No abstract

[P.S.] RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxicischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states. In eukaryotes, three different POL transcribe nuclear genes. POL I transcribes class I genes, those that code for large ribosomal RNA (rRNA). It is located in the nucleolus, represents 50 -70% of polymerase activity per cell, and as many as 40,000 copies per cell are present (1). This high number of POL I molecules transcribes as few as about 100 genes, which is appropriate as the rRNA precursor is long (i.e. 6,000 -15,000 nucleotides), and as many as 50 polymerase molecules may transcribe each rRNA gene simultaneously. This intense transcriptional activity reflects the need of the cell for rRNA; 10 million copies of rRNA are required (2) to form the 10 million ribosomes required for a single cell division. rRNA genes, transcribed by POL I, form the primary RNA transcript, the 45S-rRNA. Before release from the nucleus as ribosomal particles, 45S-rRNA is cleaved to a copy of 28S-rRNA, 18S-rRNA, and 5.8S-rRNA each. POL III transcribes class III genes, those that encode a number of small RNA molecules, including 5S-rRNA. It is located in the nucleoplasm, makes up 10% of the polymerase activity of the cell, and about 20,000 copies are present per cell (3). These...

No abstract

[P.S.] RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxicischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states. In eukaryotes, three different POL transcribe nuclear genes. POL I transcribes class I genes, those that code for large ribosomal RNA (rRNA). It is located in the nucleolus, represents 50 -70% of polymerase activity per cell, and as many as 40,000 copies per cell are present (1). This high number of POL I molecules transcribes as few as about 100 genes, which is appropriate as the rRNA precursor is long (i.e. 6,000 -15,000 nucleotides), and as many as 50 polymerase molecules may transcribe each rRNA gene simultaneously. This intense transcriptional activity reflects the need of the cell for rRNA; 10 million copies of rRNA are required (2) to form the 10 million ribosomes required for a single cell division. rRNA genes, transcribed by POL I, form the primary RNA transcript, the 45S-rRNA. Before release from the nucleus as ribosomal particles, 45S-rRNA is cleaved to a copy of 28S-rRNA, 18S-rRNA, and 5.8S-rRNA each. POL III transcribes class III genes, those that encode a number of small RNA molecules, including 5S-rRNA. It is located in the nucleoplasm, makes up 10% of the polymerase activity of the cell, and about 20,000 copies are present per cell (3). These...

No abstract