Eiichi Ohtsubo

Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110

With the goal of solving the whole‐cell problem with Escherichia coli K‐12 as a model cell, highly accurate genomes were determined for two closely related K‐12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single‐nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino‐acid residue deletion, five amino‐acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13 000 bases) were mostly within portions sequenced with out‐dated technology based on radioactive chemistry.

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post‐termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature‐sensitive Escherichia coli mutants which we isolated in this study. At non‐permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re‐initiate translation at various sites in all frames without the presence of an initiation codon. Re‐initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Isolation and characterization of IS elements repeated in the bacterial chromosome.

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.

A 37 × 103 molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1☆

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.

Identification and characterization of novel retrotransposons of the gypsy type in rice.

Abstract We found that two DNA fragments, which were obtained from Oryza sativa L cv. IR36 by PCR using degenerate primers designed for amplification of a rice gene, showed homology with the rt gene encoding reverse transcriptase of the Drosophila retrotransposon gypsy. We named the element from which they originated RIRE3 (for rice retrotransposon No. 3) and analyzed it further by isolating various clones containing segments of RIRE3. Nucleotide sequencing of the clones revealed that RIRE3 has LTRs (2316 bp) and that the internal sequence (5775 bp) includes a large ORF with gag and pol regions; the pol region includes the rt gene as well as the int gene encoding integrase in this order, as in gypsy. Interestingly, the region upstream of gag in RIRE3 contained another open reading frame, here called orf0, which does not exist in gypsy or in other retrotransposons related to it. In the course of characterizing RIRE3, we obtained a further clone, which showed less homology with the pol region of RIRE3. This clone was found to be derived from another gypsy-type retrotransposon (named RIRE8) containing the LTR sequence and orf0 both of which were only weakly homologous to that in RIRE3. Further characterization of RIRE8 revealed that there were actually two subtypes of RIRE8 (named RIRE8A and RIRE8B), which show little homology to each other in the orf0 region. Although the LTRs of RIRE3 and RIRE8 elements show very weak homology with each other, there exists a conserved sequence at their termini. We therefore carried out PCR using primers which hybridize to the rt gene of RIRE3, and total genomic DNA from various monocot and dicot plants as templates, and found that a family of RIRE3 elements was present in all plants tested.

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis

SummaryThe nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acids; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA1 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA1 either encodes a gene product or structure information that is responsible for incompatibility.

Bacterial insertion sequences.

While DNA has a property of being fundamentally stable as invariable genetic information, studies on gene expression and gene organization have revealed that the genome is often subject to dynamic changes. Some of these changes are brought about by mobile genetic elements which have been found in prokaryotic and eukaryotic genomes so far studied. Insertion sequences (ISs) are bacterial mobile DNA elements which cause various kinds of genome rearrangements, such as deletions, inversions, duplications, and replicon fusions, by their ability to transpose. These were discovered during investigation of mutations that are highly polar in the galactose and lactose operons of Escherichia coli K−12 (Jordan etal. 1968; Malamy 1966, 1970; Shapiro 1969) and in the early genes of bacteriophage λ (Brachet et al. 1970). Many of these mutations were shown by electron microscope heteroduplex analysis to be insertions of distinct segments of DNA which are hence called insertion sequences (Fiandt et al. 1972; Hirsch et al. 1972; Malamy et al. 1972). It was later shown that the transcription of flanking genes can originate from promoters located within an IS or from hybrid promoters created by the insertion event or by the IS-mediated genome rearrangements. An important note here is that the finding of IS elements as mobile elements to new loci to turn genes either off or on would re-evaluate the controlling elements described in maize by McClintock (1956, 1965) (see Starlinger and Saedler 1976).

Site Specific Recombination Involved in the Generation of Small Plasmids

SummaryThe physical structures of seven small plasmids, Rsc10, Rsc11, Rsc12, Rsc13, Rsc15, Rsc10-1 and pEM1 were analyzed. Molecular lengths of these plasmids were determined to range from 7.65 to 19.8 kilobases or kb. Electron microscope heteroduplex analysis of these plasmids show that the plasmids were all derived from pKN102 (86.3kb) in a complicated process that takes place by a series of deletion and, in some cases, transposition events. Rsc10 and Rsc11 were each formed by a simple deletion event from the parental plasmid. The physical structures of Rsc13 and pEM1 suggest that these plasmids must have been derived by a single and two successive deletion events from Rsc11. In the formation of these plasmids, all the deletions occured at the ends of the transposon, Tn3, which confers ampicillin resistance (amp) to the plasmid, or at the ends of the insertion sequence, IS1. Rsc15 was assumed to be formed in a two step process. The first step was a deletion event to form Rsc10-1 which occurs at one end of the IS1 present in pKN102. At first, the deletion event leaves out the ampicillin gene but in the second step Tn3 is transposed to the newly formed plasmid, Rsc10-1. Rsc12 is believed to have been formed in a similar fashion; first, a series of deletions and second, the transposition of Tn3.Studies on these small plasmids enabled us to also map the regions of the replication genes and ampicillin resistance on pKN102.

Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes

Abstract During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs.

The whorl‐specific action of a petunia class B floral homeotic gene

GREEN PETAL (GP) is thought to be a petunia class B floral homeotic gene, because the gp mutant flower displays a severe homeotic conversion of petals into sepals in the second whorl. However, since the third whorl stamens remain unaffected in the gp null mutant, gp is different from class B mutants in Arabidopsis and Antirrhinum, which also show a conversion of the third whorl stamens into the carpelloid tissue. BLIND (BL) is thought to be a petunia class A floral homeotic gene, because the bl mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures in the second whorl.