Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation.

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

The circadian rhythm of rat motor activity and the effect of brain monoamine inhibitors (PCPA and nialamide).

The study of the circadian motor rhythm in free-running conditions of 500 g rats during 6 successive days shows that the endogenous period of the rhythm in these conditions is 23.40 hrs. The two poisons studied (PCPA in 40 mg/100 g dose and nialamide 5 and 10 mg/100 g doses) have both a deep effect on the functioning of the endogenous rhythm. PCPA reduces the amplitude of the increase of night over day activity to a fourth of the control value. The reducation may be attributed either to increased day activity or to reduced night activity. Nialamide reduces the amplitude of this increase to 80 or 70/100 of control value; this was observed with 5 or 10 mg nialamide/100 g body weight. The most important effect of nialamide on the endogenous rhythm lies in the disappearance of the regular shortening of nearly 20 min of the endogenous rhythm in DD conditions. The abrupt transition for LD 12:12 conditions to DD disturbs the endogenous period for 24 hrs.

[Brain monoamines and circadian rhythm of spontaneous motor activity of rats]. / Monoamines cérébrales et rythme circadien de l'activité motrice spontanée du rat.

The effect of three drugs, parachlorophenylalanine, nialamide and disulfiram, drugs known by their action on the two sleep phases, slow wave sleep and I-REM-sleep, have been studied and tested by their effect on the motor circadian rhythm : PCPA and disulfiram reduce the amplitude of the rhythm by two opposed mechanisms : PCPA increases the motor activity, especially the day-time activity, disulfiram reduces the motor activity, especially the night-time activity. The former reduces the serotonine content, the latter the noradrenaline content of the central nervous system. Nialamide (5 or 10 mg/1000 g) is without any action of the rhythm. Both doses increase very much the motor activity ; but the central excitatory state undergoes the normal circadian rhythm. This monoaminoxydase inhibitor is without any action on the circadian rhythm.

Breath-to-breath variations of alveolar Po2 and Pco2 at barometric pressures of 490, 745 and 1500 Toor in resting awake dogs.

Two awake, resting dogs born and raised at low altitude were studied, breathing air (1) at 745 torr, (2) during a 12 days sojourn at 490 torr in an altitude chamber, and (3) during 5 days sojourn at 1500 torr in a hyperbaric chamber. The respired gas was continuously sampled an end-tidal PCO2 and PO2 of sequences of thirty breaths were measured by fast analyzers. The mean value of alveolar PCO2 was 29 torr at high altitude; 35 torr at 745 torr; and 40 torr in hyperbary. The changes of PCO2 indicate different alveolar ventilations which result mainly from the changes of the chemoreceptor drive which is enhanced at high altitude and decreased in hyperbary. The scattering of PCO2 is about the same at the three pressures. The scattering of PO2 is less at high altitude than at sea level, and less at sea level than in hyperbary. On a PCO2 vs PO2 diagram end-tidal PCO2 and PO2 points form elliptical clouds whose mean slopes decrease with the increase of total pressure. The characteristics of the dispersion of the alveolar pressures and of the slopes of the alveolar clouds depend on several factors among which the relevant steepness (i.e. capacitance) of the O2 and CO2 blood abosrption curves at the three pressures presumably plays the major role.

Ventilatory and circulatory O2 convection at 4000 m in pigeon at neutral or cold temperature.

Awake domestic pigeons, either maintained at 22 degrees C (series I) or acutely exposed at 2 degrees C (series II), were studied in a hypobaric chamber at 140 m and at various stages during a 4-week exposure to 4000 m. Steady-state pulmonary ventilation (Vg) and breathing pattern (VT, fr), oxygen consumption (MO2), O2 concentrations and pressures in the arterial (a) and mixed venous blood (v), hematocrit (Ht) and acid-base status in arterial blood, systolic blood pressure and heart frequency (fH) were measured. From these data cardiac output (Vb) and stroke volume (Vs), ventilatory and circulatory requirements (Vg/MO2, Vb/MO2), extraction of O2 from inspired air (EgO2) and blood EbO2), and capacitance coefficient of blood for oxygen (betabo2) were calculated. At 140 m, by comparison with predicted values for mammals of same body weight, pigeons at 22 degrees C extracted more O2 from the inspired gas, with lower fR, larger VT, similar Vg; they extracted O2 from the blood like mammals, with lower fH, larger VS, greater Vb, similar betabO2=70 mumol-L-1-torr-1. Acute exposure to 2 degrees C provoked a two-fold increase in MO2 which was achieved by doubling Vg and increasing O2 extraction from the blood. At 4000 m, in both series, pigeons hyperventilated within the first 30 min, with a resultant hypocapnic alkalosis comparable to that in mammals. Further hyperventilation with consequent greater hypocapnia and increase of arterial PO2 was complete beyond 3 hr. After a few weeks, the pH remained 0.07 above control normoxic value, Ht increased from 45 to 52%, betabO2 reached about 172 mumol-L-1-torr-1. At 2 degrees C, Vb also increased, mainly due to tachycardia.

The compressibility and the capacitance coefficient of helium-oxygen atmospheres.

The capacitance coefficient beta of an ideal gas mixture depends only on its temperature T, and its value is derived from the ideal gas law (i.e., beta = 1/RT, R being the ideal gas constant). But real gases behave as ideal gases only at low pressures, and this would not be the case in deep diving. High pressures of helium-oxygen are used in human and animal experimental dives (up to 7 or 12 MPa or more, respectively). At such pressures deviations from the ideal gas law cannot be neglected in hyperbaric atmospheres with respect to current accuracy of measuring instruments. As shown both theoretically and experimentally by this study, the non-ideal nature of helium-oxygen has a significant effect on the capacitance coefficient of hyperbaric atmospheres. The theoretical study is based on interaction energy in either homogeneous (He-He and O2-O2) or heterogeneous (He-O2) molecular pairs, and on the virial equation of state for gas mixtures. The experimental study is based on weight determination of samples of known volume of binary helium-oxygen mixtures, which are prepared in well-controlled pressure and temperature conditions. Our experimental results are in good agreement with theoretical predictions. 1) The helium compressibility factor ZHe increases linearly with pressure [ZHe = 1 + 0.0045 P (in MPa) at 30 degrees C]; and 2) in same temperature and pressure conditions (T = 303 K and P = 0.1 to 15 MPa), the same value for Z is valid for a helium-oxygen binary mixture and for pure helium. As derived from the equation of state of real gases, the capacitance coefficient is inversely related to Z (beta = 1/ZRT); therefore, for helium-oxygen mixtures, this coefficient would decrease with increasing pressure. A table is given for theoretical values of helium-oxygen capacitance coefficient, at pressures ranging from 0.1 to 15.0 MPa and at temperatures ranging from 25 degrees C to 37 degrees C.

[Adrenosympathetic system and thermoregulation in a prosimian Perodicticus potto]. / Système sympathico-surrénalien et thermorégulation chez un prosimien Perodicticus potto

The urinary excretion of catecholamines in Perodicticus potto (1.76-2.94 mug/kg 24 h) is in the same range as in other mammals and the activity of the adrenosympathetic system does not account for the low metabolic rate in this species. The adrenals contain 1.140 +/- 0.14 mug A + NA/mg fresh tissue, of which adrenaline constitutes 94.5 per cent and are thus practically identical to those in Macaca irus. In a cold environment the daily urinary excretion of catecholamines of the tropical but cold hardy potto is only moderately increased.