Massive heat-shock polypeptide synthesis in late chicken embryos: convenient system for study of protein synthesis in highly differentiated organisms.

In cultured eucaryotic cells, heat treatments specifically induced the rapid synthesis of the so-called heat-shock polypeptides. To ascertain the physiological importance of this phenomenon for highly differentiated organisms, we attempted to determine whether the heat-shock response occurs in a living endothermic organism at extreme temperatures, and if so, whether the response is organ specific. We developed a procedure to label proteins efficiently in 5- to 18-day-old chicken embryos. Heat-shock polypeptides of identical sizes of 85,000, 70,000, and 25,000 daltons were synthesized predominantly in chicken embryo fibroblasts and in many different organs of 18-day-old embryos at 42.5 to 44 degrees C.

Hybrid plasmids carrying part of the Rous sarcoma virus-specific leader sequence.

'Strong-stop' DNA, complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus genomic RNA and subgenomic mRNAs can be used to isolate RSV-specific mRNA from infected cells. To analyse leader sequences of these mRNAs and to study the mechanism of their translation, relatively large quantities of strong-stop DNA are required. The construction of four plasmids carrying useful strong-stop DNA sequences for mRNA isolation is described. One such hybrid plasmid carries a DNA insertion containing sequences derived from a region starting 42 nucleotides from the 5'-end of RSV RNA, and ending at the tRNATry primer attachment site. Since this plasmid lacks the 20-nucleotide terminal repeat sequence it can be used for the specific isolation of 5'-end fragments of RSV-specific mRNAs and of complementary DNA transcripts of these fragments.

Analysis of the secondary and tertiary structures of Rous sarcoma virus RNA.

The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated. T1 RNase partial digests have been first resolved into their components by gel electrophoresis under non denaturing conditions and then each component analyzed further by various techniques. More than one hundred structured fragments have thus been isolated and shown to consist of several individual nucleotide sequences located far apart on the genome. On the basis of the results, a cloverleaf model for the structure of RSV 35S RNA is proposed that has implications for the various biological functions of this RNA.

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.

Single and divided doses of penbutolol.

Penbutolol, a nonselective beta-adrenoreceptor antagonist, induced reduction of exercise-induced heartbeats for at least 24 hr after a single 40-mg oral dose, and was equipotent with respect to a 2 X 20-mg regimen over the same period. Ingestion for 7 days did not influence the pharmacodynamics or pharmacokinetics of penbutolol, and there was no cumulation of drug in serum. A relationship was found between the logarithms of measurable serum concentrations of penbutolol and the percentage reduction of total heartbeats. Absorption of oral penbutolol appeared to be reduced when administered in the evening. Since beta-adrenoceptor activity was relatively unchanged between 13 and 24 hr after a single 40-mg dose of penbutolol, there is a possibility that an active metabolite or metabolites may contribute to prolonged duration of action.

Characterization of the genomic RNA from a Rous sarcoma virus mutant temperature sensitive for cell transformation.

We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when passaged repeatedly at high multiplicity of infection, does not give rise to transformation defective deletion mutants comparable to those derived from RSV. In view of this fact and of the high rate of production of this mutant at 41 degrees C, we have undertaken a detailed analysis of the genome of this virus by ordering all large T1 oligonucleotides and by determining their nucleotide sequences. The results indicate a high degree of mutation in the onc gene as compared to that of Pr-A or Pr-B.

In vitro, the major ribosome binding site on Rous sarcoma virus RNA does not contain the nucleotide sequence coding for the N-terminal amino acids of the gag gene product.

Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.

Plasma cortisol levels in normal volunteers receiving either betamethasone valerate or desoximetasone by topical application.

Desoximetasone (Topisolon; Hoechst), a new topical steroid, and betamethasone 17-valerate were compared with respect to their effects on hypothalamic-pituitary-adrenal function as evidenced by plasma cortisol concentrations. Three grams of each test preparation were applied daily for 21 days to intact skin of the ventral aspects of alternate forearms of 15 normal volunteers. Five received betamethasone 17-valerate 0.1%, 5 desoximetasone 0.05%, and 5 desoximetasone 0.25%. Plasma cortisol levels were determined before and after the initial applications on days 1, 3, 10, 17, 22, 24 and 28. These values were compared with the mean control values by analysis of covariance. There was no significant difference in plasma cortisol levels. The value of performing similar studies on larger skin areas and with larger doses is discussed.

Extensive in vitro transcription of rous sarcoma virus RNA by avian myeloblastosis virus DNA polymerase and concurrent activation of the associated RNase H.

Conditions are described that promote the efficient reverse transcription of most of Rous sarcoma virus (RSV) RNA sequences by avian myeloblastosis virus DNA polymerase in vitro. A detailed analysis of the reverse transcription reaction was carried out using two procedures: in situ analysis of the RNA sequences transcribed and DNA-RNA annealing studies. Under optimal conditions, after 1 h of reaction, practically all RSV RNA sequences were transcribed with a frequency varying from 30 to 90%. The DNA product was at least 95% single stranded, had a chain length ranging from a few hundred up to 5,000 necleotide residues, half of it being larger than 1,000 residues, and, after hybridization at RNA excess, protected the entire RSV genome from RNase digestion, as monitored by the large T1 oligonucleotides of RSV RNA. Analysis of the product of a very short reaction time (5 min) showed that DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA. This in in agreement with our previous analysis of a much less efficient reverse transcription reaction. Under optimal conditions of reverse transcription, we find now that the RNase H associated with the avian myeloblastosis virus DNA polymerase is active in degrading the RNA moiety of the RNA-DNA hybrids synthesized.

New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA.

Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process. Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites. We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule. Furthermore , we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.

Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy.

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.

Quantitative determination and location of newly synthesized virus-specific ribonucleic acid in chicken cells infected with Rous sarcoma virus.

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.

In vitro product of a ribonucleic acid polymerase induced by influenza virus.

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus.