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The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Miniparamyosin, a distinct Drosophila melanogaster paramyosin isoform of 60 kDa, is shown here to be encoded by the same gene as paramyosin. The gene, located at 66D14, spans over 12.8 kilobases (kb) and is organized into 10 exons, 9 of which code for the paramyosin transcripts. An exon, located between exons 7 and 8, codes for the 5′-end of the miniparamyosin, and the two proteins share the two last exons of the gene. Mapping of the 5′-ends of these transcripts indicates that the paramyosin and miniparamyosin mRNAs arise from two overlapping transcriptional units; the miniparamyosin transcription initiation site is located inside a paramyosin intron, 8 kb downstream of the one used for paramyosin transcription. The existence of two different promoters and the conserved and nonconserved features of their sequences suggest a very complex regulation of these two muscle proteins. In fact, while paramyosin is expressed at two distinct stages of development as most other Drosophila muscle proteins, miniparamyosin appears late in development, being present only in the adult musculature. The absence of exon 1B, the specific exon of miniparamyosin, in the nematode Caenorhabditis elegans, as well as additional lines of evidence support the lack of miniparamyosin in this particular organism. However, it is present in most invertebrate species examined, including different arthropod, annelid, mollusc, and echinoderm species.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

A search of sequence data bases for a tridecamer transcription termination signal, previously described in human mtDNA as being responsible for the accumulation of mitochondrial ribosomal RNAs (rRNAs) in excess over the rest of mitochondrial genes, has revealed that this termination signal occurs in equivalent positions in a wide variety of organisms from protozoa to mammals. Due to the compact organization of the mtDNA, the tridecamer motif usually appears as part of the 3' adjacent gene sequence. Because in phylogenetically widely separated organisms the mitochondrial genome has experienced many rearrangements, it is interesting that its occurrence near the 3' end of the large rRNA is independent of the adjacent gene. The tridecamer sequence has diverged in phylogenetically widely separated organisms. Nevertheless, a well-conserved heptamer--TGGCAGA, the mitochondrial rRNA termination box--can be defined. Although extending the experimental evidence of its role as a transcription termination signal in humans will be of great interest, its evolutionary conservation strongly suggests that mitochondrial rRNA transcription termination could be a widely conserved mechanism in animals. Furthermore, the conservation of a homologous tridecamer motif in one of the last 3' secondary loops of nonmitochondrial 23S-like rRNAs suggests that the role of the sequence has changed during mitochondrial evolution.

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the C mutually implies T substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica., This work was supported by a grant of the DGICYT, Ministerio de Educación y Ciencia, Spain.

It has been suggested that mutations accumulated in mitochondrial DNA during the aging process may be causally related to the decreased physiological response of the senescent organisms. We have quantified and evaluated the integrity of the mitochondrial genome during the life span of Drosophila melanogaster. Its amount remains fairly constant representing roughly 1% of the total DNA at all ages. Southern experiments have also revealed a high stability and integrity of the mitochondrial DNA (mtDNA). However, we have detected an important decrease in the steady-state levels of all mitochondrial transcripts investigated: 16 S ribosomal RNA (16SrRNA), cytochrome c oxidase, cytochrome b, and beta H(+)-ATP synthase subunit. These changes correlate with the shape of the life span curve, preceding the decrease in survival of the male flies used in the study, and at least in the case of 16SrRNA, is tissue-specific. Although mitochondrial DNA remains unchanged in heads, thoraces, and abdomens, 16SrRNA levels decrease more severely in heads and thoraces and much less conspicuously in abdomens. On the other hand, control non-mitochondrial transcripts investigated remain essentially unaffected. These results suggest that in Drosophila the main effect of aging on the mitochondrial genetic system is downstream from mtDNA itself. The decline in the levels of beta H(+)-ATPase transcript, nuclear-encoded, suggests that not only the mitochondrial machinery, but also the nuclear one involved in mitochondrial biogenesis, is affected during aging.

The Ultrabithorax gene is required to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid region, a 40 kb DNA stretch upstream of the Ultrabithorax promoter, affect cis-regulatory elements of the Ultrabithorax gene. We now have identified specific sites in the bithoraxoid region that exhibit S1 nuclease sensitivity in vitro. These sites are not scattered along the DNA but are grouped instead in specific domains. Some of these S1-sensitive sites correlate with known breakpoint or insertional mutations. Others correspond to putative binding sites for transcription factors. The results suggest that unusual secondary structure might be important in chromosomal translocation within regulatory sequences of the Ultrabithorax product or its transcriptional regulation.

We show, in this paper, the results obtained in the sterilization process by using different irradiating structures. Starting from our earlier findings of the existence of an important synergy on the killing effect caused by the simultaneous application of oxidant agents such as hydrogen peroxide and microwave irradiation, we have now extended the work to identify the power threshold and the optimum frequency range for the effect. © 2001 John Wiley & Sons, Inc. Microwave Opt Technol Lett 30: 404–406, 2001.

Genome-wide transcriptional profiling shows that reducing gravity levels during Drosophila metamorphosis in the International Space Station (ISS) causes important alterations in gene expression: a large set of differentially expressed genes (DEGs) are observed compared to 1g controls. However, the preparation procedures for spaceflight and the nonideal environmental conditions on board the ISS subject the organisms to additional environmental stresses that demonstrably affect gene expression. Simulated microgravity experiments performed on the ground, under ideal conditions for the flies, using the random position machine (RPM), show much more subtle effects on gene expression. However, when the ground experiments are repeated under conditions designed to reproduce the additional environmental stresses imposed by spaceflight procedures, 79% of the DEGs detected in the ISS are reproduced by the RPM experiment. Gene ontology analysis of them shows they are genes that affect respiratory activity, developmental processes and stress-related changes. Here, we analyse the effects of microgravity on gene expression in relation to the environmental stresses imposed by spaceflight. Analysis using 'gene expression dynamics inspector' (GEDI) self-organizing maps reveals a subtle response of the transcriptome to microgravity. Remarkably, hypergravity simulation induces similar response of the transcriptome, but in the opposite direction, i.e. the genes promoted under microgravity are usually suppressed under hypergravity. These results suggest that the transcriptome is finely tuned to normal gravity and that microgravity, together with environmental constraints associated with space experiments, can have profound effects on gene expression.

The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.

Several cDNA clones encoding the complete Drosophila paramyosin sequence, including two potential polyadenylation sites, have been obtained. Southern analysis and in situ hybridization to polytene chromosomes indicate that in Drosophila the paramyosin gene is single copy, located on the left arm of the third chromosome at region 66D14. Northern analyses show predominantly two different RNAs which are the products of the choice between the two alternative polyadenylation sites. The two species begin to be synthesized around 10 h of development when embryonic muscles are formed, expression peaking at the end of embryogenesis. The protein is first expressed at germ band shortening in association with muscle precursor cells. A second maximum of paramyosin RNA expression occurs at late pupal stages when the higher molecular weight form becomes more abundant. In young adults this species becomes the main transcript detected. The 102 kDa polypeptide sequence is highly similar to that of Caenorhabditis elegans paramyosin. The protein has a central alpha-helical coiled-coil rod, organized in 29 groups of four typical seven-residue repeats and flanked by two short non-alpha-helical regions. Several leucine zippers are located on the hydrophobic face of the alpha-helix in paramyosin which, together with disulfide bonds between cysteines, are probably involved in the stabilization of the dimer. The structural and functional properties of Drosophila paramyosin deduced from the sequence are compared with those of known invertebrate myosins and paramyosins.

5 páginas -- PAGS nros. 169-174, The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, α-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a λgtl1 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.

From the autumn of 2001 till spring of 2005 a series of six flights to the International Space Station, ISS, were conducted using the Russian Soyuz manned launcher. These flights initially known as ‘taxi-missions’, were characterized by the participation and co-funding from both the European Space Agency, ESA, and the five national delegations from France, Italy, Belgium, Spain, and the Netherlands. The national participation was reflected both in the flight of a cosmonaut/astronaut, originating from the country co-sponsoring the flight as well as in the origin of the majority of experiments and other activities carried out during these missions. In these six Soyuz missions: Andromède (October 2001), Marco Polo (April 2002), Odissea (October 2002), Cervantes (October 2003), DELTA (April 2004) and Eneide (April 2005), some more than one hundred experiments were carried out. These experiments covered the areas of basic and applied research and technology in biology, human physiology, fluid and plasma physics, material science and Earth observation. Also a significant number of education activities were part of these missions. This paper gives a complete overview of these missions, of all science, education and related activities performed. The perspectives of these activities in the light of the space exploration programs in the XXI century and some of the uncertainties and paradoxes are discussed

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish “Cervantes” Soyuz mission and the ICE-1st experiment during the Dutch “Delta” mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight., The support of the all organizations and people involved in the Cervantes and Delta missions, the Spanish Space Program (Spanish Ministery of Education and Science), the Dutch Space Program, ESA, the CNES and NASA that made possible this work is gratefully acknowledged.

10 páginas, 7 figuras, 1 tabla -- PAGS nros. 1244-1253, Seeds of Arabidopsis thaliana were sent to the International Space Station in the “Cervantes Mission” (Spanish Soyuz Mission). Seed germination was initiated in flight by supplying culture medium. Seedlings were grown for 4 days at 22 °C, and growth was stopped by the addition of paraformaldehyde fixative. Once back on the ground, samples were immediately processed for microscopy. A ground control experiment was simultaneously replicated. Glutaraldehyde-fixed root cells from seedlings grown in the Biorack on board of the Space Shuttle (STS-84 Mission) in similar conditions were also ultrastructurally examined. The length of seedlings grown at 1g was conspicuously shorter than parallel samples grown under microgravity. We examined the morphology of the root meristematic cells, with a focus on their nucleoli in the cortex and stele. In general, root cortical cells proliferate at a higher rate and their nucleoli are more active than those of stele cells. While the stele showed longer cells with larger nucleoli in the flight samples, cortical cells from space-grown seedlings were shorter, more numerous and more densely packed than ground controls. However, nucleoli were smaller and less active in fast proliferating flight cells than in the ground controls. This reduced level of ribosome synthesis in the flight samples is probably the result of an accelerated cell cycle. An altered rate of cell proliferation may be detrimental for the plant and could be the reason for the reported smaller size of older space-grown seedlings. Finally, two-dimensional protein electrophoresis showed noticeable differences between space samples and ground controls, This work has been supported by the Spanish “Programa Nacional de Investigación Espacial” Grants Nos. ESP2002-12062-E, ESP2002-12686-E, and ESP2003-09475-C02-02

In bilaterians, the main regulator of muscle contraction is the troponin (Tpn) complex, comprising three closely interacting subunits (C, T, and I). To understand how evolutionary forces drive molecular change in protein complexes, we have compared the gene structures and expression patterns of Tpn genes in insects. In this class, while TpnC is encoded by multiple genes, TpnT and TpnI are encoded by single genes. Their isoform expression pattern is highly conserved within the Drosophilidae, and single orthologous genes were identified in the sequenced genomes of Drosophila pseudoobscura, Anopheles gambiae, and Apis mellifera. Apis expression patterns also support the equivalence of their exon organization throughout holometabolous insects. All TpnT genes include a previously unidentified indirect flight muscle (IFM)-specific exon (10A) that has evolved an expression pattern similar to that of exon 9 in TpnI. Thus, expression patterns, sequence evolution trends, and structural data indicate that Tpn genes and their isoforms have coevolved, building species- and muscle-specific troponin complexes. Furthermore, a clear case can be made for independent evolution of the IFM-specific isoforms containing alanine/proline-rich sequences. Dipteran genomes contain one tropomyosin gene that encodes one or two high-molecular weight isoforms (TmH) incorporating APPAEGA-rich sequences, specifically expressed in IFM. Corresponding exons do not exist in the Apis tropomyosin gene, but equivalent sequences occur in a high-molecular weight Apis IFM-specific TpnI isoform (TnH). Overall, our approach to comparatively analyze supramolecular complexes reveals coevolutionary trends not only in gene families but in isoforms generated by alternative splicing. © The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved., This research was supported by grants from the Spanish Government PB96-0069, ESP1999-0379-C02 and PNE-008/2001-C, BMC2001-1454.

The existence of preservation problems is one of the most important consequences of Space Biological Research. The Topical Team is critically analyzing the currently performed procedures and is establishing the bases for a recommendation on new procedures, capable of overcoming the present constraints.

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.

8 p.- 6 fig.- 1 tab, The success of the genomic sequencing programs allows the discovery of additional family members of genes encoding known functions. This is the case of the Troponin C gene repertoire in Drosophila melanogaster. We have found two new Troponin C genes, DmTpnC41F and DmTpnC25D, increasing to five the total number of Troponin C genes identified in this species. The comparative characterization of the five Troponin C genes in D. melanogaster demonstrates considerable variation in gene structure and expression pattern. Expression of one gene, DmTpnC41F, has a more restricted tissue specificity than the rest of the TpnC genes and, with the chromosomically linked DmTpnC41C, is expressed specifically in the adult thorax. The new gene, DmTpnC25D is expressed during development more broadly than the rest. In adults, it is highly expressed in the adult head. Finally, the other two genes, DmTpnC47D and DmTpnC73F, show a high embryonic/larval expression and in adults are expressed almost exclusively in the abdomens. The functional adaptive changes that may have evolved during the expansion of this gene family are briefly discussed in terms of the expression patterns, gene and protein structures leading to a simpler, more systematic nomenclature of the gene family., This research was supported by grants from the Spanish Government PB96-0069, ESP1999-0379-C02 and PNE- 008/2001-C, and by GM61738 awarded by the National Institutes of Health to RMC.

Space exploration, especially its future phase involving the International Space Station (ISS) makes possible the study of the effects on living systems of long-term expositions to such a strange environment. This phase is being initiated when Biological Sciences are crossing a no-return line into a new territory where the connection between phenotype and genotype may be finally made. We briefly review the paradoxical results obtained in Space experiments performed during the last third of the XX Century. They reveal that simple unicellular systems sense the absence of gravity changing their cytoskeletal organization and the signal transduction pathways, while animal development proceeds unaltered in these conditions, in spite of the fact that these processes are heavily involved in embryogenesis. Longer-term experiments possible in the ISS may solve this apparent contradiction. On the other hand, the current constraints on the scientific use of the ISS makes necessary the development of new hardware and the modification of current techniques to start taking advantage of this extraordinary technological facility. We discuss our advances in this direction using one of the current key biological model systems, Drosophila melanogaster. In addition, the future phase of Space exploration, possibly leading to the exploration and, may be, the colonization of another planet, will provide the means of performing interesting evolutionary experiments, studying how the terrestrial biological systems will change in their long-term adaptation to new, very different environments. In this way, Biological Research in Space may contribute to the advancement of the new Biology, in particular to the branch known as "Evo-Devo". On the other hand, as much as the Space Adventure will continue involving human beings as the main actors in the play, long-term multi-generation experiments using a fast reproducing species, such as Drosophila melanogaster, capable of producing more than 300 generations in 15 years, the useful life foreseen for ISS, will be important. Among other useful information, they will help in detecting the possible changes that a biological species may undergo in such an environment, preventing the uncontrolled occurrence of irreversible deleterious effects with catastrophic consequences on the living beings participating in this endeavour.

Space exploration, especially its future phase involving the International Space Station (ISS) makes possible the study of the effects on living systems of long-term expositions to such a strange environment. This phase is being initiated when Biological Sciences are crossing a no-return line into a new territory where the connection between phenotype and genotype may be finally made. We briefly review the paradoxical results obtained in Space experiments performed during the last third of the XX Century. They reveal that simple unicellular systems sense the absence of gravity changing their cytoskeletal organization and the signal transduction pathways, while animal development proceeds unaltered in these conditions, in spite of the fact that these processes are heavily involved in embryogenesis. Longer-term experiments possible in the ISS may solve this apparent contradiction. On the other hand, the current constraints on the scientific use of the ISS makes necessary the development of new hardware and the modification of current techniques to start taking advantage of this extraordinary technological facility. We discuss our advances in this direction using one of the current key biological model systems, Drosophila melanogaster. In addition, the future phase of Space exploration, possibly leading to the exploration and, may be, the colonization of another planet, will provide the means of performing interesting evolutionary experiments, studying how the terrestrial biological systems will change in their long-term adaptation to new, very different environments. In this way, Biological Research in Space may contribute to the advancement of the new Biology, in particular to the branch known as "Evo-Devo". On the other hand, as much as the Space Adventure will continue involving human beings as the main actors in the play, long-term multi-generation experiments using a fast reproducing species, such as Drosophila melanogaster, capable of producing more than 300 generations in 15 years, the useful life foreseen for ISS, will be important. Among other useful information, they will help in detecting the possible changes that a biological species may undergo in such an environment, preventing the uncontrolled occurrence of irreversible deleterious effects with catastrophic consequences on the living beings participating in this endeavour.

While experiments carried out in Space with isolated cells have shown that eucaryotic cells are able to sense and respond to the absence of gravity by modifying their reactions, experiments in which more complex processes have been investigated, such as Biological Systems undergoing development under Microgravity. have been surprisingly unaffected by the space environment. This can be considered a curious result since all organisms are evolutionarily adapted to the current level of the gravity force in our planet and should eventually change if this parameter will vary in a permanent manner. In fact, the small effects of the modifications in gravity on development in short term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical processes such as diffusion-controled auto-organizative reactions in currently developing biological systems. An apparent exception to this lack of effect is experiments where brine shrimp dormant gastrulae directly exposed to the space environment accumulate developmental defects as a consequence of cosmic irradiation. In this article we discuss the idea that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces such as gravity were involved in the evolutionary organization of these primitive organisms. As evolution proceed, these early mechanisms may have been obscured and/or made redundant by the appearance of new internal, environment-independent biological regulatory mechanisms. On the other hand, behavioral responses that may be important, for example, in setting the life-spans of organisms may still be more readily susceptible to manipulation by external cues as experiments carried out by our group in Space and on the ground with Drosophila melanogaster indicate., The financial support of the Spanish National Space Program (ESP97-1775) and of ESA is gratefully acknowledged.

During the last two decades an increasing amount of information has been accumulated regarding the gene structure and organization of the mitochondrial genome from various organisms. Many studies carried out mainly in mammals, have contributed to the knowledge of the basic elements involved in the replication and transcription of mitochondrial DNA. However, very little is known about these processes in invertebrates. In this review we discuss our current knowledge of the animal mitochondrial genetic system and briefly summarize the structure of the Artemia mitochondrial genome, the characteristics of its transcriptional machinery and how its expression is controlled during early development, in relation with what is known in other organisms. Artemia is the only crustacean where the mtDNA has been studied at this level of detail up to date., This work was supported by grants PB92-0059 and PB94-0088 from Dirección General de Investigación Cientı́fica y Tëcnica (Ministerio de Educación y Ciencia, Spain).

12 pages, 10 figures., The Drosophila paramyosin/miniparamyosin gene expresses two products of different molecular weight transcriptionally regulated from two different promoters. Distinct muscle types also have different relative amounts of myosin, paramyosin, and miniparamyosin, reflecting differences in the organization of their thick filaments. Immunofluorescence and EM data indicate that miniparamyosin is mainly located in the M line and at both ends of the thick filaments in Drosophila indirect flight muscles, while paramyosin is present all along the thick filaments. In the tergal depressor of the trochanter muscle, both proteins are distributed all along the A band. In contrast, in the waterbug, Lethocerus, both paramyosin and miniparamyosin are distributed along the length of the indirect flight and leg muscle thick filaments. Two-dimensional and one-dimensional Western blot analyses have revealed that miniparamyosin has several isoforms, focusing over a very wide pH range, all of which are phosphorylated in vivo. The changes in isoform patterns of miniparamyosin and paramyosin indicate a direct or indirect involvement of these proteins in muscle function and flight. This wide spectrum of potential regulatory characteristics underlines the key importance of paramyosin/miniparamyosin and its complex isoform pattern in the organization of the invertebrate thick filament., This work was supported by grants (PB91-0098 and ESP94-865) from the DGICYT (Spanish Ministry of Education and Science) and Plan Nacional de Investigación Científica, respectively, and a Human Capital and Mobility grant from the European Union (ERBCHRXCT930249). Miguel Maroto and Juan Arredondo were predoctoral fellows from the Universidad Autónoma de Madrid with funds provided by the European Space Agency and the Comunidad Autónoma de Madrid.

We have studied the process of post-embryonic segmentation in the anostracan crustacean Artemia franciscana using a specific antibody raised against the engrailed protein of this organism. Three cephalic segments are specified during embryonic development, before larval hatching, whilst trunk (thoracic) segmentation begins after the first stage free-swimming nauplius larva has emerged from the dormant cyst. Thus, cephalic and trunk segmentation seem to be at least in part independent and superimposed processes. Trunk stripes of engrailed expression are added one at a time as segments are generated from the posterior growth zone. The first detectable decision in engrailed expression is the establishment of a line of engrailed-expressing cells, interpretable as delineating the parasegmental boundary. The subsequent widening of engrailed stripes is not correlated with cell lineage events but is probably mediated by the combination of inheritance of the active state and recruitment of new cells into the engrailed-expressing stripe., The work has been supported by grants from the Spanish Government, DGICYT (PB87- 0208) and CICYT (ESP 91-627). M. M. was supported by a predoctoral fellowship from the Spanish Ministry of Education and Science and by a fellowship from the Universidad Autónoma de Madrid with funds of the European Space Agency.

Three arthropod systems are discussed in relation to their complementary and potential use in Space Biology. In a next biosatellite flight, Drosophila melanogaster pre-adapted during several months to different g levels will be flown in an automatic device that separates parental from first and second generations. In the same flight, flies will be exposed to microgravity conditions in an automatic unit in which fly motility can be recorded. In the International Microgravity Laboratory-2, several groups of Drosophila embryos will be grown in Space and the motility of a male fly population will be video-recorded. In the Biopan, an ESA exobilogy facility that can be flown attached to the exterior of a Russian biosatellite, Artemia dormant gastrulae will be exposed to the space environment in the exterior of the satellite under a normal atmosphere or in the void. Gastrulae will be separated in hit and non-hit populations. The developmental and aging response of these animals will be studied upon recovery. With these experiments we will be able to establish whether exposure to the space environment influences arthropod development and aging, and elaborate on some of the cellular mechanisms involved which should be tested in future experiments. © 1994., The financial support of the Plan Nacional del Espacio, the CNES and ESA is gratefully acknowledged, as well as the help of the ESA team in Moscow (Dr. W. Jensen, Dr. R. Demets and Mar L6pez), of the Ondatrón group and Dr. J. L. Monteagudo (Instituto Carlos III).

Taking advantage of the fact than segmentation inArtemia is largely a postembryonic process making it more susceptible to environmental influences, heat treatments ofArtemia newly-hatched nauplii were shown to induce a severe inhibition of mesodermal structures, without apparently affecting the corresponding ectodermal and endodermal derivaives. This inhibition was reversible and with enough time the missing mesodermal structures developed. These results indicate that ectoderm and endoderm development can proceed without neccessarily a concomitant mesodermal differentiation, which in turn can be largely uncoupled from that of the rest of the germ layer derivatives.

Fluorescamine is a useful reagent in monitoring protein-DNA interactions only if a convenient method of separating the complex from free protein is available. Sedimentation of the complex provides such a method at least in the case of histone-like proteins capable of extensive interaction with DNA. This approach is therefore complementary to the filter binding assay. When the interaction of protein and DNA is compared by both methods, a clear-cut distinction between two steps is obtained: (i) a nucleation step that can be measured by the filter binding assay: and (ii) the cooperative growth of the complex that can only be measured by the sedimentation assay. The method is also useful to detect small amounts of protease contamination in DNA preparations.

A quantitative model of the effect of the bithorax complex on segmentation is presented which could explain the known data of the spatiotemporal regulation of key gene complex during early Drosophila development, in relation to their effects on some of the segmentation landmarks. The model tries to put together the two different genetic levels, the genotypic and the phenotypic. At the genotypic level, a minimal cross-regulatory network of the different genes involved, Antp, Ubx, abd-A and Abd-B which explains the reported levels of expressions of these genes. At the phenotypic level, the pattern of the ventral denticle belts across the larva which are characteristics of the different segments have been compared by calculating a value of the degree of similarity in the case of the wild-type and several mutant combinations. Finally the two parts of the model are combined, showing that a satisfactory agreement between the two can be achieved. Therefore, this work is a first attempt to develop a method which will provide an explanatory solution of the old question in morphogenesis of how the phenotype is directed by the genotype of a cell or organism.

Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of a reexamination of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of the pH on the reaction and on the fluorescence of the product are presented. Polyalcohols, due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alkaline conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pHs below 7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino-acid that under these conditions will react giving a fluorescent product. Among other amino groups containing molecules tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction are also studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amount of fluorescamine in the assay under conditions in which the reaction is quantitative is described. A similar micromethod can be used with the O-phtalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alkaline and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and O-phtalaldehyde are discussed.

Mini M13 phages, about one-half as long and with one-half the DNA of wild-type filaments, contain a full amount of the gene 3 protein. This finding supports earlier evidence for an adsorption function of the gene 3 protein and its location at the end(s) rather than along the length of the filament. An unusual property of the extracted protein is its aggregation even under strongly denaturing and reducing conditions.

1. The subcellular distribution of the enzymes involved in oxaloacetate metabolism and certain related enzymes in the liver of normal fed and starved rats has been studied. A simplified fractionation procedure was developed in the course of this work. Pyruvate carboxylase and citrate synthase were found to precipitate exclusively associated to mitochondria. 2. The precise location of the mitochondrial enzymes was studied in submitochondrial fractions. The existence of two distinct oxaloacetate metabolic crossroads in rat liver has been clearly established. One is located in the mitochondrial matrix and involves the oxaloacetate forming pyruvate carboxylase, the citrate synthase and the mitochondrial malate dehydrogenase and glutamic oxaloacetic transaminase. The second one is located in the cytosol and involves the phosphoenolpyruvate carboxykinase and the cytosolic malate dehydrogenase and glutamic oxaloacetic transaminase. 3. The results of similar fractionation studies applied to rabbit liver indicate that in this animal phosphoenolpyruvate carboxykinase is located in the mitochondrial matrix. The other enzymes tested showed the same localization as in the rat. 4. The search in rat liver for a cytosolic activity from 3C to 4C gluconeogenic precursors has been negative. This result stresses the importance of the regulatory problems associated with the exit of oxaloacetate out of the mitochondria in gluconeogenesis. 5. The adaptive behaviour of the enzymes of the mitochondrial and cytoplasmic oxaloacetate crossroads has been studied in rat liver. Only phosphoenolpyruvate carboxykinase shows a marked increase in specific activity when the animals are fasted. 6. The kinetic properties of rat liver citrate synthase have been studied. NADPH inhibition is competitive against oxaloacetate while ATP inhibition is competitive against acetyl-CoA. 7. The Michaelis constants of the mitochondrial malate dehydrogenase and the kinetic constants of the mitochondrial glutamic oxaloacetic transaminase have been measured. 8. A model of the possible regulation of the metabolic flow at the oxaloacetate crossroads in gluconeogenesis in rat liver is proposed.

Gravity and radiation are undoubtedly the two major environmental factors altered in space. Gravity is a weak force, which creates a permanent potential field acting on the mass of biological systems and their cellular components, strongly reduced in space flights. Developmental systems, particularly at very early stages, provide the larger cellular compartments known, where the effects of alterations in the size of the gravity vector on living organisms can be more effectively tested. The insects, one of the more highly evolved classes of animals in which early development occurs in a syncytial embryo, are systems particularly well suited to test these effects and the specific developmental mechanisms affected. Furthermore, they share some basic features such as small size, short life cycles, relatively high radio-resistance, etc. and show a diversity of developmental strategies and tempos advantageous in experiments of this type in space. Drosophila melanogaster , the current biological paradigm to study development, with so much genetic and evolutionary background available, is clearly the reference organism for these studies. The current evidence on the effects of the physical parameters altered in space flights on insect development indicate a surprising correlation between effects seen on the fast developing and relatively small Drosophila embryo and the more slowly developing and large Carausius morosus system. In relation to the issue of the importance of developmental and environmental constraints in biological evolution, still the missing link in current evolutionary thinking, insects and space facilities for long-term experiments could provide useful experimental settings where to critically assess how development and evolution may be interconnected. Finally, it has to be pointed out that since there are experimental data indicating a possible synergism between microgravity and space radiation, possible effects of space radiation should be taken into account in the planning and evaluation of experiments designed to test the potential role of microgravity on biological development and evolution.