Partial complementation of embryo defective mutations: a general strategy to elucidate gene function.

The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP-protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.

The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos.

The EMB 506 gene of Arabidopsis, required for the normal development of the embryo beyond the globular stage, has been cloned. The gene encodes a protein of predicted size 35 kDa that contains five ankyrin (ANK) repeats within the C terminal moiety. ANK repeats are conserved domains of 33 amino acids involved in specific recognition of protein partners. The EMB 506 protein was detected at different stages of silique development but accumulated preferentially in the mature cauline leaves. The rescue of homozygous emb 506 embryos by complementation with the wild-type sequence cDNA demonstrated that the emb mutation is a consequence of the T-DNA insertion and that integration and expression of the transgene occurred during gametogenesis and/or early embryo development. In addition to the drastic effect of the emb 506 mutation during embryo development, complementation experiments revealed another effect of the gene: emb 506 plants transformed with the wild-type EMB 506 sequence were able to produce viable seeds but showed a reduction of apical dominance and the presence of adventitious buds or bracts along the stem. This result supports the idea that genes essential for embryogenesis may also be required at other stages of the plant life cycle.

An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant.

Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development. In mammals, several genes have been identified whose products are necessary to prevent entry into the apoptotic process. We have isolated a clone from an Arabidopsis thaliana cDNA library whose predicted translation product shows highly significant similarity to the mammalian defender against apoptotic death 1 (DAD1) protein. Transformation of the mutant hamster tsBN7 cells, which undergo apoptosis at restrictive temperature, demonstrates that the plant protein is as efficient as human DAD1 in rescuing these hamster cells from apoptosis. In contrast to mammals, Southern hybridisation and genomic data indicate that there are probably two genes in Arabidopsis thaliana. Northern blot analysis shows that AtDAD transcripts are present in all tissues examined, although the abundance of the transcripts is reduced in siliques during the maturation and desiccation phase of the seed. This is the first experimental proof that a homologue of an animal gene involved in apoptosis exists in plants and the first demonstration of complementation of a vertebrate mutant by a plant cDNA. Our results suggest that this process of suppression of apoptosis has been conserved in animals and plants.

The lobster parasite Anophryoides haemophila (Scuticociliatida: Orchitophryidae): nuclear 18S rDNA sequence, phylogeny and detection using oligonucleotide primers.

We have determined the nucleotide sequence of the nuclear gene encoding small-subunit ribosomal ribonucleic acid of the ciliate Anophryoides haemophila, a parasite of the American lobster Homarus americanus. The gene is 1763 bp in length, and has a guanosine-plus-cytosine content of 43.9%. Inferred phylogenetic frameworks strongly support the monophyly of the scuticociliates, and suggest that order Scuticociliatida should be elevated to at least subclass rank. Oligonucleotide probes based on A. haemophila ssurDNA can discriminate between DNAs of A. haemophila and other investigated hymenostome ciliates, and effectively prime polymerase chain reaction-based detection of A. haemophila deoxyribonucleic acid against at least a 1600-fold excess of total deoxyribonucleic acid from H. americanus. GENBANK/U51554

Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish.

Examination of gills from salmonids with bacterial gill disease using monoclonal antibody probes for Flavobacterium branchiophilum and Cytophaga columnaris.

Bacterial diseases of the gills of commercially reared salmonids in freshwater are common problems. They accounted for 18% of all diagnostic submissions to the Atlantic Veterinary College from commercial fish hatcheries. Definitive diagnosis is difficult because of the growth characteristics of the putative bacteria in culture. Research into the pathogenesis of these diseases has also been similarly limited. Monoclonal antibodies (MAbs) were developed to 2 globally significant gill bacterial pathogens, Flavobacterium branchiophilum, the causative agent of bacterial gill disease, and Cytophaga columnaris, the causative agent of columnaris disease of salmonids. These MAbs were then used as the basis for an indirect fluorescent antibody test to assess archived cases of gill disease in our study region. Of the cases tentatively diagnosed based on histopathology as bacterial gill disease, 76.2% tested positively with the MAbs to F. branchiophilum. Also present within 18.7% of these cases were bacteria which reacted positively to the MAbs for C. columnaris. We conclude that the MAbs produced are valuable diagnostic and research probes for common bacterial disease of the gills of salmon and trout in Atlantic Canada. This study also adds further proof that F. branchiophilum acting alone can be sufficient cause for bacterial gill disease.

Production of monoclonal antibodies specific for antigens derived from tissue of chinook salmon (Oncorhynchus tshawytscha) affected with plasmacytoid leukemia.

Two distinct monoclonal antibodies (MAB) were prepared for testing with kidney, spleen, and retrobulbar tissue imprints made from chinook salmon (Oncorhynchus tshawytscha) affected with plasmacytoid leukemia. (PL). Hybridomas were prepared from mice immunized with whole and lysed cells purified from renal or retrobulbar PL-positive tissues, which had been obtained from naturally and experimentally infected fish from British Columbia, Canada. The MAB reacted with at least 4 morphologically different cell types; fluorescence was associated with the plasma membrane and cytoplasm. The MAB also reacted with kidney imprints made from chinook salmon affected with a PL-like lymphoproliferative disease in California, indicating that these 2 diseases might be caused by a similar agent. The MAB did not react with any of the kidney or spleen imprints made from wild chinook salmon collected from a river in Ontario, Canada.

[Biological and clinical prevalence of pollinosis caused by ragweeds of the upper valley of the Rhône corridor]. / Prévalence biologique et clinique de la pollinose due aux ambroisies dans la vallée supérieure du couloir rhodanien.

An epidemiological study of ragweed allergy was conducted on 646 employees belonging to 6 factories located in the Rhone valley south of the city of Lyon. Information on seasonal evocative clinical symptoms was obtained through a self-administered questionnaire. Biological prevalence was assessed by measuring anti-ragweed IgE specific antibodies. Measurements were performed by immunoenzymatic assay (W1 Phadezym RAST from Pharmacia). 34 (5,4%) subjects had evocative symptoms whereas 37 (5,9%) had increased specific IgE. Persons with the highest IgE levels were symptomatic. Concordance between symptoms and biology was 35% (12/34). Results indicate that sensitization level varies according to the location of the factory and people's residence, the risk to become allergic being of 10% in the most exposed population. This data emphasize the need to promote anti-ragweed eradication policy.

A comparison of two antibiotic regimens for treatment of pelvic inflammatory disease.

Sixty-two women were randomized in a double-blind fashion to receive one of two antibiotic regimens for the treatment of clinically diagnosed pelvic inflammatory disease. Thirty of 31 patients (96.8%) receiving a combination of cefoxitin with doxycycline and 28 of 31 (90.3%) receiving a combination of clindamycin with amikacin responded to therapy (P = not significant). Chlamydia trachomatis, Neisseria gonorrhoeae, or both were isolated from 13.3, 7.0, and 4.8% of patients, respectively. Of the four patients not responding to therapy, all had inflammatory complexes. Cefoxitin/doxycycline and clindamycin/amikacin are both effective in the treatment of pelvic inflammatory disease.