Biomaterial Database

In vitro synthesis and processing of pre-rRNA in isolated macronuclei from Tetrahymena. 1988

K P Kister, and W Kaffenberger, and W A Eckert

Zoologisches Institut, Universität Heidelberg/Bundesrepublik Deutschland.

Macronuclei were isolated from logarithmically growing Tetrahymena cells in isoosmotic medium containing the weak detergent n-octanol and were purified in sucrose gradients. Electron microscopy revealed good structural preservation including intact nuclear envelopes. Initial rates of [3H]UTP incorporation into these nuclei were relatively high (2-4 pmol UMP/micrograms DNA per min), and 70 to 80% of transcription was resistant to alpha-amanitin, which is similar to the percentage of nuclear label associated with the nucleoli in electron microscopic autoradiograms. The use of transcription initiation inhibitors indicated that elongation of in vivo initiated pre-rRNA chains had essentially occurred in vitro. The radioactivity profiles of in vitro synthesized RNA in gels exhibit a heterogeneous pattern with the exception of a small peak corresponding to the length of pre-rRNA molecules. Detailed analysis of the extent and specificity of pre-rRNA processing was performed by RNA transfer hybridizations using cloned rDNA fragments as probes. The results show that the early processing events, i.e., splicing, 5'terminal and central cleavage of pre-rRNA, proceed faithfully, but at reduced rates and efficiencies. Furthermore, processing of pre-17S rRNA at the 3'end, and pre-26S rRNA at the 5'end, including the formation of immediate 5.8S rRNA precursors (ITS and 7S RNA), occurred. In contrast to previous in vivo results, a central hidden break was also introduced into part of nuclear 26S rRNA molecules. In addition to the known intermediates and by-products of processing, a large number of distinct fragments due to non-random cleavages of rRNA precursors appeared during in vitro incubation of macronuclei. Most prominent were two novel small RNA fragments from the 5'terminal end of pre-rRNA which may be products of alternative processing sites in the external transcribed spacer. Another small promoter-proximal RNA which is present in substantial amounts in vivo, was not formed under in vitro processing conditions, but degraded rapidly. This is further support to the notion that this RNA species may represent a product of premature transcription termination.

UI	MeSH Term	Description	Entries
D009693	Nucleic Acid Hybridization	Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503)	Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D002458	Cell Fractionation	Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS.	Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D002467	Cell Nucleus	Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (CELL NUCLEOLUS). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the ENDOPLASMIC RETICULUM. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed)	Cell Nuclei,Nuclei, Cell,Nucleus, Cell
D004275	DNA, Ribosomal	DNA sequences encoding RIBOSOMAL RNA and the segments of DNA separating the individual ribosomal RNA genes, referred to as RIBOSOMAL SPACER DNA.	Ribosomal DNA,rDNA
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D012323	RNA Processing, Post-Transcriptional	Post-transcriptional biological modification of messenger, transfer, or ribosomal RNAs or their precursors. It includes cleavage, methylation, thiolation, isopentenylation, pseudouridine formation, conformational changes, and association with ribosomal protein.	Post-Transcriptional RNA Modification,RNA Processing,Post-Transcriptional RNA Processing,Posttranscriptional RNA Processing,RNA Processing, Post Transcriptional,RNA Processing, Posttranscriptional,Modification, Post-Transcriptional RNA,Modifications, Post-Transcriptional RNA,Post Transcriptional RNA Modification,Post Transcriptional RNA Processing,Post-Transcriptional RNA Modifications,Processing, Posttranscriptional RNA,Processing, RNA,RNA Modification, Post-Transcriptional,RNA Modifications, Post-Transcriptional
D012335	RNA, Ribosomal	The most abundant form of RNA. Together with proteins, it forms the ribosomes, playing a structural role and also a role in ribosomal binding of mRNA and tRNAs. Individual chains are conventionally designated by their sedimentation coefficients. In eukaryotes, four large chains exist, synthesized in the nucleolus and constituting about 50% of the ribosome. (Dorland, 28th ed)	Ribosomal RNA,15S RNA,RNA, 15S
D013768	Tetrahymena	A genus of ciliate protozoa commonly used in genetic, cytological, and other research.	Tetrahymenas
D014158	Transcription, Genetic	The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.	Genetic Transcription
D014544	Uridine Triphosphate	Uridine 5'-(tetrahydrogen triphosphate). A uracil nucleotide containing three phosphate groups esterified to the sugar moiety.	UTP,Magnesium UTP,Magnesium Uridine Triphosphate,Mg-UTP,Mg UTP,Triphosphate, Magnesium Uridine,Triphosphate, Uridine,UTP, Magnesium