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PubMed1992

Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.

Animals↗
PubMed1975

Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence.

Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-or-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.

Animals↗
PubMed1974

More precise mapping of the replication origin in Escherichia coli K-12.

The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.

Amino Acids↗
PubMed1997

The emergence of form by replication.

It is shown with a simple mathematical model that if a system exhibits a given form (a spatial structure) and is put in contact with another system of the same type but in a state of spatial disorder, then under certain conditions their mutual interaction as they evolve in time allows replication of form in the disordered system with a controllable degree of faithfulness.

Biological Evolution↗
PubMed2005

Vector competence of Culicoides sonorensis (Diptera: Ceratopogonidae) for vesicular stomatitis virus.

To determine the vector competence of Culicoides sonorensis Wirth & Jones midges for vesicular stomatitis virus (VSV)-New Jersey, insects were experimentally infected per os and sampled over time. Viral replication, as determined by in situ hybridization, was seen in epithelial, neural, and hemolymph cell types throughout the insect. Spatial and temporal distribution of virus was determined by immunohistochemical examination of sequentially sampled insects. Tissues of the alimentary canal were infected in a temporal pattern that paralleled the route of digestion/absorption: foregut and midgut by day 1, surrounding hemolymph and Malpighian tubules by day 3, and finally the midgut/ hindgut junction, hindgut, and rectal region by day 5. The circulation of virus in the hemolymph by day 3 coincided with infection of the dermis and fat bodies, the salivary glands, eyes, cerebral and subthoracic ganglia, and the ovaries. Oviduct epithelium and ovarial sheaths were infected by day 3, followed by infection of the developing oocytes by day 5. Interestingly, neural infections were seen in the subabdominal ganglia innervating the midgut in 33% of insects by 1 d postfeeding in the absence of positive staining in the hemolymph or surrounding tissues. A retrograde axonal transport infection route for these ganglia is discussed. The disseminated, productive, noncytolytic infection in Culicoides is consistent with that of an efficient biological vector for VSV. Virus readily replicated throughout the insect, passing both midgut and salivary gland infection barriers and reaching transmission-related organs in 3 d. Establishing the competence of this insect vector for VSV provides the foundation for animal transmission studies in the future. The possibility of horizontal, transovarial, and mechanical transmission is discussed.

Animals↗
PubMed1981

Early initiation of deoxyribonucleic acid replication and shortening of generation time associated with inhibition of lateral wall formation by mecillinam.

The effects of mecillinam on the growth of rods of the pH-conditional morphology mutant MirM7 was studied. It has been found that mecillinam causes, coincident with transition to coccal shape, a balanced rise in the rate of viable count increase and the rate of macromolecular synthesis which lasts either until the cells enter a stationary growth phase or indefinitely, in the case of continuously diluted cultures. When the antibiotic is removed from cells which have already become coccoid, cells continue to grow at a faster rate until they resume the rod shape. No change in the per-cell rate of protein synthesis has been seen in untreated or mecillinam-treated cells before or after the change in growth rate. Studies with synchronously growing cells have shown that the antibiotic causes a shortening in the I period (initiation of deoxyribonucleic acid replication). Evaluation of the residual divisions in nalidixic acid-treated, exponential-phase cells has shown that mecillinam also shortens the D period (cell division). It is proposed that, in strain MirM7, inhibition of lateral wall elongation by the antibiotic allows the initiation of a new septum, though inhibition is still in progress. The initiation of a new septum is, in turn, responsible for both the early inibition of deoxyribonucleic acid replication and accelerated division. In the parental strain, MirA12, as well as in other sensitive gram-negative rods which divide, become cocci, and stop dividing after addition of the antibiotic, inhibition of lateral wall formation activates a feedback mechanism which prevents insertion of new septa (Satta et al., J. Bacteriol. 142:43-51, 1980). Consequently, no early initiation of deoxyribonucleic acid replication is observed, and the last division allowed by the antibiotic occurs in due time. This negative control is missing in MirM7.

Amdinocillin↗
PubMed2007

Technical note: A system for continuous recording of ruminal pH in cattle.

Continuous recording of ruminal pH in cannulated cattle has been practiced to study rumen metabolism. However, most systems reported did not permit animal mobility during pH recording. Therefore, the objective of this study was to develop a continuous rumen pH data acquisition system that permitted animal mobility during data acquisition. A further objective was to compare the pH readings obtained using the continuous recording system to readings obtained at the same time using spot sampling. The continuous recording system was composed of a heavy-duty electrode and a data logger. The electrode was attached to a 0.5-kg weight to help maintain the electrode in the ventral sac of the rumen. The electrode was connected via a 0.5-m cable to a lightweight data logger that was mounted on the animal's back using a belt wrapped around the girth. The data logger was battery powered and could hold over 13,000 pH data values. A personal digital assistant was used to configure and download data from the data logger during the experiment. Ruminal pH was continuously recorded (every 10 s) using a dry Holstein cow fed alfalfa hay ad libitum in a 3-d experiment to compare the performance of the continuous system to spot samples taken from the ventral sac of the rumen, the same location as the continuous electrode. The spot samples were collected 3 times per d for 3 d. At every sampling time, 3 replicate samples were collected, pH was determined immediately using a handheld pH meter, and readings were averaged (n = 3) and compared with the average of the 3 pH readings recorded using the continuous system at the same time. The pH recorded by spot sampling (6.63 +/- 0.04) was greater (P = 0.009) than that of the continuous system (6.56 +/- 0.03), with a correlation of r = 0.88 (P = 0.002). The continuous recording system has the potential to facilitate measurement of ruminal pH in free-roaming cattle.

Animals↗
PubMed1974

Effects of inhibition and restoration of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis.

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.

Amino Acids↗
PubMed1987

An evaluation of three biological indicator systems in flash sterilization.

An evaluation of two flash-sterilization-specific biological indicators (BI) and a traditional spore strip indicator was performed to assess sensitivity and reliability as reflected in survive/kill ratios. The BIs tested included: 3M's Attest #1261, Amsco's Proof Flash, and Castle Tec Test. Survival after "come-up" time alone, (0 exposure) and one-, two-, and three-minute exposures at 273 degrees F in a gravity displacement sterilizer was measured by media color change or turbidity after incubation at 55 degrees C. Each cycle was replicated three times on two separate days with six of each BI per run. Positive BIs were subcultured as necessary. Proof Flash presented technical difficulties due to incomplete or impossible crushing of media vials, unexpected media color changes, and evaporation of media. Tec Test was not sufficiently resistant as survivors were not detected at any exposure time. The Attest had 100% survival at zero and one-minute exposures and 94% survival after the two-minute exposure. No survivors were detected after the three-minute exposure. Although each institution should evaluate BIs for their own use independently, the data indicate that Attest #1261 monitored the three-minute flash cycles more satisfactorily than the other BIs tested.

Bacillus subtilis↗
PubMed2004

Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion.

Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T-->C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.

Amino Acid Sequence↗
PubMed2000

Feline leukemia virus envelope sequences that affect T-cell tropism and syncytium formation are not part of known receptor-binding domains.

The envelope protein is a primary pathogenic determinant for T-cell-tropic feline leukemia virus (FeLV) variants, the best studied of which is the immunodeficiency-inducing virus, 61C. We have previously demonstrated that T-cell-tropic, cytopathic, and syncytium-inducing viruses evolve in cats infected with a relatively avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T variant, which encoded scattered single-amino-acid changes throughout the envelope as well as a 4-amino-acid insertion in the C-terminal half of the surface unit (SU) of envelope, was sufficient to confer the T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser, S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol. 72:2686-2696, 1998). In the present study, we examined the role of the 4-amino-acid insertion in determining viral replication and tropism of FeLV-81T. The 4-amino-acid insertion was found to be functionally equivalent to a 6-amino-acid insertion at an identical location in the 61C variant. However, viruses expressing a chimeric 61E/81T SU, containing the insertion together with the N terminus of 61E SU, were found to be replication defective and were impaired in the processing of the envelope precursor into the functional SU and transmembrane (TM) proteins. In approximately 10% of cultured feline T cells (3201) transfected with the 61E/81T envelope chimeras and maintained over time, replication-competent tissue culture-adapted variants were isolated. Compensatory mutations in the SU of the tissue culture-adapted viruses were identified at positions 7 and 375, and each was shown to restore envelope protein processing when combined with the C-terminal 81T insertion. Unexpectedly, these viruses displayed different phenotypes in feline T cells: the virus with a change from glutamine to proline at position 7 acquired a T-cell-tropic, cytopathic phenotype, whereas the virus with a change from valine to leucine at position 375 had slower replication kinetics and caused no cytopathic effects. Given the differences in the replication properties of these viruses, it is noteworthy that the insertion as well as the two single-amino-acid changes all occur outside of predicted FeLV receptor-binding domains.

Adaptation, Physiological↗
PubMed1979

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.

Animals↗
PubMed2005

Data subsetting strategies for estimation of across-country genetic correlations.

International genetic evaluation of dairy cattle requires estimation of genetic correlations among populations to account for genotype-environment interaction. Simultaneous estimation of across-country genetic correlations among all populations of a widespread breed, such as the Holstein breed is, however, hampered by connectedness problems and computational challenges. The purpose of this study was to examine the effects of using bulls with across-country, balanced distribution of daughters on estimates of genetic correlations. For this purpose, dairy cattle populations undergoing selection in 6 countries were simulated. Two population-size settings were used. In the small population-size setting (S-populations), the 6 simulated countries had 2000 cows and 20 young progeny testing bulls per generation. In the larger population-size setting (L-populations), the 6 simulated countries had between 2000 and 64,000 cows and 20 to 640 young progeny testing bulls per generation. The simulated (true) across-country genetic correlations, depending on the country combination, varied between 0.5 and 0.9. Simulations comprised a base population and 10 generations and were replicated 16 times. Results for the S-populations were not conclusive. For the L-populations, results indicated that by use of data from a relatively small subset of bulls with distribution of daughters balanced across countries, genetic correlations could be estimated with very small bias (overall average of absolute value of bias across replicates was 0.03 for the L-populations). The suggested bull subsetting strategy would allow simultaneous estimation of across-country genetic correlations to be computed for a larger number of countries and in a shorter window of time than was possible previously.

Animals↗
PubMed2005

Panmixia in the European eel: a matter of time...

The European eel (Anguilla anguilla L.) has been a prime example of the panmixia paradigm because of its extraordinary adaptation to the North Atlantic gyral system, semelparous spawning in the Sargasso Sea and long trans-oceanic migration. Recently, this view was challenged by the suggestion of a genetic structure characterized by an isolation-by-distance (IBD) pattern. This is only likely if spawning subpopulations are spatially and/or temporally separated, followed by non-random larval dispersal. A limitation of previous genetic work on eels is the lack of replication over time to test for temporal stability of genetic structure. Here, we hypothesize that temporal genetic variation plays a significant role in explaining the spatial structure reported earlier for this species. We tested this by increasing the texture of geographical sampling and by including temporal replicates. Overall genetic differentiation among samples was low, highly significant and comparable with earlier studies (FST = 0.0014; p < 0.01). On the other hand, and in sharp contrast with current understandings, hierarchical analyses revealed no significant inter-location genetic heterogeneity and hence no IBD. Instead, genetic variation among temporal samples within sites clearly exceeded the geographical component. Our results provide support for the panmixia hypothesis and emphasize the importance of temporal replication when assessing population structure of marine fish species.

Anguilla↗
PubMed1986

Influence of dietary calcium on bone calcium utilization.

In Experiment 1, 10 microCi 45Ca/day were administered to 125 hens for 10 days. Hens were then allocated to five treatments with calcium levels ranging from .08 to 3.75% of the diet. In Experiment 2, hens with morning oviposition times were randomly allocated to 11 treatments that were periods of time postoviposition ranging from 6 hr to 24 hr, in 2-hr increments (Experiment 2). At the end of each 2-hr period, eggs from 25 hens were removed from the uterus. The 18-, 20-, and 22-hr treatments were replicated three times. In Experiment 3, hens were fed either ad libitum or feed was withheld the last 5 or 6 hr before oviposition. In Experiment 4, hens were fed 10 microCi of 45Ca for 15 days to label skeletal calcium. Hens were divided into two groups and fed a .08 or 3.75% calcium diet for 2 days. On the second day, 25 hens fed the 3.75% calcium diet were intubated with 7 g of the same diet containing .5 g calcium at 1700, 2100, 0100, 0500, and 0700 hr. The measurements used were egg weight, shell weight, and 45Ca content of the egg shell. Results indicated a significant linear or quadratic regression of dietary calcium levels on 45Ca accumulation in eggshells and eggshell weight (Experiment 1). As the calcium level of the diet increased, eggshell weight increased and 45Ca recovery decreased. Utilization of skeletal calcium for shell formation ranged from 28 to 96%. In Experiment 2, the rate of shell calcification was not constant throughout the calcification process but varied significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Predicting treatment time with the Lidcombe Program: replication and meta-analysis.

BACKGROUND: The benefits of treating stuttering close to onset have become obvious in recent years, and the Lidcombe Program has emerged as an effective and safe treatment method for children in their preschool years. The benefits of implementing the programme with young children, however, need to be weighed against the knowledge that many children recover from stuttering without treatment. In light of this, speech-language therapists need to know how long treatment is likely to take and whether treatment time can be predicted. In particular, they need to know if adopting a 'watchful waiting' approach--to see if natural recovery occurs--jeopardizes responsiveness to treatment. A recent Australian study of 250 preschool-age children found that stuttering rate was the only significant predictor of treatment time with the Lidcombe Program. In other words, children whose stuttering was more severe took longer to pass through the programme. There were other trends in the data but they did not reach significance. AIMS: The present study, conducted independently in the UK, was designed to replicate the Australian study. Direct replication enabled pooling of the data from the two studies in a meta-analysis. METHODS & PROCEDURES: The study included 66 children who began treatment before 6 years of age. They were treated with the Lidcombe Program at a specialist stuttering clinic in Norwich. Logistic regression analyses were conducted on the data. The data from both the British and Australian cohorts were pooled in a meta-analysis. OUTCOMES & RESULTS: Results indicated that Stage 1 of the Lidcombe Program was completed in a median of 11 clinic visits, which is in line with the findings of the Australian study. Stuttering rate at first clinic visit was again found to be a significant predictor of treatment time. The remaining data trends were similar to those in the Australian data. In the meta-analysis, stuttering rate was once more found to be a predictor of treatment time. Of particular interest, however, was that the increased power provided by the meta-analysis identified an additional predictor, namely onset-to-treatment interval. Contrary to what is known about the responsiveness of children to the Starkweather and Gottwald treatment, and contrary to what might be expected given what we know about natural recovery, children who had been stuttering for more than 12 months took less time to progress through the programme than children who had been stuttering for less than 12 months. CONCLUSIONS: These findings indicate that delaying intervention with the Lidcombe Program for 1 year after onset, within the preschool years, is unlikely to jeopardize responsiveness to treatment. The clinical implications of these findings are discussed.

Age Factors↗
PubMed2001

Excellence in school nursing workshop: Florida's experience in standardizing school nursing orientation and education.

Specialty preparation is needed for safe, effective school nursing practice. The Excellence in School Nursing workshop was envisioned as a means to provide a statewide orientation and education program for school nurses in Florida. As a result of a needs assessment, the Florida Departments of Health and Education formed a curriculum development committee to address the educational needs of school nurses. A 3.5-day workshop was designed for school health nursing supervisors and school nurses employed by county health departments, school districts, and community agencies. The workshop was piloted and, based on evaluations and feedback, was revised and replicated nine times throughout the state. The workshop has proved to be an effective method of providing statewide orientation and education for school nurses. The purpose of this article is to describe the workshop curriculum, planning, implementation, and evaluation. Recommendations for adaptation and replication in other states are included.

Curriculum↗
PubMed1983

Evaluation of immunity of young broiler chickens during simultaneous aflatoxicosis and ochratoxicosis.

A 2 X 2 factorial experimental design consisting of the treatments 0 and 2.5 micrograms/g aflatoxin and 0 and 2.0 micrograms/g ochratoxin A with 12 replicates of 10 birds per treatment level was used to evaluate the effects of these mycotoxins on various aspects of immunity. Male chicks (Hubbard X Hubbard) were maintained on these treatments from one day of age to 3 weeks of age at which time six replicate pens per treatment were sacrificed and various parameters measured. The additional six replicate pens per treatment were maintained on toxin feed beyond 3 weeks of age, and at 4 weeks of age, three replicate pens were immunized with sheep red blood cells (SRBC) and Brucella abortus. Antibody titers were measured up to 10 days postimmunization. Aflatoxin and ochratoxin A, individually, significantly (P less than .05) decreased body weight, and a synergistic toxicity was evident by a significant (P less than .05) decrease in body weight. Antibody titers and phagocytic activity of heterophils were not significantly (P less than .05) altered by any treatments. The relative weight of the bursa of Fabricius and the number of follicles for a given area of the folds of the bursa of Fabricius were significantly (P less than .05) decreased only by the interaction treatment. Complement activity was significantly (P less than .05) decreased by aflatoxin and the combination of aflatoxin and ochratoxin A and depressed, although not significantly (P less than .05), by ochratoxin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗