Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus.

The major threat for cassava cultivation on the Indian subcontinent is cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses which are bipartite begomoviruses with DNA A and DNA B components. Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) cause CMD in India. Two isolates of SLCMV infected the cassava cultivar Sengutchi in the fields near Malappuram and Thiruvananthapuram cities of Kerala State, India. The Malappuram isolate was persistent when maintained in the Madurai Kamaraj University (MKU, Madurai, Tamil Nadu, India) greenhouse, whereas the Thiruvananthapuram isolate did not persist. The recovered cassava plants with the non-persistent SLCMV, which were maintained vegetative in quarantine in the University of Basel (Basel, Switzerland) greenhouse, displayed re-emergence of CMD after a six-month period. Interestingly, these plants did not carry SLCMV but carried ICMV. It is interpreted that the field-collected, SLCMV-infected cassava plants were co-infected with low levels of ICMV. The loss of SLCMV in recovered cassava plants, under greenhouse conditions, then facilitated the re-emergence of ICMV. The partial dimer clones of the persistent and non-persistent isolates of SLCMV and the re-emerged isolate of ICMV were infective in Nicotiana benthamiana upon agroinoculation. Studies on pseudo-recombination between SLCMV and ICMV in N. benthamiana provided evidence for trans-replication of ICMV DNA B by SLCMV DNA A.

Sri Lankan cassava mosaic virus replication associated protein (Rep) triggers transposition of IS426 in Agrobacterium.

We report a high rate of IS426 transposition in Agrobacterium tumefaciens in the presence of the Sri Lankan cassava mosaic virus (SLCMV) replication associated protein gene (Rep). Upon conjugal transfer of the binary plasmid pCam-SLCMV-Rep with the SLCMV Rep gene in the sense orientation under the transcriptional control of the Cauliflower mosaic virus (CaMV) 35S promoter into the A. tumefaciens vir helper strain EHA105, the binary plasmid size increased in all 15 transconjugants studied. Southern blot analysis of the transconjugants with the binary plasmid probe revealed that the 35S promoter and its proximal sequences in the T-DNA were rearranged. The rearranged sequences harboured the 1.3-kb IS426 element of A. tumefaciens. Conjugal mobilisation of the binary plasmid pCam-SLCMV-asRep, with the SLCMV Rep gene in antisense orientation, did not cause DNA rearrangement in EHA105. A mutated SLCMV Rep, in which a frame shift mutation caused retention of only 27 of the 351 amino acids, did not cause IS426 transposition in A. tumefaciens. These findings show that the multifunctional begomoviral Rep protein of SLCMV triggers transposition of IS426 in Agrobacterium.

Alpha 2a-interferon-induced differentiation of human alveolar rhabdomyosarcoma cells: correlation with down-regulation of the insulin-like growth factor type I receptor.

Rhabdomyosarcoma, a tumor of skeletal muscle origin, appears developmentally arrested at an early stage in the myogenic differentiation pathway. The proliferation of an alveolar rhabdomyosarcoma cell line Rh30 is dependent on the insulin-like growth factor (IGF) II/IGF-I receptor (IGF-IR) signaling pathway and is highly sensitive to recombinant human IFN-alpha 2a, which induces growth arrest and differentiation of these malignant myoblasts. IFN-alpha 2a-induced growth arrest of Rh30 cells was observed within 48 h, and reduction in colony formation was obtained with an IC50 of 0.81 IU/ ml for 72 h exposure. Down-regulated expression of IGF-IR was apparent by 24 h after initiation of IFN-alpha 2a treatment. Furthermore, an initial increase followed by reduced expression of MyoD, in concert with elevated expression of myogenin, increased frequency of skeletal muscle myosin-positive cells, and the formation of multinucleated cells, indicated an enhancement of differentiation of Rh30 cells in the presence of IFN-alpha 2a. To probe the role of IGF-IR in the differentiation of Rh30 cells along the myogenic lineage, the effect of antisense RNA-mediated reduction of endogenous IGF-IR on growth and expression of muscle-specific proteins was determined. Rh30 cells transfected to stably express antisense IGF-IR (clone AS [symbol: see text] 23)showed significant reduction in growth rate, decreased expression of IGF-IR protein, increased expression of MyoD, myosin heavy chain, and an increased number of multinucleated cells in comparison to the parental line. These data are consistent with overexpression of IGF-IR inhibiting differentiation. IFN-alpha 2a treatment of AS [symbol: see text] 23 cells further induced both MyoD and myogenin expression, thereby allowing cells to proceed further downstream of the differentiation pathway.

Glucocorticoid antagonist RU 486 reverses agonist-induced apoptosis and c-myc repression in human leukemic CEM-C7 cells.

A synthetic glucocorticoid dexamethasone (DEX) inhibits proliferation and induces apoptosis of clone CEM-C7 cells, but not in clone CEM-C1 cells, even though they contain glucocorticoid receptors (GR). We previously showed that suppression of c-myc is a critical step in glucocorticoid-induced cell lysis of C7 cells. It is not reduced in C1 cells. In this study we review the basis for this conclusion and present evidence that the glucocorticoid antagonist RU 486 rescues DEX-treated C7 cells from cell death. An increase in DEX-repressed c-myc mRNA levels precedes the recovery of cell growth. A threshold level of Myc expression appears to be required to maintain growth and viability of C7 cells. Although C1 cells are highly resistant to lysis by glucocorticoids, addition of forskolin, an inducer of protein kinase A, synergizes to evoke complete apoptosis. This synergistic effect is prevented by RU 486, indicating direct involvement of the GR.

Suppression of c-myc is a critical step in glucocorticoid-induced human leukemic cell lysis.

Glucocorticoids evoke cytolysis in clonal human leukemic CEM-C7 cells. Suppression of c-myc mRNA by dexamethasone closely correlates with cell lysis only in CEM clones with both glucocorticoid receptor and intact lysis functions. We tested the theory that c-myc repression is essential for glucocorticoid-induced lymphocytolysis by preventing down-regulation of c-myc gene in the presence of dexamethasone and by reducing c-myc mRNA levels with antisense oligonucleotides. We find that sustained expression of c-myc provides resistance to dexamethasone-induced lysis, and antisense c-myc oligomers induce cell lysis. The lethal effects of dexamethasone in these leukemic cells appear to involve reduction of c-myc below the levels required to maintain cellular growth and integrity.

Glucocorticoids in malignant lymphoid cells: gene regulation and the minimum receptor fragment for lysis.

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocorticoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocorticoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half "Zn fingers" of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, preceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.