Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile–restorer combinations for heterosis breeding

We report in this study, an improved method for identifying male sterile–restorer combinations using the barnase–barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line × tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66–90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and ∼30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) × tester (barstar) crosses, wherein only two viable male sterile–restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T₀ generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T₁ progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation.     

Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. I. Novel method based on the resistance of sterile closure

Influence of carbon dioxide on growth and product kinetics of industrially important micro-organisms is essential for the interpretation of a bioprocess. In this research, the CO? effects on productivity and growth rate of micro-organisms have been studied by using a variety of kplug. The applied method is based on a different concentration of CO? in the headspace of ventilation flasks. The presented method is simple, inexpensive and shows similar results compared to large-scale fermentation regarding the evolution of CO? in a batch system. For the investigation of the proposed method, experiments employing Arxula adeninivorans LS3, Corynebacterium glutamicum (DM1730 and ATCC WT13032) and Hansenula polymorpha DSM70277 as model organisms in the ventilation flasks are performed. The fermentations in the RAMOS (respiratory activity monitoring system) device were carried out with a normal aeration rate (1 vvm) and under the same operating conditions as the ventilation flask f1. The modified unsteady-state model was used to predict the operation conditions of a biological system in the ventilation flasks. In the present study, a novel and easy method for the quantification of CO? sensitivity of micro-organisms in shaken bioreactors (called ventilation flask) was achieved.     

IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation

The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1β in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1β, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1β correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1β can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1β promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.     

Chromosome doubling in sterile Syringa vulgaris × S. pinnatifolia hybrids by in vitro culture of nodal explants

The aim was to produce tetraploid forms of hybrids of Syringa vulgaris × S. pinnatifolia to restore fertility and enable further breeding to be undertaken. Excised nodal sections of three selections were pre-cultured for 5 days then treated with a range of colchicine concentrations for 1, 2 or 3 days, after which they were washed three times in sterile distilled water and cultured on shoot proliferation medium. Frequent movement to fresh medium was beneficial to survival. Three successive experiments established the range of concentrations of colchicine, 0.05 mM to 0.25 mM, at which tetraploids were likely to be produced. Thus a protocol for the production of tetraploids was established. Cytometric analysis showed that tetraploid forms of two selections were produced. This revised version was published online in August 2006 with corrections to the Cover Date.     

Why don't all termite species have a sterile worker caste?

No general theory explains why a sterile worker caste is not found in all species of both Hymenoptera and Isoptera (Insecta). Recent empirical finding show that, in the termites (Isoptera), feeding outside the nest correlates well with the evolution of the sterile (true) worker caste from the non-sterile (false) worker caste. Here we explain the connection between food-nest separation and true worker evolution in termites, providing a general theory on the restricted distribution of the sterile worker caste in the Isoptera. A cost-benefit model suggests that there is a critical level of nest stability above which natural selection favours true workers over false workers, irrespective of genetical relatedness. Because food-nest separation tends to increase nest stability, this theoretical result implies that the less a termite species consumes its nest as food, the more likely is its nest stability to fall above the critical level and a true worker caste will evolve.     

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.     

Seasonal variation of carrageenans in tetrasporic,cystocarpic and ‘sterile’ stages of Gigartina skottsbergii S. et G. (Rhodophyta,Gigartinales)

Cystocarpic and sterile plants of Gigartina skottsbergii produce -carrageenans, while tetrasporophytes produce -carrageenans. No seasonal variation in the carrageenan yields in the three stages was evident. Yields were clearly higher in cystocarpic (34.1–64.7%) and sterile samples (32.3–71.8%) than in tetrasporic plants (10.1–34.0%). Chemical characteristics of the polysaccharides for any stage are the same throughout the year.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1⁺ and CD11b⁺ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

     

Haldane's rule and its legacy: Why are there so many sterile males?

A general pattern of animal hybridization, known as Haldane's rule, is that the XY (ZW) sex is more severely affected in its viability or fertility than the XX (ZZ) sex. Recent evidence suggests that three different forces have shaped this pattern: (1) the X chromosome and autosomes are in greater disharmony in the XY sex; (2) evolution of hybrid male sterility is greatly accelerated, at least in species with XY males; and (3) maternal-zygotic incompatibility preferentially affects the viability of the XX sex. In species with XY males, the rapid evolution toward hybrid male sterility may be responsible for the bulk of observations pertaining to Haldane's rule. One interesting and testable hypothesis is that sexual selection drives this rapid evolution.     

Is elytral color polymorphism in sweetpotato weevil (Coleoptera: Brentidae) a visible marker for sterile insect technique? Comparison of male mating behavior

The sterile insect technique (SIT) is widely used for suppressing or eradicating target pest insect populations. In the eradication programs using the SIT, a large number of sterile insects are marked and released in the field. In Japan, Cylas fonnrmicarius (F.) group (Coleoptera: Brentidae) weevils are marked with a fluorescent powder dye to monitor the progress of such programs. However, this monitoring technique is not fully effective because of the disappearance or contamination of the dye. Therefore, an alternative marking method is required. Currently, a rare color morph such as piceous elytra (PE) is used for visible marking of C. formicarius group weevils. A PE-monomorphic strain has previously been established by artificial selection from a small locally distributed population; this can lead to reduced survival and genetic changes in behavioral traits due to inbreeding depression. In this study, we evaluated the survival rate and mating behavior of PE males of C. formicarius group. The characteristics of the PE males were similar to those of the wild strain (WS) males. Thus, we considered that PE males were suitable for visible marking in the eradication programs using the SIT.     

Use of <Emphasis Type="Italic">2n</Emphasis> gametes for the production of sexual polyploids from sterile Oriental × Asiatic hybrids of lilies (<Emphasis Type="Italic">Lilium</Emphasis>)

Sixteen Oriental and 12 Asiatic cultivars were crossed in 158 different combinations. A total of 708 F₁ hybrids were obtained from 86 of the different combinations of 15 Oriental and 11 Asiatic cultivars. Because the Lilium cultivars (2n=2x=24) used for the production of these hybrids belong to two different taxonomic sections—Archelirion (O) and Sinomartagon (A), respectively—the F₁ hybrids (OA) could be obtained only through embryo, embryo sac rescue, ovary slice or ovule culture. Most of the F₁ hybrids were highly sterile (did not produce viable n gametes) due to the failure of chromosome pairing. However, in a few cases F₁ plants were found that produced viable 2n pollen at variable frequencies. These 2n pollen grains were successfully used for the production of backcross progenies. Using genomic in situ hybridization we found intergenomic recombinant chromosomes in the sexual polyploid progenies. These results indicate that there are effective prospects for combining important horticultural traits from the two main groups of cultivars of lilies through sexual polyploidization.     

Selection on defensive traits in a sterile caste – caste evolution: a mechanism to overcome life‐history trade‐offs?

SUMMARY During development and evolution individuals generally face a trade-off between the development of weapons and gonads. In termites, characterized by reproductive division of labor, a caste evolved—the soldiers—which is completely sterile and which might be released from developmental trade-offs between weapons and testes. These soldiers are exclusively dedicated to defense. First, we investigated whether defensive traits are under selection in sterile termite soldiers using allometric analyses. In soldiers of the genus Cryptotermes phragmotic traits such as a sculptured and foreshortened head evolve rapidly but were also lost twice. Second, we compared the scaling relationships of these weapons with those in solitary insects facing a trade-off between weapons and gonads. Defensive traits consistently had lower slopes than nondefensive traits which supports the existence of stabilizing selection on soldier phragmotic traits in order to plug galleries. Moreover, soldier head widths were colony specific and correlated with the minimum gallery diameter of a colony. This can proximately be explained by soldiers developing from different instars. The scaling relationships of these termite soldiers contrast strikingly with those of weapons of solitary insects, which are generally exaggerated (i.e., overscaling) male traits. These differences may provide important insights into trait evolution. Trade-offs constraining the development of individuals may have been uncoupled in termites by evolving different castes, each specialized for one function. When individuals in social insect are "released" from developmental constraints through the evolution of castes, this certainly contributed to the ecological and evolutionary success of social insects.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

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 to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.