A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation.

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists.

The action of ticlopidine on human platelets. Studies on aggregation, secretion, calcium mobilization and membrane glycoproteins.

The effects on platelet function of a 5-day course of Ticlopidine (Tcl) have been studied in two groups of volunteers receiving different dosage schedules. Tcl had a relatively greater inhibitory effect on aggregation induced by ADP than by other agonists, and a greater effect, in contrast to that of an ADP receptor antagonist, on the second phase than on the initial rate of aggregation. Tcl inhibited ATP secretion in response to ADP and 0.05 u/ml thrombin, but not to higher concentrations of thrombin or to calcium ionophores. No inhibitory effect was observed on Ca2+ influx or intracellular mobilization, on the binding of monoclonal antibodies to the glycoprotein IIb-IIIa complex or on the state of association of the complex. We suggest that Tcl neither inhibits the binding of ADP to its receptor nor acts directly on the fibrinogen binding site, but that it may inhibit a step in signal transduction between these two events.

Effects of a monoclonal antibody to glycoprotein IIb/IIIa (P256) and of enzymically derived fragments of P256 on human platelets.

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10(-9)-3 x 10(-8) M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10(-7) M. P256-induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In-111-labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications.

Acute lymphoblastic leukaemia: four-year survivals old and new.

Two groups of four-year survivors with acute lymphoblastic leukaemia (ALL) are compared: an 'old' series consisting of 83 patients diagnosed before 1968, and a 'new' series of 366 patients included in Medial Research Council trials (UKALL I-III) and diagnosed in 1970-4. Both series differed significantly from a group of ALL patients who survived less than four years in having lower total leucocyte and blast-cell counts at diagnosis, but the new series did not show the significant differences in organ involvement and platelet count seen in the old series. In both series, girls were more likely than boys to survive for four years and less likely to have relapsed meanwhile; in the new series, relapse rates were also lower for girls than for boys after four years, and subsequent survival was significantly better. There was no difference between the two series in survival rates of those patients who had relapsed before reaching four years. A much higher proportion of the new series, however, had reached four years without prior relapse, and these had a more favourable subsequent survival than the corresponding group of the old series. About 90% of patients achieving four years' continuous complete remission on these UKALL regimes seem likely to survive for 10 years.

Effect of oral contraceptives on factor VIII clotting activity and factor VIII related antigen in normal women.

The factor VIII clotting activity (VIIIc1 and factor VIII related antigen (VIIIRAg) were determined repeatedly in 24 pairs of age-matched normal women, one of each pair being on oral contraceptives. No significant differences in either parameter or in the VIIIc/VIIIRAg ration were found between the two groups ,although the mean factor VIII clotting activity and VIIIc/VIIIRAg ratios for women on oral contraceptives were very slightly higher than for those not on oral contraceptives.

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on platelet responses to various agonists in the presence and absence of extracellular Ca2+.

In the presence of 1 mM extracellular Ca2+ (Ca2+o), incubation of washed, quin 2 loaded platelets with a low concentration (1 nM) of 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited platelet shape change, aggregation, ATP secretion and cytosolic calcium (Ca2+i) fluxes in response to thrombin, platelet activating factor (PAF), adenosine diphosphate (ADP), vasopressin (VP) and the thromboxane mimetic U46619, but potentiated platelet activation induced by the calcium ionophores, A23187 and ionomycin, the Ca2+ flux remaining unaltered. In the presence of less than 100 nM Ca2+o, TPA again inhibited shape change but potentiated ATP secretion induced by all agonists, even those in which the cytosolic calcium flux was inhibited. Addition of TPA 30 seconds after a low dose of agonist, sufficient to elevate [Ca2+i] to about 250 nM without causing aggregation, induced substantial aggregation and dense granule secretion without affecting Ca2+ flux. These results confirm that very slight elevation of [Ca2+i] above resting levels greatly enhances the effect of low concentrations of TPA, and suggest that protein kinase C activation may exert a feedback inhibition of receptor-mediated shape change, aggregation, ATP secretion and Ca2+ mobilization.

Von Willebrand factor has more than one binding site for platelets.

By the use of a series of monoclonal antibodies against platelet membrane glycoproteins (GP) and the von Willebrand factor (vWF), it is shown that thrombin-induced binding to GP IIb/IIIa involves a different site on vWF from ristocetin-induced binding to GP Ib.