Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum).

Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Linkage of AFLP markers to lhd 1, a recessive heterochronic gene in Italian ryegrass.

A mutant, leafy head I (lhd 1), was discovered in Japan from the progeny of Italian ryegrass (Lolium multiflorum Lam.) 'Nioudachi' and local line 'Aichikei #3'. Compared with normal plants, the mutant plant is a dwarf with a larger number of intenodes per stem, shorter internodes, and smaller leaves. The plants also head later in the season. Aerial roots are usually produced from the stem nodes during rainy seasons. In characterizing lhd 1, it was found to have many branches with small leaves and many small panicles on the upper part of the plant. Panicle development was severely disturbed in lhd 1 mutants, and the number of leaves produced in the vegetative phase was nearly twice that produced in the wild-type counterpart. The lhd 1 mutant appears to be a heterochronic mutation that is able to extend the vegetative period during development. The frequency of mutants in segregating populations indicated that lhd 1 is a recessive allele. To determine the linkage relationship between the lhd 1 gene and AFLP markers,768 primer combinations were screened for polymorphisms using bulked segregant analyses in two populations with 316 and 30 plants, respectively. Five AFLP markers were linked to the lhd 1 locus. E3/M41-1 and E16/M14-2 cosegregated with lhd 1. E16/M14-1 and E30/M10-1 flanked the gene at a distance of 0.3 cM and E30/M14-2 was linked to lhd 1 at a distance of 0.6 cM.

Induction of macrophage-inflammatory protein-3alpha gene expression by TNF-dependent NF-kappaB activation.

Macrophage-inflammatory protein-3alpha (MIP-3alpha), also designated as liver and activation-regulated chemokine (LARC), Exodus, or CCL20, is a recently identified CC chemokine that is expected to play a crucial role in the initiation of immune responses. In this study, we describe that MIP-3alpha expression is under the direct control of NF-kappaB, a key transcription factor of immune and inflammatory responses. Overexpression of the p65/RelA subunit of NF-kappaB significantly increased the MIP-3alpha mRNA level. MIP-3alpha transcription was stimulated by TNF, and this stimulation was inhibited by an NF-kappaB inhibitor, I-kappaBalpha superrepressor. Analysis of the human MIP-3alpha promoter demonstrated a functional NF-kappaB site responsible for its expression. We also show that MIP-3alpha expression is induced in LPS-treated mouse livers that were primed with Propionibacterium acnes, which developed massive liver injury with infiltration of inflammatory cells. This induction was fully dependent on the TNF signaling cascade, because it was not observed in the livers of TNFR1-deficient mice. Furthermore, pretreatment with gliotoxin, an inhibitor of NF-kappaB activity, abrogated the P. acnes/LPS-induced MIP-3alpha expression of wild-type mice. These results clearly demonstrate that MIP-3alpha gene expression is dependent on NF-kappaB activity in vitro, and indicate that the TNFR1-mediated TNF signaling cascade that leads to NF-kappaB activation plays an essential role in MIP-3alpha expression in the murine liver injury model.

Human T cell leukemia virus type-I Tax activates human macrophage inflammatory protein-3 alpha/CCL20 gene transcription via the NF-kappa B pathway.

Infection by human T cell leukemia virus type (HTLV)-I is associated with several diseases, including adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. Leukocytes are attracted to the sites of inflammation by chemotactic factors. Macrophage inflammatory protein (MIP)-3 alpha/CCL20 is a recently isolated member of the CC subfamily of chemokines and has been proposed as a crucial factor to elicit inflammatory reactions. We now report that endogenous MIP-3 alpha mRNA levels are elevated in HTLV-I-infected T cell lines and in a human T cell line following the induced expression of the HTLV-I-encoded transactivator, Tax. Analysis of the human MIP-3 alpha promoter revealed that this gene is activated by Tax, via the activation of nuclear factor (NF)-kappa B, whose responsive element, -82-kappa B, is located at a position between -82 and -91 relative to the putative transcription start site. With an electromobility shift assay we further demonstrated that the -82-kappa B element was bound by the Tax-activated p50/p65 heterodimers of NF-kappa B. Expression of the specific receptor of MIP-3 alpha, CCR6, was also increased in HTLV-I-infected T cell lines, suggesting an autocrine and/or paracrine mechanism to establish the pathogenesis of HTLV-I-associated diseases.