Andrea D. Branch

Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay

Quantitative solution hybridization assays were used to determine the picogram amounts of preproenkephalin mRNA (PPenk mRNA) and the microgram quanities of total rat RNA in extracts of eight brain regions from rats which had received three daily intraperitoneal injections of cocaine (10 or 30 mg/kg/day) or saline for 14 days. The young adult male Fischer rats were sacrificed 30 min after the final injection. The highest density of PPenk mRNA (pg PPenk mRNA/micrograms total cellular RNA) was found in extracts of striatum (34.08 +/- 1.79 pg/micrograms for 11 saline-treated rats), followed by extracts of nucleus accumbens (10.08 +/- 0.81 pg/micrograms), and extracts of hypothalamus (2.99 +/- 0.31 pg/micrograms). Extracts of frontal cortex (1.78 +/- 0.24 pg/micrograms), pituitary (1.39 +/- 0.08 pg/micrograms), central grey (1.31 +/- 0.16 pg/micrograms), and cerebellum (1.24 +/- 0.09 pg/micrograms) had intermediate values. Extracts of hippocampus (0.53 +/- 0.03 pg/micrograms) had the lowest density. No significant differences were found among the treatment groups in any brain area investigated. Therefore, chronic cocaine treatment as administered in this protocol did not alter expression of the gene encoding proenkephalin.

Cell-Free Circularization of Viroid Progeny RNA by an RNA Ligase from Wheat Germ

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

Time course of enkephalin mRNA and peptides in cultured rat adrenal medulla

Explantation of rat adrenal medullae to organ culture results in dramatic changes in enkephalins and catecholamines that are similar to the changes seen in vivo in response to denervation, which eliminates transsynaptic impulse activity. We have used rapid and sensitive solution hybridization methods to measure preproenkephalin (PPenk) mRNA and total cellular RNA in samples from rat tissues and adrenal medullary explants. The profiles of adrenal medullary PPenk mRNA, enkephalin-containing (EC) peptides, total cellular RNA and catecholamines [epinephrine (epi) and norepinephrine (norepi)] were measured during 14 days of organ culture. After 8 h in culture, total RNA had declined by 60%, epi and norepi declined 80 to 85% and EC peptides by 50% while the amount of PPenk mRNA per gland increased by 400%. Between 8 h and 14 days total RNA and catecholamine levels remained constant while PPenk mRNA increased to a peak of 85 +/- 10 (S.E.M.) pg/gland at 2-4 days, a value that was 80 times greater than the zero time (preculture) values. EC peptide levels lagged behind the increase in PPenk mRNA and reached a peak of 25 +/- 4 (S.E.M.) pmol Met-enkephalin equivalents/gland at 4 days that was 80 times greater than zero time values. Both PPenk mRNA and EC peptides declined in parallel between 4 and 14 days. The ratio of the copies of proenkephalin (Penk) peptide to PPenk mRNA was estimated to be 25,000 at the time of explantation and after 4 days in culture. From steady-state kinetics half-life estimates of 9.6 h for PPenk mRNA and 14.7 h for Penk peptide were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Preproenkephalin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster : comparison with central nervous system tissues

The distribution and characteristics of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides are compared in CNS and adrenal tissues from Syrian hamsters and Sprague-Dawley rats. Total cellular RNA extracts from both rat and hamster tissues produce a single hybridization band of PPenk mRNA of approximately 1500 bases when analyzed by Northern blot hybridization. Quantitation by solution hybridization reveals that in the hamster the highest levels of PPenk mRNA are found in adrenal (16.3 +/- 1.4 pg equivalents/micrograms RNA (mean +/- S.E.M.)) and striatum (13.3 +/- 0.7), followed by hypothalamus (0.8 +/- 0.2), and hippocampus (0.4 +/- 0.2). In the rat the highest levels of PPenk mRNA are in the striatum (35 +/- 2 pg/micrograms RNA) followed by the hypothalamus (3.0 +/- 0.5), hippocampus (0.3 +/- 0.1) and adrenal (0.18 +/- 0.04). Thus, the rank order of abundance of PPenk mRNA is similar in these CNS tissues for rat and hamster. The hamster adrenal levels are more than 90-fold greater than those of the rat. The abundance of EC peptides in both hamster and rat tissues mirror the rank order found with PPenk mRNA. Hamster adrenal contains the highest level of EC peptides (441 +/- 37 pmol/mg protein (mean +/- S.E.M.)) which is more than 400-fold greater than that of the rat adrenal and 8- to 12-fold greater than that found in rat and hamster striatum or hypothalamus. Both size exclusion chromatography and Western blot analysis indicate that EC peptides in hamster adrenal are predominantly large proenkephalin-like peptides with approximately 6 copies of Met- and 1 copy of Leu-enkephalin and that included in their number is a prominent EC peptide with a molecular weight of 34 kDa. Unilateral denervation of the hamster adrenal results in a time-dependent ipsilateral decrease in EC peptide and PPenk mRNA levels. Thus, by day 8 postsurgery, PPenk mRNA levels have declined by an average of 80% while EC peptides are reduced by 68% when compared to the innervated contralateral adrenal. These results demonstrate the great abundance of PPenk mRNA and EC peptides in the hamster adrenal. They also demonstrate the apparent need for transsynaptic impulse activity to maintain the high steady-state levels of PPenk and EC peptides. These characteristics of the hamster adrenal system provide opportunities for physiological and pharmacological investigations of the regulation of proenkephalin gene expression.

Tomato DNA contains no detectable regions complementary to potato spindle tuber viroid as assayed by Southern hybridization.

DNA was isolated from crude nuclear pellets prepared from Rutgers tomato plant leaf tissue from both potato spindle tuber viroid (PSTV)-infected and uninfected plants. Following digestion by the DNA restriction enzymes EcoRI, XbaI, or BamHI, the DNA was fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and incubated with a variety of 125I-labeled RNA probes under conditions which permit hybridization. Analysis of autoradiogram patterns and fingerprints of the RNA recovered from individual bands following hybridization of the three tomato cytoplasmic ribosomal species, 5, 18, and 26 S, permitted us to construct a consistent restriction map about 8800 base pairs in length containing the 18 and 26 S species and to demonstrate that the 5 S gene clusters are not linked to the 18 and 26 S regions and themselves have a repeat length of about 350 base pairs. These studies formed the background for a search for hybridization between 125I-labeled PSTV and DNA from uninfected and PSTV-infected tomato plants. Significant hybridization was observed when conventionally prepared viroid was used as the probe in Southern hybridization experiments. However, fingerprint analysis of the RNA recovered from these bands demonstrated that this hybridization was due to the presence of ribosomal RNA species in the viroid preparation. When the viroid was subjected to further purification steps (electrophoresis in gels containing 8 M urea) prior to hybridization, all traces of hybridization due to host contaminants disappeared and no viroid-specific bands could be detected, even upon prolonged exposure of the autoradiograms. Under these same hybridization conditions, we could detect as little as one-quarter copy of a gene 550 base pairs in length.

Interference between coinoculated viroids

Experiments were carried out to seek evidence of an interaction between two viroid RNAs introduced to tomato plants in the same inoculum. At the level of symptom expression, the severe isolate of potato spindle tuber viroid (PSTV) dominated the mild isolate. Seventy-five percent of the plants inoculated with a 100-fold excess of the mild isolate developed unattenuated symptoms of severe disease. Other experiments revealed that infectious RNA molecules transcribed from cloned DNA templates containing PSTV sequences reduced the level of hop stunt viroid (HSV) RNA present in nucleic acid extracts of plants which had been inoculated with a mixture of dimeric plus-strand transcripts of these two viroids. Plants inoculated with dual transcripts--containing two copies of PSTV linked to two copies of HSV--developed characteristic symptoms of severe PSTV. Dot hybridization demonstrated that only PSTV replicated to detectable levels in these plants. A likely interpretation of these results is that the HSV portion of the dual transcripts failed to replicate because of interference from PSTV. These results raise questions about how the process of viroid replication is related to symptom expression, and lead to suggested models for the effect of viroid-like RNAs in cells under both normal and pathogenic circumstances.

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV‐crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV‐crosslinking between the same two bases as in full‐length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.