Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

Upstream open reading frames (uORFs) are often found in the 5′-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression.     

Biochemical characteristics ofS-adenosylmethionine decarboxylase from carnation (Dianthus caryophyllus L.) petals

We have partially purified S-adenosylmethionine decarboxylase (EC 4.1.1.50, SAMDC) from carnation (Dianthus caryophyllus L.) petals and generated polyclonal antibodies against CSDC 16 protein (Leeet al., 1996) overexpressed inE. coli. The protein has been purified approximately 126.8 fold through the steps involving ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephacryl S-300 gel filtration. Its molecular mass was 42 kDa in native form and we could also detect a band of 32 kDa molecular mass on SDS-PAGE in western blot analysis using the polyclonal antibodies. The Km value of this enzyme forS-adenosylmethionine was 26.3 μM. The optimum temperature and pH forS-adenosylmethionine decarboxylase activity were 35°C and pH 8.0, respectively. Putrescine and Mg²⁺ had no effects on the activation of the enzyme activity. Mg²⁺ did not have any significant effects on the enzyme activity. SAMDC activity was inhibited by putrescine, spermidine and spermine. Methylglyoxal bis-(guanylhydrazone) (MGBG), carbonyl reagents such as hydroxylamine and phenylhydrazine, and sulfhydryl reagent such as 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB) were effective inhibitors of the enzyme. However, isonicotinic acid hydrazide known as an inhibitor of 5′-pyridoxal phosphate (PLP) dependent enzyme activity had no significant effect on the enzyme activity. These results and our previously reported results (Leeet al., 1997b) suggest thatS-adenosylmethionine decarboxylase is a heterodimer, αβ, and some carbonyl group and sulfhydryl group are involved in the catalytic activity.     

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.     

5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5′-untranslated regions (5′-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, ~10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.     

Contextual Features of Higher Plant mRNA 5"-Untranslated Regions

Computer analysis of nucleotide sequences of 5"-untranslated regions (5"-UTR) of higher plant mRNA adopted from the EMBL nucleotide sequence database was carried out. It was demonstrated that the average nucleotide frequencies of the leader sequences and adjacent regions of basal promoters are similar, whereas introns and 3"-UTR have a higher content of T and a lower content of C. A particular 5"-UTR contextual feature is a misbalance in the content of complementary nucleotides, probably caused by negative influence of the stable secondary structure on the translation properties of the leader sequence. Approximately 20% of 5"-UTR possess AUG triplets, i.e., twice as much as it has been estimated earlier. The properties of the open reading frames of the leader sequence (uORF) and presumable causes of their high content in 5"-UTR of eukaryotic mRNAs are discussed. The nature of correlation between some features of uORFs and protein-coding gene sequences is analyzed. It is demonstrated that in effectively translated mRNAs the leader AUG triplets are more frequently located in a nonoptimal context, whereas the terminating codons of uORFs more frequently exist in the optimal one. A hypothesis is put forward that the efficiency of termination at the uORF stop codon might substantially interfere with the mRNA translation activity.     

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses

Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5' upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart.     

Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)⁺ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1–3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt1 but differing in the 3-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyltransferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.     

Expression of a humanS-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.     

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.