A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Supercoiling response of E. coli promoters with different spacer lengths.

The effect of negative supercoiling on a series of synthetic Escherichia coli promoters has been investigated. These promoters carry perfect consensus sequences at the -35 and -10 regions, but with different spacer lengths (Aoyama, T. et al. (1983) Nucleic Acids Res. 11, 5855-5864). Topoisomeric plasmids carrying these synthetic promoters were constructed, and their activities were compared by detecting in vitro transcripts with the probe-hybridization method. In the relaxed state, the one with 17 basepairs (bp) spacing showed the highest activity, and the activity steeply decreased both sides of the optimal spacing. Similar results have been observed by run-off transcription. By introducing negative superhelicity, the 17 bp spacing promoter showed a relatively little response to supercoiling. In contrast, the activities of those with 16 and 18 bp spacings were markedly stimulated by supercoiling, with the mean, negative superhelical density (-sigma) which gave the maximum activity being about the same for the 16-18 bp spacing promoters (-sigma = 0.03 to 0.04). The promoter with 19 bp spacing, which showed no activity in the relaxed state, exhibited a significant activity at higher superhelicities (-sigma = 0.06). Even the 20 bp spacing promoter showed some activity by increasing superhelicity, while the 15 bp spacing promoter did not. On the basis of these observations, possible mechanisms by which negative supercoiling of DNA stimulates the protomer activity are discussed.

Reconstitution of nucleosomes in vitro with a plasmid carrying the long terminal repeat of Moloney murine leukemia virus.

The potential of nucleosome assembly along the sequence of a plasmid carrying the long terminal repeat (LTR) and its flanking region of Moloney murine leukemia virus was analyzed by in vitro reconstitution experiments with histones from chicken erythrocytes. The results of electrophoretic mobility-shift and micrococcal nuclease-digestion assays indicated that the plasmid DNA contained four preferred sites for nucleosome formation. However, all of these sites were mapped on the vector moiety but not on the LTR moiety. Computer analysis of the sequences in the four preferred sites, each spanning about 150 bp, indicated that short runs of (dA,dT) containing two kinds of triplets, AAA/TTT and AAT/ATT, occurred frequently. Furthermore, many of these triplets tended to occur in the same side of the DNA helix, suggesting that DNA curvature was involved in the preferred sites for nucleosome assembly. Consistent was the observation that DNA fragments carrying these preferred sites showed anomalous electrophoretic mobilities at a low temperature.

Location of the cooperative melting regions in bacteriophage fd DNA.

Differential melting profiles of the linear replicative form (RF-III) DNA of bacteriophage fd, of the fragments obtained by the restriction endonuclease R.HinHI and of those obtained by R.Hga were investigated. With these results a physical map which locates the cooperative melting regions on the DNA was constructed, and compared with the genetic map.

Binding of the regulatory protein VirG to the phased signal sequences upstream from virulence genes on the hairy-root-inducing plasmid.

The VirG protein is a positive regulator for the virulence genes of which expression is induced by a plant factor, and is essential for Agrobacterium pathogenicity on dicotyledonous plants. The VirG protein of the hairy-root-inducing plasmid A4 was overproduced in Escherichia coli cells, and purified to homogeneity. DNase I footprinting experiments revealed that the purified VirG protein was bound to the upstream region of virulence genes including the phased vir box sequences, which had been presumed to be the VirG recognition signal from the sequence analysis. In dimethyl sulfate footprinting, the VirG protein specifically protected the guanine residues within every vir box sequence. It was concluded that the VirG protein was bound to the phased vir box sequences from the major groove along one side of double-helical DNA.

Sequence organization of replication origin of the Escherichia coli K-12 chromosome.

A sequence of 245 base-pairs (oriC) in the replication origin of the Escherichia coli K-12 chromosome has been shown to provide all the information essential for initiation of bidirectional replication. In order to elucidate the sequence organization of oriC, numerous mutants carrying a single-to-multiple transitions from G X C to A X T base-pair were constructed by localized mutagenesis in vitro, which uses sodium bisulfite, and the correlation between the mutation sites and replicating ability (Ori function) was systematically analyzed. By isolating non-defective (Ori+) mutants with multiple base changes, transitions at 71 positions among 101 G X C pairs in oriC were found to have no effect on Ori function. Investigation of defective (Ori-) mutants, on the other hand, showed that individual replacements at 18 positions were detrimental to Ori function to some extent. These irreplaceable G X C pairs fell in the positions where no substitution was detected in the Ori+ mutants. The defect of the Ori- mutants with a single base substitution was generally weaker than that of the previously constructed Ori- mutants lacking a part of oriC. The addition of two or more base changes each giving a faint Ori- phenotype, however, resulted in a more intensive Ori- phenotype. We have previously demonstrated that oriC contains several regions where deletion or insertion of oligonucleotides leads to strong Ori- phenotypes. Transitions in those areas did not cause any defect of Ori function. Combining present results on base substitution mutants with the previous observations together, we assumed that the oriC sequence provides multiple interaction sites with replication initiation factors, and the precise arrangement of these sites are required for Ori function.

Sites of dnaA protein-binding in the replication origin of the Escherichia coli K-12 chromosome.

On the basis of the observation that dnaA protein binds preferentially to DNA fragments carrying the Escherichia coli chromosomal replication origin (oriC), the binding sites were investigated by DNase I footprinting. As a result, three strong binding sites were identified in the minimal oriC sequence. The respective binding sites were 16 to 17 base-pairs long, and contained a common sequence (5') T-G-T-G-(G/T)-A-T-A-A-C (3') in the middle, although their polarities were not the same. Since mutants defective in function for autonomous replication have been isolated in the corresponding positions of the common sequence at each binding site, dnaA protein-binding at these sites seems to be significant for replication initiation.

Electron microscopic visualization of restriction sites on DNA molecules.

DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% formamide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a single EcoRI site on a lambda dv1 molecule and three HinHI sites on an fd1RF molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction of a cleavage map.

An in vitro method generating base substitutions in preselected regions of plasmid DNA: application to structural analysis of the replication origin of the Escherichia coli K-12 chromosome.

A method for introducing base substitutions in defined regions of plasmid DNA has been developed. In principle, a circular heteroduplex DNA containing a gap is constructed by annealing of two kinds of linear molecules derived from the same plasmid: One is the molecule shortened either by exonucleolytic digestion from the termini generated at a restriction site or by removal of a region flanked by two restriction sites, and the other the full-length molecule linearized at a different site. The deleted region in the shorter linear molecule becomes a single-stranded gap in the circular heteroduplex DNA. The heteroduplex is then treated with sodium bisulfite that converts specifically cytosine residues to uracil residues in single-stranded regions. After filling in the gap by repair synthesis, transformation is carried out to isolate mutant plasmids. Since two kinds of circular heteroduplexes are formed by annealing in which the sequences in the gaps are complementary to each other, mutagenesis of both strands can be accomplished in one experiment. This method was applied to construction of mutants with base substitutions in the replication origin region (oriC) of the Escherichia coli K-12 chromosome which had previously been cloned in colicin E1 plasmid vectors, and various mutants in defined regions of oriC were successfully isolated at high efficiencies. Analysis of these mutants provided evidence that oriC contains special regions, designated spacers, which separate neighboring important sequences specifying interactions with initiation factors for DNA replication at precise distances.

The nucleotide sequence of the cloned tufA gene of Escherichia coli.

The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

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.

Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein.

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.

A study of the interaction between promotor DNA and T. thermophilus DNA-dependent RNA polymerase 1,2.

As the first step in the process of RNA synthesis, RNA polymerase binds to a specific site (promoter) and forms an open complex. In this process, it is considered that the structure of DNA is changed to an unidentified form. We investigated the structure of this DNA, which consists of an open complex, by means of CD. For this purpose, we used very stable RNA polymerase (of T. thermophilus HB8) and the fd-RF-DNA fragment (Hap-Hga V), which has only one promoter. We have confirmed that the complex of the holo enzyme with Hap-Hga V fragment at 50 degrees C is an open complex. We obtained the CD spectral difference between the open complex and its constituents for the first time. The observed CD difference spectra in the UV region (250-300 nm) were compared with the theoretical difference CD. It was deduced that the DNA of the open complex may be melted around the initiation point over a rather longer range than expected.

Evaluation of therapeutic efficacy in psychogenic impotence by means of logarithmic scoring.

In an attempt to develop as objective a method for the evaluation of impotence as possible, the authors assigned numerical values to four parameters considered essential to sexual function, namely, libido, erection, ejaculation, and orgasm, and used the sum totals of these scores, collected before and after initiation of therapy, as indicators of overall sexual function. Numerical values were assigned according to a logarithmic scale, in four stages from 0 to 10, i.e., 0, 1, 3, and 10; a score of 0 signified "normal" and a score of 10, "abnormal." By comparing sum totals of scores computed before and after initiation of therapy, the authors were able to evaluate therapeutic efficacy on the basis of changes in these sum totals. Using this method, the mean total score for a control group of 24 normal subjects was 1.67 +/- 0.26 (mean +/- standard error). For the test group, which consisted of 24 patients of psychogenic impotence, the mean total score prior to initiation of therapy was 16.46 +/- 3.55, an extremely high score in comparison with the control group. After four weeks of therapy, the mean total score dropped to 9.37 +/- 1.77, indicating a statistically significant (p less than 0.05) decrease over the pre-therapy mean total score.

Primary lipogranuloma of male genitalia.

Lipogranulomas developing secondarily in the genitourinary system have been reported rather frequently, but primary lipogranulomas without any past history of etiologically related conditions are rare. We report on 2 cases recently encountered in which tumors were diagnosed as sclerosing lipogranuloma on histopathologic examination.

Content and distribution of vasoactive intestinal polypeptide (VIP) in cavernous tissue of human penis.

Penile erection is controlled by a valvular structure in the helicine artery in humans. The opening and closing of this valve are believed to be regulated by the autonomic nervous system, especially through the release of vasoactive intestinal polypeptide (VIP). We determined the content of VIP in cavernous tissue in 18 impotent patients and in 5 normal controls by radioimmunoassay, and we examined the distribution of VIP-ergic nerve fibers in cavernous tissue by an immunohistochemical method. As a result, it was found that the lower penile VIP content was more frequent among patients with organic impotence than among the controls. Furthermore, VIP-ergic nerve fibers were seen to be diffusely and loosely distributed in a large number of organic impotence patients. These findings suggest that organic impotence in some patients may be due to decreases in the VIP content and in VIP-ergic nerve fibers.

[Clinical experience of Hachimijiogan for male infertility patients].

We treated 53 patients for male infertility with TSUMURA - Hachimijiogan given as a daily dose of 7.5 g for 144 days on the average. The sperm density was improved by administration to 10 X 10(6)/ml or more in 22 patients (41.5%) and lowered to 10 X 10(6)/ml or more in only 2 patients (3.8%). The sperm motility was improved by 10% or over in 29 patients (54.7%) as compared with 5 patients (9.4%) in whom it was lowered by 10% or more. The sperm motile efficiency index ( SMEI ) was improved in 40 (75.5%) of the 53 patients. Statistically significant differences were noted in the improvement in sperm density, sperm motility and SMEI . By contrast, there was no difference in semen volume, sperm morphology or sperm agglutination between the pre- and post- treatment periods. During the period of treatment, the wives of 4 patients (7.5%) conceived children. These results suggest that TSUMURA - Hachimijiogan is effective for male infertility to a certain extent and that clinical trials on its use for male infertility may be meaningful.

[Clinical experience of bromazepam for psychogenic impotence patients].

A clinical trial was performed with the tranquilizer Bromazepam on 39 patients undergoing diagnosis of psychogenic impotence, and the drug effect was evaluated according to the criteria based on our protocol. The protocol specifies 8 tests (1 for libido, 4 for erection, 2 for ejaculation, 1 for orgasm) which are scored according to an arbitrary logarithmic scoring system. At the end of the study the points made in the 8 tests were added to obtain the total score as the basis for evaluation of the overall drug effect. After treatment all tests showed an improvement, and the improvement in erection during masturbation, reflective erection, and condition of ejaculation was statistically significant. The total score also improved from 16.77 +/- 2.62 (mean +/- S.D.) before treatment to 11.42 +/- 1.96 after treatment, and the change was again statistically significant (P less than 0.05). The rate of satisfaction as a subjective symptom of improvement also increased from 25.38 +/- 4.40% to 39.10 +/- 4.53%. The results of the present study provide evidence to indicate that Bromazepam is beneficial for psychogenic impotence.

[A clinical evaluation of AMS Hydroflex in the treatment of impotence].

The AMS Hydroflex penile prosthesis was implanted in 34 organic impotence patients between June 20, 1986 and Aug. 6, 1990. Patients ages ranged between 26 and 71 with an average of 50.5. The causes of impotences were; 17 cases of post-radical pelvic surgery, 7 cases of injury, 6 cases of diabetes and 4 of others. First evaluation was made after 12 weeks of implantation in terms of patient satisfaction with sexual intercourse and post operative complication. Sixteen patients (47.1%) were highly satisfied with intercourse, 13 (38.2%) satisfied with intercourse, 2 (5.9%) slightly dissatisfied even with successful intercourse, 1 (2.9%) with no improvement and 2 (5.9%) could not follow. Utility of the prosthesis, e.g. effectiveness and safety was; high utility in 20 (58.8%), good utility in 11 (32.4%), slight utility in 1 (2.9%), no favorable in 1 (2.9%) and no judgement in 1 (2.9%). The overall effectiveness and safety of the Hydroflex was demonstrated based on the above findings. Long term evaluation was made from the initial implantation of the device up to the termination of the trial with average of 19.2 months and was mentioned altogether.

[A clinical evaluation of AMS 700 penile prosthesis in the treatment of impotence].

The AMS 700TM & 700 CXTM penile prosthesis was implanted in 34 organic and mixed-type impotent patients between November, 1984 and July, 1988. The patients' ages ranged between 32 and 70, with an average age of 57.2. Twelve weeks after implantation, the prosthesis was evaluated in terms of patient satisfaction with sexual intercourse and postoperative complications. Twenty three patients (67.7%) were highly satisfied, 7 (20.3%) satisfied, 3 (8.8%) dissatisfied and 1 patient worsened (2.9%). Severe complication was found in two cases, both of them being complicated by infection; as a result the prosthesis was removed. Utility of the prosthesis, e.g. effectiveness and safety was; high utility in 26 cases (76.5%), moderate utility in 7 cases (20.6%), and no favorable in one case (2.9%). Thus, overall effectiveness and safety of the AMS 700 penile prosthesis, with its excellent cosmetic appearance upon implantation, were demonstrated by the above findings.