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The stability of Tetralzymena pyriformis cytoplasmic rRNAs and nuclear rRNA precursors has been studied by polyacrylamide gel electrophoresis under partly and completely denaturing conditions. Cytoplasmic 17-S rRNA ( M , = 0.66 x lo6) consists of a continuous polynucleotide chain throughout its lifetime, whereas the bulk of 26-S rRNA ( M , = 1.27 x lo6) dissociates upon denaturation. Two large fragments (Fl, F2) of somewhat different molecular weights ( M , 0.63 x lo6 and 0.58 x lo6) and the small 5.8-S rRNA fragment ( M , about 50000) are regularly observed. Some additional distinct minor fragments (F3 -F6) are noted under certain preparative conditions, suggestive of artifactual origin. The following conclusions were made from the data obtained. (a) Newly synthesized 26-S rRNA molecules do not contain the 'central' hidden break (separating Fl and F,) until about 15 min after their appearance in the cytoplasm; however, they release during denaturation the 5.8-S and/or a short-lived 7-S fragment ( M , about 75 000) which might represent a direct precursor to the 5.8-S rRNA. (b) The immediate nuclear precursor to the 26-S rRNA ( M , 1.39 x lo6) releases a small fragment of similar size (7 S). (c) The largest stable transcription product of the rDNA (prerRNA) does not contain any hidden break.The macronucleus of the ciliated protozoan Tetrahymens pyriformis contains numerous nucleoli, including extra-chromosomal, amplified rDNA units (cf. [I -4]), and shows high rates of pre-rRNA synthesis and ribosome formation during logarithmic growth of the cells (e.g. [5 -101). The general pathway of pre-rRNA processing in this organism has been found to be relatively simple [7,8,10,11]. Based on polyacrylamide/agarose gel electrophoretic analyses under non-denaturing conditions, we have shown that the largest stable rRNA precursor found under logarithmic growth conditions has an apparent molecular weight of about 2.2-2.3 x lo6 and seems to be rapidly cleaved, via two intermediates, into the mature 26-S and 1 7 4 rRNAs [9,10]. The final processing Ahhveyiuriom. rDNA, fraction of nuclear DNA containing the sequences for the rRNA precursor (prc-rRNA) plus spacer aequences; NaCLCit, standard saline citrate = 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0. All other abbreviations for nucleotides and nucleic acids are those proposed by the IUPAC-IUB commission on Biochemical Nomenclature, see Eur. J. Biocher)~. 15, 203-208 (1970).Enz,ymc.s. Deoxyribonuclease I (EC 3.1.4.5); proteinase K (EC 3,4.-,-); ribonuclease A (EC 3.1.4.22). steps, however, including those that lead to the production of the mature large rRNA together with the noncovalently bound, heat-dissociable 5.8-S rRNA, are unknown in this organism (for recent findings in other lower and higher organisms see for example [ 12 -161). Moreover, in Tetrahymena, as in various other protozoa and protostomian animals, at least one additional hidden break is introduced near or at the center of the large rRNA resulting in the formation of discrete fragments upon denaturation [17...

Several populations of the house mouse, Mus musculus, are polymorphic for the presence or absence of an inherited homogeneously staining region (HSR) in chromosome 1. The HSR consists of highly amplified DNA sequences, present in low copy numbers in the HSR-genome. A cloned HSR-derived genomic sequence detected transcripts of about 1.3 and 4.5 kb on blots of poly(A)+ RNA from liver of HSR+ mice but not from that of HSR-mice. A cDNA library was established from RNA of HSR+ mice and screened with the HSR-derived genomic clone. Positive clones were isolated and shown to be complementary to the 1.3-kb RNA species and to amplified DNA sequences in the HSR+ genome. The combined sequence of four overlapping cloned cDNAs is 959 nucleotides long and includes an open reading frame encoding a putative protein of 208 amino acids. The pertinent gene is unidentified. No homologous sequence is stored in the EMBL data base. A stretch of 109 nucleotides at the 3' end of the cDNA is homologous to the HSR-derived genomic clone used for its isolation. The putative coding region for the 4.5-kb RNAs starts only about 300 nucleotides downstream from the 3' end of the 1.3-kb RNA homology region in the same genomic fragment, as indicated by hybridization data and sequence motifs resembling promoter elements. Thus, our data suggest that at least two genes or gene families are encoded in the HSR.Homogeneously staining regions (HSRs) are chromosomal segments with various lengths and uniform staining intensity after G banding. They were originally discovered in tumor cells and cells selected for drug resistance (3). Cytochemical and molecular analyses showed that these HSRs are cytological correlates of DNA amplification (for reviews, see references 1, 13, and 24), including cellular oncogenes and genes conferring resistance to cytotoxic drugs. Overexpression of amplified genes is obviously responsible for the drug resistance or contributes to the oncogenic phenotype. In most cases, the amplified DNA units (amplicons) are much larger than the target gene region and often include coamplified genes which are also overexpressed but do not contribute to. the selected phenotype (15,21,40,42, 47,48).In contrast to these cases of HSRs originating in and restricted to specific somatic tissues or cell lines, an HSR found as a polymorphism in chromosome 1 in several populations of the house mouse, Mus musculus, is not only present in somatic tissues but also inherited through the germ line (46). An HSR from wild mice in Switzerland was introduced into a laboratory mouse strain by meiotic recombination (46). From metaphase spreads of chromosomes of these animals, the HSR was microdissected and microcloned (49). Analyses of several of these clones showed that the HSR includes highly amplified DNA sequences compared with the HSR-free (HSR-) genome (49). With in situ hybridization, sequences homologous to those amplified in the HSR were detected at or near the HSR integration site in HSR-chromosome 1. This indicates that an in situ amplifi-* Correspondin...

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We have analysed nuclear RNA from T. thermophila by RNA transfer hybridization using cloned rDNA fragments. A very high number of in vivo intermediates and by-products of rRNA processing were identified. These include putative intermediates of the splicing process and alternative products resulting from temporal variability in various endonucleolytic cleavages. In addition, four small RNA species including only transcribed spacer sequences were detected. These are (1) the IVS RNA (approximately 400 bases), the by-product of the splicing process, (2) a fragment from the internal transcribed spacer (approximately 360 bases), possibly resulting from 3'-end processing of pre-17S rRNA, (3) a fragment comprising most or all of the external transcribed spacer (approximately 600 bases) obviously representing the major by-product of 5'-end processing, and, in addition, (4) a small fragment from the initiation region (approximately 230 bases) which might be a product of premature transcription termination.