Improved version of the red fluorescent protein (drFP583/DsRed/RFP).

GFP has established itself as a highly useful tool throughout many areas of modern biology. Recently, the novel fluorescent protein drFP583, also termed DsRed or RFP, was clonedfrom a coral of the Discosoma genus. The protein is only weakly homologous to GFP and has a red emission spectrum, which makes drFP583 an attractive candidate for in vivo double labeling together with GFP variants. However, wildtype drFP583 has several drawbacks, including inefficient folding of the protein, extremely slow maturation of the chromophore, and tetramerization even in dilute solutions. Here we report on important improvements to this reporter that lead to higher levels of fluorescent drFP583 species in the cell. We further characterized our best mutant for applications in yeast and mammalian cell biology.

The maize streak virus coat protein transcription unit exhibits tissue-specific expression in transgenic rice.

Maize streak geminivirus (MSV) is a single-stranded DNA virus that infects cereals and other grasses. A promoter region incorporating the MSV large intergenic region and movement protein gene sequence was ligated to the gus (beta-glucuronidase) reporter gene which replaced the virus coat protein (CP) gene. The CP promoter activity was analysed in transgenic rice plants (Oryza sativa L.) and was compared with that obtained in plants transformed with the gus gene downstream of the cauliflower mosaic virus (CaMV) 35S promoter. The MSV CP promoter activity varied in the five plant lines tested, but was always less than that of the CaMV promoter. Histochemistry showed that the MSV CP promoter was active in cells of regenerating callus but in regenerated plants it provided an expression pattern restricted to the vascular tissues of the root, stem, leaf and floral organs. Expression was highest in phloem-associated tissues of the vegetative organs and was absent from the tip and elongation region of seedling roots. Thus, the MSV CP promoter shows a degree of developmental regulation and can be used to confer tissue-specific expression in transgenic rice plants.

Novel phenotypes and developmental arrest in early embryo specific mutants of maize

Embryo specific (emb) mutants exhibit aberrant embryo development without deleterious effects on endosperm development. We have analyzed five emb mutants of maize, which, based on their developmental profiles can be divided into two groups: mutants arrested at early stages and mutants with novel phenotypes. The members of the first group resemble wild-type proembryos and never reach other developmental stages. In the second group the tube-shaped mutants emb*-8522 and emb*-8535 completely lack apical-basal differentiation, while in mutant emb*-8516 a second embryo-like structure arises from the suspensor. The five emb mutations analyzed are non-allelic and two of the mutations are very likely caused by insertion of the transposon mutator, opening the door for their molecular analysis.

Splicing features in maize streak virus virion- and complementary-sense gene expression.

The single-stranded DNA geminiviruses produce transcripts from both strands (virion- and complementary-sense) of a nuclear double-stranded DNA molecule. In maize streak virus (MSV)-infected maize plants, approximately 80% of the complementary-sense transcripts produce the C1 protein, whilst the remaining 20% are spliced to remove a 92 nt intron and produce a C1:C2 fusion protein (Rep). Disruption of the complementary-sense 3' splice site abolished virus replication. The majority of the virion-sense transcripts initiated one nucleotide upstream of the V1 (movement protein) gene and a minority a further 141 nucleotides upstream. A 76 nt intron, with features typical of plant introns, was identified within the V1 gene, upstream of the coat protein gene. Spliced and unspliced forms of each virion-sense transcript were produced, but they differed in splicing efficiency. Approximately 50% of the major transcript and less than 10% of the minor transcript were processed. Mutagenesis of the consensus 5' splice site in the V1 gene resulted in the use of alternative cryptic splice sites, confirming the importance of splicing for MSV infection. Spliced virion-sense transcripts were also identified in tissue infected with the closely-related Digitaria streak virus (DSV) but not with another subgroup I geminivirus, wheat dwarf virus. Collectively, the multiple transcript initiation sites and different splicing efficiencies suggest that splicing is an important feature in the regulation of both early and late gene expression in MSV and DSV.

[Interlocking nail according to Seidel in recent diaphyseal fractures of the humerus. Review of 41 cases of 48 fractures]. / L'enclouage verrouillé selon Seidel des fractures diaphysaires humérales récentes. 41 cas revus sur 48 fractures.

The Seidel's humeral interlocking nail is used in our department since december 1986. We report about the 48 first cases, 41 of them have been reviewed with a mean time follow up of 14 months. The indications were humeral mid-shaft fractures with associated lesions (20 cases), failures of non operative treatment (10 cases) and compound fractures (7 cases). Primary radialis nerve lesions has to be explored before nailing. In 41 cases we used a static procedure; post-operative immobilisation average time: were 13 days. Consolidation occurred in all cases within an average time of 10.5 weeks. Post-operative complications consisted in 1 case of infection healed after removal of the nail, and 1 case of secondary displacement after dynamic nailing with secondary radio-circumflex paralysis. The results were appreciated concording to the criteria of Stewart and Hundlay. We noted 64 per cent excellent and good results for fractures of the upper third, 80 per cent for fractures of the middle third and 85 per cent for the distal third of the diaphysis. All transverse fractures had a very good result but also the transverse and spiral fractures with third fragment which represent very unstable fractures especially at the upper third. The closed interlocking nailing of the humeral fractures according to Seidel represents a reliable and stable fixation method. Consolidation occurs in all cases whatever the type or the level of fracture.

[Ender nail with interlocking mechanism or dynamic hip screw in pertrochanteric fractures? A prospective study extending its limits]. / Ender-Nagel mit Verriegelung oder dynamische Hüftschraube bei pertrochantären Frakturen? Eine prospektive, grenzüberschreitende Studie.

In a prospective study on 148 patients with trochanteric fractures of the hip we compared the results of two implant-systems: the Ender-nailing modified by the dynamical interlocking method of Kempf and Bitar, and the dynamic hip screw (DHS) of the AO-ASIF. The Ender-method had the shorter operation time, earlier weight-bearing and less septic complications. Fracture consolidation was complete after three months in all cases. But in 8% reosteosynthesis because of hip joint perforations of nails was necessary. The method leads in less cases to anatomical reduction (85%), more often relevant varus (9%) and rotationary (14%) malpositions and functional deficits in hip (25%) and knee joints (9%), compared with the DHS. The DHS had less implant complications, reosteosynthesis was necessary in 4%. Technical failures were seldom. In 96% anatomical reduction could be achieved and the function of the hip was in 87% good to excellent. The Ender-nailing with dynamic interlocking is a system for internal fixation of trochanteric fractures in elder patients. The DHS-system has better functional results. Both systems need to be performed in a very careful, exact surgical procedure, to avoid complications.

Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica.

The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism. These tumours cannot be regenerated into normal plants. To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N. rustica. Progeny of an hsp70-T-6b transformant developed into normal plants. The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings. Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems. Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse. Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions. However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period. Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.