Actinocatenispora comari sp. nov., an endophytic actinomycete isolated from aerial parts of Comarum salesowianum.

A novel actinomycete, designated NUM-2625T, was isolated as an endophytic bacterium in aerial parts of Comarum salesowianum, an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625T showed the highest similarity to Actinocatenispora thailandica TT2-10T (99.4 %), Actinocatenispora sera KV-744T (99.3 %), and Actinocatenispora rupis CS5-AC17T (97.7 %). Chemotaxonomic properties of strain NUM-2625T were essentially consistent with those of the genus Actinocatenispora, such as the presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-9(H4) and MK-9(H6) as the major menaquinones, and iso-C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, and anteiso-C17 : 0 as the major fatty acids. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values revealed a low relatedness between strain NUM-2625T and the other type strains of the genus Actinocatenispora. In addition, strain NUM-2625T exhibited several phenotypic properties that could be used to distinguish it from its closest relatives. Based on the results of polyphasic analyses, strain NUM-2625T represents a novel species in the genus Actinocatenispora, for which the name Actinocatenispora comari sp. nov. is proposed. The type strain is NUM-2625T (=NBRC 114660T=TBRC 13496T).

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.

New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

A new mycinosyl rosamicin derivative produced by an engineered Micromonospora rosaria mutant with a cytochrome P450 gene disruption introducing the D-mycinose biosynthetic gene.

Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The D-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.

Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.

Function of cytochrome P450 enzymes RosC and RosD in the biosynthesis of rosamicin macrolide antibiotic produced by Micromonospora rosaria.

The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin.

Function of cytochrome P450 enzymes MycCI and MycG in Micromonospora griseorubida, a producer of the macrolide antibiotic mycinamicin.

The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.

Production of a hybrid 16-membered macrolide antibiotic by genetic engineering of Micromonospora sp. TPMA0041.

Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. We previously engineered an expression plasmid pSETmycinose containing the D-mycinose biosynthesis genes from M. griseorubida A11725. This plasmid was introduced into Micromonospora sp. FERM BP-1076 cells, which produce the 16-membered macrolide antibiotic izenamicin. The resulting engineered strain TPMA0041 produced 23-O-mycinosyl-20-deoxy-izenamicin B(1) and 22-O-mycinosyl-izenamicin B(2). 23-O-mycinosyl-20-deoxy-izenamicin B(1) has been produced by the engineered strain M. rosaria TPMA0001 containing pSETmycinose as 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (=IZI) in our recent study, and 22-O-mycinosyl-izenamicin B(2) has previously been synthesized as a macrolide antibiotic TMC-016 with strong antibacterial activity. The production of 22-O-mycinosyl-izenamicin B(2) (=TMC-016) was increased when propionate, a precursor of methylmalonyl-CoA, was added to the culture broth.

Functional analysis of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin macrolide antibiotics.

Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg(2+). With the reconstituted in vitro assays, the order of mycinamicin VI-->III-->IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.

Macrolides are a class of valuable antibiotics that include a macrolactone ring, at least one appended sugar unit, and, in most cases, additional hydroxyl or epoxide groups installed by cytochrome P450 enzymes. These functional groups contribute to structural diversification and serve to improve the bioactivity profiles of natural products. Here, we have characterized in vitro two P450 enzymes from the mycinamicin biosynthetic pathway of Micromonospora griseorubida. First, MycCI was characterized as the C21 methyl hydroxylase of mycinamicin VIII, the earliest macrolide form in the postpolyketide synthase tailoring pathway. Moreover, we established that optimal activity of MycCI depends on the native ferredoxin MycCII. Second, MycG P450 catalyzes consecutive hydroxylation and epoxidation reactions with mycinamicin IV as initial substrate. These reactions require prior dimethylation of 6-deoxyallose to mycinose for effective conversion by the dual function MycG enzyme.

Production of rosamicin derivatives in Micromonospora rosaria by introduction of D-mycinose biosynthetic gene with PhiC31-derived integration vector pSET152.

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. The D-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and D-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site PhiC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of "unnatural" natural mycinosyl compounds by various actinomycete strains using the bacteriophage PhiC31 att/int system.

Avulsion fracture of the lateral ankle ligament complex in severe inversion injury: incidence and clinical outcome.

BACKGROUND: Avulsion fracture of the lateral ankle ligaments is often undetected on early radiographs. The epidemiology and treatment of such avulsion fractures have received much less attention than the epidemiology and treatment of rupture of these ligaments. HYPOTHESIS: The clinical characteristics of avulsion fracture are different from those of ligament rupture. Unlike nonoperative treatment of lateral ligament rupture, nonoperative treatment of avulsion fracture does not yield satisfactory results. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 169 consecutive patients with severe inversion injury were classified into a ligament rupture group or avulsion fracture group on the basis of physical examination findings and anterior talofibular ligament and calcaneofibular ligament radiographic views. Age, sex, activity level, and the mechanism of injury were analyzed. Patients in both groups were treated by casting. Follow-up examination of 152 patients included clinical assessment and functional evaluation based on the Karlsson system. RESULTS: Avulsion fracture was diagnosed in 44 (26%) of the 169 patients and was most common among children and patients over 40 years of age. Sedentary level activity and low-energy injury were more common in the avulsion fracture group than in the ligament rupture group (77% vs 37%, respectively, P = .001; 68% vs 43%, respectively, P =.004). Nonoperative treatment of avulsion fracture (mean Karlsson score, 89.1 points) yielded satisfactory results that were comparable with those of nonoperative treatment of ligament rupture (mean Karlsson score, 88.4 points) (P = .123). Osseous union was achieved in 65% of the patients with avulsion fracture. CONCLUSION: Avulsion fracture of the lateral ankle ligaments in cases of severe inversion injury is more common than previously believed. Because of the high incidence and difficulty of detection in children, a high level of suspicion is necessary in order to obtain an accurate diagnosis of avulsion fracture in cases of severe inversion injury and to achieve adequate stability.

The delayed manifestation of T-cell receptor (TCR) variants in X-irradiated mice depends on Trp53 status.

The influence of Trp53 on the radiation-induced elevation of T-cell receptor (TCR) variant fractions was examined in splenic T lymphocytes of Trp53-proficient and -deficient mice. Wild-type Trp53+/+, heterozygous Trp53+/- and null Trp53-/- mice were exposed to 3 Gy of X rays at 8 weeks of age. The fraction of TCR-defective variants was measured at various times after irradiation. Initially, the TCR variant fraction increased rapidly and reached its maximum level at 9 days after irradiation before decreasing gradually. In Trp53+/+ and Trp53+/- mice, the TCR variant fraction fell to normal background levels at 16 and 20 weeks of age, respectively. In contrast, the TCR variant fraction of Trp53-/- mice failed to decrease to background levels during the observation period. Baseline levels were then maintained for approximately 60 weeks in the Trp53+/+ mice and approximately 40 weeks in the Trp53+/- mice. After the long flat period, a significant re-increase in the fraction of TCR variants was found after 72 weeks of age in the irradiated Trp53+/+ mice and after 44 weeks of age in the irradiated Trp53+/- mice. Measurement of the fraction of apoptotic cells in the spleen and thymus 4 h after X irradiation at these ages in Trp53+/+ and Trp53+/- mice demonstrated a reduction in apoptosis in the irradiated mice compared to the nonirradiated mice. This suggests that the delayed increase in TCR variants after irradiation is due to a reduction in Trp53-dependent apoptosis.

Pathoanatomy of posterior malleolar fractures of the ankle.

BACKGROUND: The functional outcome following ankle fractures that involve a posterior malleolar fragment is often not satisfactory, and treatment of this type of fracture remains controversial. Thorough knowledge of the pathologic anatomy of the posterior malleolar fracture is essential for planning appropriate treatment. Thus, we conducted a computed tomographic study to clarify the pathologic anatomy of the posterior malleolar fracture. METHODS: Between 1999 and 2003, fifty-seven consecutive patients with a unilateral ankle fracture with one or more posterior fragments were managed at our hospital. We reviewed the patients' preoperative computed tomographic scans to determine (1) the ratio of the posterior fragment area to the total cross-sectional area of the tibial plafond and (2) the angle between the bimalleolar axis and the major fracture line of the posterior malleolus. Each fracture was categorized according to the location of the major fracture line on the computed tomographic image at the level of the tibial plafond. RESULTS: The fifty-seven fractures were categorized into three types: (1) the posterolateral-oblique type (thirty-eight fractures; 67%), (2) the medial-extension type (eleven fractures; 19%), and (3) the small-shell type (eight fractures; 14%). Two of the eleven medial-extension fractures extended to the anterior part of the medial malleolus. A total of nine of the eleven medial-extension fractures actually consisted of two fragments [corrected] The conditions are not exclusive of one another; for example, in the case of one of the fractures exhibiting two fragments, the fracture also extended to the anterior part of the medial malleolus [corrected] The average area of the fragment comprised 11.7% of the cross-sectional area of the tibial plafond for posterolateral-oblique fractures and 29.8% for medial-extension fractures. In the cases of seven of the nine fractures that comprised >25% of the tibial plafond, the fracture line extended to the medial malleolus. The angles between the bimalleolar axis and the major fracture line of the posterior malleolus varied. CONCLUSIONS: The fracture lines associated with posterior malleolar fractures appear to be highly variable. A large fragment extending to the medial malleolus existed in almost 20% of the posterior malleolar fractures in the current study, and some fragments involved almost the entire medial malleolus. Because of the great variation in fracture configurations, preoperative use of computed tomography may be justified. The information obtained from this study will be helpful for conducting basic research of this condition and for determining appropriate surgical approaches.

Maniwamycins: new quorum-sensing inhibitors against Chromobacterium violaceum CV026 were isolated from Streptomyces sp. TOHO-M025.

Quorum sensing is an important microbial signaling system that controls the expression of many virulence genes. Maniwamycins C-F, new compounds and quorum-sensing inhibitors, were isolated from the culture broth of Streptomyces sp. TOHO-M025 using a silica gel column and preparative HPLC. The structures of maniwamycins were elucidated by spectroscopic analyses, including NMR. The compounds each have an azoxy moiety. All maniwamycins inhibited violacein synthesis, which is controlled by quorum sensing, in Chromobacterium violaceum CV026.

Accumulation of radiocesium in wild mushrooms collected from a Japanese forest and cesium uptake by microorganisms isolated from the mushroom-growing soils.

Mushrooms and soils samples collected from a sub-alpine forest of Mt. Fuji in Japan were measured for 137Cs and stable Cs. The ranges of 137Cs specific activities and stable Cs concentrations in the mushrooms were 291-7950 Bq kg(-1) dry weight and 4.69-58.1 mg kg(-1) dry weight, respectively. Both 137Cs specific activities and stable Cs concentrations in the mushrooms were higher than those in common agricultural plants. The 137Cs specific activities and stable Cs concentrations in the soils were 3.18-149 Bq kg(-1) dry weight and 0.618-2.18 mg kg(-1) dry weight, respectively. The appearance frequencies of filamentous actinomycetes and planktonic bacteria from the soils decreased according to increasing Cs contents in the medium. No relationship was observed between the appearance frequencies of those and the stable Cs concentrations in the soils. The filamentous actinomycetes from any soil sample could not grow in the presence of 25 mM Cs, although the planktonic bacteria from the soil samples could grow with up to 50 mM Cs in YM agar. In addition, the planktonic bacteria from approximately 70% of the soil samples could grow even in the presence of 100 mM Cs. Filamentous actinomycetes were more sensitive to Cs than planktonic bacteria. In in vitro experiments, Cs uptake by these strains of filamentous actinomycetes and planktonic bacteria was high in the presence of 5 mM CsCl and the strains accumulated Cs, the same as in mushrooms. Our results indicate that filamentous actinomycetes in the soils have higher sensitivity to Cs than planktonic bacteria, and several strains of filamentous actinomycetes have a high Cs accumulation in the presence of 5 mM Cs.

Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.

The targeted inactivation of polyketide synthase mycAV in the mycinamicin producer, Micromonospora griseorubida, and a complementation study.

Mycinamicin is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against gram-positive bacteria. Recently, the nucleotide sequences of the mycinamicn biosynthetic gene cluster in M. griseorubida have been completely determined. Mycinamicin non-producer M7A21 was isolated by mycAV inactivation, which encodes the module 7 of mycinamicin polyketide synthase (PKS) required for the biosynthesis of the mycinamicin biosynthetic intermediate protomycinolide-IV (PML-IV). When the bioconversion to mycinamicin II (M-II) from PML-IV was performed using M7A21 and the feeding culture method, the productivity of M-II was the same as that of M-II in wild-type strain A11725. p446M7 containing mycAV was constructed using the Escherichia coli-Streptomyces shuttle vector pGM446. The mycinamicin productivity of M7A21 was restored by the introduction of p446M7 into the M7A21 cell, but almost all p446M7 was integrated into the chromosome of M7A21 because the plasmid was unstable in M7A21. The feeding culture and the introduction of the complement gene for M7A21 would be powerful tools to perform combinatorial biosynthesis for the production of new macrolide antibiotics.

Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida.

Mycinamicin, composed of a branched lactone and two sugars, desosamine and mycinose, at the C-5 and C-21 positions, is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against Gram-positive bacteria. The nucleotide sequence (62 kb) of the mycinamicin biosynthetic gene cluster, in which there were 22 open reading frames (ORFs), was completely determined. All of the products from the 22 ORFs are responsible for the biosynthesis of mycinamicin II and self-protection against the compounds synthesized. Central to the cluster is a polyketide synthase locus (mycA), which encodes a seven-module system comprised of five multifunctional proteins. Immediately downstream of mycA, there is a set of genes for desosamine biosynthesis (mydA-G and mycB). Moreover, mydH, whose product is responsible for the biosynthesis of mycinose, lies between mydA and B. On the other hand, eight ORFs were detected upstream of the mycinamicin PKS gene. The myrB, mycG, and mycF genes had already been characterized by Inouye et al. The other five ORFs (mycCI, mycCII, mydI, mycE, and mycD) lie between mycA1 and mycF, and these five genes and mycF are responsible for the biosynthesis of mycinose. In the PKS gene, four regions of KS and AT domains in modules 1, 4, 5, and 6 indicated that it does not show the high GC content typical for Streptomyces genes, nor the unusual frame plot patterns for Streptomyces genes. Methylmalonyl-CoA was used as substrate in the functional units of those four modules. The relationship between the substrate and the unusual frame plot pattern of the KS and AT domains was observed in the other PKS genes, and it is suggested that the KS-AT original region was horizontally transferred into the PKS genes on the chromosomal DNA of several actinomycetes strains.

Role of p53 gene in apoptotic repair of genotoxic tissue damage in mice.

When DNA is damaged by exposure to a small amount of radiation, it is repaired efficiently by innate mechanisms. However, if cellular damage is more extensive, DNA repair cannot be adequately completed. To clarify the role of the p53 gene in apoptotic tissue repair, the incidence of in-vivo radiation-induced somatic mutation was evaluated by measuring the T cell receptor (TCR) gene expression in p53(+/+) and p53(-/-) mice. After gamma-irradiation with 3 Gy, the TCR mutation frequency (MF) was higher in p53(+/+) mice than in the controls. However, when the mice were exposed to 3 Gy at a low dose rate, the TCR MF did not increase in the p53(+/+) mice, whereas it increased and remained elevated in p53(-/-) mice, which are unable to induce apoptosis. In p53(+/+) mice, the TCR MF peaked 9 days after gamma-irradiation with 3 Gy at a high dose rate, and then gradually decreased with a half-life of about 13 days. However, in p53(-/-) mice, the peak level of the TCR MF did not decline significantly with time. Hence, complete repair of mutagenic damage in irradiated tissues requires the integration of DNA repair and p53-dependent apoptotic tissue repair.