Haruo Ozeki

Structure and organization of Marchantia polymorpha chloroplast genome: I. Cloning and gene identification

We have determined the complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha, using a clone bank of chloroplast DNA fragments. The circular genome consists of 121,024 base-pairs and includes two large inverted repeats (IRA and IRB, each 10,058 base-pairs), a large single-copy region (LSC, 81,095 base-pairs), and a small single-copy region (SSC, 19,813 base-pairs). The nucleotide sequence was analysed with a computer to deduce the entire gene organization, assuming the universal genetic code and the presence of introns in the coding sequences. We detected 136 possible genes. 103 gene products of which are related to known stable RNA or protein molecules. Stable RNA genes for four species of ribosomal RNA and 32 species of tRNA were located, although one of the tRNA genes may be defective. Twenty genes encoding polypeptides involved in photosynthesis and electron transport were identified by comparison with known chloroplast genes. Twenty-five open reading frames (ORFs) show structural similarities to Escherichia coli RNA polymerase subunits, 19 ribosomal proteins and two related proteins. Seven ORFs are comparable with human mitochondrial NADH dehydrogenase genes. A computer-aided homology search predicted possible chloroplast homologues of bacterial proteins; two ORFs for bacterial 4Fe-4S-type ferredoxin, two for distinct subunits of a protein-dependent transport system, one ORF for a component of nitrogenase, and one for an antenna protein of a light-harvesting complex. The other 33 ORFs, consisting of 29 to 2136 codons, remain to be identified, but some of them seem to be conserved in evolution. Detailed information on gene identification is presented in the accompanying papers. We postulated that there were 22 introns in 20 genes (8 tRNA genes and 12 ORFs), which may be classified into the groups I and II found in fungal mitochondrial genes. The structural gene for ribosomal protein S12 is trans-split on the opposite DNA strand. The universal genetic code was confirmed by the substitution pattern of simultaneous codons, and by possible codon recognition of the chloroplast-encoded tRNA molecules, assuming no importation of tRNA molecules from the cytoplasm. The nucleotide residue A or T is preferred at the third position of the codons (G+C, 11.9%) and in intergenic spacers (G+C, 19.5%), resulting in an overall G+C content that is low (28.8%) throughout the liverwort chloroplast genome. Possible gene expression signals such as promoters and terminators for transcription, predicted locations of gene products, and DNA replicative origins are discussed.

The 8 kDa polypeptide in photosystem I is a probable candidate of an iron-sulfur center protein coded by the chloroplast gene frxA

The N‐terminal sequence of the 8 kDa polypeptide isolated from spinach photosystem I (PS I) particles was determined by a gas‐phase sequencer. The sequence showed the characteristic distribution of cysteine residues in the bacterial‐type ferredoxins and was highly homologous to that deduced from the chloroplast gene frxA of liverwort, Marchantia polymorpha. It is strongly suggested that the 8 kDa polypeptide has to be an apoprotein of one of the iron‐sulfur center proteins in PS I particles.

Suppressor-sensitive mutants of coliphage ∅80

Abstract About fifty suppressor sensitive (sus) mutants of phage ∅80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in ∅80. Some presumed “early” mutants were also found. Defective lysogens were isolated from a ∅80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage ∅80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage ∅80 was determined. Clustering of all head genes and also of tail genes, as in phage λ, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of ∅80 was also similar to that of phage λ: namely, the cluster of head genes was found to be located at one end of the vegetative map of ∅80, being followed by that of tail genes, and presumed “early” genes are located at the other end of the map.

Chloroplast gene organization in plants

Abstract Complete sequences are now available for the chloroplast genomes of two green plants. The information that can be gleaned from these sequences should help us to understand not only how chloroplasts function within present-day plants, but may also yield insights into the evolutionary relationships of photosynthetic organisms and into the gene movement that has occurred among the varius genetic compartments of eukaryotic cells.

Mutant tyrosine tRNA of altered amino acid specificity

Each set of tRNA molecules accepting a particular amino acid are recognized and acylated by an amino acyl tRNA synthetase. This enzyme must recognize some feature in this set of tRNA molecules which distinguishes them from all other tRNAs. In this communication we describe the isolation and initial characterization of a set of E. coli tyr tRNA mutants that have altered amino acid specificity. These mutants should eventually lead to an understanding of which features of the tRNA are recognized by the tyrosine tRNA synthetase. Sequence analysis of one of the mutants (SU+III~-“~) has revealed that a single base change (A82 + G) near the amino acid acceptor end of the molecule is sufficient to alter its specificity.

Structure and organization of Marchantia polymorpha chloroplast genome. IV: Inverted repeat and small single copy regions

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

Precursor molecules of Escherichia coli transfer RNAs accumulated in a temperature-sensitive mutant☆

Abstract Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su+ 3 tRNA1Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.

Identification of transfer RNA suppressors in Escherichia coli: I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln

Abstract In order to isolate the gene for amber suppressor su + 2 ( SupE ) in Escherichia coli , a non-defective su + 2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′ su + 2 gal (λ) were selected, linking su + 2 to the right-hand prophage attachment site, att λ PB′ ; second, these F′-factors were relysogenized by λ and defective transducing phages, λd su + 2, were produced by induction; and third, non-defective λp su + 2 transducing phages were produced by recombination of λd su + 2 isolates with λ. Upon infection by λp su + 2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA 2 Gln , mutant tRNA 2 Gln and tRNA m Met . The mutant tRNA 2 Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA 1 Gln was not stimulated by the infection of λp su + 2. We conclude that the wild-type allele of su + 2 ( SupE ) is the structural gene for tRNA 2 Gln , and the su + 2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA 2 Gln . The fact that λp su + 2 also induces the production of tRNA m Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA 2 Gln .

Gross map location of Escherichia coli transfer RNA genes

Abstract Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F− strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNAf1Met gene newly determined, the λ-transducing phage carrying the tRNAf1Met gene was found.

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.