Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

A novel vector for positive selection of inserts harboring an open reading frame by translational coupling

We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can then be selected by exposure to colicin E3. Our method differs from previous methods in that the marker gene is activated without protein fusion, and that selection occurs irrespective of the reading frame of the insert.     

An open reading frame upstream from the nifH gene of Klebsiella pneumoniae

An open reading frame upstream from nifHDK operon of Klebsiella pneumoniae had been described. The orientation of this open reading frame is opposite to that of nifHDK and sequence homology was found between the open reading frame promoter and the promoter of nifHDK operon. A recombinant plasmid carrying the promoter region of the open reading frame fused to the beta-galactosidase gene was constructed. Strains of E.coli were transformed with the plasmid containing this open reading frame promoter-lacZ fusion or co-transformed with it and a plasmid carrying the nifA gene. An appreciable activity of beta-galactosidase was found in strains which received both plasmids, indicating that the promoter of the open reading frame can be activated by the product of nifA gene. Thus, the open reading frame found between nifHDK operon and nifJ behaves just like other nif genes of K.pneumoniae in requiring the product of nifA as the positive effector for expression.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus

Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin alpha- and beta-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin alpha fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin alpha. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.     

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Summary An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 M atrazine. Its chromosome number was 36–38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.     

Evidence from photosynthetic characteristics for the hybrid origin of Diplotaxis muralis from a C3-C4 intermediate and a C3 species

Artificial hybridization studies have been carried out between plants with different photosynthetic types to study the genetic mechanism of photosynthetic types. However, there are only few reports describing the possibility of natural hybridization between plants with different photosynthetic types. A previous cytological and morphological study suggested that a cruciferous allotetraploid species, Diplotaxis muralis (L.) DC. (2n = 42), originated from natural hybridization between D. tenuifolia (L.) DC. (2n = 22) and D. viminea (L.) DC. (2n = 20). These putative parents have recently been reported to be a C (3)-C (4) intermediate and a C (3) species, respectively. If this hybridization occurred, D. muralis should have characteristics intermediate between those of the C (3)-C (4) intermediate and C (3) types. We compared leaf structures and photosynthetic characteristics of the three species. The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of D. viminea had only a few organelles. The BS cells in D. muralis displayed intermediate features between the putative parents. Glycine decarboxylase P protein was confined to the BS mitochondria in D. tenuifolia, but accumulated mainly in the mesophyll mitochondria in D. viminea. In D. muralis, it accumulated in both the BS and the mesophyll mitochondria. Values of CO (2) compensation point and its response to changing light intensity were also intermediate between the putative parents. These data support the theory that D. muralis was created by natural hybridization between species with different photosynthetic types.     

A plasmid from a non-insect-transmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line

Two novel rolling circle replication (RCR) plasmids, pOYM (3932 nt) and pOYNIM (3062 nt), were isolated from a mildly pathogenic variant line (OY-M) and a mildly pathogenic plus non-insect-transmissible line (OY-NIM), respectively, of onion yellows (OY) phytoplasma, a plant and insect endocellular mollicute. OY-M was isolated from an original wild-type line (OY-W) after regular maintenance using alternate plant/insect infections, while OY-NIM was further isolated from OY-M after maintenance by plant grafting without insect vectors. The RCR-initiator proteins (Rep) of both plasmids, which have a characteristic structure with both plasmid- and virus-like domains, were highly homologous to that of a previously described OY-W plasmid, pOYW (3933 nt), and were expressed in OY-M- and OY-NIM-infected plants, indicating that this replicon is stably maintained in the phytoplasma. Interestingly, pOYNIM lacked two ORFs that exist in both pOYW and pOYM, which encode a single-stranded DNA binding protein (SSB) and an uncharacterized putative membrane protein, indicating that these two proteins are not necessary for the phytoplasma to live in plant cells. These are the first candidates as phytoplasma proteins possibly related to host specificity.     

Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame

Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.