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.     

Two attached non-rigor crossbridge forms in insect flight muscle

We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.     

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.     

Letter: Crossbridge angle when ADP is bound to myosin

Recent experiments have shown that ADP binds to myosin in glycerol-extracted muscle fibers without causing detachment of the crossbridges. In order to determine if ADP binding caused an alteration in the angle of the attached crossbridges, X-ray diffraction photographs were taken of glycerol-extracted insect muscle fibres in the presence of ADP. The diffraction patterns obtained were identical to the rigor pattern, and it was concluded that the angle of attachment was the same as in rigor muscle.     

Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.     

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.