotrimine, which has attracted considerable interest from the synthetic and medicinal chemistry communities owing to its unusual connectivity between tryptamine subunits. Here, we examine the potential antibacterial activity of this unique indole alkaloid.
In the biotin system biotin is incorporated in the probe by using biotinylated dNTPs during probe synthesis. The incorporated biotin is detected directly by avidin or streptavidin or an anti-biotin antibody conjugated to a fluorochrome or an enzyme such as alkaline phosphatase or horseradish peroxidase. Avidin is a 68 kD glycoprotein derived from egg white and streptavidin is a 60 kD protein from .