Processing of mammalian rRNA precursors at the 3' end of 18S rRNA. Identification of cis-acting signals suggests the involvement of U13 small nucleolar RNA.

Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis-acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence.

Actinomycin D stimulates the transcription of rRNA minigenes transfected into mouse cells. Implications for the in vivo hypersensitivity of rRNA gene transcription.

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long been known, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.

Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells.

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells.

Alternative pre-rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis.

rRNA processing pathways in humans have been reinvestigated through systematic Northern-blot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenin-labeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41S, 32S and 21S pre-rRNA, two major pre-rRNA fractions were identified; a '30S' (about 5800 nucleotides) precursor to 18S rRNA containing an external transcribed spacer at the 5' end (ETS) and internal transcribed spacer (ITS) 1 sequences and a '12S' (about 950 nucleotides) precursor to 5.8S rRNA containing ITS 2 sequences.

Cloning and high level expression of a synthetic gene for human basic fibroblast growth factor.

A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein. The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter. This promoter directed the synthesis of a fusion protein between a 31 amino acids fragment of the lambda phage cII protein and bFGF.