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lacA coding for beta-galactosidase (beta-gal) was cloned from the genomic DNA of Aspergillus oryzae RIB40. There were 9 exons in lacA and the coding region of 3,015 bp encoded a protein of 1,005 aa with a deduced molecular mass of 109,898. A. oryzae lacA was highly homologous to fungal beta-gals, with the highest aa identity of 70.7% to A. niger lacA, and also showed significant identity to acid beta-gals belonging to family 35 glycosyl hydrolases. Approximately 10 copies of lacA under control of A. oryzae glaA promoter were integrated into the chromosome of A. oryzae M-2-3. The recombinant strain expressed more than 700-fold of the beta-gal activity as compared to the wild type strain under induction by maltose.

Complementary and genomic DNAs encoding an acid-stable α-amylase (asAA) were cloned from Aspergillus kawachii IFO4308 and their nucleotide sequences were determined. The structural gene (asaA) consisted of a 1,920 bp open reading frame that encoded 640 amino acids. The gene consisted of 9 exons and 8 introns. The signal peptide was composed of 21 amino acid residues. The amino acid sequence from the N-terminus to the 479th residue shoeed 97% homology with the Aspergillus niger acid α-amylase and that from the 480th residue to the C-terminus, including the raw-starch-affinity site, i.e., the TS region, the amino acid sequence of which was rich in threonine and serine, was characteristic of the asAA. A yeast transformant carrying the cDNA produced and secreted an acid-stable α-amylase which was capable of digesting raw starch.

A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene.

Organic acids are essential flavor components in alcoholic beverages such as wine and sake (Japanese rice wine). The effects of fumarase deficiency on the productivity of malic, fumaric and succinic acids in Saccharomyces cerevisiae were examined using the FUM1 gene disruptant. The single nuclear gene FUM1 encoding fumarase in S. cerevisiae, DBY746, was disrupted by a site-directed one-step gene disruption technique. The fumarase activity of the isolated FUM1 disruptant was abolished. The FUM1 disruptant having a pet phenotype showed no growth on minimal medium containing glycerol as a carbon source even after prolonged incubation. The productions of organic acids by the FUM1 disruptant in YPD liquid culture containing 10% glucose and in sake mash were analyzed by HPLC. The analytical results of the FUM1 disruptant showed there were no differences in extracellular malate productivity, fumarate productivity had increased, and that succinate productivity was reduced compared with those of the control strain. In the production of sake using the FUM1 disruptant, fermentation ability as monitored by CO2 evolution was not different from that of the control strain. These results indicate that site-directed FUM1 disruption alters organic acid production without any loss of fermentation ability in yeast.

In Aspergillus oryzae wild-type strains, the expression of the agdA gene encoding alpha-glucosidase (AGL) is induced by maltose at the transcriptional level in a similar manner to the amyB gene encoding Takaamylase A (TAA) and the glaA gene encoding glucoamylase (GLA). In A. oryzae transformants containing multiple copies of the agdA gene, a high-level of AGL activity was observed. This was accompanied by a significant reduction in TAA and GLA activities. Moreover, transformants with the highest AGL activity showed the lowest degree of TAA and GLA activities. Northern blot analyses showed that the transcriptional levels of amyB and glaA in the AGL-overproducing transformant were drastically reduced when large amounts of agdA mRNA were detected in maltose-grown mycelia. In addition, the glucose concentration of the maltose-containing medium that was used to grow the AGL-overproducing transformant for RNA extraction was higher than that of the control transformant. These results suggest that the reduced expression of the amyB and glaA genes in the AGL-overproducing transformant was due to either titration of a common regulatory protein(s) involved in maltose induction or carbon catabolite repression.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase, on multicopy plasmids caused the accumulation of the precursor form of alkaline phosphatase. The cells lost their viability by a half-life of 60 min and exhibited high sensitivity to 1% sodium dodecyl sulfate (SDS), suggesting that the assembly of the surface proteins were affected by overexpression of the phoA gene. From the cells exhibiting resistance to 1% SDS, we obtained 20 mutants that secrete more alkaline phosphatase into the periplasmic space. Three representatives of the mutants accumulated no precursor molecules and secreted alkaline phosphatase by five- to six-fold that of the wild-type cells carrying multicopy phoA. In all of the three mutants, the amount of phoA transcripts were two to four times less than those in the wild-type cells, indicating that the ability to secrete a large amount of alkaline phosphatase was conferred by decreasing the synthetic rates of the phoA gene product. When the promoter of phoA was replaced with the tacI promoter and the expression level of the phoA gene was regulated with isopropyl-1-thio-β-d-galactoside, the secretion of alkaline phosphatase into the periplasm decreased as the synthetic rate of the phoA gene product increased over a threshold. All these results indicate that overproduction of the phoA gene product causes defects in the secretion of alkaline phosphatase and that the regulation of the expression level is essential for efficient translocation.

In our previous work we cloned on λ Charon4A phage vector a 6.4-kb EcoRI fragment, which had the transforming activity of inducing gene amplification with a 16-kb repeating unit on the Bacillus subtilis chromosome by competence transformation; those transformants were selected as tunicamycin resistant (Tm(r)) transformants. We found that this DNA fragment can induce another type of gene amplification with a 8.7-kb repeating unit by transformation when we use an amyE07 mutant as a recipient cell and select both Tm(r) and amyE(+) transformants. In the latter (8.7-kb type) gene amplification, the copy number was increased up to 20 in contrast to about 10 copies in the former 16-kb type. We replaced a part of the 6.4kb EcoRI fragment with a cat gene as a model of a foreign gene, and succeeded in the amplification of the cat gene on the chromosome by the latter type of transformation.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding β-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between − 377 to − 290 (the number indicates the distance in base pairs from the translation initiation point (+ 1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to − 377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between − 377 to − 290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.

alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.

Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78–94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.

We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.

The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries. The genomic gene was located in the 5.4 kb EcoRI fragment. The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68,000 daltons (non-glycosylated form). The genomic gene of A. shirousami GAase was introduced into Aspergillus oryzae. These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts. Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A. oryzae.

Cladosporium cladosporioides No.9の無細胞抽出液より硫安沈殿,ブチルートヨパール, DEAE-トヨパールおよびトヨパールHW55のクロマトグラフィーにより酢酸エステルを特異的に分解する酢酸エステル分解酵素(Est I, Est II)を単離精製した. 各精製酵素の分子量はゲルクロマトグラフィーによりEst Iは約6.3万, Est II は約6.6万と推定された. また,各酢酸エステル分解酵素の至適反応温度はEst Iは35°C, Est IIは30°Cであった.熱安定性はともに50°C, 30分処理で完全に失活した.至適pHはEst IはpH 9.1, Est IIはpH 9.3であった. pH安定範囲は,前者がpH 7～9,後者がpH 5～6であった.両酵素ともにDFP, PMSFに阻害され,エゼリンで阻害されないことによりセリン残基を有するカルボキシエステラーゼと推定された.SH基阻害剤であるPCMB, DTNBおよび金属イオンキレート剤であるEDTAでは阻害されなかった.また, Ag2+および2価の重金属イオンのCu2+, Hg2+で強く反応を阻害された. エチルエステルに対する基質特異性は,酢酸エステルに比較して最大でも約10～20%の相対活性しかなかった.トリグリセリド,モノグリセリドの長鎖脂肪酸を持つものにはまったく作用せず,短鎖脂肪酸を持つものにわずかに活性を示した.酢酸エステルに対してはEst Iは酢酸イソアミルに対してEst IIは酢酸イソブチルに対して最大の活性を示し,酢酸β-フェネチルに対しても高い分解活性を示した.本酵素はアルコールの酢酸エステルを特異的に分解するカルボキシエステラーゼであった.

Nuclear receptor family proteins are structurally related transcription factors activated by specific lipophilic compounds. Because they are activated by a variety of hormonal molecules, including retinoic acid, vitamin D, and steroid hormones, they are assumed to be promising targets for clinical drugs. We previously found that one ascochlorin (1) derivative, 4-O-carboxymethyl-ascochlorin (2), is a potent agonist of peroxisome proliferator activated receptor gamma (PPARgamma). Here, we synthesized derivatives of 1, designated as a lead compound, to create new modulators of nuclear hormone receptors. Two derivatives, 4-O-carboxymethyl-2-O-methylascochlorin (9) and 4-O-isonicotinoyl-2-O-methylascochlorin (10), showed improved agonistic activity for PPARgamma and induced differentiation of a progenitor cell line, C3H10T1/2. We also found that 1, dehydroascofuranon (29), and a 2,4-O-diacetyl-1-carboxylic acid derivative of 1 (5) specifically activated estrogen receptors, PPARalpha, and an androgen receptor. All of the derivatives (1-29) activated the pregnane X receptor. These results suggest that the chemical structure of 1 is useful in designing novel modulators of nuclear receptors.

The prenyl-phenol antibiotics ascochlorin-related compounds, are known to reduce serum cholesterol and triglyceride, suppress hypertension, and ameliorate types-I and II diabetes. However, little is known about the molecular mechanism for these physiological effects. Here we report that the ascochlorin derivative, 4-O-carboxymethyl ascochlorin (AS-6) acts as a potent activator of the nuclear hormone receptor, PPARgamma, although it does not activate the related receptors, PPARalpha, PPARdelta or RARalpha. AS-6 interacts directly with the PPARgamma molecule in vitro, and induces differentiation of the mouse preadipocyte cell line 3T3-L1. Our results suggest that AS-6 is a partial agonist for PPARgamma with a novel chemical structure.

Test sake fermentation was carried out using an Aspergillus oryzae transformant (TF2–5) which had the glucoamylase gene from Aspergillus shirousamii RIB2504. The fermentation progressed rapidly due to high glucoamylase activity, and the steamed rice rapidly dissolved in the moromi-mash. Consequently, the total alcohol yield increased. In addition, the obtained sake had a moderate sweetness and a rich fruity flavor.

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.

The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1-603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1-511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07– 0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the intergration position of the plasmid on the chromosome.

Aspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed of a higher level compared with the amyB gene because of its multi-copy intergration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

We isolated complexes containing oriC region DNA and outer membrane, named origin complex heavy and origin complex light, from the cells of Escherichia coli cultured in media with poor or rich of nutrients, and found the different nature of association between origin DNA and outer membrane. The ratio of origin complex light to origin complex heavy prepared from the cells cultured in rich media was lower than that of those from minimal medium culture. Outer membrane preparations from the cells grown in nutritious media had high abilities of association with origin complex light in the presence of magnesium. These results indicated that the number of binding sites on outer membrane with origin region DNA increase, or the binding between outer membrane and origin region DNA become more rigid, when cells grow faster and DNA replication initiate more frequently in a nutritious medium.

Analysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose. The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding beta-glucuronidase (GUS), and the resultant plasmid was introduced into A. oryzae. Expression of GUS protein in the A. oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose. The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined. A comparison of the nucleotide sequence of the A. oryzae glaA promoter with those of A. oryzae amyB, encoding alpha-amylase, and A. niger glaA showed two regions with similar sequences. Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose.

Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.

Adenylate cyclase-deficient (cya) mutants of Escherichia coli K-12 were selectively and highly resistant to mecillinam (FL1060) among several beta-lactam antibiotics in the absence of cyclic adenosine 3',5'-monophosphate (cAMP). They became sensitive to the drug in the presence of cAMP. Also, cAMP receptor protein-negative (crp) mutants, with the exception of strain 5333, were highly resistant to mecillinam in the presence and in the absence of cAMP. Mecillinam exerted two distinct and sequential effects in both cya+ strains and cya strains supplemented with cAMP: (i) rounding of cells and (ii) cessation of cell division. The first effect was accompanied by a decrease in growth rate, whereas the second effect was accompanied by enlargement and lysis of the rounded cells. The second effect of mecillinam was dependent on inoculum size and cAMP. When the cell density was above about 10(6) cells per ml, the rounded cells stopped dividing but did not lyse. In the absence of cAMP, cya strains neither stopped dividing nor lysed; they were resistant to the second, lethal effect of mecillinam.

Brefeldin A (BFA) is an antibiotic having diverse biological effects such as antifungal, antiviral and antitumor activities. The effect of BFA on biosynthesis of cellular components was examined to elucidate the mode of action of BFA using C. albicans IAM 4888.When C. albicans was grown in the presence of BFA, cells became rounded and enlarged several times larger than the untreated control cells. Cell walls of the treated cells became irregular and a number of Sudan III-stainable lipid droplets was formed in the cytoplasm. Accompanying these morphological changes, a marked alteration occurred in the cellular lipid composition; neutral lipids increased whereas phospholipid decreased. [14C]Acetate incorporation into the lipid fraction proceeded in accordance with the growth in the presence of BFA. On the other hand, [32P]orthophosphate incorporation into phospholipid was severely inhibited. Incorporation of radiolabeled precursors into DNA, RNA and protein was not affected on a cell weight basis.

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.

The effect of 4-0-methylascochlorin (MAC), an experimental hypocholesterolemic agent, on cholesterol metabolism was investigated in rats in two separate experiments. The administration of MAC for 2 and 6 consecutive weeks at daily doses of 100–135 mg/kg resulted in reduction in serum cholesterol levels of 16% after 2 weeks of treatment in the first experiment, and 13% after 6 weeks in the second experiment in comparison to the corresponding controls. MAC administered at a daily dose of 100 mg/kg for 2 weeks showed a significant increase in the biliary excretion of bile acids and cholesterol in bile-duct cannulated rats with or without the administration of taurocholate. In the second experiment, MAC treatment for 6 weeks produced a marked increase in the fecal output of acidic sterols during a 2 to 6-week period. MAC treatment also further enhanced hepatic cholesterol 7α-hydroxylase in the rats. Therefore, it appears that the mechanism of serum cholesterol lowering due to MAC is related to the enhancement of hepatic bile acid synthesis and the increase in biliary and fecal excretion of bile acids.

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp2 ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-14C into RNA still continued even after the incorporation of N-acetyl-3H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC ...

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.

The effect of tunicamycin on the secretion of extracellular α-amylase by Bacillus subtilis was studied. Tunicamycin at 5μg/ml did not inhibit the synthesis of the enzyme, but in the presence of the drug the cells accumulated to about half of the amount of α-amylase which would have otherwise been secreted into the medium in the absence of the antibiotic. Cell-bound α-amylase accumulated in the presence of tunicamycin reached an amount as much as 10 to 30 times more than that of the control, and was found to accumulate between the cytoplasmic membrane and the cell wall. This effect of accumulation seemed to be specific of tunicamycin.

Two diketopiperazines have been isolated from the culture broth of a soil Actinomycetales, Streptomyces sp. strain ML1532. They were cyclo(L-Val-L-Pro) and cyclo(L-Tyr-L-Pro). The selective cytotoxicity of cyclo(L-Tyr-L-Pro) was slightly higher than that of cyclo(L-Val-L-Pro) against SV40-transformed cells.