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Over the last few years, intensive agriculture has often been denounced as a source of negative effects, particularly at the environmental and health level (overexploitation of natural resources, degradation of their quality, appearance and development of several diseases, etc.). Reducing the excessive use of agricultural inputs for the protection of the environment and the preservation of human and animal health is a social requirement nowadays. Investing in more sustainable agricultural models which make it possible to reduce, or even eliminate the risks, has become urgent. A possible solution may be to resort to agroecological systems. In order to be sustainable, these new systems must be performant at the agronomic, economic, social, and environmental levels. There is a multitude of tools for assessing the sustainability of agricultural systems. These tools are inappropriate for organic and agroecological systems, and do not make it possible to measure the agroecological transition performance of farms (Trabelsi et al. Environ Sci Pollut Res 23:139-156, 2016; Trabelsi 2017). This research project aims to design a decision support tool in order to help farms throughout the agroecological transition process, to assess the performance of this transition, and to put forward improvement scenarios. Contrary to other assessment methods, ESSIMAGE (Evaluation and Simulation of Agroecological Systems) is based on both pressure and impact indicators, and takes the specificities of agricultural production systems into account. It is a dynamic tool which not only makes it possible to assess farm performance at the present moment but also to consider the future by putting forward possible alternative improvement scenarios and by simulating their consequences at a later stage. ESSIMAGE is based on the interaction of two elements: agro-environmental, social, and economic indicators, and the GIS (Geographic Information System) software. This tool has been tested as part of a CASDAR "Post-MAET Gimone" ( agriculture.gouv.fr/ministere/mobilisation-collective-pour-lagroecologie. ) project on the subject of "Collective mobilization for agroecology" by using farm data, most of the farms having been involved in an agro-environmental measure for the progressive reduction of phytosanitary treatments since 2008. It has made it possible to compare the agroecological performances of these farms with an optimal situation, as well as with each other. Considering the research objectives and the approaches discussed, this study is an original step in the development of agricultural management strategies in favor of agroecology.

Faced with health, environmental, and socio-economic issues related to the heavy use of pesticides, diffuse phytosanitary pollution becomes a major concern shared by all the field actors. These actors, namely the farmers and territorial managers, have expressed the need to implement decision support tools for the territorial management of diffuse pollution resulting from the plant protection practices and their impacts. To meet these steadily increasing requests, a cartographic analysis approach was implemented based on GIS which allows the spatialization of the diffuse pollution impacts related to plant protection practices on the Etang de l'Or catchment area in the South of France. Risk mapping represents a support-decision tool that enables the different field actors to identify and locate vulnerable areas, so as to determine action plans and agri-environmental measures depending on the context of the natural environment. This work shows that mapping is helpful for managing risks related to the use of pesticides in agriculture by employing indicators of pressure (TFI) and risk on the applicator's health (IRSA) and on the environment (IRTE). These indicators were designed to assess the impact of plant protection practices at various spatial scales (field, farm, etc.). The cartographic analysis of risks related to plant protection practices shows that diffuse pollution is unequally located in the North (known for its abundant garrigues and vineyards) and in the South of the Etang de l'Or catchment area (the Mauguio-Lunel agricultural plain known for its diversified cropping systems). This spatial inequity is essentially related to land use and agricultural production system. Indeed, the agricultural lands cover about 60% of the total catchment area. Consequently, this cartographic analysis helps the territorial actors with the implementation of strategies for managing risks of diffuse pollution related to pesticides use in agriculture, based on environmental and socio-economic issues and the characteristics of the natural environment.

The health, environmental and socio-economic issues related to the massive use of plant protection products are a concern for all the stakeholders involved in the agricultural sector. These stakeholders, including farmers and territorial actors, have expressed a need for decision-support tools for the management of diffuse pollution related to plant protection practices and their impacts. To meet the needs expressed by the public authorities and the territorial actors for such decision-support tools, we have developed a technical-economic model "OptiPhy" for risk mitigation based on indicators of pesticide toxicity risk to applicator health (IRSA) and to the environment (IRTE), under the constraint of suitable economic outcomes. This technical-economic optimisation model is based on linear programming techniques and offers various scenarios to help the different actors in choosing plant protection products, depending on their different levels of constraints and aspirations. The health and environmental risk indicators can be broken down into sub-indicators so that management can be tailored to the context. This model for technical-economic optimisation and management of plant protection practices can analyse scenarios for the reduction of pesticide-related risks by proposing combinations of substitution PPPs, according to criteria of efficiency, economic performance and vulnerability of the natural environment. The results of the scenarios obtained on real ITKs in different cropping systems show that it is possible to reduce the PPP pressure (TFI) and reduce toxicity risks to applicator health (IRSA) and to the environment (IRTE) by up to approximately 50 %.

The use of plant protection products enables farmers to maximize economic performance and yields, but in return, the environment and human health can be greatly affected because of their toxicity. There are currently strong calls for farmers to reduce the use of these toxic products for the preservation of the environment and the human health, and it has become urgent to invest in more sustainable models that help reduce these risks. One possible solution is the transition toward agroecological production systems. These new systems must be beneficial economically, socially, and environmentally in terms of human health. There are many tools available, based on a range of indicators, for assessing the sustainability of agricultural systems on conventional farm holdings. These methods are little suitable to agroecological farms and do not measure the performance of agroecological transition farms. In this article, we therefore develop a model for the strategic definition, guidance, and assistance for a transition to agroecological practices, capable of assessing performance of this transition and simulating the consequences of possible changes. This model was built by coupling (i) a decision-support tool and a technico-economic simulator with (ii) a conceptual model built from the dynamics of agroecological practices. This tool is currently being tested in the framework of a Compte d'Affectation Spéciale pour le Développement Agricole et Rural (CASDAR) project (CASDAR: project launched in 2013 by the French Ministry of Agriculture, Food and Forestry, on the theme "collective mobilisation for agroecology," http://agriculture.gouv.fr/Appel-a-projets-CASDAR ) using data from farms, most of which are engaged in agroenvironmental process and reducing plant protection treatments since 2008.

Diffuse phytosanitary pollution is a complex phenomenon to manage. Reducing this type of pollution is one of today's key socio-economic and environmental challenges. At the regional level, few approaches enable the actors concerned to implement agricultural management strategies to reduce the use and impact of phytosanitary products. Our research problem focused on the consequences of intensive agriculture and, in particular, how to evaluate the impact of phytosanitary products on human health and the environment. In this article, we present the SimPhy simulation game which places the actors from a given region directly into a situation in which they manage farms whilst under pressure to reduce phytosanitaries (quantity and toxicity). The application focused on the Merja Zerga catchment area in Morocco. The region is dominated by intensive agriculture which is located upstream from a Ramsar-classified wetland area. The SimPhy simulation game is based on a decision support system-type tool. It allows us to anticipate the impact of regulations on farming systems. It also enables us to analyse the consequences of the actors' strategies on farm economies, human health and the quality of ecosystems. Initial results from the SimPhy simulation game enabled the technicians from Agricultural Development Center (CDA) themselves to learn about managing agricultural production systems in a dynamic and interactive fashion. With the simulation game, it was possible to learn about the farmer's ability to adapt to new regulatory constraints, and the involved consequences for toxicity risks for human health and the environment.

During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.

The Aurora kinase family has been involved both in vivo and in vitro in the stability of the metaphase plate and chromosome segregation. However, to date only one member of this family, the protein kinase Aurora B, has been implicated in the regulation of meiotic division in Caenorhabditis elegans. In this species, disruption of Aurora B results in the failure of polar body extrusion. To investigate whether Aurora A is also required in meiosis, we microinjected highly specific alpha-Aurora A antibodies in Xenopus oocytes. We demonstrated that microinjected oocytes fail to extrude the first polar body and are arrested with condensed chromosomes on a typical metaphase I plate, which has not performed its normal 90 degrees rotation. We additionally found that, although the failure of first polar body extrusion observed in alpha-Aurora A-microinjected oocytes is likely mediated by Eg5, the impairment of the metaphase plate rotation does not involve this kinesin-like protein. Surprisingly, although chromosomes remain condensed at a metaphase I stage in alpha-Aurora A-microinjected oocytes, the cytoplasmic cell cycle events progress normally through meiosis until metaphase II arrest. Moreover, these oocytes are able to undergo parthenogenetic activation. We conclude that Aurora A and Eg5 are involved in meiosis I to meiosis II transition in Xenopus oocytes.

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.

During Xenopus oocyte maturation, the Mos protein kinase is synthesized and activates the MAP kinase cascade. In this report, we demonstrate that the synthesis and activation of Mos are two separable processes. We find that Hsp90 function is required for activation and phosphorylation of Mos and full activation of the MAP kinase cascade. Once Mos is activated, Hsp90 function is no longer required. We show that Mos interacts with both Hsp90 and Hsp70, and that there is an inverse relationship between association of Mos with these two chaperones. We propose that Mos protein kinase is activated by a novel mechanism involving sequential association with Hsp70 and Hsp90 as well as phosphorylation. We also present evidence for a two-phase activation of MAP kinase in Xenopus oocytes.

In the yeast Saccharomyces cerevisiae, the RNA14 and RNA15 gene products have been implicated in RNA cleavage and polyadenylation in vitro and in the choice of polyadenylation site of ACT1 mRNA in vivo. The RNA14 gene produces three transcripts that differ in their 3' end, suggesting the use of different polyadenylation sites. The appearance of the three RNA14 transcripts was examined in different rna14 and/or rna15 mutant strains. In the rna14-1 or rna15-2 mutant strains, only the large transcript is present at the non-permissive temperature, showing that the rna14-1 and rna15-2 mutations lead to the use of the most distal RNA14 polyadenylation site, which turns out to be the most efficient. The rna14-5 mutation, which does not primarily modify the choice of poly(A) site, increases the global amount of RNA14 transcript. Surprisingly, this RNA14 mRNA overproduction is also observed in the double rna14-1 rna15-2 mutant strain. Moreover, in the strains in which the RNA14 transcripts are overproduced, short heterogeneous polyadenylated antisense RNAs are detected in the 3' region of the RNA14 large transcript. Taken together these observations suggest that, in addition to poly(A) site choice, Rna14 protein has another function involved in the control of global RNA14 mRNA level.

SSM4 was isolated as a suppressor of rna14-1, a mutant involved in nuclear mRNA maturation. In order to isolate genes interacting with SSM4, we have searched for mutants that are syntheticlethal in association with an SSM4 deletion. Among the mutants obtained, one, named sls1-1, shows a pet- phenotype. We have cloned and sequenced this gene. It encodes a protein with a calculated molecular mass of 73 kDa. This protein contains a mitochondrial targeting presequence but does not show homology with other known proteins. Deletion of SLS1 does not affect cell viability on glucose but is lethal on a non-fermentable medium. The Sls1p protein does not appear to be involved in mitochondrial DNA replication, transcription, or in RNA splicing maturation or stability. We have also tagged this protein and localized it in mitochondria. Treatment with alkaline carbonate does not extract this protein from mitochondria, suggesting strongly that it is a mitochondrial integral membrane protein. Thus, the SLS1 gene, encodes a mitochondrial integral membrane protein and is paradoxically synlethal in association with a deletion of the SSM4 gene, which encodes an integral nuclear membrane protein.

The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes. Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro. These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo. To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains. We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene. This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate. In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants. Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation. These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice. This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation.

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.

Using a plasmid (pSWS) similar to one that has been successfully used for large-scale production of hepatitis B virus (HBV) envelope protein particles (pSVS) but containing the corresponding woodchuck hepatitis virus (WHV) envelope gene sequences, we have stably transformed the rodent dihydrofolate reductase-deficient cell line CHO dhfr-. Although production of WHV envelope particles in CHO/pSWS cell lines was low, it was sufficient to test whether these particles could bind to polymerized serum albumin. Whereas binding of HBV particles produced in CHO/pSVS cells to polymerized human serum albumin could readily be detected, we found no evidence that the WHV envelope protein particles produced in vitro bind to either human or woodchuck polymerized serum albumin.

We have cloned and expressed in Escherichia coli three different parts of the HBx open reading frame, the N- and C-termini and the interior or central portion, using two vector systems. The sera of 43 hepatitis B virus patients representing three clinical categories--asymptomatic carriers, chronic active hepatitis, and hepatitis B patients with cirrhosis--known to be anti-HBx positive, were tested for reactivity against these constructs by Western blotting. The great majority of sera, regardless of the clinical categories, clearly recognise all three parts of HBx, strongly suggesting that the normal mechanism of biosynthesis of the HBx gene product is a straight-forward translation of the open reading frame starting from the first ATG. However, asymptomatic carriers show a marked, often almost exclusive, preference for recognition of the central portion of HBx, while patients with chronic hepatitis and patients with cirrhosis generally recognise all three parts of HBx to a similar extent.

Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.

To clarify the significance of the X gene of hepatitis B virus, we have tested for anti-HBx in the serum and HBxAg in the liver at different stages of the natural history of hepatitis B virus infection. Sera were screened by enzyme-linked immunosorbent assay and positive results confirmed by immunoblot. Purified recombinant MS2 Pol-HBx fusion protein was used as target for both assays. Among serial sera of patients with nonfulminant acute hepatitis, 24 of 64 patients (37.5%) were positive for anti-HBx. In fulminant cases, 15 of 36 patients (42%) had anti-HBx. In chronic hepatitis patients with high rates of hepatitis B virus replication, we found a significantly (p less than 0.01) higher prevalence of anti-HBx, 14 of 25 patients (56%), than in those with low replication, 14 of 66 patients (21%), or among asymptomatic HBsAg carrier blood donors (20 of 126 = 16%) without detectable hepatitis B virus replication (p less than 0.0001). The highest prevalence of anti-HBx was found in HBsAg carriers with cirrhosis (41 of 54 patients = 76%) and/or with hepatocellular carcinoma (18 of 33 patients = 54%). The findings suggest that anti-HBx appears as a common and early marker of hepatitis B virus infection, transient in self-limited hepatitis but persisting with progression to chronicity. In chronic hepatitis, the prevalence of anti-HBx correlated with the intensity and duration of hepatitis B virus replication but neither with the severity of the liver disease nor with malignant transformation per se.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of an EcoRI duck hepatitis B virus (DHBV) clone was elucidated by using the Maxam and Gilbert method. This sequence, which is 3,021 nucleotides long, was compared with the two previously analyzed hepatitis B-like viruses (human and woodchuck). From this comparison, it was shown that DHBV is derived from an ancestor common to the two others but has a slightly different genomic organization. There was no intergenic region between genes 5 and 8, which were fused into a single open reading frame in DHBV. Genes for the surface and core proteins were assigned to open reading frames 7 and 5/8. Amino acid comparisons showed some structural relationship between gene 6 product and avian reverse transcriptase, suggesting either evolution from a common ancestor or convergence to some particular structure to fulfill a specific function. This should be correlated with the synthesis of an RNA intermediate during DNA replication. This is also taken as an argument in favor of the hypothesis that gene 6 codes for the DNA polymerase that is found within the virion. DNA sequence comparison also showed that the two mammalian hepatitis B viruses are more homologous to each other than they are to DHBV, indicating that DHBV starts to evolve on its own earlier than the two other viruses, as do birds compared with mammals. From this it is proposed that the viruses evolved in a fashion parallel to the species they infect.

The structural gene coding for both polypeptides I and II which are the two major polypeptides of the Hepatitis B surface antigen, is found to be localized on the viral genome. This gene, referred to as gene S, is located in the partially single stranded region. It maps between positions 73.6 and 95.1% of the genome length. It is composed of 678 nucleotides, which correspond to a theoretical polypeptide of 25,422 molecular weight.

We have cloned the X gene (HBx) and the HBc antigen (HBc Ag) gene of human hepatitis B virus (HBV) in Escherichia coli as fusion products with beta-galactosidase. Both HBV genes are expressed in E. coli strain CSR 603. Expression is detected by u.v. irradiation of the bacteria, metabolic labelling and electrophoresis of the labelled extracts on SDS-polyacrylamide gels. The HBc Ag protein produced in bacteria can be recognised by anti-HBc sera and peptides derived from the protein are also recognised by anti-HBe sera. The HBx protein is recognised by some, but not all, sera which are anti-HBe positive. HBx Ag is also recognised by a woodchuck antibody similar to anti-HBe (anti-WHe). These results constitute the first proof that the open reading frame X is a true viral gene and is expressed during HBV (and WHV) infection and that an HBx/anti-HBx system, which may have important biological implications, can exist in parallel with the classic HBe/anti-HBe system.

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.

Nucleotide sequence analysis was performed with the Maxam--Gilbert method on a cloned woodchuck hepatitis virus DNA (Eco WHV DNA). The structural gene coding for the envelope protein of the virus was localized on the viral genome in the partially single-stranded region between map positions 91.2 and 71. This gene is composed of 669 nucleotides and can code for a polypeptide of 25,645 daltons. The DNA sequence and the deduced amino acid sequence were compared with those of the corresponding gene and surface antigen of the related hepatitis B virus, allowing some insight into the localization of the antigenic site.

The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the S chain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.