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We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of the Escherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed by E. coli primase in vivo and in vitro, we propose that (i) E. coli primase itself recognizes a 3'GTC 5' sequence on the template strand, (ii) DnaB helicase releases the specificity of E. coli primase and, (iii) the consensus recognition sequence for E. coli primase associated with DnaB helicase is 3'PuPyPy 5'.

The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.

1. The structures, including stereochemistry, of the two major metabolites of pravastatin sodium in an isolated rat hepatocyte system, i.e. the 4'a alpha-glutathione conjugate (CM-1) and the 3',5'-dihydrodiol (CM-2), were determined by one- and two-dimensional n.m.r. spectroscopy. 2. The structures of two synthetic pravastatin epoxides, possible precursors of the metabolites, were also established. 3. One of the synthetic epoxides, 4'a beta, 5' beta-epoxide was converted to the pravastatin metabolite, 4'a alpha-glutathione conjugate (CM-1) by a rat liver cytosol system and is proposed as the common metabolic intermediate between pravastatin sodium and the metabolites, CM-1 and CM-2.

Prostaglandin E(1)-induced hypotension (25% reduction from the preadministration level in mean arterial pressure) was applied to thirteen patients. Eight patients among them were operated in the supine position (group I) and other five in the prone position (group II). The maintenance dose of PGE(1) was considerably lower in group II than in group I (0.067 micro g.kg(-1).min(-1) vs. 0.119 micro g.kg(-1).min(-1)). In group I, there was a significant increase in CI, with a significant decrease in SVRI and PVRI during PGE(1)-induced hypotension. Such a high dose of PGE(1) (0.119 micro g.kg(-1).min(-1)) was considered to have a direct dilating action on the systemic resistance bed as well as on the pulmonary vasculature. It was considered that the suppression of hypoxic pulmonary vasoconstriction could be a mechanism to increase venous admixture during PGE(1)-induced hypotension. In group II, there was no significant increase in CI, and no significant decrease in SVRI and PVRI. PGE(1)-induced hypotension can be safely applied to the anesthetized patients, but we should be careful to apply it to the patients in the prone position, because lower dose of PGE(1) can induce severe hypotension, which is not accompanied by the increase in CI as occurres in the patients in the supine position.

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.

A decoupling structure using an additional LC resonant configuration for NMR transmit coils is analysed and the probe sensitivity of the receive coil with the decoupled transmit coil is calculated using simple circuit models. An adjustment-free technique for the decoupling circuit under different loading conditions is proposed using the calculated results and demonstrated by experiments. The proposed method can simplify the probe operation.

A carboxypeptidase of St. griseus K-1 (CPase S) was found to possess the specificities of both mammalian pancreatic CPase A and B. Three adsorbents for affinity chromatography were prepared by coupling l-Leu, d-Leu, and d-Arg with CH-Sepharose 4B. d-Arg-CH-Sepharose and l-Leu-CH-Sepharose retained the purified CPase S but d-Leu-CH-Sepharose did not. The activities of CPase S toward CGL and BGA were eluted in the same position. CPase S migrated as a single band on polyacrylamide gel electrophoresis and the two activities were both extracted from this band on the gel.

A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76.2% in nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.

Large amounts of extra proteins, X (in the envelope fraction) and X' (in the cytoplasmic fraction) were detected by SDS-polyacrylamide gel electrophoresis when DNA of Escherichia coli was damaged. These two proteins had the same apparent molecular weight (appproximately 40,000) and were produced under identical conditions, including requirement for the recA" and lexA+ genotype. Sucrose density gradient centrifugation revealed that protein X' consisted of relatively large and heterogeneous aggregates in the cytoplasmic fraction; the distribution of protein X in the envelope was not determined. As proteins X and X' were shown to be equivalent, it is suggested that they are identical. Co-precipitation of the aggregates of protein X' with the envelope led to the appearance of protein X in the envelope fraction.

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.

Deuterium oxide affected all types of transport processes of ions in rice seedlings. The processes of the active transport of Rb+, the passive diffusion of Ca2+, and the preferential and specific absorption of Ge by the seedlings in D2O solutions, were suppressively distorted by D2O. The suppressive effect on the translocation of all ions studied and the absorption of Rb+, was greater with higher concentrations of D2O. However, clear-cut relations of the effect of increasing D2O concentrations on the ion transport were not obtained. The extent of the replacement of H2O by D2O in the plant appeared to affect the movement of ions in the plant.

To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [gamma-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono- to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [alpha-32P]GTP, indicating that they were the intact primer RNA with tri- (or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.

For studying microcirculation of intact tissues or organs, a new noninvasive, heat-transient method using laser photoexcitation is described. As a heating source an argon ion laser or R6G dye laser was used via a quartz fiber optic guide. Infrared radiation from heated tissue was detected by a thermography apparatus. After switching the laser irradiation on and off, a thermal transient curve of a model tissue, the skin, was biphasic: a component with a fast time constant and the other with a slow time constant. The temperature increases of both phases following laser irradiation were linear to the applied power from the laser onto the tissue. The temperature rises as a function of the wavelength of irradiated light have shown that the absorption of the light by tissue hemoglobin is a main heat-generation source. Furthermore, the temperature rises as a function of tissue blood volume and flow have shown that the component with a slow time constant is more related to the tissue hemoglobin concentration and tissue blood flow and heat conductivity. Thus, the analysis of heat-transient curves following laser irradiation gives information as to regional tissue blood volume, blood flow, and tissue heat conductivity.

Increases in afterload cause marked decreases in cardiac output and left ventricular stroke volume in patients with congestive heart failure. We studied the effects of afterload reduction therapy with prostaglandin E1 in three patients with intraoperative heart failure and two patients with low output syndrome after coronary artery bypass grafting. After 120 minutes, mean pulmonary arterial pressure decreased from 25.6 to 17.4 mmHg, pulmonary wedge pressure from 20.0 to 10.2 mmHg and right atrial pressure from 10.2 to 6.8 mmHg. Cardiac index increased from 1.73 to 2.80 l.min-1.m-2. The improvement of peripheral circulatory failure was remarkable and all patients recovered from heart failure. This study suggests that PGE1 is effective for afterload reduction therapy.

It is important to examine the presence of preformed antibodies in hemodialysis patients before renal transplantation, because it causes the graft rejection in organ transplantation. In the 239 hemodialysis patients we analysed 3 preformed antibodies divided TW, BW and BC. Sensitized rate (TW+, BW+ and BC+) was 0% at no blood transfusions (BT), 9.3% at 1-5 BT and 28.9% at 6 more BT. The number of blood transfusions increased the sensitized rate (TW). Sensitized rate (BW+, BC+) was 6.2% at no BT, 9.3% at 1-5BT and 12.0% at 6 more BT, while sensitized rate (BC+) was 33.3% at no BT, 26.7% at 1-5BT and 14.5% at 6 more BT. There was no correlation between the number of blood transfusions and the sensitized rate (BW+, BC+). At one year after the first check, 69.2% of TW positive patients was converted to TW negative and 53.8% of BW positive patients was converted to BW negative each. Further studies of the preformed antibodies are required to assess the clinical course of long surviving patients.

Profound hypothermia below 20 degrees C achieved by surface cooling using simple ice water bath equipment and deep ether anaesthesia is used with the aid of autonomic nerve blocking agents to obtain cardiac arrest for periods of over one hour for open-heart surgery. Blood levels of ether were between 40.6 mg/dl and 285.7 mg/dl during anaesthesia. No arrhythmia occurred and vital signs were quite stable. Hypocarbia throughout the procedure, severe base deficit after circulatory arrest, spontaneous recovery of metabolic acidosis, and a nearly normal cH+ (pH) were observed. Catecholamine increased moderately after circulatory arrest, but was far below shock levels. Plasma renin activity was markedly elevated but angiotensin II stayed at non-significant levels throughout the procedure. Excess lactate showed no significant change. Hyperglycaemia was noted. The mortality rate was 7.7 per cent and neurological disorders occurred in less than 5.8 per cent of the recent 52 cases.

The patient with spinal progressive muscle atrophy (SPMA) has many anesthetic problems; muscle atrophy, respiratory complications, hypersensitivity to non-depolarizing muscle relaxants and succinylcholine-induced hyperkalemia. Although neurological complications following spinal and epidural anesthesia have been reported in various neurologic diseases, we chose epidural anesthesia for the patient with SPMA and experienced no problems during and after the operation. We conclude that epidural anesthesia is a useful technique for the patient with SPMA.

Prostaglandin E1 (PGE1) has a direct vasodilating effect to smooth muscles of resistant vessel much more than that of capacitance one. In afterload mismatch, vasodilator which acts on resistant vessel mainly should be selected. We used PGE1 for vasodilator therapy to two patients with low cardiac output syndrome (LOS) in intensive care unit. One patient undergoing coronary artery bypass grafting developed LOS and disseminated intravascular coagulation which derived from post-transfusional anaphylactoid reaction. Another patient undergoing mitral valve replacement, tricuspid valve annuloplasty and coronary artery bypass grafting also developed LOS with massive thrombosis caused by cold agglutinin during cardio-pulmonary bypass. In both patients, PGE1 decreased systemic vascular resistance index and pulmonary arterial resistance index, and increased cardiac output. PGE1 inhibits release of lysosomal enzymes from polymorphonuclear leukocytes and increases splanchnic blood flow. These effects lead to inhibit production of myocardial depressant factor and might relate to the improvement of LOS partially. We suggest that PGE1 was effective for LOS after cardiac surgery, especially in afterload mismatch.

The effect of anesthesia and surgery on cell-mediated immunity was investigated in patients with chronic renal failure (CRF) having latent immunological abnormalities compared with patients with normal renal function (NRF) as control. The patient had minor surgery with enflurane anesthesia. During the perioperative period the numbers of peripheral lymphocyte and its subsets were measured by single and two-color analysis, by applying monoclonal antibodies against lymphocyte membrane surface markers. While the number of peripheral lymphocyte was lower in CRF group than in NRF group throughout the whole period, it decreased significantly on the first and third post-operative days in CRF group. No change in peripheral lymphocyte subsets and CD 4/CD 8 ratio was observed except for increase in CD 8 one hour after incision and decrease in CD 8 on the first postoperative day. Significant difference from NRF group was observed neither in peripheral lymphocyte subset nor in CD 4/CD 8 ratio. In minor surgery under enflurane anesthesia no disturbance on immunological system was observed even in the patients with chronic renal failure.

Thallium-201 has been suggested as a useful diagnostic agent for myocardial scanning. We report herewith the clinical usefulness of Tl-201 as a result of our animal studies with rats as well as clinical trial for human being. After intravenous injection, we killed rats at regular intervals, then measured the activity in each anatomized organ by gamma-well counter and observed the internal distribution and examined autoradiograms with the naked eye. As a result, the accumulation of Tl=201 in the myocardium observed in 5 min after injection, reaching the maximum level in 30 min and gradually decreased hereafter. We performed 22 myocardial clinical scans. In adults we could clearly detect the lesion of myocardial infarction, but in children found it difficult to obtain full sufficient information due to small heart size and high pulse rate. Judging from the foregoing we think it possible to obtain much clearer images by using a data analyzer.

An enzyme electrode system for the determination of creatinine and creatine was developed by utilizing three enzymes: creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl), and sarcosine oxidase (SO). These enzymes were co-immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure, which has selective permeability to hydrogen peroxide. Two kinds of multi-enzyme electrodes were constructed by combining a polarographic electrode for sensing hydrogen peroxide and an immobilized CA/Cl/SO membrane or Cl/SO membrane for creatinine plus creatine or creatine, respectively. The multi-enzyme electrodes responded linearly up to 100 mg of creatinine and creatine per liter in human serum. Response time was 20 s in the rate method and the detection limit was 1 mg/L. Only 25 microL of serum sample is required. Analytical recoveries, precisions, and correlations with the Jaffé method were excellent, and the multi-enzyme electrodes were sufficiently stable to perform more than 500 assays. No loss of activity of immobilized enzymes was observed after nine months of storage at 4 degrees C in air.

A 46-year-old man, with TTP and renal failure, received splenectomy because complete remission was not achieved after the treatments such as plasma exchange, antiplatelet agents and corticosteroid. After the administration of methylprednisolone, anesthesia was induced with thiamylal and succinylcholine chloride and maintained with nitrous oxide, enflurane, fentanyl and vecuronium. Total blood loss was 1710 gram. Four units of packed red cell and 9 units of fresh frozen plasma were infused. The anesthetic course was uneventful. TTP is a rare and usually fatal disease and its etiology is unknown. Careless infusion of platelet or fresh blood may accelerate the disease process and multiorgan dysfunction may present other anesthetic problems. In this report, problems of anesthetic management for TTP were discussed.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.

Potassium absorption by the root of rice seedlings, equilibrated with D2O before K+-absorption, decreased proportionally with D2O concentration and the translocation of the absorbed cations by roots to shoots decreased markedly above 40% D2O. An inhibition curve for the absorption process drawn as a relative rate vs. D2O concentration exhibits no threshold in concentration of D2O. While K+-absorption of the seedlings unequilibrated with D2O did not change up to 60% D2O and decreased above 80%. The concentration of D2O of root tissue water reached a balance with the external concentration within 30 min, but the concentration of shoot water required 5-6 h to balance with the external. The concentration of both root and shoot water did not coincide with the external. The suppressive effect of D2O on potassium absorption links not only to D/H ratio in root water but also in shoot water.

The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG. Upstream to these sequences, there is a typical procaryotic promoter. TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.

Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 microM dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro.

PGE1 has a beneficial effect on cardiac failure with mitral valve regurgitation by decreasing the "after load". An 82-year-old female had a total cystectomy of a bladder tumor. The preoperative standard 12 lead electrocardiogram showed atrial fibrillation and incomplete right bundle branch block. The preoperative echocardiogram showed regurgitation of both mitral valve and tricuspid valve. Under heavy premedication, we intubated with fentanyl and pancuronium bromide, maintained anesthesia with enflurane. After incision, both pulmonary artery pressure and pulmonary capillary wedge pressure increased, and cardiac index decreased. Continuous injection of 100ng.kg-1.min-1 PGE1 made pulmonary artery pressure and pulmonary capillary wedge pressure to decrease, and cardiac index to increase. PaO2, however, decreased apparently. PGE1 was effective for cardiac failure with mitral valve regurgitation associated with pulmonary hypertension. But attention must be given to the decrease in PaO2.

The concentrations of deuterium oxide (D2O) in the root tissue water of winter rye seedlings equilibrated with the external D2O concentration within 30 min and in the shoot tissue water after 5-6 h. The equilibrated value in the root water was 87% and in the shoot water, 55% of the external concentration. The K+ absorption rate of the seedlings decreased from a value of 39 to 18 mumol g-1 h-1 when the D2O concentration was changed through a range from 0% to 97%. D2O suppressed the absorption of water and K+ by the seedlings. The higher the D2O concentration the greater the suppression, but it was less than with similarly treated rice plants. However, the process of D2O absorption by the seedlings was similar to that of rice seedlings (Envir. exp. Bot., 24, 369).

A series of N-alkyl-N-nitrosoureas, which produce both alkylation and carbamoylation, was found to induce neurite formation in mouse neuroblastoma N-18 cells just as does N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Related compounds, which produce only carbamoylation or nitroguanidination, did not induce morphological differentiation, while methyl methanesulfonate (MMS) and dimethyl sulfate, which produce only alkylation, did. MNNG, methyl nitrosourea and MMS were more effective in the induction of differentiation than their corresponding ethyl derivatives. These data suggested that alkylation contributed to the induction of morphological differentiation in N-18 cells.

Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda). Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right. A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG. Properties of the transition sites are discussed in relation to the primer synthesis.

A fifth and new DFP-sensitive alkaline proteinase E, with strong esterase activity toward Ac-(Ala)3-OMe was found in pronase, a protease mixture from St. griseus K-1. Proteinase E was shown to be different from the elastase [EC 3.4.21.11]-like enzyme or subtilisin [EC 3.4.21.14]like enzyme, and alkaline proteinase A, B, and C in pronase. Proteinase E was purified to a state appearing homogeneous on polyacrylamide gel electrophoresis. Its molecular weight was estimated as 26,600 by gel filtration. It was unstable below pH 5.6. Studies on its actions on acyl-amino acid esters showed that it hydrolyzed the ester bonds of esters of tryptophan, tyrosine, phenylalanine, leucine, and alanine in decreasing order of ease.