Ultrastructural and biochemical studies on the precursor ribosomal particles isolated from rat liver nucleoli.

Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80-100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80-100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.

Increased anxiety and impaired pain response in puromycin-sensitive aminopeptidase gene-deficient mice obtained by a mouse gene-trap method.

A mouse mutation, termed goku, was generated by a gene-trap strategy. goku homozygous mice showed dwarfism, a marked increase in anxiety, and an analgesic effect. Molecular analysis indicated that the mutated gene encodes a puromycin-sensitive aminopeptidase (Psa; EC 3. 4.11.14), whose functions in vivo are unknown. Transcriptional arrest of the Psa gene and a drastic decrease of aminopeptidase activity indicated that the function of Psa is disrupted in homozygous mice. Together with the finding that the Psa gene is strongly expressed in the brain, especially in the striatum and hippocampus, these results suggest that the Psa gene is required for normal growth and the behavior associated with anxiety and pain.

Isolation and characterization of cDNA encoding a spicule matrix protein in Hemicentrotus pulcherrimus micromeres.

A cDNA clone, termed pHPSMC, was obtained from the Japanese sea urchin Hemicentrotus pulcherrimus, and it was found to be highly homologous in sequence to the spicule matrix protein cDNA of Strongylocentrotus purpuratus (Sucov et al., Dev. Biol. 120: 507-519, 1987). During early embryogenesis, mRNA complementary to pHPSMC appeared in gastrulae and remained at a similar level until the pluteus stage. In situ hybridization revealed that the mRNA was localized exclusively in primary mesenchyme cells in gastrulae. pHPSMC mRNA was detected in micromeres in vitro after 48 h of culture, but it was not found in blastomeres immediately after isolation. These features suggested that pHPSMC represents the spicule matrix protein cDNA cognate in Hemicentrotus pulcherrimus. In the derived polypeptide, we detected a domain containing a tandemly repeated 13-amino acid sequence as did Sucov et al. (1987). Unexpectedly, the sequence of the repeated element was completely different from that originally reported for Strongylocentrotus purpuratus, but it was very similar to the corrected sequence that appeared recently.

Faithful initiation of the in vitro transcription of a cloned rDNA from Tetrahymena pyriformis.

A cloned rDNA fragment containing the transcriptional start point of Tetrahymena pyriformis was transcribed in vitro with a crude extract from homologous Tetrahymena cells. When a KpnI-HindIII fragment from the cloned rDNA plasmid was used as the template, the runoff transcription gave rise to two major RNA products about 490 and 460 nucleotides (nt) in length. An RNA of about 490 nt long was found to correspond to the expected transcript starting from the in vivo start point determined at our laboratory [Saiga et al., Nucl. Acids Res. 10 (1982) 4223-4235]. Precise size determination was performed using an RNA size marker prepared by hybridizing the template DNA with in vitro-capped 35S pre-rRNA followed by treatment with single-strand specific P1 nuclease. The size of the runoff transcript changed as predicted, according to downstream truncation points. The effects of alpha-amanitin and actinomycin D showed the transcription to be dependent on the added template DNA and to be catalyzed by form-I RNA polymerase. Possible reasons for the discrepancy between our determination of the size of the transcription product and that of Sutiphong et al. [Biochemistry 23 (1984) 6319-6326] are discussed.

Common sequence elements are important for transcription and replication of the extrachromosomal rRNA-encoding genes of Tetrahymena.

Three types of conserved sequence elements have previously been identified in the generally non-conserved, 5' non-transcribed spacer region of the rRNA-encoding gene (rDNA) of Tetrahymena species. In vivo and in vitro experiments have identified the start point (tsp) for rDNA transcription. Comparative sequence analysis suggested that the type-I sequence element was likely to function as the promoter for rDNA transcription. We have used in vitro transcription assays to demonstrate that sequences in the 3' half of the type-I repeat located proximal to the tsp are essential for the accurate initiation of rRNA synthesis. These sequences define the 5' boundary of the core or minimal promoter element and include the sequences which function in rDNA replication and maintenance in the homologous regions in two upstream type-I sequence elements. These sequences may bind factors important for both the transcription and replication of rDNA.

Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-beta subunit.

A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using lambda gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-beta subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0.8 and 1.0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 degrees C. In situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-beta cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-beta subunit supports the hypothesis that the number of proline residues increases in the LH-beta subunit the closer phylogenetically the vertebrate is to mammals.

DNA-dependent RNA polymerase from a protozoan, Tetrahymena pyriformis. Extraction and partial characterization.

Three peaks of DNA-dependent RNA polymerase (EC 2.7.7.6) activity were resolved when the enzyme was prepared from the isolated macronuclei of Tetrahymena pyriformis GL(amicronucleate strain) and chromatographed on DEAE-Sephadex A25. They were eluted at around 0.05, 0.15, and 0.2 M of ammonium sulfate, and termed TIa, TIb, and TII, respectively. All three enzymes transcribed heat-denatured DNA more efficiently, especially the peak TII, detecable only when heat-denatured DNA was used as a template. Further characterization of each enzyme, after they were rechromatographed on DEAE-Sephadex, demonstrated the similarity in many respects of TIa and TIb, and the distinct nature of the TII enzyme. TIa, TIb, and TII were all insensitive to rifampicin, while only TII was substantially inhibited by alpha-amanitin. On the other hand, the activity of TII was progressively lowered by increasing the concentration of ammonium sulfate in the assay mixture, a finding incompatible with those obtained thus far. It is concluded from the data that the Tetrahymena polymerase is of eukaryotic and not of bacterial type in spite of the findings indicating the bacterial nature of this organism.

Structural organization of the rae28 gene, a putative murine homologue of the Drosophila polyhomeotic gene.

A putative murine homologue of the Drosophila polyhomeotic gene, named rae28, has been isolated from a genomic library of 129/SV mouse and its structural organization has been analyzed. rae28 is a single gene of approximately 22 kb long and consists of 15 exons. Its 5'-flanking region lacks typical transcriptional regulatory sequences, such as TATA and CCAAT boxes, but contains GC-rich sequences and seven putative binding sites for a transcription factor, Sp1. One major transcription start point has been determined. The overall exon-intron organization suggested that three different Rae28 mRNAs are generated through alternative splicing. Furthermore, the rae28 gene has been located on the R-positive F3 band of mouse chromosome 6 by the direct R-banding fluorescence in situ hybridization methods.

Properties of in vitro transcription by isolated Xenopus oocyte nucleoli.

Some properties of in vitro transcription by isolated Xenopus oocyte nucleoli were described. When incubated with labeled RNA precursors, Xenopus oocyte nucleoli exhibited prolonged incorporation of radioactivity into RNA. The synthetic activity was exclusively due to type I RNA polymerase as revealed by its insensitivity to low and high doses of alpha-amanitin. The size of the in vitro transcript was mostly larger than 28S at 10 minute incubation and became smaller as incubation proceeded. When [gamma-32P]ATP was included in the reaction mixture, 32P radioactivity was incorporated into RNA suggesting the possible initiation of transcription in this system. However, analysis of the terminal nucleotide of the transcript revealed that the incorporation of radioactivity from [gamma-32P]ATP was not due to the initiation of transcription but due to polynucleotide kinase activity in the nucleolar preparation. These results demonstrate that the incorporation of radioactivity from [gamma-32P] labeled nucleoside triphosphates cannot necessarily be regarded as an index of the initiation of transcription.

In vitro transcription in isolated nucleoli of Tetrahymena pyriformis.

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation.