Shear stress effects on cell growth and <Emphasis Type="SmallCaps">L</Emphasis>-DOPA production by suspension culture of <Emphasis Type="Italic">Stizolobium hassjoo</Emphasis> cells in an agitated bioreactor

Suspension cultures of Stizolobium hassjoo cells were cultivated in a 7l bioreactor. The growth rate and intracellular L-DOPA content of the cells using two different turbine impellers were compared. There were distinct differences in growth behavior and L-DOPA productivity in the range of 100 to 500 rpm for flat-blade turbine impeller. Disk turbine retarded significantly the cell growth but not so significantly for L-DOPA production in the range of 200 to 300 rpm. The shear force intensity of the two impellers at various rotational rates was compared with shear force index (SFI), and power input per unit mass and eddy length scale. There was good consistency among the three indexes for shear force intensity. Thus with SFI the shear force intensity of bioreactor can be indirectly estimated. A critical shear stress that may cause sublytic effect in cells was identified for flat-blade turbine operated at 400 rpm. The common effect between the shear stress and the proton elicitation in the bioreactor was elucidated with a hypothesis of signal transduction by second messenger, H⁺. Our results suggested that H⁺ transduced the signal to protoplast when S. hassjoo cells were stimulated by shear stress. This resulted in an increase of H⁺ which triggered a similar reaction to the pH control of culture broth and enhanced the L-DOPA production.     

The greater seedling high-light tolerance of <Emphasis Type="Italic">Quercus robur</Emphasis> over <Emphasis Type="Italic">Fagus sylvatica </Emphasis>is linked to a greater physiological plasticity

The responses of Quercus robur (oak) and Fagus sylvatica (beech) seedlings to four different light environments (full, 50%, 40% and 15% sunlight) and to a rapid increase in irradiance were explored during the summer, after 2 years of growth in a forest nursery at Nancy (France). Significant differences between the two species were found for most variables. Phenotypic plasticity for morphological variables (root-shoot ratio, leaf size, leaf weight ratio) was higher in beech than in oak, while the reverse was true for anatomical (stomatal density, epidermis thickness, exchange surface area of the palisade parenchyma) and physiological (maximum photosynthetic rate, stomatal conductance, Rubisco activity) variables. Predawn photochemical efficiency (F_v/F_m) was higher in oak than in beech in all light environments except in 15% sunlight. F_v/F_m was significantly lower in 100% sunlight than in the other light environments in beech but not in oak. Maximum photosynthetic rates (A_max) increased with increasing light availability in the two species but they were always higher in oak than in beech. Oak exhibited higher Rubisco activity than beech in full sunlight. The transfer of shade-adapted seedlings to the open caused a decrease of F_v/F_m, which was larger for beech than for oak. Transferred oak but not beech plants recovered gradually to the control F_v/F_m values. The decreased chlorophyll content and the increased non-photochemical quenching observed in high-light beech seedlings were not enough to avoid photoinhibition. The results suggest that a greater tolerance of strong irradiance is linked to an enhanced physiological plasticity (variables related to photosynthesis), while shade tolerance relies on an enhanced plasticity in light-harvesting variables (crown morphology and chlorophyll content).     

Tissue-culture enhanced transposition of the maize transposable element <Emphasis Type="Italic">Dissociation</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis> var. '<Emphasis Type="Italic">Italica</Emphasis>'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli (Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct (Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated production of transgenic plants of<Emphasis Type="Italic"> Muscari armeniacum</Emphasis> Leichtl. ex Bak.

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l^-1 cefotaxime for 1 week followed by a medium containing 75 mg l^-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg^r) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg^r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg^r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.     

Cytological investigation of male sterility in sorghum caused by a dominant mutation (<Emphasis Type="Italic">Ms</Emphasis><Subscript><Emphasis Type="Italic">tc</Emphasis></Subscript>) derived from tissue culture

Male sterility mutations are an important tool in the investigation of anther and pollen development and for obtaining hybrid seeds in plant breeding. Cytological analysis of microsporo- and microgameto-genesis in sorghum plants with dominant mutation of male sterility (Ms_tc) derived from tissue culture has been carried out. Using substitution backcrosses, this mutation was introduced first into the nuclear background of the fertile sorghum line SK-723 and from this line into Volzhskoe-4w (V-4w). The mechanism of Ms_tc action on anther and pollen development differed in different nuclear backgrounds. In SK-723, phenotypic expression of Ms_tc began before the beginning of meiosis, which resulted in degeneration of sporogenous tissue in some anthers and in significant disturbances of anther morphology. In microsporocytes that did not degenerate, the frequency of non-specific meiotic abnormalities characteristic of the fertile line SK-723 significantly increased. In addition, in the mutant plants, a number of specific meiotic abnormalities--almost complete desynapsis, and formation of syncytial structures--were observed, apparently the consequence of Ms_tc action. In mono- or bi-nucleate microspores, degenerative processes resulting in formation of empty or anomalously coloured pollen grains led to almost complete male sterility. In the V-4w nuclear background, changes in anther structure and meiotic disturbances were infrequent. The degenerative processes began at the uni- or binucleate microspore stage and resulted in formation of empty or abnormally coloured pollen grains, and in partial pollen sterility. Thus, the same nuclear male sterility-inducing mutation in different nuclear backgrounds affects different stages of pollen development.     

Simple and versatile selection of <Emphasis Type="Italic">Arabidopsis </Emphasis>transformants

The selection of large amounts of seeds of Arabidopsis thaliana obtained after in planta transformation under sterile conditions and in tissue culture is relatively labour-intensive, time-consuming and expensive. We describe here an alternative selection procedure that uses regular greenhouse conditions and functions with a wide variety of selective agents. The selection system is based on the inert growth substrate Grodan and was intensively tested for the selection of transgenic Arabidopsis seeds with kanamycin, hygromycin, BASTA, sulfadiazine and methotrexate. However, the system has the capacity to be applicable for more selective agents and to a wider range of plant species. For Arabidopsis, the effectiveness of selective agents is comparable to or better than that obtained with tissue culture procedures.     

Mapping of a thermo-sensitive earliness <Emphasis Type="Italic">per se</Emphasis> gene on <Emphasis Type="Italic">Triticum monococcum</Emphasis> chromosome 1A<Superscript>m</Superscript>

An earliness per se gene, designated Eps-A^m1, was mapped in diploid wheat in F₂ and single-seed descent mapping populations from the cross between cultivated (DV92) and wild (G3116) Triticum monococcum accessions. A QTL with a peak on RFLP loci Xcdo393 and Xwg241, the most distal markers on the long arm of chromosome 1A^m, explained 47% of the variation in heading date (LOD score 8.3). Progeny tests for the two F_2:3 families with critical recombination events between Xcdo393 and Xwg241 showed that the gene was distal to Xcdo393 and linked to Xwg241. Progeny tests and replicated experiments with line #3 suggested that Eps-A^m1 was distal to Xwg241. This gene showed a large effect on heading date in the controlled environment experiments, and a smaller, but significant, effect under natural conditions. Eps-A^m1 showed significant epistatic interactions with photoperiod and vernalization treatments, suggesting that the different classes of genes affecting heading date interact as part of a complex network that controls the timing of flowering induction. Besides its interactions with other genes affecting heading date, Eps-A^m1 showed a significant interaction with temperature. The effect of temperature was larger in plants carrying the DV92 allele for late flowering than in those carrying the G3116 allele for early flowering. Average differences in heading date between the experiments performed at 16 °C and 23 °C were approximately 11 days (P < 0.001) for the lines carrying the Eps-A^m1 allele for early flowering but approximately 50 days (P < 0.0001) for the lines carrying the allele for late flowering. The large differences in heading time (average 80 days) observed between plants carrying the G3116 and DV92 alleles when grown at 16 °C, suggest that it would be possible to produce very detailed maps for this gene to facilitate its future positional cloning.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of North American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.)

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.     

Tissue structure and respiration of stems of <Emphasis Type="Italic">Betula pendula</Emphasis> under contrasting ozone exposure and nutrition

Tissue structure and respiration (Rs) of stems were analyzed in Betula pendula grown throughout the growing season in either filtered air (control) or 90/40 nl O₃ l^-1 (day/night). Both regimes were split into high and low nutrient supply. High nutrition increased tissue and cell sizes within the stem xylem, phloem and periderm, whereas ozone (O₃) tended to reduce tissue widths, inhibiting in particular the cambial activity of xylem growth in low-fertilized, O₃-exposed plants (O₃/LF). Callose deposition was enhanced in the phloem sieve plates and tannins tended to condense in vacuoles of parenchyma cells under O₃ stress. Decline occurred close to lenticels, related to O₃ impact during shoot differentiation and was probably exacerbated by the limited assimilate translocation. Radial stem growth ceased 4 weeks earlier than in control plants; however, the area-based Rs was enhanced during intense growth in high-fertilized, O₃-exposed plants. Photosynthetic CO₂ refixation of stems reached about 50% of their dark respiration rate and the relative growth rate (RGR) did not differ between treatments. At high nutrition, RGR enhanced Rs to levels twice as high as the maintenance level. Unit construction costs appeared to be similar in each treatment, although CO₂ release on a volume-increment basis was lowered by 45% in O₃/LF plants. This latter effect is ascribed to lowered maintenance demands of a xylem remaining reduced in width by 50%. The high respiratory costs in the carbon balance of O₃/LF plants result from an enhanced leaf rather than stem respiration, given the high demand for stress compensation in the foliage.     

Physiological energetics of the protobranch bivalve <Emphasis Type="Italic">Yoldia hyperborea</Emphasis> in a cold ocean environment

Yoldia hyperborea is a deposit-feeding circumpolar protobranch that also inhabits muddy sediments of the cold water boreal system of Conception Bay, Newfoundland, Canada. Little is known about this species, despite its wide distribution and frequent high density in the benthos. The present work deals with oxygen consumption and ammonia excretion under cold ambient conditions. Y. hyperborea showed low basal metabolism [0.051 ml O₂ h^у·(g dry weight)^у, T=у°C] and low ammonia excretion rates [4.212 µg·NH₄-N·h^у·(g dry weight)^у, T=у°C]. Low metabolic activity could prove a useful strategy during periods of low food availability. In addition, Y. hyperborea was able to regulate its O₂ consumption rate at very low pO₂ levels, which may be advantageous for a species that may experience periods of hypoxia.     

Nutrient partitioning and storage in arid-zone forests of the mangroves Rhizophora <Emphasis Type="Italic">stylosa</Emphasis> and <Emphasis Type="Italic">Avicennia</Emphasis><Emphasis Type="Italic">marina</Emphasis>

Mangrove partitioning and storage of macronutrients and trace metals were examined in different arid coastal settings of Western Australia. Total living biomass in three Rhizophora stylosa forests, which ranged from 233 to 289 t DW ha^-1, was significantly greater than biomass in three Avicennia marina forests (range: 79-155 t DW ha^-1). Although prop roots and stems were the largest single tree components for R. stylosa and A. marina, respectively, most nutrients were stored in leaves and living roots of both species. However, only a small fraction of the total nutrient pool was vested in tree biomass; the vast bulk was in soils. A large below-ground pool of dead fine roots was identified at all stands, equivalent to 36-88% DW of total living tree biomass. The amount of Ca, S, Cl, Na, Si, Fe, Mn, Zn, B, Mo and Cu vested in dead roots of both species was greater than in the total living tree biomass. The proportion of Fe and S vested in live and dead roots was exceptionally large, consistent with previous evidence of metal plaques on mangrove roots. Sulphur, iron and zinc in dead roots of both species constituted the bulk of these metals. R. stylosa trees preferentially accumulated more Mg, S, Cl, Na, Si, Fe, Mn, B and Mo than A. marina trees. Proportionally greater storage of P, N, Ca, K, Cu and Zn occurred in two of the three A. marina forests. Foliar concentrations of Mg, S, Mn, B and Mo in mangrove leaves were at the high end of the range reported for other tropical trees, but other elemental concentrations were at the low or mid-range. Nitrogen limitation in these forests is implied by a positive correlation between total tree N and net canopy production and by a lower percentage of ecosystem N in tree biomass as compared with other forests. Unlike terrestrial forests where a large proportion of nutrient capital is vested in floor litter, most elements in these mangrove forests are stored in dead roots. A large reservoir of dead roots below the forest floor may serve as a conservation mechanism, particularly in such arid oligotrophic environments.     

Structural and molecular analysis of self-incompatibility in almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis>)

. A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S₈) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.     

Exogenous passive heating during torpor arousal in free-ranging rock elephant shrews, <Emphasis Type="Italic">Elephantulus myurus</Emphasis>

In the laboratory rock elephant shrews (Elephantulus myurus; mean body mass 56.6 g) displayed the lowest torpor T_{b min} yet recorded (ca. 5°C) in a placental daily heterotherm. It was unknown whether these low T_bs were characteristic of daily heterothermy in free-ranging animals. It was also unclear how cost effective these low T_bs were since considerable energy is required to arouse from low T_bs on a daily basis. We continuously measured body temperature once every hour for 85 days in 13 free-ranging E. myurus from May to August 2001 (winter) in Weenen Game Reserve, KwaZulu-Natal, South Africa. We recorded a total of 412 torpor bouts. Free-ranging E. myurus had a high propensity for torpor with females displaying higher torpor frequency than males. The lowest T_b recorded was 7.5°C at T_a=2.7°C and the minimum torpor T_b was strongly correlated with ambient temperature. Torpor arousal was tightly coupled with ambient temperature cycles. Low torpor T_{b min} at low T_as was therefore cost-effective because the animals offset the high cost of arousal through exogenous passive heating. Laboratory studies under constant ambient temperatures may therefore underestimate the energetic benefits of torpor in free-ranging small mammals that inhabit regions where seasonality is moderate.     

High-resolution mapping of the bolting gene <Emphasis Type="Italic">B</Emphasis> of sugar beet

Sugar beet (Beta vulgaris L.) is a biennial species. Shoot elongation (bolting) starts after a period of low temperature. The dominant allele of locus B causes early bolting without cold treatment. This allele is abundant in wild beets whereas cultivated beets carry the recessive allele. Fifteen AFLP markers, tightly linked to the bolting locus, have been identified using bulked segregant analysis. The F₂-population consisted of 2,134 individuals derived after selfing a single F₁-plant (Bb). In a first step, a linkage map was established with 249 markers based on 775 F₂-individuals with a coverage of 822.3 cM. The loci are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Seventeen marker loci were placed at a distance less than 3.2 cM around the bolting gene. In a second step, four of those markers most closely linked to B were mapped with the entire F₂-population. Two of the markers were mapped flanking the B gene at distances of 0.14 and 0.23 cM. The other two markers were mapped at a distance of 0.5 cM from the gene. The tight linkage could be verified by testing 88 unrelated plants from a breeding program. The closely linked markers will enable breeders to select for the non-bolting character without laborious test crossings. Moreover, these markers are being used for map-based cloning of the bolting gene.     

Bioreactor shoot cultures of <Emphasis Type="Italic">Rhododendron tomentosum</Emphasis> (<Emphasis Type="Italic">Ledum palustre</Emphasis>) for a large-scale production of bioactive volatile compounds

Rhododendron tomentosum Harmaja (Ledum palustre), a peat bog plant from Ericaceae family, has been used in traditional medicine as the anti-arthritis agent. Although modern researches confirm its anti-inflammatory properties, it remains threatened by habitat degradation and possibilities to collect this endangered species from its natural environment for further biological activity studies are limited. Therefore, R. tomentosum liquid in vitro cultures were established as the alternative source of that valuable plant material. Schenk–Hildebrandt medium with 24.60 μM 2-isopentenyladenine and 592.02 μM adenine provides intensive growth and proper morphology of the obtained microshoots. The R. tomentosum biomass was scaled up using the various bioreactors (immersion, temporary immersion and spraying systems) for better growth and improved volatile oil production. The largest biomass accumulation (fresh weight?=?250 g l^?1, growth index?=?280, dry weight?=?20 g l^?1) and essential oil content (0.5% v/m) were achieved with application of commercially available RITA^® bioreactor. GC/MS analysis revealed the high content of p-cymene (6.9%), alloaromadendrene (5.5%), shyobunone (8.2%) and ledene oxide (II) (13.0%) in the volatile fraction obtained from RITA^® system. The biomass growth parameters and production profile in terms of essential oil and selected terpenoid compounds were determined during the 2 month period. The influence of culture conditions and bioreactor construction on the growth and volatile oil production in R. tomentosum biomasses was discussed.     

Complete sequence of <Emphasis Type="Italic">Tvv1</Emphasis>, a family of Ty<Emphasis Type="Italic">1 copia</Emphasis>-like retrotransposons of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L., reconstituted by chromosome walking

A chromosome-walking strategy was used to sequence and characterize retrotransposons in the grapevine genome. The reconstitution of a family of retroelements, named Tvv1, was achieved by six successive steps. These elements share a single, highly conserved open reading frame 4,153 nucleotides-long, putatively encoding the gag, pro, int, rt and rh proteins. Comparison of the Tvv1 open reading frame coding potential with those of drosophila copia and tobacco Tnt1, revealed that Tvv1 is closely related to Ty1 copia-like retrotransposons. A highly variable untranslated leader region, upstream of the open reading frame, allowed us to differentiate Tvv1 variants, which represent a family of at least 28 copies, in varying sizes. This internal region is flanked by two long terminal repeats in direct orientation, sized between 149 and 157 bp. Among elements theoretically sized from 4,970 to 5,550 bp, we describe the full-length sequence of a reference element Tvv1-1, 5,343 nucleotides-long. The full-length sequence of Tvv1-1 compared to pea PDR1 shows a 53.3% identity. In addition, both elements contain long terminal repeats of nearly the same size in which the U5 region could be entirely absent. Therefore, we assume that Tvv1 and PDR1 could constitute a particular class of short LTRs retroelements.     

Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH₄OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins. Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Clearance rates in the Arctic bivalves <Emphasis Type="Italic">Hiatella arctica</Emphasis> and <Emphasis Type="Italic">Mya </Emphasis>sp.

Filtration was studied in two Arctic clams, Hiatella arctica and Mya sp., collected in Young Sound, Northeast Greenland. Clearance rates were determined as a function of ambient temperature and algal cell concentration, using the clearance method and feeding with a unicellular flagellate. For both species, clearance rates increased with increasing temperature from <у up to 4-8°C. At higher temperatures, filtration ceased and the clams closed their valves. Clearance rates were also determined in temperate specimens of H. arctica collected on the west coast of Sweden. For these specimens, clearance rates increased with increasing temperature from 0 to 18-20°C. When weight-specific clearance rates were compared between the two populations and between species, there were no differences at 1°C. Clearance rates in Arctic H. arctica were maximal at algal cell concentrations corresponding to 2.5-8 µg chlorophyll a l^-1. Temperature compensation in Arctic bivalves is discussed and it is concluded that adaptations to constant low temperatures consist of a lower minimum temperature, for active filtration. Low clearance rates due to low temperatures did not seem to limit growth, under the prevailing conditions in Young Sound.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.