Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

THE BIOCHEMISTRY OF NITRIFYING MICROORGANISMS

• 1 Biological nitrification is mediated primarily by two genera of bacteria, Nitrosomonas and its marine form Nitrosocystis, oxidizing ammonia to nitrite, and Nitrobacter, converting nitrite into nitrate. These are chemoautotrophic organisms since they usually derive their energy for growth by oxidizing these inorganic nitrogen compounds and their carbon from carbon dioxide, carbonates or bicarbonates.
• 2 The morphology and structure of these Gram-negative bacteria studied by electron microscopy show numerous intracellular membranes reminiscent of those in photosynthetic bacteria and blue-green algae. These structures may therefore be associated with the production of ATP.
• 3 The bacteria are difficult to grow in pure cultures in sufficient amounts for biochemical work since their generation time is around 10 hr. and the yields are only about one hundredth of those obtained with heterotrophic bacteria. Thus in continuous cultures great care must be taken to avoid ‘wash-out’ of the cells. Since Nitrosomonas and Nitrosocystis produce copious amounts of nitrous acid, which would eventually retard growth, pH stat units are used to titrate the cultures continuously with a solution of sodium carbonate, to hold the pH around 7–8.
• 4 The respiratory chain which is associated with cell membranes, contains flavin, quinones and many cytochromes linking to oxygen as a terminal acceptor. In Nitro-somonas-Nitrosocytis hydroxylamine is oxidized by the electron transfer chain and in Nitrobacter nitrous acid is utilized. The ammonia-oxidizing system, which in Nitrosomonas probably resides near the cell surface, does not appear to survive cell breakage. During the oxidation of hydroxylamine and nitrous acid by the respiratory chains, a phosphorylation occurs but the P/O ratios around 0–30 are low. There is little energy reserve material in the cells, possibly β-hydroxybutyrate and some metaphosphates and as soon as the oxidative processes are impaired the cells cease dividing.
• 5 Chemoautotrophic bacteria have a novel way of producing reduced nicotinamide adenine dinucleotide (NADH). This involves a reversal of electron flow from reduced cytochrome c to nicotinamide adenine dinucleotide (NAD) that is energy-dependent, thus requiring adenosine triphosphate.
• 6 Reductase enzymes, nitrate, nitrite and hydroxylamine reductases in Nitrobacter and nitrite and hydroxylamine reductases in Nitrosomonas, have been described. They appear to be readily extracted in soluble form and are probably assimilatory enzymes since ¹⁶N labelled nitrate, nitrite and hydroxylamine respectively in Nitrobacter and the last two in Nitrosomonas are readily incorporated into cell nitrogen. It has been suggested that a particulate nitrate reductase in Nitrobacter is coupled to the synthesis of adenosine triphosphate but adequate experimental evidence for this concept has not been produced.
• 7 Some recent observations with Nitrobacter suggest that it grows on acetate, deriving all its energy and carbon skeletons from this source but the mean generation time for the bacterium is unchanged. Under these conditions the carbon dioxide fixing enzymes of the pentose pathway are suppressed. This then is a case of facultative chemoautotrophy but there is no increase in the biosynthesis of the TCA enzymes. Whether this is a widespread phenomenon in other chemoautotrophic bacteria remains to be established. If this does prove to be the case it would aid their survival in a variety of habitats and extend their distribution in soils and seas.
• 8 The carbon dioxide fixing enzymes of the pentose pathway are found in the soluble parts of the cells. The major route is via the carboxydismutase system with only a small incorporation via the phosphoenolpyruvate carboxylase enzyme. Enzymes of the tricarboxylic acid cycle have low activities compared with those in heterotrophs and this overall slow metabolism, rather than the lack of a specific enzyme such as NADH oxidase, may well account for the slow growth of these bacteria. Although there is very active glutamic dehydrogenase in Nitrosomonas that utilizes ammonia, the enzyme has a very small activity in Nitrobacter. This poses a problem of the route of incorporation of nitrite nitrogen into cell nitrogen in the latter bacterium.
• 9 A few heterotrophic fungi have been described which oxidize ammonia to nitrate but their activity is small compared with that of the nitrifying bacteria.
• 10 It is concluded that the nitrifying bacteria which have many novel biochemical features not met with in other organisms merit further study.

     

MOVEMENT OF EELWORMS: V. OBSERVATIONS ON APHELENCHOIDES RITZEMA-BOSI (SCHWARTZ, 1912) STEINER, 1932 ON FLORISTS' CHRYSANTHEMUMS

Adults of Aphelenchoides ritzema-bosi tend to migrate up the stems of chrysanthemum plants in stationary water films possibly by a negatively geotropic response. A current of water down the stem opposes such an upward movement. Greatest mobility occurred in thick films of water in places with a high concentration of epidermal hairs as at the top of the stem and on the undersurface of leaves. Ciné films of movement in thick and thin films showed that there were fundamental differences in the type of locomotion in these two environments. Invasion of leaves via stomata was observed and the method of movement is described. The presence of A. ritzema-bosi in leaves appears to render the epidermis permeable to water. During dry weather there is little movement inside the leaf, but after rainfall activity increases as water enters the leaf. Spread of eelworm infestation in the leaf occurs in the mesophyll and across veins although initially these act as barriers. Emergence occurs via the stomata, chiefly on the undersurface. When the leaf is wet, about 50% of the eelworms emerge in the first hour. During wet weather many eelworms were recovered from the surfaces of leaves and it is suggested that eelworms spread mostly under these conditions.     

Secondary production of chironomid communities in insecticide-treated and untreated headwater streams

SUMMARY.

• 1 Production of chironomid communities of three first order, Appalachian Mountain streams was estimated and the effects of an insecticide-induced disturbance on chironomid production was examined.
• 2 Annual production of non-Tanypodinae chironomids in the streams during the first study year (no treatment) ranged from 1366 to 3636 mg m^?2, while production of Tanypodinae chironomids ranged from 48 to 116 mg m ^?2. Production/biomass ratios ranged between 19 and 23 for non-Tanypodinae and from 6 to 7 for Tanypodinae chironomids.
• 3 Insecticide applications resulted in significantly lower chironomid densities and biomass in the treated stream relative to the pretreatment year and reference stream. Annual production of non-Tanypodinae (703 mg m^?2) and Tanypodinae (32 mg m ^?2) chironomids in the treated stream decreased by 64% and 67%, respectively, compared with the pretreatment year. In contrast, production of non-Tanypodinae (2084 mg m^?2) increased by 34% and production of Tanypodinae (96 mg m^?2) by 57% in the reference stream.

     

Cavity size, clutch-size and the breeding ecology of Tree Swallows Tachycineta bicolor

Previous studies provide evidence that cavity size influences clutch-size and reproductive success in some hole-nesting birds, because overcrowding in cavities may cause brood mortality due to trampling or hyperthermia. We tested this hypothesis with two experiments at nestbox populations of Tree Swallows Tachycineta bicolor in southeastern Ontario. Female Tree Swallows showed a preference for nesting in large boxes over small ones in the first experiment, and they laid significantly smaller clutches in small nestboxes during both experiments. Differences in clutch–size between large and small nestboxes could not be attributed to other factors known to influence clutch-size in birds (e.g. parental quality, habitat quality, laying date). Reproductive success, however, did not differ between pairs using the two box types during either experiment, nor did it differ during within-clutch-size comparisons between box types. Some measures of nestling quality were significantly lower for broods in large boxes, but during most comparisons there were no differences. These results do not support the adaptive reason for why cavity size affects clutch-size. We suggest that broods in our experiments did not experience the microhabitat conditions necessary to induce the expected differences in brood mortality.     

Production of a stream shredder, Peltoperla maria (Plecoptera: Peltoperlidae) in disturbed and undisturbed hardwood catchments

SUMMARY. (1) The average benthic density of Peltoperla maria in an undisturbed southern Appalachian stream was more than twice that of a nearby stream draining a previously clear-cul catchment in its tenth year of natural secondary succession.
(2) Peltoperla production estimates, using three methods, do not show a significant difference in production between streams draining the two catchments. We attribute these results to quicker growth and slightly higher densities of larger nymphs in the disturbed stream. Production estimates for the disturbed stream ranged from 498 to 560 mg (ash free dry weight) m⁻²y⁻¹ while those for the undisturbed stream were 41–4–515 mg m⁻² y⁻¹.
(3) Our results reinforce the view that conclusions based solely upon numerical densities may lead to erroneus interpretations about the roles organisms play in ecosystems.
(4) Annual frass production by this shredder is about 20 times (10 g m⁻² y⁻¹) the secondary production of P. maria.     

Field Studies of Cereal Leaf Growth: IV. WINTER WHEAT LEAF EXTENSION IN RELATION TO TEMPERATURE AND LEAF WATER STATUS

Throughout winter and early spring, rule and auxanometer measurementsshowed that leaf extension rate (R_E) was directly related totemperature and stopped at about 0°C. During this period,both night and day time R_E responded similarly to temperature.Bright sunshine in late April and May caused fast transpirationwhich was associated with low leaf water potential () and slowR_E. When bright sun was obscured by cloud, R_E increased butthis did not compensate for previous slow R_E. Leaf turgor potential,calculated as the difference between and leaf osmotic potential,was large (0.6–1.8 MPa) and bore little relation to R_E.Low was associated with slower R_E than would have been expectedwithout water stress, but the relation was not unique. On abright day in May, adaptation to low occurred and during theafternoon R_E was faster than at similar values of and meristemtemperatures before noon. The response of R_E and duration ofleaf extension to temperature suggested that for any particularleaf grown under field conditions, variation in mean growingtemperature would affect final leaf length only slightly. Becausesevere water stress slows R_E without affecting the durationof leaf extension markedly, it decreases final leaf size.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.