Interaction ofFLC and late-flowering mutations inArabidopsis thaliana

The phenotype caused by mutations that affect the timing of flowering inArabidopsis thaliana has been most extensively analyzed in the Landsbergerecta (Ler) genetic background. In Ler, the late-flowering phenotype ofFRIGIDA and mutations inLUMINIDEPENDENS is suppressed by the Ler allele ofFLC. In this study, the interactions of nine mutations conferring late flowering with theFLC allele of the Columbia ecotype (FLC-Col), which does not suppress late flowering, were examined. The effect on flowering time of combining six of the mutations withFLC-Col was additive; plants containingFLC-Col withfd, gi, fwa, fha, ft, andfe flowered slightly later than plants containing these mutations with theLer allele ofFLC. In contrast, a synergistic effect was observed betweenFLC-Col and three mutations;fca, fpa, andfve plants became extremely late flowering when combined withFLC-Col. Maximum delay in flowering for the majority of the mutant strains requiredFLC-Col in a homozygous state, although forfpa andfe a single copy ofFLC-Col allowed maximum lateness. In addition, thefd andfe mutations became more dominant in the presence ofFLC-Col.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Construction of improved vectors for gene cloning inMicromonospora melanosporea

Two vectors forMicromonospora melanosporea have been constructed with theStreptomyces plasmid pIJ702 along with thesgm gene (sisomicin-gentamicin resistance gene fromM. zionensis) as the second antibiotic resistance marker. These plasmids, containingsgm gene as the second selectable marker, may be an attractive alternative to pIJ702, which is incapable of conferring melanin production inM. melanosporea and consequently is not useful for insertional inactivation in this bacterium. The constructions remove restriction site forM. melanosporea restriction endonuclease and provide additional unique sites for the insertional inactivation of selectable markers, which enhance the use of these plasmids as general cloning vectors in bothM. melanosporea andS. lividans. On the other side, inS. lividans, plasmid pMK33-1 facilitates isolation and studies of promoters based on detection of extremely convenient phenotype of melanin production. This has been proved by shotgun cloning of chromosomal DNA fragments ofM. melanosporea and chromogenic identification ofS. lividans transformants which were capable of producing a melanin.     

Evolution of the immunoglobulin heavy chain variable region (Igh-V) locus in the genusMus

The evolution of the mouse immunoglobulin heavy chain variable region (Igh-V) locus was investigated by the comprehensive analysis of variable region (Vh) gene family content and restriction fragment polymorphism in the genusMus. The examination of naturalMus domesticus populations suggests an important role for recombination in the generation of the considerable restriction fragment polymorphism found at theIgh-V locus. Although the sizes of individualVh gene families vary widely both within and between differentMus species, evolutionary trends ofVh gene family copy number are revealed by the analysis of homologues of mouseVh gene families inRattus andPeromyscus. Processes of duplication, deletion, and sequence divergence all contribute to the evolution ofVh gene copy number. CertainVh gene families have expanded or contracted differently in the various muroid lineages examined. Collectively, these findings suggest that the evolution of individualVh family size is not driven by strong selective pressure but is relatively neutral, and that gene flow, rather than selection, serves to maintain the high level of restriction fragment polymorphism seen inM. domesticus.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

Serologische Untersuchungen zur Gliederung derBrassicaceae

The serological investigations support the opinion ofJanchen (1942) to combine the generaBunias, Isatis, andSisymbrium in the tribeSisymbrieae; Cheiranthus, Erysimum, andMatthiola in the tribeHesperideae; andBrassica, Crambe, Sinapis, andSuccowia in the tribeBrassiceae. They further underline the central position of theSisymbrieae and the isolated position of theHeliophileae. In accordance withEigner (1973) theBrassiceae are placed closer to theSisymbrieae than inJanchen; the same holds for thePringleeae. No serological justification could be found to uniteArabis andBarbarea in the tribeArabideae, andAlyssum andLunaria in theAlysseae. From the antigen-systems used among the representatives ofJanchen's Lepidieae the generaLepidium andNeslia show remarkable correspondence both toCamelina andThlaspi, but not toCochlearia which appears distant fromCamelina andThlaspi also.

Teil 1/Part 1.     

Chromosome numbers of asteranthos and the putatively related Lecythidaceae

An updated review of chromosome numbers in the Lecythidaceae is presented, which includes new counts forGustavia angustifolia andLecythis lanceolata (both withn=17) and gives identifications to species for five previously published counts based on unidentified material. The earlier conclusion that the largely Asian Planchonioideae havex=13, the African Napoleonaeoideae havex=16, and the New World Lecythidoideae havex=17 still holds. The new count ofn=21 forAsteranthos brasiliensis does not support the previous treatment ofAsteranthos, a monotypic genus of the upper Amazon drainage, as the only New World representative of the otherwise tropical west African Napoleonaeoideae. Mounting evidence favors recognition of a monotypic family, the Asteranthaceae, of yet unclear relationships.     

Photonastic and thermonastic opening of capitulum in dandelion,Taraxacum officinale andTaraxacum japonicum

The capitula ofTaraxacum officinale andT. japonicum open in response to temperature rise at lower temperatures (thermonasty), and in response to light at higher temperatures (photonasty), as was the case inT. albidum. The capitula ofT. officinale could respond to the same temperature rise more sensitively than those ofT. albidum orT. japonicum. The minimum temperature for photonastic opening is as low as 13 C forT. officinale, while that forT. albidum andT. japonicum is about 18 C. That is why the capitula ofT. officinale opened earlier than those ofT. albidum andT. japonicum in the morning in April under natural conditions. The capitulum continued to be open for about 13–14 hr inT. officinale and about 8–11 hr inT. japonicum and inT. albidum both under natural conditions in April and even under constant light-temperature conditions, suggesting that the time of capitula-closing in these three species is not controlled by changes in environmental factors (light and/or temperature).     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Contrasting effects of habitat structure on the recruitment and mortality of an epibiotic macroalga

Field surveys over 2 years in contiguous beds of the seagrassesZostera capricorni andPosidonia australis showed that the green algaCodium duthieae was consistently more abundant inZ. capricorni than inP. australis. In 1 year, mature plants were also more abundant at the boundary between the seagrass beds than in either bed. Field experiments and programmes of sampling were used to investigate three potential explanations for the unusual distribution of this alga: (1) that the availability of substrata suitable for attachment of the alga differed between the two seagrass beds; (2) that mortality of matureC. duthieae differed between the seagrass beds; and/or (3) that the intensity of recruitment was different in the two seagrass beds.C. duthieae plants were exclusively epibionts of the bivalveAnadara trapezia. Detailed sampling showed that the abundance ofA. trapezia was similar in both seagrass beds and that the distribution of bivalves suitable as substrata forC. duthieae plants was not obviously related to proximity to the boundary between the beds. Two experiments investigated the survival ofC. duthieae plants in each bed. In the first, matureC. duthieae plants transplanted into theP. australis bed suffered similar rates of mortality to plants which were disturbed and moved within theZ. capricorni bed or which were left undisturbed in theZ. capricorni bed. Fewer of the host bivalves were recovered from theZ. capricorni bed, however, indicating that the mechanism of mortality differed between the beds, hosts being more frequently dislodged in theZ. capricorni bed. Removal of the leaves of the seagrasses had consistently greater effects on near-bottom current velocities in theZ. capricorni bed than in theP. australis bed and significantly increased mortality ofC. duthieae in theZ. capricorni bed. Survival of plants was greater in plots of artificial leaves ofP. australis placed in theZ. capricorni bed than in plots of the naturalZ. capricorni leaves or plots where the natural leaves were removed. Most mortality in theZ. capricorni bed was due to dislodgement of the alga and its bivalve substratum. Corresponding manipulations of leaves in theP. australis bed had consistently smaller effects on survivorship of both the alga and its host. Patterns in the recruitment of the alga most clearly reflected the distribution of adults.C. duthieae recruits were 5 times more abundant in theZ. capricorni bed and at the boundary between the two beds than in theP. australis bed. The results demonstrate how habitat structure, provided by the canopy of leaves of the two species of seagrass, can have contrasting effects on the recruitment and mortality of a macroalga. In the case ofC. duthieae, it appears that the differential pattern of recruitment is the primary determinant of the distribution of adult plants.     

rbcL sequences support exclusion ofRetzia,Desfontainia, andNicodemia from theGentianales

The taxonomic positions ofRetzia, Desfontainia, andNicodemia have been much discussed, and all three genera have been included inLoganiaceae (Gentianales). We have made a cladistic analysis ofrbcL gene sequences to determine the relationships of these taxa toGentianales. Four newrbcL sequences are presented; i.e., ofRetzia, Desfontainia, Diervilla (Caprifoliaceae), andEuthystachys (Stilbaceae). Our results show thatRetzia, Desfontainia, andNicodemia are not closely related toLoganiaceae or theGentianales. Retzia is most closely related toEuthystachys and is better included inStilbaceae. The positions ofDesfontainia andNicodemia are not settled, butDesfontainia shows affinity for theDipsacales s.l. andNicodemia for theLamiales s.l.     

Comparison of the cell surface barrier and enzymatic modification system inBrevibacterium flavum andB. lactofermentum

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC⁺ strains ofEscherichia coli with lower efficiency than McrBC⁻.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR⁺ strains ofE. coli with lower efficiency than EcoKR⁻, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.     

The appetence behaviour of two South African paralysis-inducing ixodid ticks

The appetence responses of the ixodid ticksIxodes rubicundus andRhipicephalus punctatus to various stimuli were quantified under laboratory conditions. A greater percentage ofI. rubicundus generally responded to the different stimuli at lower than at higher temperatures while the converse was usually true forR. punctatus. Radiation heat and shadowing evoked a response in the greatest percentage ofI. rubicundus, whereas CO₂ and host odour were responsible for a similar reaction inR. punctatus. A single perturbation of the substrate caused more than 50% ofI. rubicundus to respond while constant perturbation resulted in a progressive decrease in the proportion of ticks reacting. Significantly moreR. punctatus thanI. rubicundus were attracted to a CO₂ trap and theR. punctatus were also attracted to the trap over a greater distance. Significantly more older than younger ticks of both species responded to the various stimuli.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

Structural genesvirC1 andvirC2 of the host range determiningvirC operon are determinants of virulence inAgrobacterium tumefaciens

The host range determiningvir C operon ofAgrobacterium tumefaciens is known to consist of two open rea’ding frames designatedvirC1 andvirC2. Earlier work that employed insertional mutations invirC1 andvirC2 established the role of thevirC2 component in the determination of virulence. In this work a plasmid with an internal deletion invirCl was constructed. This deletion derivative restored virulence to bacteria carrying a mutation in thevirC2 region but not to bacteria carrying avirC1 mutation. This evidence establishes that bothvirC1 andvirC2 are required for efficient host plant transformation byAgrobacterium tumefaciens.