Tolylfluanide + tebuconazole in the control of some ornamental foliage diseases

In the control of Sphaerotheca pannosa var. rosae on rose Tolylfluanide + tebuconazole (Folicur Multi 50 WG at concentration 0.1%) was used for spray 2 -times at 14-day-intervals or 4-times at 7-day-intervals. After 4 weeks of plants protection effectiveness of tested product was about 75%. In the control of Diplocarpon rosae, the product was applied when first disease symptoms appeared on rose shrubs. Application was repeated 5-times at 14-day-intervals or 9-times at 7-day-intervals. After 9 week-protection effectiveness of tested product was about 85% and depended on frequency of sprayings. In the control of Puccinia horiana on chrysanthemum, Tolylfluanide + tebuconazole was used as plant spray twice at 14-day-intervals or 4-times at 7-day-intervals. After 4 week-protection the product suppressed of new telia formation about 55%. Application of the product for willow rust (Melampsora epitea) control suppressed formation of new uredia about 86% and half of them were dried. In the control of pelargonium rust (Puccinia pelargonii-zonalis) the product was used as plant spray 4-times at 7-day-intervals. It suppressed formation of new uredia about 90% and 1/3 of them were dried. It was found that 1 or 7 days after rose spray, spores of D. rosae collected from leaf blades only in 6% germinated. Spores taken from nonspraying leaves germinated in 90%. In case of P. pelargonii-zonalis, after 1 or 7 days after spraying, spores collected from protected plants germinated at 3%, compared to 90% on untreated plants. Spores of B. cinerea, collected from protected plants germinated at about 10%, whereas on control leaves at 90%.     

Optimization of L-phenylalanine production of Corynebacterium glutamicum under product feedback inhibition by elevated oxygen transfer rate.

Production feedback inhibition both on cell growth and on product formation of phenylalanine fermentation might be alleviated by elevated oxygen supply. Batch fermentations by a high phenylalanine producing strain Corynebacterium glutamicum CCRC 18335 at various initial phenylalanine concentrations (P(0)) ranging from 0 to 20 g/L and different oxygen transfer rate coefficients (K(L)a) ranging from 23 to 76 h(-1) were studied. The fermentation parameters with respect to P(0) were strongly dependent on K(L)a. Cell yield favored higher K(L)a and lower P(0). Product yield with respect to varying phenylalanine concentration was evaluated by the relative oxygen availability (ROA). The optimal ROA for phenylalanine formation was strongly dependent on the product concentration. While P(0) was low, the product inhibition was less significant and the maximum product yield occurred while ROA was at 0.5-0.6. While P(0) was high, the product inhibition was significant and the maximum product yield occurred while ROA was at 0.8-0.9. These results suggest that the product feedback inhibition of phenylalanine fermentation processes can be alleviated by a gradual increase in oxygen supply rate while the increasing product concentration is taken into account. The strategy is demonstrated in a fed-batch culture with elevated oxygen supply. The final phenylalanine concentration was 23.2 g/L, which was 45% better than that of the fed-batch fermentation without elevated oxygen supply. Likewise, the maximum productivity was improved by 42% at 0.37 g/(L x h).     

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.     

Evaluation of manometric temperature measurement, a process analytical technology tool for freeze-drying: Part I, product temperature measurement

This study examines the factors that may cause systematic errors in the manometric temperature measurement (MTM) procedure used to evaluate product temperature during primary drying. MTM was conducted during primary drying using different vial loads, and the MTM product temperatures were compared with temperatures directly measured by thermocouples. To clarify the impact of freeze-drying load on MTM product temperatures, simulation of the MTM vapor pressure rise was performed, and the results were compared with the experimental results. The effect of product temperature heterogeneity in MTM product temperature determination was investigated by comparing the MTM product temperatures with directly measured thermocouple product temperatures in systems differing in temperature heterogeneity. Both the simulated and experimental results showed that at least 50 vials (5 mL) were needed to give sufficiently rapid pressure rise during the MTM data collection period (25 seconds) in the freeze dryer, to allow accurate determination of the product temperature. The product temperature is location dependent, with higher temperature for vials on the edge of the array and lower temperature for the vials in the center of the array. The product temperature heterogeneity is also dependent upon the freeze-drying conditions. In product temperature heterogeneous systems, MTM measures a temperature close to the coldest product temperature, even, if only a small fraction of the samples have the coldest product temperature. The MTM method is valid even at very low product temperature (−45°C). Published: February 10, 2006     

Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l -1 , a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l -1 h -1 , using 2.1 kg pressed yeast kg product -1 and 0.52 kg glucose kg product -1 . During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

Assessing the Replacement of Electrical Home Appliances for the Environment

To evaluate whether replacing an existing product with a new, more energy‐efficient product is environmentally preferable, we used an assessment approach based on life cycle assessment. With this approach, consumers can assess various replacement products, including products of different sizes or environmental performance in addition to consideration of various conditions of product use. The approach utilizes a diagram in which replacement conditions of products are compared with iso‐environmental‐load lines to determine the appropriateness of replacement. The approach also allows the assessment of energy and resource consumption and environmental impacts not only during the use stage, but also at other product stages. Iso‐environmental‐load lines to assess delayed replacement were also examined and derived. We then applied the approach in a case study of energy consumption by replacing three types of electric home appliances in Japan: TVs, air conditioners, and refrigerators. The results of assessment showed that replacing refrigerators after 8–10 years of use was preferable even if the replacement product was larger. The appropriateness of replacing TVs and air conditioners based on energy consumption depended on the replacement product and on the duration of daily use, and in several cases, delayed replacement was preferable. Replacement of air conditioners after 8–10 years of use was not preferable if the consumer already owned the most energy‐efficient product at the time of the purchase. The necessity of accounting for a variety of available replacement products was confirmed.     

Identification of quantitative trait loci for growth and carcass composition in cattle

A genomic screening to detect quantitative trait loci (QTL) affecting growth, carcass composition and meat quality traits was pursued. Two hundred nineteen microsatellite markers were genotyped on 176 of 620 (28%) progeny from a Brahman x Angus sire mated to mostly MARC III dams. Selective genotyping, based on retail product yield (%) and fat yield (%), was used to select individuals to be genotyped. Traits included in the study were birth weight (kg), hot carcass weight (kg), retail product yield, fat yield, marbling score (400 = slight00 and 500 = small00), USDA yield grade, and estimated kidney, heart and pelvic fat (%). The QTL were classified as significant when the expected number of false positives (ENFP) was less than 0.05 (F-statistic greater than 17.3), and suggestive when the ENFP was <1 (F-statistic between 10.2 and 17.3). A significant QTL (F = 19; ENFP = 0.02) was detected for marbling score at centimorgan (cM) 54 on chromosome 2. Suggestive QTL were detected for fat yield at 50 cM, for retail product yield at 53 cM, and for USDA yield grade at 63 cM on chromosome 1, for marbling score at 56 cM, for retail product yield at 70 cM, and for estimated kidney, heart and pelvic fat at 79 cM on chromosome 3, for marbling score at 44 cM, for hot carcass weight at 49 cM, and for estimated kidney, heart and pelvic fat at 62 cM on chromosome 16, and for fat yield at 35 cM on chromosome 17. Two suggestive QTL for birth weight were identified, one at 12 cM on chromosome 20 and the other at 56 cM on chromosome 21. An additional suggestive QTL was detected for retail product yield, for fat yield, and for USDA yield grade at 26 cM on chromosome 26. Results presented here represent the initial search for quantitative trait loci in this family. Validation of detected QTL in other populations will be necessary.     

Addressing a whole bioprocess in real-time using an optical biosensor-formation,recovery and purification of antibody fragments from a recombinant<Emphasis Type="Italic"> E. coli</Emphasis> host

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at –70°C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.     

Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l &#109 1, a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l &#109 1 h &#109 1, using 2.1 kg pressed yeast kg product &#109 1 and 0.52 kg glucose kg product &#109 1. During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.     

Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.     

Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.     

Fluorescent tracer method for protein SH groups: III. Use ofN-(7-dimethylamino-4-methyleoumarinyl) malelmide as a tracer of cysteine-containing peptides

We studied whetherN-(7-dimethylamino-4-methyleoumarinyl) maleimide (DACM) could be used as a fluorescent tracer for the purification and analysis of cysteine-containing peptides. An addition product of DACM with SH compound was stable at room temperature in the solution of pH 9.0 and in 50% acetic acid. However, it was hydrolyzed when heated at 110°C for 48 h in 6n HCl. On filter paper, 1 pmol of the addition product in a spot 3 mm in diameter was visible under ultraviolet illumination. The addition product was also stable on filter paper for at least several months after spotting. The elution velocity of the addition products with low molecular weight SH compounds in the Sephadex G-25 column was low considering their molecular weights. However, in general, the elution velocity increased with an increase in the molecular weight of the addition product. The addition product with a peptide having cysteinyl residue at an N-terminal showed another abnormally retarded peak in elution profile which presumably corresponded to a cyclic compound, a thiazane derivative. However, it was shown on the C-terminal tryptic peptide of actin (Cys Phe) that the conversion to a thiazane derivative could be avoided by hydrolyzing the succinimide portion of DACM at pH 9.0 before digestion. The R_f values on paper chromatography for the addition products also depended on their molecular weights. However, the hydrophobicity of the coumarin portion of DACM and of the side chain of amino acid residues also affected the value. It was concluded that DACM was a valuable reagent for the purification and analysis of cysteine-containing peptides.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

FRESH-CUT ANNURCA APPLES: ACCEPTABILITY STUDY AND SHELF-LIFE DETERMINATION

To establish the likelihood of purchase and acceptability level of new fresh-cut apples, the responses of two categories of consumers, children and adults, were evaluated. The effect of storage time on acceptability was investigated and the shelf life of fresh-cut apples was determined by using a Weibull hazard model.
The samples were distributed at different storage time (from second to fifth day) at the Science Museum of Naples as part of an educational program for children to evaluate the likelihood of buying and the acceptability level of the product. The data were collected over 5 weeks, and the test was performed in 2,421 children and 224 adults. The product was distributed at the University of Naples at different storage time (time 0 to 10 days) to perform the Weibull test using 63 consumers.
The adults and children appreciated the product, and the adults were generally willing to buy it. The Weibull analysis proved an appropriate method to evaluate product shelf life, which was estimated at 7.5 days at 4C.

PRACTICAL APPLICATIONS

This study contributes to enhance the competitiveness of a traditional food as Protected Geographical Indication Annurca apples. In fact, an attractive way to improve the consumption of this variety of apple is to market it as a ready-to-eat product. Also, the research allows for a better understanding of the children's acceptability level of new fresh-cut apples in order to improve the intake of apples among primary schoolchildren. In fact, an interesting way to increase the children's consumption of fruit is to distribute it at school level and also to increase the convenience for children. Finally, the fact that the product was distributed to the Science Museum of Naples can be considered as a premarket test for a new product launch.     

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3′-diaminobenzidine (DAB)-Mn²⁺ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co²⁺- containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB--cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehydeoxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB--cobalt complex. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.     

Histochemical localization of acetylcholinesterase in the cerebral ganglia of Fasciola hepatica, a parasitic flatworm

Acetylcholinesterase activity was found in the cell bodies and extracellularly in the neuropile of the cerebral ganglia of the adult trematode parasite, Fasciola hepatica. Within neuronal cell bodies of the cerebral ganglion, acetylcholinesterase reaction product was found in the endoplasmic reticulum, in the cisternae of the Golgi apparatus, and in secretory vesicles near the inner (releasing face) cisternae. Acetylcholinesterase reaction product was not seen intracellularly within any nerve processes. The reaction product was found around the somatic cell membranes and in the extracellular space between closely apposed nerve processes in the neuropile. Acetylcholinesterase reaction product was associated with synaptic endings that contained clear spheroidal synaptic vesicles, and the reaction product was localized at the site of synaptic contact between the zone of apposition of the pre- and postsynaptic terminals. This intracellular and extracellular distribution of the enzyme is consistent with its function as the degrading enzyme in cholinergic transmission.     

A two-step acid-catalyzed process for the production of biodiesel from rice bran oil

A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceride was hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzed methanolysis process was employed for the efficient conversion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 degrees C. Depending on the initial FFA content of oil, 55-90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzed methanolysis at 100 degrees C. By this two-step methanolysis reaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as gamma-oryzanol (16-18%), mixture of phytosterol, tocol and steryl ester (19-21%).     

Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.