Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification.

Within a 7 kb segment of the mtDNA molecule of the root knot nematode, Meloidogyne javanica, that lacks standard mitochondrial genes, are three sets of strictly tandemly arranged, direct repeat sequences: approximately 36 copies of a 102 ntp sequence that contains a TaqI site; 11 copies of a 63 ntp sequence, and 5 copies of an 8 ntp sequence. The 7 kb repeat-containing segment is bounded by putative tRNAasp and tRNAf-met genes and the arrangement of sequences within this segment is: the tRNAasp gene; a unique 1,528 ntp segment that contains two highly stable hairpin-forming sequences; the 102 ntp repeat set; the 8 ntp repeat set; a unique 1,068 ntp segment; the 63 ntp repeat set; and the tRNAf-met gene. The nucleotide sequences of the 102 ntp copies and the 63 ntp copies have been conserved among the species examined. Data from Southern hybridization experiments indicate that 102 ntp and 63 ntp repeats occur in the mtDNAs of three, two and two races of M.incognita, M.hapla and M.arenaria, respectively. Nucleotide sequences of the M.incognita Race-3 102 ntp repeat were found to be either identical or highly similar to those of the M.javanica 102 ntp repeat. Differences in migration distance and number of 102 ntp repeat-containing bands seen in Southern hybridization autoradiographs of restriction-digested mtDNAs of M.javanica and the different host races of M.incognita, M.hapla and M.arenaria are sufficient to distinguish the different host races of each species.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Westerdykella globosa,a proposal for a new combination

The surface ornamentation of ascospores ofPreussia globosa was compared in an isolate from paddy soil in Japan and a culture derived from the holotype. The ascospores of two cultures were characterized by the surface ornamentation of a single, semicircular spiral ridge. This new finding strongly suggested that the fungus should be transferred to the genusWesterdykella. Therefore, the morphological and cultural characters of the fungus were re-examined, and the new combinationWesterdykella globosa is herein proposed.     

Stellatospora,a new genus of the Sordariaceae

In the course of study of fungi from soil, a new genus and species,Stellatospora terricola, was isolated. The fungus is distinguished from other known genera in having star- or comfit-shaped ascospores with a distinct germ pore. The morphological characters of the genus are considered to resemble those of the Sordariaceae in Ascomycotina.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Growth and respiratory activity of mold fungus (Trichoderma lignorum)

The growth ofTrichoderma lignorum was studied in relation to the carbon balance of the culture system and the respiratory activity at different growth phases. Conidia, after inoculation into the medium, swelled and germinated rapidly. The growth rate of the hyphae at the exponential phase was 0.46 hr⁻¹ (2.2 hr for mass doubling) at 25 C. The yield efficiency of hyphal biomass-C at the cost of glucose-C was 67%, while those of the waste products-C excreted and of CO₂-C evolved were, 6.5% and 26.5%, respectively. The yield efficiency of conidia-C to the decrease of hyphae-C was 34%. The germination, growth rate and carbon balance were not affected by different concentrations of glucose from 10 to 2×10³ mg glucose-C/l. Carbon dioxide was needed as the growth factor for the initiation of the germination of conidia, but there was no increase in yield efficiency as a result of CO₂ fixation. The respiratory rate of the fungus changed drastically as the growth proceeded. The rate of endogenous respiration of conidia was less than 0.2 mg CO₂-C/g conidia-C/hr which increased immediately after inoculation into the medium. The highest respiratory rate of hyphae (100–110 mg CO₂-C/g hyphae-C/hr) was obtained throughout the exponential phase. Thereafter, decreasing rapidly, the respiratory rate of submerged hyphae of 1-week-old showed only 1.8 mg CO₂-C/g biomass-C/hr, whereas the rate of aerial hyphae forming conidia increased again, but did not exceed 10 mg CO₂-C/g biomass-C/hr.