Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Water-use efficiency as a means of modelling net assimilation in boreal forests

Although the processes governing photosynthesis are well understood, scaling from shoot to canopy in coniferous forests is complex. Development of different sap-flow techniques has made it possible to measure transpiration of whole trees and thereby also of whole canopies. There is a strong link between photosynthesis and transpiration, for which reason it would be interesting to test whether measurements of canopy transpiration could also be used to estimate canopy photosynthesis. As a first step towards this, water-use efficiency (WUE) was studied at branch and canopy scales on the basis of branch gas-exchange measurements, with half-hourly and daily temporal resolution. Half-hourly and daily WUE at both branch and canopy scales showed a strong dependency on vapour-pressure deficit ('e). Branch photosynthesis modelled from branch transpiration and 'e mimicked well measured branch photosynthesis. Also, modelled photosynthesis, scaled to canopy and compared to net forest CO₂ exchange measured by the eddy-covariance technique, occasionally showed good agreement. In spite of these seemingly promising results, there was a difference in the response to 'e between branches and between years, which needs to be better understood.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Seroresponse (IgG) after Vaccination and Natural Infection of Cattle with Babesia Divergens

The antibody response against Babesia divergens in vaccinated calves and in unvaccinated sentinels on farms where vaccination had been practiced routinely, was investigated using a live vaccine. Sera were obtained before and 3 weeks after vaccination in March and April, approximately 1 month before the animals were put out on pasture. Additional blood samples were collected at the end of the grazing season and again the next spring. At that time previously unvaccinated sentinel calves were vaccinated and their antibody response was tested 3 weeks later. All sera were analysed by an IF-technique. All of the vaccinated calves (100%) were seropositive 3 weeks after vaccination. The seroresponse did not differ signifacantly between animals vaccinated before their first or second grazing season although the age difference was about 12 months. No clinical symptoms of babesiosis were seen in vaccinated animals. The titres were, however, significantly higher 3 weeks after vaccination than 6 months later. After the grazing season about 42% of the unvaccinated sentinel calves were sero–positve. Two of these calves had clinical babesiosis on pasture in July and September respectively. The number of sentinel calves which became infected on pasture showed a large farm-to-farm variation although all cattle on the farms once had been infected-/vaccinated with B. divergens. Probably the different number of calves infected was a reflection of a variation in tick density on the different pastures. All calves, which were seropositive after the grazing season, were also seropositive after 6 months indoors. The titres declined during the winter period, but they were still within the range of 2 doubling dilution steps.     

TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences

TGF-beta occurs in a latent complex of high Mr. We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-beta 1 complex, denoted TGF-beta 1 binding protein (TGF-beta 1-BP). Most of the sequence of fibroblast TGF-beta 1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. beta-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-beta 1-BP purified from human platelets is considerably smaller than the fibroblast form (125-160 kd vs. 170-190 kd), suggesting that there is alternative splicing of the TGF-beta 1-BP gene or that TGF-beta 1-BP undergoes cell-specific proteolysis. TGF-beta 1-BP was found not to bind and inactive TGF-beta 1; its role in the latent complex is discussed.     

Biological basis for the management of ‘lugs negra’ (Sarcothalia crispata Gigartinales,Rhodophyta) in southern Chile

The present paper describes growth dynamics in a natural bed of the resource luga negra (Sarcothalia crispata) in Guapilinao, southern Chile (41°57 S, 73°31 W). This resource is currently harvested and exported as raw material for the production of carrageenan. Seasonal variation in biomass, frond size, density and phenology was determined by periodic sampling. Natural recruitment was evaluated on different substrata added to the field; at the same time, substrata were inoculated under greenhouse conditions. Results showed that luga negra has seasonal growth: biomass increased from a minimum in spring to a maximum in mid to late summer. On the other hand, density was minimal in winter (200 ind. m^–2) and increased to 2000 ind. m^–2 in late spring. Peak abundance of mature tetrasporic fronds occurred in late summer, whereas that of cystocarpic fronds occurred in winter. Recruitment began in summer and extended into winter. Survival on different substrata were compared. Gametophytes had better survival rates on clam shells and 5 mm rope while tetrasporophytes had the best survival rate on clam shells and secondarily on boulders.