The Yeast Omnipotent Suppressor Sup46 Encodes a Ribosomal Protein Which Is a Functional and Structural Homolog of the Escherichia Coli S4 Ram Protein

The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.     

Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense： Identification and Copy Number

The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 （AmphiS23） and L35 （AmphiL35） from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates. Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in therat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.     

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。     

A 32 kDa protein--whose phosphorylation correlates with oncogenic Ras-induced cell cycle arrest in activated Xenopus egg extracts--is identified as ribosomal protein S6

Oncogenic Ras induces cell-cycle arrest in mammalian cells and in fertilized Xenopus eggs. How oncogenic Ras induces cell-cycle arrest remains unclear. We previously showed that oncogenic Ras induces cell-cycle arrest in activated Xenopus egg extracts (cycling extracts) and that the induced cell-cycle arrest correlates with hyperphosphorylation of a 32 kDa protein. However, the identity of the 32 kDa protein was not known. By using a sucrose density-gradient centrifugation, Triton X-100-acetic acid-urea (TAU)-gel electrophoresis, composite agarose-polyacrylamide gel electrophoresis (CAPAGE), SDS-PAGE, and partial tryptic peptide sequence analysis, the 32 kDa protein has now been identified as S6, a 40S subunit ribosomal protein. Hence, our results indicate that the oncogenic Ras-induced cell-cycle arrest is correlated with hyperphosphorylation of S6, suggesting that phosphorylation of S6 plays an important role in the induced cell-cycle arrest. It has been shown that conditional deletion of gene encoding S6 in mammalian cells prevents proliferation, demonstrating the importance of S6 in cell proliferation. The exact role S6 plays in cell proliferation is unclear. However, phosphorylation of S6 has been implicated in the regulation of protein synthesis. Thus, our results are consistent with the concept that oncogenic Ras induces S6 phosphorylation to influence protein synthesis, thereby contributing to the cell-cycle arrest. In addition, our results also demonstrate that composite agarose-polyacrylamide gel electrophoresis is suitable for the separation of large molecular complexes.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Structure and regulation of a Candida albicans RP10 gene which encodes an immunogenic protein homologous to Saccharomyces cerevisiae ribosomal protein 10.

The Candida albicans clone cDNA10 was isolated on the basis that it encodes a protein which is immunogenic during infections in humans (R. K. Swoboda, G. Bertram, H. Hollander, D. Greenspan, J. S. Greenspan, N. A. R. Gow, G. W. Gooday, and A. J. P. Brown, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45 degrees C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.     

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast)

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.     

The cloning and sequencing of the UDP-galactose 4-epimerase gene (galE) from Avibacterium paragallinarum.

The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES-2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase.     

Complete mitochondrial genome of three Branchiostegus (Perciformes, Malacanthidae) species: genome description and phylogenetic considerations

We cloned and sequenced the complete mitochondrial DNA (mtDNA) of three tilefishes (Branchiostegus albus, Branchiostegus argentatus, and Branchiostegus japonicus) to characterize and compare their mitochondrial genomes (mitogenomes). The mitogenomes of B. albus, B. argentatus, and B. japonicus were 16,532, 16,550, and 16,541 bp long, respectively, and all consisted of 37 genes (13 protein-coding genes, 2 ribosomal RNA, and 22 transfer RNA (tRNAs)), which are typical for vertebrate mtDNA. As in other bony fishes, most genes were encoded on the H-strand, except for the nad6 and eight tRNA genes that were encoded on the L-strand. Among the 13 protein-coding genes of all three tilefishes, 2 reading-frame overlaps were found on the same strand: atp8 and atp6 overlapped by 10 nucleotides, and nad4L and nad4 overlapped by 7 nucleotides. The identity of the nad4 gene between B. albus and B. argentatus was the lowest at 87%. Conversely, the identity of the nad6 gene between B. albus and B. japonicus was the highest at 99%. Most tRNA genes were similar in length among the three species, while the tRNA-Ser((AGY)) of B. japonicus was 9 bp longer than those of B. albus and B. argentatus. The control region of the mitogenome spanned 853, 862, and 856 bp in B. albus, B. argentatus, and B. japonicus, respectively. A maximum likelihood tree constructed using 11,035 sites contained five independent groups with bootstrap values of 100% in support of their divergence. All three tilefishes examined were clustered with the Pomacanthidae species in Group II.     

4个棉花ADF基因的分子鉴定及其差异表达

肌动蛋白解聚合因子(actin-depolymerizing factor, ADF)是一种在真核生物中广泛存在的低分子量的肌动蛋白结合蛋白,它在调控细胞内肌动蛋白纤丝的解聚合和再聚合中起着关键作用。我们在棉纤维cDNA文库中分离克隆了4个ADF基因(cDNAs),分别命名为GhADF2,GhADF3,GhADF4,GhADF5。GhADF2 cDNA 长度为705 bp,编码139个氨基酸;GhADF3 cDNA长度为819 bp,编码139个氨基酸;GhADF4 cDNA长度为804 bp,编码143个氨基酸;GhADF5 cDNA长度为644 bp,编码141个氨基酸。分析表明,GhADF2与GhADF3的氨基酸序列同源性为99%。而且,GhADF2/3与矮牵牛PeADF2之间的氨基酸序列同源性也高达89%。GhADF4与拟南芥AtADF6的亲缘关系较近,二者的氨基酸序列同源性为78%。GhADF5与拟南芥AtADF5的亲缘关系较近,氨基酸序列的同源性为83%。上述结果表明植物ADF基因在进化中具有高度保守性。RT-PCR分析表明,GhADF2在纤维中优势表达,而GhADF5基因则在子叶中表达量最高。另一方面,GhADF3和GhADF4似乎不具有组织特异性或偏爱性表达。同一组织中不同GhADF基因表达量有较大的差异,表明它们可能涉及棉花不同组织生长发育过程的调节。而且,在进化过程中,各ADF同分异构体之间可能发展形成某种功能上的差异性。     

The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.