Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Sequential pathological studies in the udder of goats intramammarily infected withAspergillus fumigatus

Intramammary inoculation of goats withAspergillus fumigatus spores resulted in the development of mastitis with characteristic gross and microscopic lesions. The mastitis and the lesions were restricted to the infected udder halves only and there was no dissemination of infection to other tissues of the body. The experiment was continued for 45 days. Gross changes in the infected udder were observed up to the 45th day post-infection. The lesions, in general, included variable sized abscesses in the first 15 days followed by development of varying sized greyish-white nodules in the infected udders. Microscopic changes consisted of granulomatous reaction with well developed granulomas in the infected udders. Hyphae and spores ofAspergillus fumigatus could be demonstrated in sections of the infected udders up to 45 days after infection. Reisolation of the fungus consistently was achieved up to 45 days. It is concluded that intramammary inoculation ofAspergillus fumigatus spores in goats leads to chronic granulomatous mastitis.     

Cloning of the hsp70 (dnaK) genes from Rhizobium meliloti and Pseudomonas cepacia: phylogenetic analyses of mitochondrial origin based on a highly conserved protein sequence.

The genes for hsp70 (or dnaK) have been cloned and sequenced from Rhizobium meliloti and Pseudomonas cepacia, two bacterial species belonging to the alpha- and beta-subdivisions of gram-negative proteobacteria, respectively. On the basis of global alignment of HSP70 proteins, several sequence signatures have been identified that are distinctive of mitochondrial homologs and gram-negative proteobacteria on the one hand and the chloroplasts and cyanobacteria on the other. Detailed phylogenetic analyses of HSP70 sequences from various eubacteria and eukaryotic organellar and cytosolic homologs support the inference regarding the origin of mitochondria from a member of the alpha-proteobacteria and of chloroplasts from cyanobacteria. The analysis presented here also suggests a monophyletic origin of the mitochondrial homologs.     

Changes in mitochondrial shape and distribution induced by ethacrynic acid and the transient formation of a mitochondrial reticulum

We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B-SC-1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 μM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3′-dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the druginduced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug-induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed. © 1994 wiley-Liss, Inc.