Discovery and characterization of Acanthamoeba castellanii mitochondrial 5S rRNA

Although 5S rRNA is a highly conserved and universal component of eubacterial, archaeal, chloroplast, and eukaryotic cytoplasmic ribosomes, a mitochondrial DNA-encoded 5S rRNA has so far been identified only in land plants and certain protists. This raises the question of whether 5S rRNA is actually required for and used in mitochondrial translation. In the protist Acanthamoeba castellanii, BLAST searches fail to reveal a 5S rRNA gene in the complete mitochondrial genome sequence, nor is a 5S-sized RNA species detectable in ethidium bromide-stained gels of highly purified mitochondrial RNA preparations. Here we show that an alternative visualization technique, UV shadowing, readily detects a novel, mitochondrion-specific small RNA in A. castellanii mitochondrial RNA preparations, and that this RNA species is, in fact, a 5S rRNA encoded by the A. castellanii mitochondrial genome. These results emphasize the need for caution when interpreting negative results that suggest the absence of 5S rRNA and/or a mitochondrial DNA-encoded 5S rRNA sequence in other (particularly protist) mitochondrial systems.     

Oocyte and somatic 5S ribosomal RNA and 5S RNA encoding genes in Xenopus tropicalis.

We have investigated the structure of oocyte and somatic 5S ribosomal RNA and of 5S RNA encoding genes in Xenopus tropicalis. The sequences of the two 5S RNA families differ in four positions, but only one of these substitutions, a C to U transition in position 79 within the internal control region of the corresponding 5S RNA encoding genes, is a distinguishing characteristic of all Xenopus somatic and oocyte 5S RNAs characterized to date, including those from Xenopus laevis and Xenopus borealis. 5S RNA genes in Xenopus tropicalis are organized in clusters of multiple repeats of a 264 base pair unit; the structural and functional organization of the Xenopus tropicalis oocyte 5S gene is similar to the somatic but distinct from the oocyte 5S DNA in Xenopus laevis and Xenopus borealis. A comparative sequence analysis reveals the presence of a strictly conserved pentamer motif AAAGT in the 5'-flanking region of Xenopus 5S genes which we demonstrate in a separate communication to serve as a binding signal for an upstream stimulatory factor.     

Isolation and characterization of the bovine corticotropin/beta-lipotropin precursor gene

The entire bovine corticotropin/beta-lipotropin precursor gene has been isolated as a set of overlapping genomic DNA fragments which extend over a length of approximately 17000 base pairs. Restriction mapping of the cloned DNA fragments and nucleotide sequence analysis of the whole mRNA-coding segments and their surrounding regions have established that the corticotropin/beta-lipotropin precursor gene is approximately 7300-base-pairs long and contains two intervening sequences; one with an approximate length of 4000 base pairs is located within the segment encoding the 5'-untranslated region of the mRNA, and the other with an approximate length of 220 base pairs interrupts the protein-coding sequence near the signal peptide region. Sequence analysis of more than 200 base pairs preceding the proximal end of the corticotropin/beta-lipotropin precursor gene has revealed a 'Hogness box' and a variant of the model sequence d(G-G-TC-C-A-A-T-C-T) as well as palindrome structures as observed in other eukaryotic genes. Furthermore, some sequence similarities in the 5'-flanking region are found between the corticotropin/beta-lipotropin precursor gene and the mouse alpha-globin and beta-globin genes, all of which are negatively regulated by glucocorticoids. At least four homologous repetitive sequences are distributed at 3000-5000-base-pair distances in the corticotropin/beta-lipotropin precursor gene region; two such sequences are located in the 5'-flanking region, and one within each intervening sequence. Blot hybridization analysis of bovine pituitary nuclear RNA has indicated that the entire corticotropin/beta-lipotropin precursor gene is transcribed into a primary hnRNA product, which is then spliced to form the mature mRNA.     

Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development

A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined. The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb. Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons. There are eight introns in both the human and the hamster des genes. Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved. A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns. These sequences all belong to the human AluI family. When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al. [Cell 43 (1985) 327-338] for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes [Minty and Kedes, Mol. Cell. Biol. 6 (1986) 2125-2136] in the 5'-flanking region. The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene. Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.     

Loci for human U1 RNA: structural and evolutionary implications

Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.     

Complete DNA sequence coding for the large ribosomal RNA of yeast mitochondria.

The mitochondrial gene coding for the large ribosomal RNA (21S) has been isolated from a rho- clone of Saccharomyces cerevisiae. A DNA segment of about 5500 base pairs has been sequenced which included the totality of the sequence coding for the mature ribosomal RNA and the intron. The mature RNA sequence corresponds to a length of 3273 nucleotides. Despite the very low guanine-cytosine content (20.5%), many stretches of sequence are homologous to the corresponding Escherichia coli 23S ribosomal RNA. The sequence can be folded into a secondary structure according to the general models for prokaryotic and eukaryotic large ribosomal RNAs. Like the E.coli gene, the mitochondrial gene contains the sequences that look like the eukaryotic 5.8S and the chloroplastic 4.5S ribosomal RNAs. The 5' and 3' end regions show a complementarity over fourteen nucleotides.