Characterization of the 3′ untranslated region of mouse DNA topoisomerase IIα mRNA

Expression of DNA topoisomerase IIα protein varies through the cell cycle with its peak in G₂/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIα mRNA stability as well as promoter activity. We isolated the 3′ genomic region of the mouse topoisomerase IIα gene and investigated whether or not the 3′ untranslated region (UTR) of the topoisomerase IIα mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIα 3′ UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3′ region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5′ splice site. The minority of the non-spliced topoisomerase IIα 3′ UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIα 3′ genomic region, or the major spliced form of the 3′ UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIα promoter. Our results strongly suggest that the mouse topoisomerase IIα 3′ UTR by itself is not involved in the cell-cycle-dependent mRNA stability.     

No role of the 5′ untranslated region of ornithine decarboxylase mRNA in the feedback control of the enzyme

The polyamines are ubiquitous in nature and appear to fulfil several important functions, mostly related to growth, in the cell. The first, and often rate-limiting, step in the biosynthesis of the polyamines is catalysed by ornithine decarboxylase (ODC), which is subject to a variety of control mechanisms. The polyamines exert a strong feedback regulation of the expression - as well as the degradation of the enzyme. The regulation of ODC expression appears to occur at the translational level. The ODC mRNA contains a long GC-rich 5 untranslated region (UTR), which has been demonstrated to hamper the translation of the mRNA. However, it has not yet been conclusively established whether this part of the mRNA fulfils any function in relation to the polyamine-mediated control of ODC synthesis. In the present study, we have used stable transgenic CHO cells, expressing either full-length ODC mRNA or 5 UTR-truncated ODC mRNA, to elucidate the role, if any, of the 5 UTR in the translational regulation of the enzyme by polyamines. No differences in regulatory properties were observed between the cells expressing the full-length ODC mRNA and those expressing the ODC mRNA devoid of most the 5 UTR. The cell lines down-regulated ODC (synthesis as well as activity) to the same extent upon exposure to an excess of polyamines, demonstrating that the feedback control of ODC mRNA translation occurs by a mechanism independent of the major part of the 5 UTR of the ODC mRNA.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

Expression of the glutathione peroxidase gene lacking its 3′ untranslated region

In order to investigate the expression of human cellular glutathione peroxidase (GPx), we mutated the gene encoding GPx by deleting either the 5 or 3 untranslated region (utr), subcloned the deleted fragments into plasmid pSVL followed by transfection into COS-7 cells and measured the amount of GPx expressed. When the 5 utr of the gene was deleted, GPx was not expressed. However, the deletion of the 3 utr resulted in some expression of GPx. Deletion of the poly A region of the GPx gene resulted in the expression of GPx but the level was lower than that of the full-length cGPx. The complete deletion of the 3 utr resulted in a half of the expression of the poly A deletion mutant. Thus, the expression of GPx increased according to the length of the 3 utr. These results suggest that the GPx gene carrying one SECIS on 5 utr (FEBS Lett. 312(1992)10-14) is essential for GPx expression. SECIS on 3 utr might not play a key role of GPx expression. Expression of GPx by COS-7 cells was not observed when a plasmid harboring an antisense gene was transfected.     

Sequence simplicity and evolution of the 3′ untranslated region of the histone H1° Gene

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site. Correspondence to: P. Suau     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

A novel NcoI polymorphism creates a fifth haplotype in the 3′ untranslated region of CKM

A novel NcoI polymorphism has been detected in the 3untranslated region of the creatine kinase (CKM) gene. The additional NcoI restriction site creates a fifth haplotype for the NcoI and TaqI restriction fragments length polymorphisms at this locus, and segregates with the myotonic dystrophy gene in 3 generations of an affected family.     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

Construction of a deletion-scanning library for the 5′-proximal promoter region of the bean β-phaseolin gene

A library of internal deletion mutants was constructed in the 5′-proximal promoter region of the gene encoding the bean seed storage protein β-phaseolin. A nick was introduced randomly in the target DNA sequence by depurination and treatment with exonuclease III, and served as the initiating point for deleting adjacent DNA sequences by S1 nuclease. A syntheticPst I linker was ligated to the blunt-ended DNA to serve as a restriction marker for mapping the approximate position of deletion mutants. Subcloning of a kanamycin marker gene into the linker site facilitated selection of plasmid DNA in which internal deletions were introduced in the target DNA sequence. Distribution of internal deletion mutants was mapped by determining the locations of thePst I site in the target sequence. DNA sequence analysis of mutants indicated that the extent of internal deletions ranged from 6 to 100 bp with a mean of 39 bp. In addition, the CAAT and TATA boxes in the promoter region of the β-phaseolin gene were effectively dissected in these mutants.     

A novel polymorphism in the 5′ untranslated region of theporcine cytochrome b5 (CYB5) gene is associated with decreased fat androstenone level

Raising intact male pigs would have a significant economic impact on the pork industry; however, the presence of 16-androstene (a major cause of boar taint) in meat from male pigs would be highly objectionable to consumers. In pigs, a positive correlation has been found between cytochrome b5 (CYB5) and production of 16-androstene. The search for polymorphism of CYB5 and functional analysis of polymorphism found should have an important impact on the efforts to develop genetic markers to select for low androstenone levels in fat from pigs. The aim of this study was to search the porcine CYB5 gene for mutations, examine its expression, identify genetic polymorphisms, and study how a genetic variation in this enzyme translates into interindividual variation in androstenone levels in fat from pig testis. We have identified a single nucleotide polymorphism (SNP) (G→T) at base 8 upstream of ATG in the CYB5 5′ untranslated region which is associated with a lower fat androstenone level. Of the 229 testis samples tested, 84.8% were homozygous for the variant G, 12.4% were heterozygous, and 2.8% were homozygous for the variant T. Functional analysis of this mutation revealed that an individual homozygous for the T allele showed significantly lower CYB5 activity than an individual homozygous for the G allele. Thus, this may be at least partially responsible for a lower level of androstenone in pigs. Our findings provide an important genetic basis toward the goal of predicting the androstenone status in pigs and developing genetic markers for low androstenone.