María T. Franze-Fernández

Transcription and RNA Replication of Tacaribe Virus Genome and Antigenome Analogs Require N and L Proteins: Z Protein Is an Inhibitor of These Processes

ABSTRACT Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5′ and 3′ termini by sequences corresponding to those of the 5′ and 3′ noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.

Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina.n Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas.n En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We report the embryonic and adult phenotypes of a number of mutations of the abd-A gene of the bithorax complex. Some of them result in loss of abd-A function in the whole abd-A domain and are usually lethal. These probably eliminate or inactivate abd-A protein products. Other mutations affect only part of the abd-A domain. These are viable, appear to map outside the abd-A transcription unit, and presumably alter the normal spatial regulation of abd-A products. We propose a model of abd-A structure based on a protein-coding region and two cis-regulatory regions. Regulatory region 1, 3 to the transcription unit, contains positive and negative regulatory elements. Regulatory region 2, 5 to the transcription unit, establishes the correct level of abd-A activity in the abdominal metameres.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.Resumen del trabajo presentado al III Meeting Red de Excelencia Tematica: RNA Life, celebrado en Salamanca del 24 al 26 de septiembre de 2018.Thyroid hormone is an important epigenetic factor in brain development, acting by modulating rates of gene expression. The active form of thyroid hormone, 3,5,3-triiodothyronine (T3) is produced in part by the thyroid gland but also after 5-deiodination of thyroxine (T4) in target tissues. In brain, approximately 80% of T3 is formed locally from T4 through the activity of the 5-deiodinase type 2 (D2), an enzyme that is expressed mostly by glial cells, tanycytes in the third ventricle, and astrocytes throughout the brain. D2 activity is an important point of control of thyroid hormone action because it increases in situations of low T4, thus preserving brain T3 concentrations. In this work, we have studied the expression of D2 by quantitative in situ hybridization in hypothyroid animals during postnatal development. Our hypothesis was that those regions that are most dependent on thyroid hormone should present selective increases of D2 as a protection against hypothyroidism. D2 mRNA concentration was increased severalfold over normal levels in relay nuclei and cortical targets of the primary somatosensory and auditory pathways. The results suggest that these pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some interneurons of the cerebral cortex were also labeled. Therefore, the T3 target cells, mostly neurons, are dependent on local astrocytes for T3 supply.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado a la Vth International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis celebrada en Primosten (Croacia) del 6 al 9 de Junio de 2012.-- Tambien presentado por Carlos R. Vazquez de Aldana al International Symposium: Biology and Communications celebrado en Madrid del 26 al 27 de marzo de 2012.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

The 5′ region of Tacaribe virus L RNA encodes a protein with a potential metal binding domain

We have just completed the Tacaribe arenavirus (TV) genome structure by sequencing the 5 region of the L RNA. Analysis of the sequence has indicated the existence of an open reading frame (ORF) in the viral sense RNA encoding a 95 amino acid polypeptide. The first in phase AUG codon is in positions 70-72 from the 5 end of the viral RNA surrounded by a sequence favorable for the initiation of protein synthesis. The ORF ends at positions 355-357. The predicted polypeptide (P11) contains a cysteine-rich sequence bearing a remarkable similarity to the zinc finger sequences found in a number of proteins. We have recently reported that the 3 region of the TV L RNA encodes a polypeptide comprising 2210 amino acids in the viral-complementary sequence. This latter gene, i.e., the L gene, terminates at positions 442-440 from the 5 end of the viral RNA. The two genes encoded by the L RNA (L and P11) are in opposite strands of the RNA in sequences that do not overlap, but are separated by a noncoding intergenic region of 82 nucleotides. The nucleotide sequence of the intergenic region leads to the prediction of a strong secondary structure.

The 3' end termini of the Tacaribe arenavirus subgenomic RNAs

Tacaribe virus (TV), a member of the Arenaviridae family, contains two single-stranded RNA genome segments called S and L. Two proteins, in an ambisense coding strategy, are encoded in both the S RNA and the L RNA. The 3 ends of the TV four putative mRNAs have been characterized using S1 nuclease mapping. The experiments revealed that the transcripts terminate within the intergenic region in each RNA segment. No special sequences that might function as termination signals were evident. The 3 end sequences of the four putative mRNAs can be predicted to adopt GC-rich stable hairpin configurations (delta G greater than or equal to -25 kcal). These observations suggest that the transcript structure rather than particular sequences might be the signal involved in the termination of arenavirus transcription.

Tacaribe virus L gene encodes a protein of 2210 amino acid residues

The nucleotide sequence of Tacaribe virus (TV) L gene was obtained from two sets of overlapping cDNA clones constructed by walking along the virus L RNA using two successive synthetic DNA primers. Analysis of the sequence indicated the existence of a unique long open reading frame in the viral complementary strand. The first in-phase AUG codon is in positions 31-33 from the 5 end of the viral complementary L RNA surrounded by a sequence favorable for initiation of protein synthesis. The open reading frame ends at positions 6661-6663. The predicted TV L protein is a 2210 amino acid long polypeptide with an estimated molecular weight of 251,942. Comparison of the amino acid sequence of TV L protein with peptide sequences predicted from L-derived cDNA clones of lymphocytic choriomeningitis virus shows an overall 42% of homology.

Effect of protein synthesis inhibitors and low concentrations of actinomycin D on ribosomal RNA synthesis

It is known that low concentrations (0.001-0.05 pg/ml) of actinomycin D selectively inhibit ribosomal RNA (rRNA) synthesis when administered ‘in vivo’ [ 1,2]. This effect has been explained on the basis of a direct action of the drug on nucleolar transcription [3]. However, the same doses of actinomycin D that are effective in vivo do not inhibit RNA polymerase I (EC 2.7.7.6) activity when added to isolated nuclei although they do slightly decrease RNA polymerase II activity; this result suggested that the specific action of the antibiotic on rRNA synthesis might be indirect [4]. The observations that after in vivo administration of low doses of actinomycin D there is a decrease in the RNA polymerase II with a concomitant inhibition in the RNA polymerase I activity in the isolated nuclei and furthermore, that the time course of this inhibition is similar to that found after suppression of protein synthesis, lend support to the proposal that low doses of actinomycin D affect rRNA synthesis through inhibition of the synthesis of messenger RNAs (mRNAs) of high turnover which code for proteins required for nucleolar activity [S]. In this proposal the idea is implied that rRNA synthesis is under the control of mRNA synthesis. In the forementioned studies rRNA synthesis was measured in isolated nuclei or nucleoli [4,5]. In these in vitro systems the problem of determining the specific activity of the nucleotide precursor pools [6,7] is overcome, and therefore they are widely used for studying transcription of ribosomal genes [4,5,8-131. However, the rate of elongation by RNA polymerase I is only l-2% that in vivo [9]. On the

Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

ABSTRACT Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Zs inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z.

Effect of amino acids on the α-amanitin-insensitive RNA polymerase activity in the isolated nuclei of ehrlich ascites cells

Abstract The effect of amino acids on the α-amanitin-insensitive RNA polymerase activity in the isolated nuclei of Ehrlich ascites cells was studied. The following results were obtained: 1. 1. The RNA polymerase activity was stimulated by increasing the amino acid concentration in the incubation medium of the cells. 2. 2. The amino acid requirement for RNA polymerase activation differed from the requirement that for the stimulation of protein synthesis. 3. 3. No particular amino acid affected the RNA polymerase activity. The combined effect from the reduction of the concentration of several amino acids was higher than the response given by the deprivation of individual amino acids. 4. 4. The RNA polymerase activation by amino acids has not been obtained in the presence of protein synthesis inhibitors, indicating that protein synthesis is involved in the process. 5. 5. After protein synthesis inhibition by antibiotics there was a rapid decay in the RNA polymerase activity. A similar decay has been obtained after reducing the amino acid concentration. It is postulated that the α-amanitin-sensitive RNA polymerase activity in the isolated nuclei of Ehrlich ascites cells is regulated by a short-lived protein(s). Amino acids could stimulate the synthesis or decrease the degradation rate of this protein(s).

Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs.

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.

EHRLICH ASCITES CELLS DNA-DEPENDENT RNA POLYMERASES: EFFECT OF AMINO ACIDS AND PROTEIN SYNTHESIS INHIBITION

. The biochemical mechanisms underlying this re- gulation, however, are in general unknown. We have found that in Ehrlich ascites cells, the rRNA synthesis and the activity of the enzyme most probably involved in its transcription (cY-amanitin resistant RNA poly merase) are regulated by the amino acids concentra- tion in the incubation medium [2] and that this con- trol is mediated by the synthesis and decay of a short lived protein [3]. In the previous work, RNA poly- merase activity had been assayed in isolated nuclei; consequently it has been possible to decide whether the enzyme itself or some protein component of the RNA polymerase system have a short half life. As an approach to studying this problem we have isolated the RNA polymerases from Ehrlich ascites cells and compared the enzymes obtained from cells incubated in different conditions. 2. Methods The preparation of Ehrlich ascites tumour cells and of the incubation media with and without amino acids, and incubation of the cells have been done as described previously [3]. Nuclei were purified as previously in- dicated [3] with the modification that all the solutions contained 0.1 mM spermidine. Nuclei isolated from 1 g of cells were resuspended in 3.5-5 ml of a solu- tion containing: 0.02 M Tris-HCl, pH 7.90; 2 mM dithioerythritol (DTE); 5 mM MgCl 2 ; 1 M saccharose. The nuclear suspension was immediately used for RNA polymerase assays and for solubilization of the en- zymes. 2.1.