Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.     

Effects of linear cooling rate on motion characteristics of stallion spermatozoa

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37 degrees C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20 degrees C was evaluated. Sperm motion was not affected by cooling at plunge, -0.42 or -0.28 degrees C/minute. However, storage of spermatozoa at 5 degrees C after slow cooling below 20 degrees C was superior to storage at 20 degrees C. In Experiment 2, 3 cooling rates from 37 degrees to 5 degrees C were evaluated. Cooling at either -0.05 or -0.7 degrees C/minute was superior (P<0.05) to plunging spermatozoa to 5 degrees C. Cooling at -0.05 degrees C/minute rather than -0.7 degrees C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5 degrees C were evaluated, with all samples cooled at -0.7 degrees C/minute from 37 to 20 degrees C. Sperm motion was similar (P>0.05) after cooling below 20 degrees C at -0.012, -0.05 or -0.10 degrees C/minute, and the 2 slower rates were superior (P<0.05) to cooling at -0.3 degrees C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20 degrees C, but should be cooled at 相似文献   

Tac promoter vectors incorporating the bacteriophage T7 gene 10 translational enhancer sequence for improved expression of cloned genes in Escherichia coli.

Two new plasmid expression vectors, pTacT7 and pTacT7L, have been constructed, which incorporate between the tac promoter and a downstream NcoI-HindIII polylinker sequence a synthetic sequence derived from the region upstream from gene 10 of bacteriophage T7 (g10-L). This sequence was recently shown to act as a translational enhancer (Olins et al., 1988) and was termed "Epsilon" (Enhancer of Protein Synthesis Initiation) element (Olins and Rangwala, 1989). In this communication we describe in detail the construction of ptacT7 and ptacT7L. Furthermore, we present evidence that the "Epsilon" element is able to enhance 3 to 20-fold the expression levels of two poorly expressed test genes encoding the human placental proteins PP9 and PP15. On the other hand, the expression levels of two highly expressed test genes encoding the human placental proteins PP4 and FXIIIa could not be further enhanced by the presence of the "Epsilon" element. These experiments show that the T7 gene 10 leader sequence can be utilized to improve the expression yields of otherwise poorly expressed heterologous genes in Escherichia coli.     

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

Unusual behaviour of SPO1 DNA with respect to restriction and modification enzymes recognizing the sequence 5''-G-G-C-C

Summary SPO1 DNA contains only 5 cleavage sites for restriction enzymes which recognize and cleave the sequence 5-G-G-C-C (HaeIII or BsuR). Fragments of SPO1 DNA cloned in E. coli to substitute 5-hydroxymethyluracil (HMU) by thymine (T) remain resistant to HaeIII indicating that this unexpectedly small number of cleavages by HaeIII is not correlated with the presence of HMU in the normal phage DNA. It was previously shown that SPO1 is neither subject to B. subtilis R restriction (Trautner et al., 1974) nor modification in vivo (Günthert et al., 1975). We now show that SPO1 DNA can however be restricted and modified in vitro.     

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes

Summary The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent. ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not apear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.     

Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.     

Extraction and fractionation of complex lipid mixtures with dense carbon dioxide on a micro scale

The effects of pressure, temperature and some organic solvents on the recovery of various lipid classes from plant and animal tissues can be assessed by fractional extraction with dense carbon dioxide and consecutive analysis by thin-layer chromatography.