Mituru Takanami

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.

Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication.

SummaryA DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA+ cells, whereas pBR322 replicates only in polA+ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori+ and Ori- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.

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.

A stable ribonucleoprotein for amino acid incorporation

Abstract A ribonucleoprotein (RN-P), which is stable and active for incorporation in vitro of [ 14 C]amino acid, has been prepared from rat liver in an almost pure form. The method of purification is based on the fact that the RN-P complex is reversibly precipitated by an increasing concentration of divalent cations, such as Mg and Ca. In the purified preparation, the ratio of u.v. absorption maximum (260 mμ) to minimum (235 mμ) was 1.58 and the N:P ratio approximately 3.34. Maximum incorporation in vitro of [ 14 C]amino acid into the purified RN-P was obtained by addition of ATP, an ATP-generating system, GTP, and a soluble protein fraction. The optimal pH for incorporation was in the range of 7.8–8.0, and the reaction was accelerated by a low concentration of KCl.

Characterization and sequence determination of the replicator region in the hairy-root-inducing plasmid pRiA 4b

SummaryStarting from a cosmid library of the 250 kb hairy root inducing plasmid pRiA 4b, a mini-pRiA 4b replicon of 4.6 kb was isolated. This mini-plasmid was stably maintained in Agrobacterium species and its replication characteristics, such as temperature-sensitive replication, copy number and incompatibility, were similar to those of the parental pRiA 4b. Analysis of deletion mutants indicated that almost the entire 4.6 kb region was required for autonomous replication. The nucleotide sequence of mini-pRiA 4b was determined by the chain-termination method. Three large open reading frames, which are likely to contribute to the replication of pRiA 4b, were identified in the same orientation along the sequence.

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.

Observations on the structure of the termination factor rho and its attachment to DNA

Abstract The termination factor “rho” appears as a hexagonal arrangement of six subunits of identical size. The approximate diameters of the hexagonal particle and each subunit are 115 A and 40 A, respectively. On mixing with the replicative form DNA of bacteriophage fd, a limited number of the rho factor is seen attached to the circular DNA molecule.

Starting nucleotide sequences of RNA synthesized on the replicative form DNA of coliphage fd

Abstract The replicative form DNA, with no single-strand break (RF-I DNA), was purified from Escherichia coli cells infected with a filamentous coliphage fd, and the starting nucleotide sequences of RNA formed by E. coli RNA-polymerase under the direction of RF-I DNA as template were analysed. Incorporation of four nucleoside [γ-32P]triphosphates into the 5′-end of RNA was studied, and it was found that RNA chains synthesized on RF-I DNA were initiated predominantly with ATP- and GTP-ends. The ratio of the ATP-end to GTP-end was roughly 1:1.6 to 2.0 under the conditions used. RNA was synthesized in the reaction mixture containing [γ-32P]ATP and [3H]ATP. The labelled RNA was hydrolysed with pancreatic and T1 RNases, and radioactive fragments produced were analysed. The main terminal fragment identified was pppApUpGp. RNA synthesized with [γ-32P]GTP and [3H]GTP was hydrolyzed with pancreatic RNase, and resulting radioactive fragments were analysed. The predominant terminal fragment produced was pppGpUp. In order to identify the third nucleotide of RNA starting with the GTP-end, RNA was synthesized with nucleoside [α-32P]triphosphates, and nearest-neighbour analysis was carried out. A and U were found in the third position. It was concluded that at least three species of RNA starting with the following sequences were synthesized under the direction of RF-I DNA of phage fd: pppApUpGp-----, pppGpUpAp-----, pppGpUpUp-----.