Mireille Dorizzi

Temperature-dependent sex determination and gonadal differentiation in reptiles

In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

TEMPERATURE-DEPENDENT SEX DETERMINATION AND GONADAL DIFFERENTIATION IN REPTILES

Abstract. In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

Expression of AMH, SF1, and SOX9 in gonads of genetic female chickens during sex reversal induced by an aromatase inhibitor

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti‐Müllerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex‐reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular‐like cords, sex‐reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular‐like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli‐cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.

Purification of tRNATrp, tRNAVal, and partial purification of tRNAIle and tRNAMetf from beef liver

tRNATrp from beef lever has been purified by classical chromatographical methods. Total tRNA, prepared on a large scale (total aminoacid acceptance 1280 pmol/A260 unit) was submitted to chromatography on benzoylated-DEAE cellulose, then DEAE Sephadex. The major species accepting tryptophan issued from the second chromatography was aminoacylated with [14C] tryptophan and chromatographed on benzoylated-DEAE cellulose. The tRNA carrying the radioactive label was eluted in the ethanolic region. After stripping, the resulting tRNATrp has an acceptance of 1800 pmol/A260 unit. No isoacceptors could be demonstrated by chromatography of the pure species on RCP 5 in 6 M urea. The yield in pure tRNATrp was currently in the range of 25 to 30 percent of the total tryptophan acceptance of the starting curde tRNA.

A new approach to DNA polymerase kinetics.

Abstract Little is known of the detailed mechanisms of the polymerization reactions carried out by RNA and DNA polymerases. Besides technical reasons, there are mathematical difficulties not encountered in traditional enzymology. The product of the reaction after one polymerization step is also the substrate of the next step. A number of polymerases, isolated from various sources, have an exonuclease activity. The chain which is being synthesized may be either elongated or trimmed, and its growth has the character of a random walk. In this case, although the overall reaction scheme is more complex, the experiments are more informative, as every dNTP may be transformed into two distinct products: incorporated, or free dNMP. Having solved some of the mathematical difficulties of the random walk problem, we are able to propose a strategy for the study of the polymerization/excision kinetics. We measure the amount y ( t ) of nucleotide that is polymerized at time t and the amount x ( t ) of nucleoside monophosphate that has accumulated. When d y d x is plotted against the concentration of dNTP, a curve is obtained with a characteristic shape, a straight line in a large number of cases. From there, kinetic constants can be estimated. The analysis is made in terms of four possible kinetic schemes. In the most elementary model there are only two rate constants, one for incorporation and one for excision. This model is a limiting case of all other models. The frayed-unfrayed model of Brutlag & Kornberg (1972), Hopfields kinetic proofreading scheme (Hopfield, 1974), and the delayed-escape scheme (Ninio, 1975) are examined in detail, and we show how the kinetic experiments may in principle distinguish between the schemes. Our approach is illustrated with three experiments in which Escherichia coli DNA polymerase I acts on poly(dC), and poly(dT) · oligo(dA) 10 .

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

A memory effect in DNA replication

A study of the polymerization/excision ratio in the replication of poly(dA), primed with oligo(dT), was carried out with E. coli DNA polymerase I, at various primer and enzyme concentrations. The variations in this ratio suggest that 1) the DNA polymerase is able to switch between two states of low and high exonuclease activities and 2) after dissociating from the template, the DNA polymerase drifts towards the low exonuclease state. The recovery of the high exonuclease state would require several successive incorporations.