Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation AciDs plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences signifcantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.Arabidopsis thaliana is an excellent model plant for classical and molecular genetic studies. Current efforts toward characterizing the genome of Arabidopsis include restriction fragment length polymorphism analysis (1, 2), production and physical mapping of yeast artificial chromosome libraries (3-5), and Agrobacterium tumefaciens T-DNA insertional mutagenesis (6-8). An alternative means of isolating genes, which has been effective in many other model systems, is the use of transposable elements. In this paper, we report the development of a two-element Activator/Dissociation (Ac/ Ds) transposable element system suitable for gene tagging in Arabidopsis.The Ac/Ds transposable element system was originally discovered in maize by McClintock (9). It has since been shown that Ac transposes in Nicotiana tabacum (10) and numerous other dicotyledonous plant species (for review, see ref. 11). Its behavior in heterologous plant species has been most intensively studied in tobacco (10, 12-21), tomato (22-24), and A. thaliana (25-28). Many transposition-related properties associated with Ac activity in maize are also found in heterologous systems. For instance, the 4.6-kilobase (kb) autonomous element forms 8-base-pair duplications upon insertion (10), is capable of mobilizing receptor Ds elements (12,13,25,29), and preferentially transposes to nearby locations (16,28).To develop an efficient transposon tagging system for Arabidopsis and other plants, we have constructed a twoelement Ac/Ds system containing plant selectable markers designed to monitor the presence of Ac, Ds, and Ds-related excision events (25,30). A marked Ds element (DsDHFR) was constructed by inserting a plant selectable marker cassette, including a dihydrofolate reductase (DHFR) gene under the control of a cauliflower mosaic virus (CaMV) 35S promoter, into the central region of a Ds element. We have previously shown that the DsDHFR element transposes in tobacco and Arabidopsis, and the DHFR cassette does not decrease the frequency of Ds transposition (25). Furthermore, it was determined that approximately two-thirds of excised DsDHFR elements reinserted in transgenic tobacco protoplasts while the oth...

A recessive mutant with white leaves was identified in a screen of a population of T-DNA-tagged Arabidopsis thaliana plants. The mutation is lethal, but plants develop almost to maturity under sterile conditions. The white areas in leaves are devoid of developed chloroplasts, but the plants frequently develop green sectors which contain green chloroplasts. Molecular characterisation of the affected gene revealed that the mutant is allelic to pale cress (pac), a recently described mutation, and was therefore named pac-2. Sequencing of cDNAs and the genomic region revealed several noteworthy features of this genetic locus. In pac-2 the T-DNA had inserted in the region of the promoter and abolished transcription of the PAC gene completely. Cytokinin induced greening in mature, white homozygous pac-2 plants, and therefore is likely to be responsible for the greening observed in callus and shoots induced on roots from such plants. However, the PAC transcript was found to be absent in both white leaves and green callus. Thus, since cytokinin induced greening in the absence of PAC RNA this plant hormone appears to be able to bypass PAC function.

Allo-MHC specific antigen recognition might not only be involved in acute, but also in chronic rejection. The clonotypic specificity of the T-cell receptor to recognize all-MHC is located in the variable (V) alpha and beta chain. A restricted T-cell receptor repertoire could support an immunological basis for chronic rejection. The novel feature of this study is that V beta repertoire was assessed in ongoing chronic rejection before end-stage renal failure and in acute rejection. V beta s 1 to 20 were quantitated by PCR in PBMC and biopsies of rejecting renal allografts. The V beta pattern in PBMC demonstrated a polyclonal distribution. However, the intragraft V beta repertoire was restricted to 1 to 3 dominant V betas and highly individual in 9 of 12 patients. Number and type of the HLA mismatch and the time interval between transplantation and biopsy did not correlate to the V beta distribution. The individual response is attributed to genetic predisposition factors of the recipient. Therefore, the restriction of the V beta repertoire indicates allo-MHC dependent immune processes not only in acute, but also in ongoing chronic rejection. Tailor-made antibodies against dominant V betas might offer specific individual immunosuppression in treating both acute and ongoing chronic rejection.

Critically ill patients in the intensive care unit (ICU) may develop eye problems, due to impaired ocular protective mechanisms or direct involvement of the eye in severe systemic diseases. If eye infections or ocular surface disorders are not identified in time, endophthalmitis or corneal ulcer may develop and can cause permanent functional injuries of the eye. A retrospective analysis was performed and a total of 283 complete intensive care courses of treatment were evaluated, taking into account ophthalmic medical consultations for frequent cardinal symptoms. The most common cardinal symptoms were lagophthalmus (exposure keratopathy), chemosis, redness and periorbital haematoma. The following predisposing risk factors for the onset of ocular complications during intensive care treatment were detected: chemosis (p < 0.001), redness (p = 0.007), lagophthalmus (p = 0.001), ventilation (p < 0.001), use of muscle relaxants (p < 0.001), cardiovascular (p < 0.001), and neurological diseases (p < 0.001). In 71.7 % of ICU patients, additional treatment was prescribed during the eye consultation. This includes special eye care treatment (6.0 %) and/or drug therapy (64.0 %), as well as oculoplastic surgery in 4,3 % of critically ill patients. The most common oculoplastic-surgical procedure in the ICU was lid adhesion to achieve adequate protection of the corneal surface in patients with severe exposure keratopathy. Oculoplastic surgery is the method of choice for protecting the cornea in critically ill patients, when conservative options such as hypoallergenic adhesive tape or a moisture chamber are not sufficient to protect the ocular surface. The main challenges are to pay attention to the indication and performance in due time, and to avoid permanent loss of function through transparency reduction or irregular astigmatism in post-recovery patients.

In a patient with metastatic melanoma transmitted by the renal allograft, HLA serves as an alloantigen per se and is associated with tumor antigens at the same time. The influence of this antigeneic pattern on the Vbeta T-cell repertoire in an allogeneic melanoma, allograft, and peripheral blood mononuclear cells (PBMC) was assessed by polymerase chain reaction. Vbeta13.1 and 19 were found in both the melanoma and the graft. Vbeta14 was detected only in the melanoma and Vbeta6 was detected only in the kidney. PBMC revealed an unrestricted Vbeta pattern. Markers for cytotoxic activity of T cells--granzyme B and perforin--were not expressed during immunosuppressive therapy as clinically reflected in a nonrejecting allograft and in a progressing melanoma. In vitro PBMC proliferated to recombinant interleukin-2, whereas recombinant interferon-gamma did not augment this response. Initiation of immune therapy, in addition to discontinuation of immunosuppression, might support the rejection of the allogeneic tumor by dominant Vbeta T cells.