Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela.

BACKGROUND: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. OBJECTIVE: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. METHODS: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. RESULTS: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. CONCLUSIONS: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.

Shine-Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG.

During translation, usually only one in approximately 400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation.

Prior interaction of ATP with yeast mRNAs enhances protein synthesis at the initiation step.

ATP hydrolysis is important for different stages of the protein synthesis process. A novel effect of this nucleotide was detected using mRNAs isolated from S. cerevisiae after phenol extraction of polysomes. When polysomal mRNA (pmRNA) or poly(A)(+) RNA were preincubated with ATP (approximately 3 mM, near physiological concentration), their translational activity in a cell-free system from yeast was stimulated 2-3 fold. This increased translational activity is specific for the poly(A)(+) RNA fraction, correlates with facilitated assembly of 80S initiation complexes, and is associated to increased synthesis of high molecular weight polypeptides. TCA precipitation assays of RNA incubated with [(14)C]ATP suggested an association of the nucleotide with the nucleic acid. The amount of [(14)C]ATP co-precipitated was dependent on magnesium (optimum at 5-6 mM), was partially inhibited by monovalent ions, and was maximal with poli(A)(+) RNA. Existence of RNA-associated kinases or ATPases appear unlikely since neither phosphorylation nor nucleotide hydrolysis were observed during preincubation of pmRNA with ATP. Another evidence of ATP-RNA interaction was an increased absorbance at 260 nm after incubation suggesting unwinding of the RNA secondary structure. Therefore, preincubation with ATP may affect the conformation of mRNAs and thereby facilitate the initiation of protein synthesis. This event could be part of an in vivo energy-dependent mechanism for translational control.

Efficient and faithful in vitro translation of natural and synthetic mRNA with human ribosomes.

Efficient systems for in vitro translation are of importance for biochemical and gene expression studies as well as for biotechnological developments. We optimized a cell-free translation system using subcellular fractions from human placenta and high quality placental tRNAs isolated using a simple and fast procedure. The postmitochondrial fraction or a reconstituted system containing soluble proteins plus polysomes were able to efficiently translate endogenous and exogenous mRNAs. Optima for ions, enzymes, tRNA and energy mix components were determined for a poly(U)-directed poly(Phe) synthesis test. The use of homologous tRNAPhe, omission of commercial creatine kinase, and addition of 3.5 mM spermidine at near physiological magnesium concentration (2.5 mM), were the most significant improvements. Under optimal conditions, poly(Phe) synthesis proceeded at a maximal initial rate of 1.2 Phe/80S/min at 37 degrees C, while natural mRNA translation by S-30 started at a near in vivo estimated rate of 0.3-0.5 amino acid/80S/sec. Furthermore, natural mRNA directed the synthesis of a family of polypeptides closely resembling the pattern of cytoplasmic proteins in both, molecular weight and relative amounts. This efficient and faithful system is of interest for biochemical studies of the human translational machinery, as well as a basis for screening new drugs affecting protein synthesis in pathogenic microorganisms.