The functional properties of hemoglobins from Triturus rristutus curnij2.x have been characterized both from an equilibrium and a kinetic point of view. A special attention has been given to the effect of organic phosphates in view of their role in the modulation of the hemoglobin function. Under stripped conditions newt hemoglobins display a negative Bohr effect which becomes much less pronounced in the presence of 0.2 M inorganic phosphates.The effect of organic phosphates involves not only a drop in the 0, affinity but also a large changc in the shape of the 0, Bohr effect which becomes markedly positive in the presence of my-inositol hexakis(ph0sphate). The experimental data reported make it possible to give a complete picture of the functional behaviour of newt hemoglobins, making a contribution to clarifying the dependence of the sign of the Bohr effect on the acclimatization temuerature of the animxls that has been Dreviouslv reported [Morpurgo, G., Battaglia, P. A. and Leggio, T. (1970) N a t k (Lond.) 225, 76 -771.Respiratory proteins exhibit a great deal of variation in their absolute ligand affinities and in their susceptibility to control by metabolic effectors [I]. These functional differences are generally thought to bc a reflection of the variable environincntal conditions in which organisms live and of the different physiological requirements of their tissues. I t is therefore of great interest to study how two mechanisins of control of hemoglobin function, namely homotropic and heterotropic interactions [2], have evolved in different species in order to meet the specific metabolic demands of the corresponding species.As previously reported 133 (see [4] for review) newt hemolysate shows a Bohr effect that is reversed as compared with mammalian hemoglobins. Moreover the Bohr effect was reported by Morpurgo et al. to bc temperature-dependent, having a negative slope at 4 ' C and a positive one at 30 'C.
Messenger RNAs for mouse embryonic globins were purified from yolk sac erythroid cells by oligodeoxythymidilate-cellulose Chromatography and sucrose density centrifugation. Full-sized complementary DNA copies of embryonic globin mRNAs were synthesized.Acrylamide gel electrophoresis of these RNAs indicate an average molecular weight of 220 000. including a polyadenylated sequence of about 35 residues, as determined by hybridization to t3 H jpolyuridylate.The wheat-germ translation products of mRNAs have the size and the ionic characteristics of the four embryonic globin chains a, x, y and z.Hybridization kinetics in vast RNA excess were performed and compared to standard rot curves of adult globin messengers, demonstrating a total base sequence complexity of about 880000 daltons, that is four different RNA sequences of 220000 molecular weight. The titration of embryonic globin cDNAs with increasing amounts of their complementary RNA templates indicates that the embryonic globin messengers were isolated at a high degree of purity.The expression of embryonic globin genes in yolk sac erythroid cells is a model suitable for molecular studies of terminal differentiation and of messenger RNA (mRNA) ageing in eukaryotes. Indeed, yolksac-derived erythroid cells proliferate and differentiate in vivo and can be easily obtained as a pure and homogeneous population at early and late developmental stages of nucleated erythroblasts [l]. Yolk sac erythroid cells synthesize three embryonic hemoglobins, namely hemoglobin El, EI1 and Elll, formed by four globin chains, i.e. the embryonic x (a-like chain), y and z @-like chain) and the adult a chain [2,3].These cytological features, together with other biochemical properties (see for review [4]) may allow the quantification of the expression of embryonic globin genes at each differentiative stage, that is it permits the characterization of the timing and modulation of gene transcription, the intermediate steps of nuclear RNA processing and the modality of messenger RNA degradation due to molecular ageing.The isolation of specific mRNAs has proved so far to be the prerequisite tool for studying the organization and expression of the eukaryote genome. We describe here the isolation and partial characterization of messenger RNAs coding for mouse embryonic globin Ahhreimtion. Hepes, 4-(2-hydroxyethyl)-l-pipera7ineethane sulphonic acid.chains. Messengers were prepared from yolk sac erythroid cells at the 13th day of foetal development, since at this stage the synthesis of hemoglobins accounts for at least 9.5% of the total synthesized proteins [5,6]. Embryonic globin messenger RNAs can thus be isolated without appreciable contamination by other minor species of mRNA. MATERIALS AND METHODS Isolation of Embryonic Globin mRNAsErythroid cells derived from the yolk sac of mouse embryos (strain C57 B1 Cas) were obtained from the circulating blood at day 13 of foetal development after silicon oil density purification of nucleated erythroid cells, as described previously [5]. Cells were lysed b...
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