Expression in Spiroplasma citri of an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for cloning and expressing foreign genes in S. citri, an organism which reads UGA as a tryptophan codon (C. Stamburski, J. Renaudin, and J.M. Bové, J. Bacteriol. 173:2225-2230, 1991). We now report cloning and expression in S. citri of the G fragment of cytadhesin P1 gene from Mycoplasma pneumoniae. The G fragment was inserted in the SpV1 RF downstream of a synthetic ribosome binding site and introduced into S. citri by electroporation. Northern (RNA) blot analyses showed that in S. citri, the G fragment was transcribed from an SpV1 RF promoter as a 1.2-kb mRNA. The translation product was detected by Western blotting (immunoblotting) with a rabbit antiserum raised against total proteins from M. pneumoniae (strain FH) and was proved to be P1 specific by using monoclonal antibodies specific for the G region of the P1 protein. The apparent molecular mass of the polypeptide (24.5 kDa) indicates that in S. citri, the G fragment was fully translated in spite of the seven UGA codons present in the reading frame.     

Lessons from sequenced genomes. Overlapping genes in Methanococcus jannaschii?

This paper describes our finding on overlapping genes in Methanococcus jannaschii genome. We found that one of the open reading frames (ORFs) within the M. jannaschii genome contains the nucleotide sequence of tRNA(Ser), which raises a serious question of the correctness of the initiation codon assignment for that ORF. We suggest that there are two other possible AUG initiation codons downstream from the TTG triplet, which was initially considered as a translation start site. Only one of the AUG triplets is preceded by the Shine-Dalgarno sequence that seems to be required for binding the ribosome and initiation of translation.     

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria.     

Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency

Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.     

Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5′-terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.     

Control of start codon choice on a plant viral RNA encoding overlapping genes.

The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.     

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.     

赤麂线粒体全基因组的序列和结构

提取赤麂细胞株总DNA,参照我们实验室已测定的同属动物小麂线粒体全基因组序列设计引物,PCR扩增、测序、拼接,获得赤麂线粒体全基因组序列并进行生物信息学分析。赤麂线粒体全基因组序列全长16354bp。定位了22个tRNA基因、2个rRNA基因、13个蛋白编码基因和1个D-loop区。赤麂与小麂及其它哺乳动物线粒体的基因组结构相同,它们的序列同源性都较高。     

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.     

Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.     

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.     

G+C content variation along and among Saccharomyces cerevisiae chromosomes.

Past analyses of the genome of the yeast Saccharomyces cerevisiae have revealed substantial regional variation in G+C content. Important questions remain, though, as to the origin, nature, significance, and generality of this variation. We conducted an extensive analysis of the yeast genome to try to answer these questions. Our results indicate that open reading frames (ORFs) with similar G+C contents at silent codon positions are significantly clustered on chromosomes. This clustering can be explained by very short range correlations of silent-site G+C contents at neighboring ORFs. ORFs of high silent-site G+C content are disproportionately concentrated on shorter chromosomes, which causes a negative relationship between chromosome length and G+C content. Contrary to previous reports, there is no correlation between gene density and silent-site G+C content in yeast. Chromosome III is atypical in many regards, and possible reasons for this are discussed.