The effect of prostaglandin E1 on in vitro transcription of sperm chromatin, isolated from patients with azoospermia, teratospermia and chronic prostatitis.

We have investigated the influence of Prostaglandin E1 on the in vitro transcription of chromatin, isolated from spermatozoa of patients suffering from different pathologies, leading to infertility, namely, azoospermia, teratospermia and chronic prostatitis. Our studies indicate that prostaglandin E1 has a stimulatory effect both on in vitro transcription, on the number of RNA polymerase molecules and the polyribonucleotide elongation rates as compared to sperm chromatin from healthy patients. The results on the incorporation of alpha-32P-ATP in to RNA in the presence and absence of Prostaglandin E1 correlate well with the data on the number of actively transcribing RNA polymerase molecules and the rate of RNA elongation, which might be due to low levels of prostaglandin E1 in human semen.

Effect of mimosine on DNA synthesis in mammalian cells.

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.

Quantification of actively transcribing RNA polymerase B molecules in human sperm chromatin and the effect of prostaglandin E1.

Run-on transcription experiments with sonicated chromatin from spermatozoa of healthy men revealed the presence of in vitro transcription. The number of actively transcribing RNA polymerase B molecules in sperm chromatin in vitro, isolated from healthy individuals, and the effect of prostaglandin El, were investigated. The results indicate that the number of RNA polymerase B molecules actively engaged in transcription increased one and a half times when 5 units of prostaglandin E1 was added to the reaction mixtures. These results correlate well with the authors' previous findings that the incorporation of labelled uridine triphosphate into pre-mRNA was higher when prostaglandin E1 was added to the reaction mixtures in run-on transcription experiments, and when sperm chromatin from healthy individuals was used as a template.

Effects of gamma-irradiation on biosynthesis of different types of ribonucleic acids in normal and regenerating rat liver.

1. The effect of gamma-irradiation (4000rd) on the synthesis of ribosomal (pre-rRNA) and heterogeneous nuclear RNA (pre-mRNA) in normal and in regenerating rat liver was studied by using 40 min labelling with [6(-14)C]orotic acid. 2. Partial hepatectomy caused a sharp transient increase in the specific radioactivity of the endogenous low-molecular-weight RNA precursors in the livers of both normal and irradiated rats. Irradiation of intact animals did not affect the pool. 3. Irradiation enhanced the synthesis of pre-rRNA for at least 12h. The synthesis of pre-mRNA was also enhanced, but only in the first 3h after irradiation. 4. Partial hepatectomy strongly stimulated the synthesis of both pre-rRNA and pre-mRNA. 5. The synthesis of pre-rRNA was enhanced also in regenerating liver of animals irradiated before or after the operation. The conclusion can be drawn that the early increase in the synthesis of ribosomal RNA is a non-specific cellular response to different injuring factors. 6. The only case where irradiation caused an early inhibition of RNA synthesis was that of pre-mRNA in regenerating liver. This supports the hypothesis that ionizing radiation does not suppress the transcription per se but affects the mechanisms of activation of new genes (cellular programming).