Peter J. Wejksnora

An RNA polymerase I promoter located in the CHO and mouse ribosomal DNA spacers: functional analysis and factor and sequence requirements

We report results of experiments in which we demonstrated the existence of a polymerase I promoter within the ribosomal DNA spacer upstream from the rRNA initiation site in Chinese hamsters and mice. Transcription of the CHO spacer promoter was achieved by the same protein factors, C and D, that catalyzed transcription of the gene promoter, and these factors bound stably to the CHO spacer promoter in a preinitiation complex, just as they did to the gene promoter. In contrast to the CHO spacer promoter, which was transcribed in vitro nearly as efficiently as the gene promoter, the mouse spacer promoter was far less active; this low activity was attributable to the fact that the mouse spacer promoter bound factor D inefficiently. It is striking that the active CHO spacer promoter violated the otherwise universal rule that metazoan RNA polymerase I promoters all have a G residue at position -16. Sequence comparisons also revealed a great similarity between the CHO and mouse spacer promoter regions, yet there was much less similarity between the flanking sequences. There was also only limited homology between the spacer and gene promoter regions, but despite this the two kinds of initiation regions were organized similarly, both consisting of an essential core promoter domain and a stimulatory domain that extended upstream to approximately residue -135. Evolutionary considerations argue strongly that the presence of ribosomal DNA spacer promoters offers a significant selective advantage.

The ribosomal RNA genes of three salmonid species

The ribosomal RNA cistrons of three species of trout: Salvelinus namaycush (lake trout), Salvelinus fontinalis (brook trout), and Salmo gairdneri (rainbow trout) were examined by Southern blot analysis of genomic DNA. The repeat length of the cistron of S. namaycush is 26 kb. A repeat-length polymorphism was observed in some of the individual fish examined. These individuals showed 24-kb repeats. In some individuals both forms were present. The restriction maps of the transcribed regions of all three species were similar and showed a site homology with other vertebrate ribosomal RNA genes. Interspecific comparison showed restriction-site differences within the spacer regions examined. A restriction-site polymorphism within the 28 S gene was observed in S. fontinalis. The rDNA of S. namaycush liver showed a high degree of methylation as determined by digestion with the restriction endonucleases MspI and HpaII.

Characterization of the region around the start point of transcription of ribosomal RNA in the Chinese hamster

The initiation site for ribosomal RNA transcription in the Chinese hamster was identified and the sequence around and upstream determined. The start point region shows considerable homology with comparable regions in the mouse and rat. In the Chinese hamster, the region between bp -700 and -200 consists of imperfect repeats approximately 120-130 bp in length which are flanked by T-rich regions. The region within each repeat which is homologous with an adjacent repeat decreases in length as the start point is approached. The final promoter-proximal repeat preserves only an 11-bp region of the promoter-distal repeats. This short sequence, termed the repeat core, appears with a periodicity of about 120-130 bp in the Chinese hamster, and is conserved in both mouse and rat. In humans, a short repeated sequence occupies similar positions, suggesting that while complete 120-130-bp repeats are not a feature of all mammalian RNA polymerase I promoter-proximal r X DNA spacers, a short sequence repeating with approximate 120-130-bp periodicity may be such a feature.

Effect of acrylamide monomer on hepatic CYP1A1 monooxygenase induction in rainbow trout.

1. Rainbow trout hepatic microsomes were pre-incubated in vitro with 1 pM, 1 nM, and 1 microM acrylamide for 20 or 30 min and ethoxyresorufin-O-deethylase (EROD) was measured. In vitro pre-incubation did not produce a significant decrease in EROD catalytic activity. 2. The effects of 50 ppm acrylamide monomer on hepatic CYP1A1 mRNA and CYP1A1 isozyme steady state levels in rainbow trout were determined after 6, 10, and 14 days of exposure. 3. Acrylamide monomer produced a 35% increase, 51% decrease and 140% increase in CYP1A1 mRNA at 6, 10 and 14 days, respectively, while at the same time CYP1A1 isozyme levels were decreased 12%, 67%, and 62% and EROD activity was decreased 33%, 90%, and 86%, respectively. 4. This indicates that acrylamide treatment may result in either a change in the translation of CYP1A1 mRNA or an isozyme selective inactivation of CYP1A1 resulting in loss of CYP1A1 apoprotein. 5. The effect of acrylamide treatment on hepatic CYP1A1 induction was determined using 10 or 14 day treatment with 50 ppm acrylamide monomer in a flowthrough exposure and induction with beta-naphthoflavone (beta-NF; 100 mg/kg i.p.) for 1 or 4 days. 6. Acrylamide and 1-day beta-NF had no effect on CYP1A1 mRNA levels when compared to 1-day beta-NF treatment alone, but acrylamide and 4-day beta-NF resulted in a 74% decrease in CYP1A1 mRNA compared to 4-day beta-NF treatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of increase in ploidy on the activation of nucleolar organizer regions in Chinese hamster ovary (CHO) cells

The effect of increased ploidy on the activation of specific nucleolar organizer regions (NORs) was examined by comparing the distribution and frequency of active NORs in pseudodiploid Chinese hamster ovary (CHO) cells with a quasi-tetraploid hybrid line. Active NORs were identified on both unrearranged chromosomes and isochromosomes of the Z group by silver staining. The increase in cell ploidy in the hybrid did not result in the complete inactivation of specific NORs or the activation of a previously silent NOR. However, for several chromosome pairs identified as carrying NORs, apparent translocations and deletions which produced the karyotype of the pseudodiploid cells deleted or inactivated the NOR of one member of a homologous pair. When two copies of such chromosomes were present in the quasi-tetraploid hybrid line, the activity of their NORs showed apparent coordination. Furthermore, the frequency of activity of individual NORs in two CHO lines and in a quasi-tetraploid hybrid line suggests that active NORs are not inherited directly.

Expression and characterization of the τ subunit of phosphorylase kinase

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.

Fragments of rDNA within the Chinese hamster genome

We report the isolation and partial characterization of distinctEcoRI fragments of the Chinese hamster genome which contain regions complementary to a 1-kb portion of the mature 18 S ribosomal RNA molecule. This previously undescribed 18 S rDNA-like region, which we have termed a “fragment of ribosomal DNA” (frDNA), has been shown by sequence analysis to correspond to a region extending 1 kb upstream from the 3′ terminus of the mature 18 S rRNA. Within the five frDNA-containing clones described here, no other region of the ribosomal RNA cistron was detected, making it unlikely that these are polymorphic forms of the ribosomal DNA repeat. The 18 S rDNA-complementary region appears to be flanked by an imperfect direct repeat, which could have been the result of the retroinsertion of a fragment of ribosomal RNA. Directly adjacent to the 18 S rDNA-like region we have identified nonribosomal sequences which appear common to all of the frDNA-containing clones we examined. At least eight different-sizedEcoRI fragments contain frDNAs and the abundance of the frDNAs appears to be of the order of 30 per genome. The occurrence of multiple copies of this ribosomal-nonribosomal chimera suggests that, once formed, the chimera was duplicated within the genome.