The genetic code in bovine mitochondria: sequence of genes for the cytochrome oxidase subunit II and two tRNAs

Bovine-heart mitochondrial DNA from a single animal was isolated and fragments representative of the entire genome cloned into multicopy plasmid vectors to facilitate determination of its complete nucleotide sequence. We present here the sequence of the region covering the gene for cytochrome oxidase subunit II. Comparison of this sequence with the amino acid sequence of the homologous beef-heart protein has enabled the determination of most of the bovine mitochondrial genetic code. The code differs from the "universal" genetic code in that UGA codes for tryptophan and not termination, and AUA codes for methionine and not isoleucine. The only codon family not represented is the AGA/AGG pair normally used for arginine; evidence from other genes suggests that these code for termination in bovine mitochondria. The sequence presented also includes the adjacent tRNAAsp and tRNALys genes. The tRNAAsp gene is separated by one nucleotide from the 5' end of the COII gene and only three bases separate the 3' end of this gene and the adjacent tRNALys gene. This highly compact gene organisation is very similar to that found in the corresponding region of the human mitochondrial genome and the gene arrangement is identical. The structure of the respective bovine and human tRNAs vary primarily the "D-" and "T psi C-loops".     

In vitro construction of yeast tRNAAsp variants: nucleotide substitutions and additions in T-stem and T-loop.

A procedure for the construction of 3'-end labelled yeast tRNAAsp harboring substitutions or additions of any desired nucleotide in T-stem and T-loop (position 57 to 61) has been developed. This was done by in vitro enzymatic manipulations of the yeast tRNAAsp involving specific hydrolysis with RNases, phosphorylation and dephosphorylation with T4 polynucleotide kinase and ligation with T4 RNA ligase. Using this procedure we have replaced conserved or semi-conserved nucleotides located in position 57 to 61 of yeast tRNAAsp. We have also constructed different yeast tRNAAsp with eight bases instead of seven in T-loop. Further use of these tRNAAsp variants will be discussed with the help of the crystallographic three-dimensional structure.     

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.     

Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.     

Characterization of a rat tRNA gene cluster: comparison of nucleotide sequences of the gene for tRNALeu newly found in repeating units

In the rat, DNA carrying a cluster of the genes for tRNAAsp, tRNAGly, and tRNAGlu, aligned in that order, is repeated about 10 times. Seven DNA clones corresponding to the independent repeating units were isolated from a rat gene library. Nucleotide sequence analysis of these clones revealed the presence of a fourth tRNA gene, the gene for tRNALeu, in the cluster. The tRNALeu gene is located about 600 base pairs (bp) upstream from the tRNAAsp gene and its polarity differs from those of the other three tRNA genes. Among the repeating units, the nucleotide sequence of tRNALeu is conserved to a relatively high degree.     

ColIb plasmid genes that inhibit the replication of T5 and T7 bacteriophage

The colicin Ib (ColIb) plasmid genes that inhibit the replication of the T5-like and T7 bacteriophage have been cloned on an approximately 7200-bp ClaI fragment and their sites relative to each other and to the colicin immunity (imm) gene have been mapped. The inhibition of wild-type T7 by the clone is shown to be caused by the same gene or genes (pic) that cause the inhibition of T7 kinase-negative mutants and is a different gene than the one that causes inhibition of T5 (ibf or abi). The pic gene does not hybridize to the pif genes of the F plasmid that also cause the replication of T7 to be inhibited. The abi gene and the pic gene map very closely together but are under the control of different promoters. The abi gene has a maximum size of 900 bp and lies approximately 3000 bp away from the immunity gene, distal to the colicin gene. A site which maps in or near the gene binds very tightly to Escherichia coli RNA polymerase. The pic gene or genes lie between the abi gene and the imm gene and are contiguous with abi. Promoters for pic have been mapped and hypotheses to explain the inhibition of T7 by a cloned gene but not the whole ColIb plasmid are presented.     

Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes.

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

The nucleotide sequence of mannosyl-Q-containing tRNAAsp from Xenopus laevis oocytes

The nucleotide sequence of tRNAAsp from X. laevis oocytes was determined as being: (sequence in text) The tRNA is 75 nucleotides long. This sequence is very similar (75% to 97% identity) to all other eukaryotic tRNAAsp sequenced so far, except for the bovine liver tRNAAsp (32% identity). The relation between the presence of a mannosyl group on queuosine (Q) at position 34 and the nucleotide sequence of the anticodon loop is discussed.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: I. Batch cultures and kinetic modeling

A novel Eschericha coli expression system directed by bacteriophage T7 RNA Polymerase utilized for overexpression of the cloned gene. The recombinant cell contains the plasmid with a bacteriophage promoter, the T7 promoter, to regulate the expression of the target gene. This promoter is recongnized only by T7 RNA polymerase, whose gene has been fused into the host chromosome and is under control of the lacUV5 promoter. Therefore, the target gene on the plasmid can be expressed only in the presence of T7 RNA polymerase, which is induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The batch cultures were performed to investigate the effect of induction on kinetics of cell growth and foreign protein formation and to determine the optimal induction strategy. It was observed that the specific growth rates of the recombinant cells dramatically decrease after induction, and that there is an optimal induction time for maximizing the accumulated intracellular foreign protein. This optimal induction time varies singificantly with inducer concentration. To better understand the optimal behavior, a lumped mechanistic model was constructed to analyze the induced cell growth and foreign protein formation rates. (c) 1992 John Wiley & Sons, Inc.     

Expression of the gene for the polyoma small T antigen in Escherichia coli.

A cloned segment of the polyoma virus genome encoding the small T antigen has been fused, in the correct phase for translation, to the 5' end of the beta-galactosidase gene. The hybrid gene, cloned in Escherichia coli, produces a protein resembling the small T antigen.