Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis.

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.

Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint.

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity.

RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.

Prolongation of survival of primary renal allografts by feeding of donor spleen cells.

We have found that feeding Brown Norway (BN) rat spleen cells to Lewis rats prior to transplanting BN kidneys prolongs allograft survival (mean: 8.8 days in unfed rats, 21 days in the BN cell-fed rats; longest survival: 11 days without allo-feeding vs. 37 days with feeding). We have also found that feeding BN cells both before and after transplantation further extends survival (mean: 38 days; longest survival: 105 days). We also examined the cells infiltrating the grafts during the early stages of the allograft response (day 5). Using flow cytometry, we found a significant decrease in the number of leukocytes infiltrating the transplanted kidneys of fed animals. This decrease was mainly due to a drop in the number of infiltrating T cells. We also found that cytokine mRNA production by the graft-infiltrating lymphocytes, assessed by reverse transcription polymerase chain reaction, showed a significant increase in interleukin-4 and transforming-growth factor-beta mRNA in the graft-infiltrating lymphocytes of fed animals compared with the controls.

Medial smooth muscle cell loss in arterial allografts occurs by cytolytic cell induced apoptosis.

OBJECTIVE: Experimental arterial allografts, used as models of chronic rejection, undergo marked loss of smooth muscle cells (SMC) from their media prior to the development of occlusive, intimal proliferative lesions. Medial SMC loss has been described in human heart transplants, and may be related to the development of occlusive coronary lesions which are the hallmark of chronic rejection. This SMC loss does not exhibit the characteristics of necrotic cell death. We sought to determine whether medial SMC loss in arterial allografts occurs by apoptosis. We further investigated these allografts for cytolytic cell-derived inducers of apoptosis, Finally, we compared two different strain combinations to assess the impact of varying histoincompatability on medial SMC loss. METHODS: Evidence for internucleosomal DNA degradation, which is characteristic of apoptosis, was sought by the in situ terminal deoxynucleotidyl transferase nick end labelling (TUNEL) method carried out on Lewis to Fisher rat femoral artery transplants (disparate at minor loci only) and Brown Norway to Lewis aortic transplants (fully disparate at major and minor loci). Isografts (Lewis to Lewis) served as controls. In a separate series of experiments graft mRNA was extracted and analysed by reverse transcription-polymerase chain reaction (RT-PCR) with primers for molecular inducers of apoptosis (TNF-alpha, Fas ligand, perforin, and granzyme-B) which are derived from cytolytic cells known to be present in allografts. RESULTS: Allograft media contained large numbers of TUNEL stained nuclei in both strain combinations. Neither isografts nor ungrafted femoral artery segments stained positive for apoptosis. RT-PCR on whole allografts in both strain combinations revealed sustained upregulation of perforin, granzyme-B, Fas-ligand and TNF-alpha mRNA concomitant with medial SMC loss. Autografts demonstrated sustained up regulation of TNF-alpha, and perforin, but only brief upregulation of granzyme-B, and no upregulation of Fas-ligand. CONCLUSIONS: These data strongly suggest that medial SMC loss in allograft arteriopathy occurs by apoptosis. Further, RT-PCR data indicate that cytolytic cell-derived inducers of apoptosis are upregulated in these grafts and may be accountable for medial SMC apoptotic cell death. Finally, fully-disparate (Brown Norway to Lewis) and minor-only incompatible (Lewis to Fisher) strain combinations both resulted in marked intimal proliferation, medial SMC loss by apoptosis, and similar patterns of expression of cytolytic cell derived inducers of apoptosis. Insofar as intimal proliferative lesion-formation may be dependent on medial damage (as in arterial-injury models), understanding the mechanism of medial SMC loss may provide a novel therapeutic approach to human cardiac transplant arteriopathy.

Class II MHC expression on rat intestinal intraepithelial lymphocytes.

The expression of class II major histocompatibility complex (MHC) on rat intestinal intraepithelial lymphocytes (IELs) and mesenteric lymph node cells (MLNCs) were examined by immunofluorescence and flow cytometry. As expected, MLNCs contain eight small populations of CD4+ class II MHC+ T-cells in addition to classical antigen presenting cells. In contrast, rat IELs include a significant population of class II MHC+ T-cells, predominantly in the CD8+ CD4-alpha beta TCR+ subset. IEL samples with a relatively high percentage of class II MHC+ cells also include some CD4+ class II MHC+ cells; IEL samples with a low percentage of class II MHC+ cells also include some CD4- CD8- class II MHC+ cells. The role of lymphocyte subpopulations in the intestinal epithelium may need to be revisited in consideration of these findings.

Pattern of proinflammatory cytokine mRNA expression during Trichinella spiralis infection of the rat.

Trichinella spiralis occupies an intramulticellular niche in the small intestinal epithelium, and thus we examined the intestine and gut-associated tissues for proinflammatory cytokines during the infection. We document the patterns of interleukin-1 (IL-1), IL-6, gamma interferon, and tumor necrosis factor alpha mRNA expression in the duodenum, jejunum, Peyer's patches, mesenteric lymph node, spleen, and liver in T. spiralis-infected rats. By reverse transcription-PCR detection of mRNAs, IL-1beta was found increased in the jejunum but only on day 2. The jejunal IL-1beta increase was attributed to the epithelium by isolating epithelial cells and then depleting them of intraepithelial lymphocytes prior to analysis. The only cytokine for which mRNA was substantially increased in tissues later in infection was tumor necrosis factor alpha in the spleen and, to a lesser extent, in the mesenteric lymph node. In fact mRNA levels for some cytokines declined below uninfected levels in some organs during the infection. IL-1 may be important in the initiation of the intestinal inflammatory response to this infection.

Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes.

The study of intestinal intraepithelial lymphocytes (IEL) has been hindered by the difficulty of isolating a population of lymphocytes which is free of epithelial cell or lamina propria cell contaminants and representative of the in vivo population of IEL in both phenotype and function. We describe an improved technique for the extraction and purification of IEL from the proximal small intestine of the rat. This technique rapidly and reproducibly isolates 5-10 x 10(6) IEL/rat with 90-95% purity and viability without the use of enzymes which affect lymphocyte function. The resulting cell population, which is 75% alpha beta T cell receptor (TCR)+, 70% CD8+, and 33% CD4+ T cells, and only 5% B cells and 2% macrophages, is of suitable purity to allow for flow cytometric analysis of the entire population of cells without requiring gating on lymphocytes. IEL are comprised of a unique T cell repertoire in that 27% of cells co-express the CD4 and CD8 molecules, but only 11% of CD4+ cells co-express CD45RC. All CD4+ cells express the alpha beta TCR, but 9% of IEL are CD8+ CD4- alpha beta TCR-. The adhesion molecules alpha 4 integrin and L-selectin are expressed on 57% and less than 1% of IEL, respectively. The isolated IEL population contains mRNA for IL-1 alpha, IL-1 beta, IL-1R, IL-1RA, IL-2, IL-6R, IFN-gamma, TGF-alpha, TGF-beta 1, and TNF-alpha. Mesenteric lymph node cells (MLNC) were examined in parallel. This technique allows for the isolation of rat IEL appropriate for phenotypic analysis by flow cytometry and for cytokine analysis by reverse transcription/polymerase chain reaction.

Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1.

GADD45 was originally identified as a cDNA clone induced by growth arrest and DNA damage. We show that Gadd45 is a nuclear protein, widely expressed in normal tissues, particularly in quiescent cellular populations. Using cell synchronisation methods we show that Gadd45 levels are highest in the G1 phase of the cell cycle, and are greatly reduced during S phase. Immunoprecipitation of Gadd45 from mammalian cells reveals that it is tightly associated with a protein which reacts with antibodies to the cyclin dependent kinase inhibitor p21Cip1. Binding of recombinant Gadd45 protein to overlapping p21Cip1 peptides in ELISA assays and use of the yeast two hybrid assay show that Gadd45 directly interacts with this cell cycle inhibitor. These data suggest that Gadd45 may act in the regulation of the cell cycle. It is postulated that the interactions of Gadd45 with both p21Cip1 and PCNA are important for the modulation of cell cycles, and for the inhibition of DNA replication.

Characterisation of the interaction between PCNA and Gadd45.

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.