Hiroyuki Sugisaki

Nucleotide sequence of the kanamycin resistance transposon Tn903.

Abstract The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.

Nucleotide sequence of small ColE1 derivatives: Structure of the regions essential for autonomous replication and colicin E1 immunity

SummaryA small ColE1 derivative, pAO2, which replicates like the original ColE1 and confers immunity to colicin E1 on its host cell has been constructed from a quarter region of ColE1 DNA (Oka, 1978). The entire nucleotide sequence of pAO2 (1,613 base pairs) was determined based on its fine cleavage map. The sequence of a similar plasmid, pAO3, carrying additional 70 base pairs was also deduced.The sequence in the region covering the replication initiation site on these plasmids was consistent with those reported for ColE1 by Tomizawa et al. (1977) and by Bastia (1977). DNA sequences indispensable for autonomous replication were examined by constructing plasmids from various restriction fragments of pAO2 DNA. As a result, a region of 436 base pairs was found to contain sufficient information to permit replication. The occurrence of initiation and termination codons and of the ribosome-binding sequence on pAO2 DNA suggests that a polypeptide chain consisting of 113 amino acid residues may be encoded by the region in which the colicin E1 immunity gene has been mapped.

Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid

The nucleotide sequence of a 2248 bp portion of the plasmid mini-F has been determined. This region includes the replication origin and all of the plasmid-coded information required for replication. The same region is also capable of expressing incompatibility. A striking feature of the sequence is the presence of nine 19-bp repeating units. Four of these repeats, all arranged in one direction, comprise a cluster, and the remaining five, all arranged in the opposite direction, comprise another cluster. These clusters are separated by a region of about 850 bp that encodes a hypothetical 29-kd polypeptide. This region has sequences highly homologous to those found in the origin regions of the Escherichia coli (Sugimoto et al., 1979; Meijer et al., 1979) and Salmonella typhimurium (Zyskind and Smith, 1980) genomes.

Studies on bacteriophage fd DNA. IV. The sequence of messenger RNA for the major coat protein gene.

One of the RNA species transcribed in vitro on phage fd replicative form DNA is initiated at a site preceding the major coat protein gene and terminated immediately after this gene. The total sequence of this RNA seecies was determined. The transcript was 369 bases long, and contained the sequence identical to the ribosome-binding site for phage f1 coat protein gene (Pieczenik et al., 1974) at positions 88 to 119 and the sequence for coat protein at positions 175 to 324. The coat protein sequence was immediately followed by two termination codons UGA and UAA. The AUG codon appeared at the fifth and 23rd tripletframe upstream from the codon for the first amino acid (Ala) of coat protein. The latter AUG codon was located in the middle of the ribosome-binding site. The result strongly suggests that coat protein is formed from a precursor containing 23 extra amino acid residues at the N-terminus. The transcript was usually terminated with a sequence of eight U residues. It was also noted that there is a region of strong secondary structure near the 3′-end.

Structure and gene organization in the transforming Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.

Nucleotide sequence at the insertion sites of a kanamycin transposon

THE TRANSPOSON Tn903 carrying a gene for kanamycin resistance, is 3,100 base pairs in size and contains an inverted repeat sequence of 1,050 base pairs at both ends1–4. We report here the generation of a 9-base pair repeated sequence at the insertion site of Tn903. Tn903 can be transposed to at least nine different sites on the coliphage fd DNA1,2. We have also transposed Tn903 to three sites on small colicin E1 plasmid (ColE1) derivatives of about 1,600 base pairs. The presence of these many insertion sites on small recipient DNA molecules implies that no specific sequence is involved in the target sites on the recipient. To investigate the mechanism of transposition of Tn903 at the molecular level, we have now analysed three independent insertions of Tn903 on the small ColE1 DNA molecules. The nucleotide sequences of the three target sites and of the corresponding junctions between Tn903 and the small ColE1 have been determined.

Studies on bacteriophage fd DNA: II. Localization of RNA initiation sites on the cleavage map of the fd genome

Abstract In an in vitro RNA synthesizing system, a single size of A-start RNA and three different sizes of G-start RNA are predominantly transcribed on the doubly closed replicative form (RFI) DNA of phage fd. When the RFI DNA was cleaved into three fragments ( HinH-A, HinH-B and HinH-C ) by a restriction endonuclease from Haemophilus influenzae H-I, the A-start RNA was predominantly initiated on HinH-B and the three G-start RNAs on HinH-A . RFI DNA was further cleaved into smaller pieces by two other restriction endonucleases from H. aphirophilus and H. gallinarum . Upon mixing the digests with RNA polymerase, two specific fragments derived from HinH-A were bound to the polymerase with GTP present. G-start RNA was efficiently initiated on the fragments isolated by this procedure. On the basis of these observations and estimates of the size of RNA formed on each fragment, the initiation sites for major RNA species were localized on the cleavage map of the phage fd genome previously constructed.

Studies on bacteriophage fd DNA: I. A cleavage map of the fd genome☆

Abstract In order to construct a physical map of the bacteriophage fd genome, the doubly closed replicative form (RFI) DNA of phage fd was cleaved into unique fragments by four different restriction endonucleases (Hap, Hga, HinH and Hind) prepared from Haemophilus strains H. aphirophilus, H. gallinarum, H. influenzae H-I and H. influenzae Rd, respectively. As Hind cleaved RFI DNA at a single site, this site was used as a reference point for mapping. HinH cleaved RFI DNA at three sites, Hga at six sites and Hap at 13 sites, respectively. The 5′-termini of the fragments produced by either HinH or Hga were labelled with 32P in the polynucleotide kinase reaction. The labelled fragments were separated and further cleaved by other enzymes. The re-digestion products of partially digested fragments were also analysed. On the basis of these data and estimates of the size of each fragment, a cleavage map of the phage fd genome was constructed.

Cloning and Characterization of the Genes Encoding Enzymes for the Protocatechuate Meta-degradation Pathway of Pseudomonas ochraceae NGJ1

The 2-pyrone-4,6-dicarboxylate lactonase gene (proL), the protocatechuate 4,5-dioxygenase α and β subunits genes (proOa and proOb), and the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (proD) were cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1. These genes were in the order proLOaObD on the DNA, and a possible transcription terminator sequence followed. The proL and proD genes were over-expressed in Escherichia coli, and their gene products were purified for identification, while the expression of proOaOb was at a lower level. The protocatechuate meta-degradation operon was reconstituted with the recombinant plasmids and expressed successfully in E. coli.

Molecular and Catalytic Properties of Monoacetylphloroglucinol Acetyltransferase from Pseudomonas sp. YGJ3

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl−. Cl− and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M r=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG]2) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.