Identification of a highly conserved 3′ UTR in the translationally regulated mRNA for prostaglandin synthase

Prostaglandin H synthase (E.C. 1.14.99.1) is induced by growth factors and lymphokines such as EGF and IL-1, and is suppressed by anti-inflammatory glucocorticoids. Inhibition of enzyme synthesis by glucocorticoids is mediated by a novel translational control that appears to involve conversion of the PG synthase mRNA into a cryptic non-hybridizable form.

In order to understand expression of the enzyme in more detail, a full length 2.8 Kb cDNA was cloned from a human embryonic lung cell cDNA library and the complete mRNA including the 3′ untranslated region (3′ UTR), was sequenced. The coding sequence for the human PG synthase shows greater than 90% homology with the sheep and mouse enzymes. A high degree of conservation (70%), however, was also observed in the approximately 750 nucleotide sequence that comprises the 3′ non-coding domain of both sheep and human PG synthase mRNA's and with the approximately 900 nucleotide 3′ UTR of the mouse RNA (68% sheep vs mouse; 47% human vs mouse). Extensive microregions of 10–30 nucleotides are distributed throughout the 3′ UTR where homology between species is 95–100%.

This high degree of conservation in a non-coding region and recent evidence from other genes suggests that these 3′ UTR sequences have important regulatory functions possibly related to translational control of this mRNA by growth factors and glucocorticoids.     

Coding and 3' non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme

The nucleotide sequence of an almost complete cDNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) has been determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A-rich stretch of the 5' non-coding region and the complete 3' non-coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N-terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent mol. wt. of the subunit of the purified enzyme.     

Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.     

Analytical procedures for a cryptic messenger RNA that mediates translational control of prostaglandin synthase by glucocorticoids

Expression of the enzyme prostaglandin H synthase in cultured vascular smooth muscle cells required epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) and was inhibited by cycloheximide but not actinomycin D. Preincubation with the glucocorticoid dexamethasone (0.5 microM) blocked the EGF-induced expression of prostaglandin H (PGH) synthase. Following dexamethasone addition, levels of hybridizable mRNA for PG synthase were reduced by over 90% within 1 h. After dexamethasone was removed, PG synthase mRNA recovered within 3 h by a process that was not inhibited by actinomycin D. These observations, together with other findings, suggested that the mRNA was being converted into some nonextractable and nontranslated form, probably by binding of a glucocorticoid-induced protein to the conserved 3' untranslated region. In order to investigate further the nature of this phenomenon, seven different literature procedures were evaluated for extracting and determining the PG synthase mRNA. Five of the seven procedures failed to detect hybridizable PG synthase mRNA in glucocorticoid-treated cells. Two procedures, however, recovered mRNA in both glucocorticoid-treated and control cells. A comparison of the protocols indicated that only those methods that incorporate a cationic detergent (sodium N-lauroylsarcosine), instead of anionic detergents in the lysis or homogenization buffers, successfully extract the glucocorticoid-suppressed PG synthase mRNA. Based upon these results two procedures are described, one that optimizes the extraction and determination of the glucocorticoid-suppressed (cryptic) form of the mRNA, and another which optimizes the analysis of normal mRNA without extracting the cryptic form. The results indicate that translational control of PG synthase by glucocorticoids is regulated by converting the mRNA into a cryptic form that is more firmly tissue bound than normal mRNA.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein.

Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.     

Molecular cloning of murine p68, a Ca2+-binding protein of the lipocortin family

A plasmid cDNA library prepared from a T-lymphocyte clone of murine strain B10.A origin was screened by cross-species DNA hybridisation using a partial human p68 cDNA clone, identified as containing coding sequences for previously determined amino-acid sequences. The longest p68 cDNA insert from this library and a full-length cDNA insert from a second similar library were fully sequenced. A comparison of the derived amino-acid sequence with that of human p68 revealed extensive homology (95% overall). Homology at the nucleotide level was 89% in the open reading frame and 85% and 50% in the 5' (33 nucleotides) and 3' (347 nucleotides) non-coding regions respectively. Eight segments of internal homology were observed, each containing a highly conserved consensus region of 17 amino acids correlating with that described for several membrane associated calcium-binding proteins [Geisow, M. J., Fritsche, U., Hexham, J. M. & Johnson, T. (1986) Nature (Lond.) 320, 636-638]. These results provide further evidence that p68 is a member of the same gene family as p32,p36 and lipocortin I and demonstrate an unusually high level of inter-species sequence conservation of p68 between mouse and human.     

Structural characterization and tissue-specific expression of the mRNAs encoding isoenzymes from two rat mitochondrial creatine kinase genes.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy transduction. Mitochondrial CK (MtCK) reversibly catalyzes the transfer of high energy phosphate to creatine and exists, in the human, as two isoenzymes encoded by separate genes. We report here the cDNA sequences of the two isoenzymes of MtCK in the rat. Rat sarcomeric MtCK has 87% nucleotide identity in the 1257 bp coding region and 82% in the 154 bp 3' untranslated region as compared with human sarcomeric MtCK. Rat ubiquitous MtCK has 92% nucleotide identity over the 1254 bp coding region with human ubiquitous MtCK and 81% identity of the 148 by 3' untranslated region. Nucleotide identity between the rat sarcomeric and ubiquitous MtCK coding regions is 70%, with no conservation of their 3' untranslated regions. Thus, MtCK sequence is conserved in a tissue-specific, rather than species-specific, manner. Conservation of the 3' untranslated regions is highly unusual and suggests a regulatory function for this region. The NH2-terminal transit peptide sequences share 82% amino acid homology between rat and human sarcomeric MtCKs and 92% homology between rat and human ubiquitous MtCKs, but have only 41% homology to each other. This tissue-specific conservation of the transit peptides suggests receptor specificity in mitochondrial uptake. Rat sarcomeric MtCK mRNA is expressed only in skeletal muscle and heart, but rat ubiquitous MtCK mRNA is expressed in many tissues, with highest levels in brain, gut and kidney. Ubiquitous MtCK mRNA levels are dramatically regulated in uterus and placenta during pregnancy. Coexpression of sarcomeric and ubiquitous MtCK with their cytosolic counterparts, MCK and BCK, respectively, supports the creatine phosphate shuttle hypothesis and suggests that expression of these genes is coordinately regulated.