Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Characterization and functional analysis of hsp21.8b: An orthologous small heat shock protein gene in Tribolium castaneum

Small heat shock proteins (sHSPs) act as molecular chaperones and are widely distributed in all kinds of organisms. Comparative analysis revealed that an orthologous shsp was present during insect evolution. Here, hsp21.8b, one insect orthologous shsp, had been identified in Tribolium castaneum. Quantitative real‐time PCR illustrated that Tchsp21.8b was expressed in all developmental stages, along with the lowest expression at early embryonic stage and relative high expression at other stages especially in late eggs and late pupae. In the adult period, Tchsp21.8b exhibited the highest expression level in central nervous system and followed in elytron, epidermis, ovary and fat body. Moreover, it was upregulated 3.39‐fold in response to enhanced heat stress (45°C) for 4 hr but not to cold stress (4°C) and was upregulated by 1.73‐ to 1.94‐fold under ultraviolet (UV) exposure during 4–6 hr. It was also downregulated by 20.8%–41.8% under starvation in 3 days and had a “down‐up‐down” trend under the pathogen stresses. Larval RNA interference of Tchsp21.8b caused 40.6% insects mortality and reduced the oviposition amount by 66.0% and only 21.0% of the ds‐Tchsp21.8b eggs could hatch into larvae. These results suggested that as an orthologous shsp, Tchsp21.8b not only plays important roles in the growth, development and fecundity of T. castaneum but with the competence to resist the environment stresses, although the response is relatively weak compared to other hsps. Results from this study also uncovered the functions of the orthologous shsp in the development and anti‐stresses ability of T. castanuem. It provided more scientific evidence for revealing the physiological mechanisms of shsps of the insects and enhanced the capabilities to control different pests.     

Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

Analysis of blastomogenic activity of mammal carcinogens in <Emphasis Type="Italic">Drosophila</Emphasis> using the <Emphasis Type="Italic">wts</Emphasis><Superscript><Emphasis Type="Italic">P4</Emphasis></Superscript> allele and RNA interference-induced P53 silencing

The test for somatic mutagenesis and recombination in Drosophila is one of the widely used approaches for determination of possible carcinogenic effects of chemical compounds. The use of heterozygotes for mutant tumor suppressor gene wts enables more direct evaluation of the blastomogenic effects of chemical compounds, by tumor formation in the adult flies. This study presents evaluation of the SMART effectiveness upon the use of Drosophila heterozygotes for the wts ^P4 gene, first included into the test system. The increase of the test resolution capacity compared to the literature data for the wts ^P2 allele was observed. Using wts ^P4 heterozygotes, a total of 20 carcinogenic compounds, and their slightly carcinogenic and non-carcinogenic analogs were tested. Specificity of the method was about 100%, and sensitivity depended on the type of the agent tested. The latter was absolute for the direct action carcinogens, with respect to carcinogens, requiring the metabolic activation. The sensitivity was elective and was limited by the presence of the enzymes capable of activating of these compounds. To increase the test sensitivity, the RNA interference-mediated silencing of the Drosophila p53 functional activity was successfully applied. Moreover, the frequency of wts tumor induction considerably increased both in spontaneous and induced mutagenesis conditions.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Phylogeny of <Emphasis Type="Italic">Gunnera</Emphasis>

 The phylogeny of the genus Gunnera is investigated for the first time. Twelve species representing the six currently recognised subgenera are analysed. Two chloroplast DNA regions, the rbcL gene and the rps16 intron, together provide 46 informative characters out of 2335. A combined analysis of both genes gives four most parsimonious trees, firmly establishing the east South American G. herteri as sister group to the rest of the genus. The African G. perpensa is sister group to two well-supported clades, one including the South American subgenera Misandra and Panke, the other the Australian/New Zealand/Malayan species of subgenera Milligania and Pseudogunnera. Thus, South America is a composite area for Gunnera, showing up at two different levels in the cladogram. Our analysis supports a close biogeographic relationship between Australia and New Zealand. The evolution of some morphological characters is discussed. Lastly, the unusual structure of some of the rbcL sequences is reported. Received July 6, 2000 Accepted October 24, 2000     

Evolutionary alkaline transition in human cytochrome <Emphasis Type="Italic">c</Emphasis>

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pK_a value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.     

Morphological and molecular characterization of two ITS groups of <Emphasis Type="Italic">Erysiphe</Emphasis> (Erysiphales) occurring on <Emphasis Type="Italic">Syringa</Emphasis> and <Emphasis Type="Italic">Ligustrum</Emphasis> (Oleaceae)

ITS sequences determined for 53 Erysiphe specimens on Syringa and Ligustrum collected in Europe, East Asia, and North and South America were divided into two ITS groups, S and K types. Phylogenetic analysis showed that these two ITS types do not share a common ancestor and form separate clades. The K type on Ligustrum was identified as Erysiphe ligustri based on the three-dimensional branching pattern of appendages. Morphological observations showed that there are some morphological differences—pigmentation of appendages and number of ascospores per ascus—between the S and K types on Syringa. Based on these morphological observations, the S and K types on Syringa were identified as E. syringae and E. syringae-japonicae, respectively. The recent abundant production of chasmothecia by lilac powdery mildew in Europe was caused by E. syringae-japonicae introduced from East Asia. DNA sequence analyses of the rDNA ITS region and the 28S rDNA, tub2, CYP51, and Chs1 genes did not support an interspecific hybrid origin for E. syringae-japonicae. Haplotype analysis suggested that E. syringae originated in North America and independently migrated to East Asia and Europe/South America.     

Biology of <Emphasis Type="Italic">Eretmocerus sudanensis</Emphasis> n. sp. Zolnerowich and Rose,parasitoid of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> Gennadius

Eretmocerus sudanensis = E. nr. emirates was released in 1992 into South Florida from Sudan to control Bemisia tabaci. It has since become the dominant Eretmocerus species in southwest Florida. Specimens were recovered at the Southwest Florida Research and Education Center in Collier County from a population of B. tabaci on collard Brassica oleracea, and the parasitoid described as new species, though as yet unpublished. Here we report results of studies conducted to evaluate the biology of this parasitoid of B. tabaci biotype B under laboratory controlled conditions (26°C ± 0.5, 13 h light, 62% ± 6 HR). No preoviposition period was observed for E. sudanensis. Females lived an average of 13.2 days and in this time deposited an average of 161 eggs at a mean oviposition rate of 13 eggs per day. The total compared favorably with published values for E. melanoscutus (123 eggs) and was similar to E. mundus (171 eggs). E. sudanensis showed the same tendencies of preference and suitability for the intermediate host stages (2nd and 3rd) as reported for these same 2 species. However, parameters such as development time from egg to adult (16 days) and net reproductive rate (Ro = 91.3 eggs/female) were shorter and greater, respectively, compared to published results for either E. melanoscutus or E. mundus under similar conditions. Such differences in demographic parameters could indicate a significantly greater reproductive potential for E. sudanensis.     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.     

Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Fast genome editing in Bacillus subtilis

Bacillus subtilis is a model organism for Gram‐positive bacteria and widely used in the study of cellular functions and processes including protein secretion, sporulation, and signal transduction. It is also an important industrial host for the production of proteins and chemicals. Generally, genome editing of B. subtilis often needs the construction of integration vectors in Escherichia coli, linearizing the constructed plasmids, and subsequent transformation of the linear deoxyribonucleic acid via natural competence or electroporation. In this work, we examined the feasibility to directly transform and integrate B. subtilis using linear deoxyribonucleic acid from Gibson assembly without the need for cloning in E. coli. Linear deoxyribonucleic acid of 8–10 kb showed the highest transformation efficiency which was similar to that of using linearized plasmids constructed in E. coli. This method shortens the overall process from 1 week to 1 day and allows the integration of multiple genes in one step, providing a simple and fast method for genome editing in B. subtilis.