Transcription of the repetitive DNA sequences in polyoma-transformed and nontransformed mouse cells in culture.

RNA-DNA saturation hybridization experiments were used to estimate the extent of transcription of repetitive DNA sequences in polyoma-transformed and nontransformed mouse cells in culture. Measurements were made with RNA from nontransformed cells in both the subconfluent and confluent stages of growth, transformed cells in normal growth medium, and transformed cells grown in medium containing 5-bromodeoxyuridine, 3 mu-g/ml, a treatment which reduces their tumorigenic potential. No differences were observed in the amount of repetitive DNA transcribed or in the families of sequences expressed, except in transformed cells grown in the presence of 5-bromodeoxyuridine, in which case the extent of transcription was reduced.

Cost-effectiveness of a worksite hypertension treatment program.

The cost-effectiveness of treating hypertension at the patient's place of work was compared in a randomized controlled trial with care delivered in a community. The average total cost per patient for worksite care in this 12-month study was not significantly different from that for regular care ($242.86 +/- 6.94 vs $211.34 +/- 18.66, mean +/- SEM). The worksite health system cost was significantly more expensive ($197.36 +/- 4.99 vs $129.33 +/- 13.34, p less than 0.001) but the patient cost was significantly less ($45.40 +/- 3.23 vs $82.00 +/- 6.20, p less than 0.01). The mean reduction in diastolic blood pressure (BP) at the year-end assessment was significantly greater in the worksite group (12.1 +/- 0.6 vs 6.5 +/- 0.6 mm Hg, p less than 0.001). The incremental cost-effectiveness ratio of $5.63 per mm Hg for worksite care was less than the base cost-effectiveness ratio of $32.51 per mm Hg for regular care, indicating that the worksite program was substantially more cost-effective. Our findings support health policies that favor allocating resources to work-based hypertension treatment programs for the target group identified in this study.

Ageing studies in rat liver. I. Complexity of RNA from two to ten months of age.

A highly sensitive nucleic acid hybridization assay was used to compare the extent of nonrepetitive DNA transcription in rat liver between the ages of two and ten months. The basic approach consisted on initially purifying the DNA expressed in liver at these ages and then using it in reactions with homologous and heterologous RNAs. Such experiments failed to reveal any differences in nonrepetitive DNA transcription as a function of age. The possibility was also explored that there might be an age-associated variation in the proportion of the total RNA complexity attributable to poly(A+) RNA. These experiments, too, were negative in that the poly(A+) RNA was found in all cases to account for approximately 50% of the total sequence diversity. Overall, these data strongly suggest that ageing is not accompanied by a steady, progressive change in the regions of the genome transcribed, either quantitatively or qualitatively.

Sequence comparisons of medium RNA segment among 15 California serogroup viruses.

The complete nucleotide sequences have been determined for the M segment of 12 California (CAL) serogroup bunyaviruses. A method is described here of long reverse transcription-polymerase chain reaction (RT-PCR) that yields the full-length medium (M) RNA genomic segment. A phylogenetic tree was constructed by comparison of the open reading frames (ORFs) in the M RNA segment of 15 CAL serogroup viruses. Three distinct branches were identified and they are represented by the California encephalitis (CE), Melao (MEL), and Trivittatus (TVT) complexes. These groups correspond to those previously established by small (S) RNA genomic sequences. In addition, except for Inkoo virus, the predicted relationship among these viruses agreed with those found by serology.

Comparison of the M RNA genome segments of two human isolates of La Crosse virus.

The M genomic RNA segments of La Crosse (LAC) virus isolates from the brains of two children autopsied 18 years apart in Wisconsin were molecularly cloned using a reverse transcriptase-PCR assay and the nucleotide sequences of the cDNAs determined. The M RNA of each virus contains 4526 nucleotides, similar to that reported previously for a New York mosquito isolate of LAC. There were 20 nucleotide differences between the two human isolates, which results in the prediction of 7 amino acid changes in the proteins encoded in the single, long open reading frame of the M segment. One of these predicted differences occurs in the G2 glycoprotein and six in the G1 glycoprotein. The two viruses were identical in terms of predicted amino acid sequence in the region believed to represent a nonstructural protein. These data have been further compared to those available for two other California serogroup isolates.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes.

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Detection of California serogroup viruses using universal primers and reverse transcription-polymerase chain reaction.

Universal primers have been identified and a protocol developed that are suitable for rapid detection of California encephalitis (CE) complex viruses in a reverse transcription-polymerase chain reaction (RT-PCR) assay. These primers correspond to sequences in the coding regions of the G2 glycoprotein of the middle-size RNA segment. The identities of the amplified products were confirmed by sequencing on the clones or PCR products. The technique is capable of detecting 40 plaque-forming units (PFU) directly on an ethidium bromide-stained agarose gel and the sensitivity increases to 0.4-1 PFU when a radiolabeled probe is used as the detector.

Detection of California serogroup Bunyaviruses in tissue culture and mosquito pools by PCR.

A reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for rapid detection of 14 California serogroup viruses using universal primer pairs. These primers are specific for the small RNA (S RNA) and middle RNA (M RNA) segments. The method has been employed to detect Jamestown Canyon (JC) virus in naturally infected mosquitoes. With this technique, it is possible to detect virus in an amount of material equivalent to as little as 0.03 mosquito.

The sequence of the M RNA of an isolate of La Crosse virus.

The middle-size (M) genomic RNA of a New York State, U.S.A. isolate of La Crosse (LAC) virus has been cloned by a random priming procedure and its nucleotide sequence determined by the dideoxy method. The RNA was found to be 4526 nucleotides in length and to have a base composition of 34.2% U, 27.8% A, 20.6% C and 17.4% G. There is a single, long open reading frame in the viral complementary RNA that contains sufficient information to code for a protein of 1441 amino acids. In these respects, as in many others, the LAC virus M RNA and its encoded protein were very similar, if not identical, to those previously reported by other investigators for the closely related snowshoe hare virus. The M RNAs of the two viruses show 79% nucleotide sequence homology and 89% homology in the amino acid sequence of their encoded proteins. Several algorithms for predicting surface residues, as well as the Chou-Fasman rules for predicting secondary structure, were used to compare the LAC virus and snowshoe hare virus M gene proteins. These analyses identified 39 sites on the proteins as those most likely to be linear antigenic determinants that might contribute to the differences between the two viruses.

Electrophoresis of small proteins in highly concentrated and crosslinked polyacrylamide gradient gels.

A high concentration (40%) of acrylamide plus N,N'-methylenebisacrylamide combined with a high level of crosslinking (12.5%) yielded clear gels capable of restricting the passage of small proteins. This gel composition was chosen in preference to other combinations, in particular those producing opaque gels which have larger pore sizes and which provide a reduced sieving effect. Gradient gels were prepared in which the gel concentration rose from 3 to 40% and the degree of crosslinking increased from 4 to 12.5%. Such gels were suitable for fractionating crude, unreduced, and uncharacterized extracts containing proteins ranging in molecular size from 10,000 to several million daltons under conditions where all proteins are retained on the gel even after prolonged electrophoresis. The gels yielded zones which were of improved sharpness and resolution compared with gels of lower concentration and degree of crosslinking, and can be used to provide an estimate of molecular size. Examples of the use of HX gradient gels included both anodic and cathodic electrophoresis at pH 8.3 and 3.1, respectively, of serum and cereal-grain proteins and a partial enzymic hydrolysate of serum albumin.

Evidence for three separate antigenic sites on the G1 protein of La Crosse Virus.

Competitive binding assays with monoclonal antibodies have been used to show that there are a minimum of three nonoverlapping antigenic sites on the G1 glycoprotein of La Crosse virus. One of these sites contains the epitopes for three of the five monoclonal antibodies employed and is involved with both hemagglutination and neutralization. A second site contains the epitope for an antibody that inhibits hemagglutination but has no neutralizing activity. The third site encompasses the epitope for an antibody that at present has no identified biological role.

Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.

Nonrepetitive DNA transcription in normal and regenerating rat liver.

Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNA-DNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcrips present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA.

Synovial chondrometaplasia. A case report.

Synovial chondrometaplasia is an uncommon benign lesion most commonly affecting males in the 20 to 60 age range. The youngest heretofore reported patient is 14 years of age. In a male patient of 7 years, 2 months, with a bone age of 5 years with left elbow involvement, there was no history of antecedent trauma. The presenting complaint was enlargement of the elbow with limitation of motion. At surgery 54 osteocartilaginous bodies were removed, some of which were still covered with synovium. Eighteen months following an anterior synovectomy, there was 5 degrees to 128 degrees of pain-free elbow flexion and full painless forearm pronation and supination with no recurrence of the disease.

Sequence diversity of nuclear and polysomal polyadenylated and non-polyadenylated RNA in normal and regenerating rat liver.

A DNA probe purified from RNA . DNA hybrids of total sham-operated liver RNA and non-repetitive DNA was used to show that nuclear poly(A)-rich RNA from sham-operated liver and from 2.5-h and 48-h regenerating liver contains about 50% of the complexity of total liver RNA. It was further shown that the differences between normal and regenerating liver, reported earlier from this laboratory, occur in the poly(A)-free fraction of nuclear RNA. At the polysome level it was found that polysomal RNA has one-third of the sequence diversity of total RNA and of this, approximately 65% can be accounted for by poly(A)-rich and 55% by poly(A)-free molecules. When DNA probes were prepared from hybrids formed using polysomal RNA from sham-operated liver and regenerating liver at 2.5 h and 48 h post-hepatectomy and then employed in reactions with homologous and heterologous RNAs, no differences were detectable between either normal and regenerating liver or regenerating liver at times during hypertrophy and hyperplasia.

Deoxyribonucleic acid hybridization analysis of the defective bacteriophage carried by strain 15 of Escherichia coli.

Evidence from several laboratories indicates that strain 15 of Escherichia coli is lysogenic for a defective phage. When lysates from induced cultures were centrifuged in CsCl, three bands were obtained. In order of decreasing density, these bands contained tailless particles, complete phages, and a second band of complete phages, in a ratio of 65.7:28.6:5.7. Reassociation rate measurements were used to establish that the molecular weights of the deoxyribonucleic acid (DNA) species from the phages in the first two bands are similar. A smaller genome is postulated in the complete phages from the minor band. Hybridization experiments revealed extensive homology between the DNA species from all three phage bands, thus suggesting that the complete and tailless particles are not different at the genetic level. The DNA from each phage band was also shown to hybridize almost completely with DNA from either E. coli 15T(-) or a reportedly cured derivative of 15T(-). In contrast, only about 25% of each phage DNA was able to react with DNA from E. coli strains B and K-12 C-600.

Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice.

The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase. Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions. Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated. In contrast, while no significant late strand transcription was detected in transformed cells in culture, the tumors induced by these cells contained transcripts complementary to about 12% of this strand. The results are discussed in terms of current knowledge of the amount of the virus genome required to transform cells.

Replication of non-repetitive DNA during early, middle and late S phase. The general class and the subset transcribed.

Synchronized cultures of Chinese hamster ovary cells were pulse-labelled with 5-bromodeoxyuridine (BrdU) during early (0-2.0 h), middle (2.5-4.0 h) and late (4.5-6.0 h) S phase in two successive cell cycles. In each case, the DNA containing BrdU in both strands was duplicated at the same time in both cycles and was isolated for further characterization by centrifugation in CsCl gradients. These DNAs were then radiolabelled by nick-translation and used in either DNA-DNA or RNA-DNA hybridization experiments. In the DNA-DNA experiments, advantage was taken of the substantial rate increases attainable in high concentrations of dextran sulfate to obtain complete reassociation curves with relatively small amounts of material. Assuming that no unresolved low repetition frequency components exist, renaturation kinetics suggest that early replicating DNA contains a greater proportion of non-repetitive sequences than DNA synthesized at later times, the order being early greater than middle greater than late. However, in terms of complexity the non-repeated DNA duplicated early had only 74% of the diverse sequences present in log-phase cells, whereas that replicated in middle and late S phase had 82 and 79.5%, respectively. It therefore appears that while DNA synthesized at different times in S phase may contain varying proportions of non-repetitive sequences, when their diversity is taken into account very few of these sequences (25% or less) exhibit temporal control of replication. Finally, measurements with total cell RNA indicated that the transcribed fraction of non-repeated DNA showed a slight preference for replication in early S phase.