Bacterial survival studies to assess the efficacy of static pile composting and above ground burial for disposal of bovine carcases

Aim: To assess the survival of bacteria during two alternative means of cattle carcase disposal in windrows: static pile composting (SPC) and above ground burial in soil (AGB), under temperate climate conditions on agricultural land, compared to surface disposal as the control method. Methods and Results: Bacteriological reference materials (pooled bovine faeces in permeable nylon bags and lyophilized cultures of Escherichia coli in glass ampoules) were positioned above and below each of 33 beef cattle carcases (250–300 kg). Temperatures at these sites were monitored with data loggers, while temperature and CO₂ probes were applied repeatedly at varying depths along the windrows. Aliquots of each reference material were cultured from three randomly selected animals from the SPC and AGB group and from all three control animals on five occasions (at 28, 56, 84, 126 and 182 days). SPC was highly efficacious in the destruction of coliforms in faeces and E. coli in ampoules within 28 days, while AGB was not significantly better than controls until 84 days, and bacteria in reference materials above the AGB carcases were still viable after 182 days. Temperature probes and loggers showed SPC provided sustained temperatures of 55–70°C, while AGB did not reach temperatures of 30°C, and the temperature differences correlated with bacteriological findings. Conclusions: In relation to emergency disease management, SPC can be successfully applied to eliminate pathogenic bacteria in cattle carcases, but AGB is unsuitable for carcase disposal. Significance and Impact of the Study: In emergency, animal disease outbreaks in temperate climates requiring large‐scale ruminant carcase disposal, SPC can be successfully applied for the destruction of micro‐organisms.     

Bacterial Content in Samples from Different Sites in the Rumen of Sheep and Cows as Determined in Two Culture Media

Samples of material from the rumen of cattle and of sheep were cultured in both Gall Broth (GB) and Reinforced Clostridium Medium (RCM). Samples were taken, at monthly intervals, from five sites in cattle and two sites in sheep in two series and, at weekly intervals, from two sites in cattle and in sheep in two series. Comparisons were made of the growth of organisms in the media, using the number of fertile tubes per set of six dilutions as a measure of the viable count. RCM gave a greater number of fertile tubes than GB with samples from cattle, but the reverse was the case with samples from sheep. Real differences in the number of fertile tubes from different sites in cattle were found at different times, but there was no consistent pattern. Simultaneous sampling from different sites in the rumen is, therefore, essential to provide an estimate of sampling error within animals. Although no site to site differences were detectable with rumen material from sheep, it is advisable to take more than one sample at a given time to assess sampling error.     

套袋苹果不同类型果袋内温、湿度变化特征及其对果实外观品质的影响

研究了套袋苹果果实发育过程中不同类型果袋内温、湿度的变化以及果袋对果实外观品质的影响.结果表明:果袋内的温度和湿度变化呈负相关,一天中袋内温度从9:00开始迅速升高,最高温度在春秋季一般出现在13:00-14:00,盛夏一般出现在13:00-17:00,最低温度出现在5:00前后,最高湿度出现在6:00前后.不同类型的果袋温、湿度变化不同,特别在高温和阴雨天差异明显,最高温度表现为双层袋》单层袋,而最高湿度表现为单层袋》双层袋.高温往往伴随极度干燥,当袋内温度超过35 ℃时,微域湿度一般低于40%.套袋对苹果果实品质有重要影响,果实外观品质以双层袋较好、单层袋较差;而双层袋中又以XL袋效果最好.     

National survey for Salmonella in pigs, cattle and sheep at slaughter in Great Britain (1999-2000)

AIMS: The objective of these surveys was to estimate the prevalence of faecal carriage of Salmonella in healthy pigs, cattle and sheep at slaughter, and of pig carcase contamination with Salmonella. These data can be used as a baseline against which future change in Salmonella prevalence in these species at slaughter can be monitored. METHODS AND RESULTS: In this first randomized National Survey for faecal carriage of Salmonella in slaughter pigs, cattle and sheep in Great Britain, 2509 pigs, 891 cattle and 973 sheep were sampled in 34 pig abattoirs and 117 red meat abattoirs in England, Scotland and Wales. Carriage of Salmonella in 25 g caecal contents was identified in 578 (23.0% pigs) but in only 134 (5.3%) of carcase swabs. The predominant Salmonella serovars found in both types of sample were S. Typhimurium (11.1% caeca, 2.1% carcases) and S. Derby (6.3% caeca, 1.6% carcases). The main definitive phage types (DT) of S. Typhimurium found were DT104 (21.9% of caecal S. Typhimurium isolates), DT193 (18.7%), untypable strains (17.6%), DT208 (13.3%) and U302 (13.3%). Three isolates of S. Enteritidis (PTs 13A and 4) and one enrofloxacin-resistant S. Choleraesuis were also isolated. A positive 'meat-juice ELISA' was obtained from 15.2% of pigs at 40% optical density (O.D.) cut-off level and 35.7% at 10% cut-off. There was poor correlation between positive ELISA results or carcase contamination and the caecal carriage of Salmonella. The ratio of carcase contamination to caecal carriage rates was highest in abattoirs from the midland region of England and in smaller abattoirs. In cattle and sheep 1 g samples of rectal faeces were tested. Two isolates (i.e. 0.2%) were recovered from cattle, one each of S. Typhimurium, DT193 and DT12. One sheep sample (0.1%) contained a Salmonella, S. Typhimurium DT41. In a small subsidiary validation exercise using 25 g of rectal faeces from 174 cattle samples, three (1.7%) isolates of Salmonella (S. Typhimurium DT104, S. Agama, S. Derby) were found. CONCLUSIONS: The carriage rate of Salmonella in prime slaughter cattle and sheep in Great Britain was very low compared with pigs. This suggests that future control measures should be focused on reduction of Salmonella infection on pig farms and minimizing contamination of carcases at slaughter. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has set baseline figures for Salmonella carriage in these species slaughtered for human consumption in Great Britain. These figures were collected in a representative way, which enables them to be used for monitoring trends and setting control targets.     

Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log₁₀/100 cm² of hides and 0.75 log₁₀/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.     

Survival Times of Selected Enteropathogenic Bacteria in Frozen Passionfruit Nectar Base

Five test organisms were used: Escherichia coli, Salmonella typhosa, Salmonella schottmuelleri, Salmonella enteritidis, and Shigella paradysenteriae. Even when large inocula of these test cultures were introduced into fresh passionfruit nectar base, all test organisms were killed within 1 to 2 hr, provided the nectar base was held at room temperature for more than 1 hr before freezing. If the nectar base was frozen immediately after inoculation, four of the five test organisms were eliminated almost as quickly. But the fifth, Salmonella enteritidis, proved to be exceptional: it was being recovered after 90 days of storage at -20 C, when the last available sample was analyzed.     

Variability of Temperature, pH, and Moisture in an Aerobic Composting Process

This study measured the environmental variability which exists in a commercial aerobic composting process. The specific process studied is carried out in six decomposition cells which present six different phases of the process. Temperature, pH, and moisture content were determined in several randomly chosen sample sites in each cell, both at the beginning and at the end of the time the material was left in the cell. Temperature and pH varied greatly from one sample site to another in each cell, whereas moisture content was less varied. A significant rise in both temperature and pH was observed at two stages of degradation.     

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

Prevalence and antimicrobial susceptibility of Salmonella spp. isolates from US cattle in feedlots in 1999 and 2000

AIMS: Faecal samples from cattle in US feedlots were evaluated for the presence of Salmonella. When Salmonella isolates were recovered the antimicrobial resistance patterns were determined. METHODS AND RESULTS: Faecal samples were collected from pen floors in 73 feedlots in 12 states during the period from October 1999 to September 2000. Pens of cattle selected for sampling were those that had been in the feedlot for the shortest period of time, the longest period of time and a randomly selected pen from the remaining pens. Faecal samples were cultured for Salmonella spp. and all Salmonella isolates were categorized by serotype. The susceptibilities of all isolates were determined using a panel of 17 antimicrobials. Overall, 6.3% (654/10,417) of the samples cultured positive for Salmonella spp. and 22.2% (94/422) of pens and 50.7% (37/73) of feedlots had one or more positive samples. There was little difference in the proportion of positive samples from short-fed (6.1%, 212/3482), random (6.4%, 217/3400) and long-fed (6.4%, 224/3485) pens of cattle. One of two pens of cattle that could not be attributed to a pen type had a single positive sample (2.0%, 1/50). Samples collected during the period of April to June (6.8%, 209/3054) and July to September (11.4%, 286/2500) were more likely to be positive than those collected during October to December (4.0%, 73/1838) and January to March (2.8%, 86/3025). The most common serotypes of Salmonella were dissimilar from those that are typically seen in human illness and cattle illness. The majority of isolates (62.8%, 441/702) were sensitive to all of the antimicrobials tested. Resistance was most frequently observed to tetracycline (35.9%, 252/702) followed by streptomycin (11.1%, 78/702), ampicillin (10.4%, 73/702) and chloramphenicol (10.4%, 73/702). Multiple resistance (resistance to > or =2 antimicrobials) was observed for 11.7% (82/702) of the isolates. CONCLUSIONS: Salmonella was isolated at low frequency from faeces of feedlot cattle and the serotypes were not those commonly associated with human illness. In addition most of the Salmonella isolates were sensitive to all the antimicrobials tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to understanding the ecology of Salmonella in cattle feedlots and the prevalence of resistance among potential food-borne pathogens.     

Survival of Salmonella enterica serovar Newport in manure and manure-amended soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log(10), 28 to 64 days for 2 log(10), and 42 to 96 days for 3 log(10) declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Immunoassays to detect the serum antibody response of cattle to infection with Salmonella Typhimurium definitive type 104 and following vaccination with Bovivac S

AIMS: To use ELISA and immunoblotting assays to examine the serum antibody response of cattle infected with Salmonella Typhimurium DT104 and following vaccination with Bovivac S. METHODS AND RESULTS: Three hundred and twenty-nine cattle, including 16 shedding multiresistant Salmonella Typhimurium DT104, were screened for serum antibodies binding to O=1, 4, 5, 12 lipopolysaccharide (LPS) antigens before and after vaccination with Bovivac S. Sera with an ELISA reading of 0.9A405 or above were shown to contain antibodies, of the IgG-class only, to the LPS of Salmonella Typhimurium using immunoblotting. Prior to vaccination, only 11 cattle had serum IgG-class antibodies to the O=4, 5 LPS antigens, and of these one also had antibodies to outer membrane proteins and H=i flagellar antigens. Following vaccination, 87 out of 315 cattle developed serum antibodies to the LPS of Salmonella Typhimurium. CONCLUSIONS: Evidence of infection of cattle with Salmonella Typhimurium was readily obtained with an LPS-based ELISA in association with an immunoblotting procedure, supplementing existing bacteriological procedures. This enabled the detection of an increase in the number of cattle with serum antibodies to Salmonella Typhimurium LPS following vaccination with Bovivac S. SIGNIFICANCE AND IMPACT OF THE STUDY: The immunoassays described provided evidence of infection with Salmonella Typhimurium and served as a valuable adjunct to established bacteriology.     

Effects of acclimation temperature on the growth of Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio

Temperature affects growth rate in aquatic organisms. This can be evaluated in short term using biochemical indexes (RNA/DNA and Protein/DNA). The effect of acclimatization temperature on the instantaneous growth and physiological condition of Perna viridis was studied in organisms collected in La Esmeralda, Sucre State (Venezuela) and taken to the laboratory, where groups of 100 organisms (size 3.0 - 3.5 cm, anteroposterior measurement) were acclimatized at 15, 20, 26 or 28 degrees C during four weeks. Later they were kept in a 60 liters aquarium for another six weeks under the same conditions. Each week, ten organisms per group were extracted to measure concentrations of RNA, DNA (by a fluorometric method with ethidium bromide) and proteins (by a colorimetric method), in tissues (digestive gland, adductor muscle and gills). Protein concentration was greater and highly significant at 15 degrees C for all studied tissues. The opposite was obtained with the RNA/DNA and Protein/DNA ratios: the greatest increase was observed at the highest temperature (28 degrees C) for all tissues. At the lowest temperature there was a tendency to reduce both indexes with time. Greater instantaneous growth can be expected at higher temperatures and 28 degrees C was optimal for growth in these specimens.     

Ecology of Vibrio cholerae non-O1 and Salmonella spp. and role of zooplankton in their seasonal distribution in Fukuyama coastal waters, Japan

Seasonal variation of human pathogens such as Vibrio Cholerae non-01 and Salmonella spp. in Fukuyama coastal waters and the role of zooplankton in their distribution were studies for a period of 1 year. Comparison of two established methods, viz., the elevated temperature method and the two-step enrichment method of enumerating V. cholerae, showed that the former is superior in the recoveries of V. cholerae non-01. Isolation of this pathogen on a wider range of salinities (0.4 to 32.5%) revealed that these organisms are apparently an autochthonous component of the aquatic environment. Temperature appears to be the most crucial element in governing the distribution of V. cholerae non-01. Among the 69 isolates serotyped, 22 different serovars were identified, while one isolate failed to react with any of the known Louisiana State University antisera tested. Zooplankton samples did not harbor more V. Cholerae non-01 than the water column did. Better isolation of an allochthonous pathogen, viz., Salmonella spp., was noticed from the water samples when swabs were employed. Of the 251 isolates serotyped, 18 serotypes with three variants of Salmonella spp. were identified. A high amount of nutrients in the water column increased the survival rate of these pathogens in saline waters as evidenced by a higher incidence of various serotypes in polluted Fukuyama port than in clean marine waters. Salmonella spp. association between V. cholerae non-01 of Salmonella spp. with zooplankton could be noticed as influencing their seasonal distribution.     

Ecology of Vibrio cholerae non-O1 and Salmonella spp. and role of zooplankton in their seasonal distribution in Fukuyama coastal waters, Japan.

Seasonal variation of human pathogens such as Vibrio Cholerae non-01 and Salmonella spp. in Fukuyama coastal waters and the role of zooplankton in their distribution were studies for a period of 1 year. Comparison of two established methods, viz., the elevated temperature method and the two-step enrichment method of enumerating V. cholerae, showed that the former is superior in the recoveries of V. cholerae non-01. Isolation of this pathogen on a wider range of salinities (0.4 to 32.5%) revealed that these organisms are apparently an autochthonous component of the aquatic environment. Temperature appears to be the most crucial element in governing the distribution of V. cholerae non-01. Among the 69 isolates serotyped, 22 different serovars were identified, while one isolate failed to react with any of the known Louisiana State University antisera tested. Zooplankton samples did not harbor more V. Cholerae non-01 than the water column did. Better isolation of an allochthonous pathogen, viz., Salmonella spp., was noticed from the water samples when swabs were employed. Of the 251 isolates serotyped, 18 serotypes with three variants of Salmonella spp. were identified. A high amount of nutrients in the water column increased the survival rate of these pathogens in saline waters as evidenced by a higher incidence of various serotypes in polluted Fukuyama port than in clean marine waters. Salmonella spp. association between V. cholerae non-01 of Salmonella spp. with zooplankton could be noticed as influencing their seasonal distribution.     

Does a warmer lake mean smaller benthic algae? Evidence against the importance of temperature–size relationships in natural systems

Ectotherms decrease in size with increasing ambient temperature. Temperature–size relationships (TSR) have been observed experimentally in a wide range of animals, algae, protozoans and bacteria. However, it is still unclear whether temperature is an important factor controlling the size of organisms in natural populations. In this study, we used natural variability in water temperature in the nearshore areas of a single lake to test TSR in populations of benthic diatoms. We deployed standard tile substrates at 5 m depth (similar light availability) at cold and warm sites that were exposed to different hydrodynamic forces. We compared cell sizes of three species of diatoms (Achnanthidium minutissimum, Gomphonema acuminatum and Gyrosigma acuminatum) at these sites. Counter to the TSR, diatom cells at warm sites were either larger (Achnanthidium, Gomphonema) or similar in size (Gyrosigma) compared to those at colder sites. Diatom size was also related to site exposure (hydrodynamic forces), but differently for species with different architectures. TSR were not detectable in the field for these three species of benthic diatom, even when tested within a single ecosystem at a given time of the year. The size of benthic diatoms, however, varied in a predictable way between sites, and such differences could affect the functioning of these primary producers in different parts of the littoral zone.     

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.