Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.

The expression of the genome requires the precise and controlled removal of intervening sequences within premessenger RNAs (pre-mRNA splicing). Assembly of the active spliceosome entails successive rearrangements, including the entry and exit of molecular partners (reviewed in [1]). Phosphorylation events are probably molecular switches to control these conformational changes. Indeed, experiments with phosphatase inhibitors, purified phosphatases and nonhydrolysable ATP analogues have shown that multiple phosphorylation and dephosphorylation events are required for spliceosome assembly and splicing [2][3][4]. Among the best-characterized of the phosphorylated splicing factors are the serine-arginine rich (SR) proteins (reviewed in [5]), whose intranuclear distribution and activity are influenced by phosphorylation by specific kinases including SRPK1, SRPK2 [6,7], and Clk ⁄ Sty [8].SF3b155 ⁄ SAP155, an integral spliceosome component and substrate of cyclin E ⁄ CDK2 [9], is a non-SR protein whose phosphorylation state is also regulated during the splicing process [10]. In addition, other factors that regulate splicing in a phosphorylation-dependent manner have been identified (reviewed in [11,12]).Splicing factor 1 (SF1) was identified as necessary for spliceosome assembly by in vitro reconstitution assays with protein fractions from HeLa cell nuclear extracts [13], and in a synthetic lethality screen with Mud2p, the yeast homologue of the splicing factor U2 auxiliary factor large subunit ( U2AF 65 ) Protein phosphorylation ensures the accurate and controlled expression of the genome, for instance by regulating the activities of pre-mRNA splicing factors. Here we report that splicing factor 1 (SF1), which is involved in an early step of intronic sequence recognition, is highly phosphorylated in mammalian cells on two serines within an SPSP motif at the junction between its U2AF 65 and RNA binding domains. We show that SF1 interacts in vitro with the protein kinase KIS, which possesses a 'U2AF homology motif' (UHM) domain. The UHM domain of KIS is required for KIS and SF1 to interact, and for KIS to efficiently phosphorylate SF1 on the SPSP motif. Importantly, SPSP phosphorylation by KIS increases binding of SF1 to U2AF 65 , and enhances formation of the ternary SF1-U2AF 65 -RNA complex. These results further suggest that this phosphorylation event has an important role for the function of SF1, and possibly for the structural rearrangements associated with spliceosome assembly and function.Abbreviations BPS, branch point sequence; CIP, calf intestinal phosphatase; DTT, dithiothreitol; GST, glutathione-S-transferase; SF1, splicing factor 1; siRNA, small interfering RNA; snRNP, small nuclear ribonucleoprotein particle; RRM, RNA recognition motif; U2AF, U2 auxiliary factor; UHM, U2AF homology motif.