Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis,Nipponia nippon

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.     

The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression

Endothelin-1 is a 21-amino acid potent vasoconstrictor peptide produced by vascular endothelial cells. We have cloned the whole length of the human preproendothelin-1 (PPET-1) gene and the corresponding cDNA and determined the complete nucleotide sequences. The 2026-nucleotide human mRNA for PPET-1 (excluding the polY(A) tail) is encoded in five exons distributed over 6836 base pairs of the genome. The 5'-flanking region of the gene contains (i) octanucleotide sequences for the phorbol ester-responsive elements, also known as the binding elements for FOS.JUN complex; (ii) consensus motifs for the binding site of nuclear factor 1, which may mediate the induction described previously of PPET-1 mRNA by transforming growth factor-beta; (iii) hexanucleotide sequences for the acute phase reactant regulatory elements that may be involved in the induction of endothelin-1 under acute physical stress in vivo. Further, the 3'-nontranslated sequence of human PPET-1 mRNA contains three AUUUA motifs, which may mediate selective translation-dependent destabilization of the mRNA. Northern blot analysis in cultured endothelial cells from human umbilical veins shows that PPET-1 mRNA is in fact rapidly induced by the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate within 10 min. Analysis of mRNA life span by using actinomycin D demonstrates that PPET-1 mRNA has a short intracellular half-life of about 15 min and is superinduced by cycloheximide. This superinduction is found to be due to the stabilization of the mRNA by cycloheximide, as in the case of other known AUUUA-containing mRNAs. These findings suggest that the regulation of expression of PPET-1 mRNA may be mediated in part by these sequence elements.     

Stability control of MTL1 mRNA by the RNA-binding protein Khd1p in yeast

Khd1p (KH-domain protein 1) is a yeast RNA-binding protein highly homologous to mammalian hnRNP K. Khd1p associates with hundreds of potential mRNA targets including a bud-localized ASH1 mRNA and mRNAs encoding membrane-associated proteins such as Mid2p and Mtl1p. While Khd1p negatively regulates gene expression of Ash1p by translational repression, Khd1p positively regulates gene expression of Mtl1p by mRNA stabilization. To investigate how Khd1p regulates the stability of MTL1 mRNA, we searched for cis-acting elements and trans-acting factors controlling MTL1 mRNA stability. Regional analysis revealed that partial deletion of the coding sequences of MTL1 mRNA restored the decreased MTL1 mRNA and protein levels in khd1Δ mutants. This region, encompassing nucleotides 532 to 1032 of the Mtl1p coding sequence, contains CNN repeats that direct Khd1p-binding. Insertion of this sequence into other mRNAs conferred mRNA instability in khd1Δ mutants. We further searched for factors involved in the destabilization of MTL1 mRNA. Mutations in CCR4 and CAF1/POP2, encoding major cytoplasmic deadenylases, or of SKI genes, which code for components of a complex involved in 3' to 5' degradation, did not restore the decreased MTL1 mRNA levels caused by khd1Δ mutation. However, mutations in DCP1 and DCP2, encoding a decapping enzyme complex, and XRN1, encoding a 5'-3' exonuclease, restored the decreased MTL1 mRNA levels. Furthermore, Khd1p colocalized with Dcp1p in processing bodies, cytoplasmic sites for mRNA degradation. Our results suggest that MTL1 mRNA bears a cis-acting element involved in destabilization by the decapping enzyme and the 5'-3' exonuclease, and Khd1p stabilizes MTL1 mRNA through binding to this element.     

Determinants of Drosophila fushi tarazu mRNA instability.

The fushi tarazu gene is essential for the establishment of the Drosophila embryonic body plan. When first expressed in early embryogenesis, fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, fushi tarazu mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of fushi tarazu mRNA is probably crucial for attaining this localized pattern of expression. mRNA stability in transgenic embryos was measured by a new method that does not use drugs or external interference. Experiments using hybrid genes that fuse fushi tarazu sequences to those of the stable ribosomal protein A1 mRNA provide evidence for at least two destabilizing elements in the fushi tarazu mRNA, one located within the 5' one-third of the mRNA and the other near the 3' end (termed FIE3 for ftz instability element 3'). The FIE3 lies within a 201-nucleotide sequence just upstream of the polyadenylation signal and can act autonomously to destabilize a heterologous mRNA. Further deletion constructs identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other previously identified destabilization sequences suggests that FIE3 contains a novel RNA destabilization element.     

二甲基苯并蒽对肺肿瘤细胞P1—450基因表达的调节

Dimethylbenzanthracene (DMBA) stimulates expression of P1-450 gene in human lung tumor cells (ChaGo). A concentration and time-dependent increase in the level of P1-450 specific mRNA sequences has been observed in ChaGo cells treated with sublethal concentrations of DMBA. The methylation pattern of "-CCGG-" sequence of most of the coding region and of the 3' end of P1-450 gene is not affected by such DMBA treatment; DMBA-treatment of the cell induces hypomethylation of only the 5' end "-CCGG-" sequences of the gene. These results demonstrate that DMBA-treatment of ChaGo cells induced increased expression of gene can be correlated to the increased degree of site specific hypomethylation of the internal "-C-" residues of the "-CCGG-" sequences located at the 5' end of the P1-450 gene. However, it is also possible to have other molecular mechanism of regulation.     

The 5''-proximal region of the wheat Cab-1 gene contains a 268-bp enhancer-like sequence for phytochrome response.

We have previously reported that the expression of the wheat Cab-1 gene is subject to phytochrome regulation and a 1.8-kb 5' upstream sequence of this gene is sufficient for the regulated expression. To delineate sequences for the phytochrome response we analyzed a series of 5' deletion mutants as well as chimeric gene constructs comprising different sequences of the Cab-1 upstream region in transgenic tobacco seedlings. We found that a deletion mutant containing a 357-bp 5' upstream sequence still exhibits maximal levels of phytochrome-regulated expression. A 268-bp enhancer-like element, located between -89 and -357, is responsible for the phytochrome response of the Cab-1 gene; sequences upstream from -357 to -843 and downstream from -124 to +1100 are probably not involved. Finally, we show that the Cab-1 mRNA stability is not regulated by phytochrome.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.