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BackgroundThe oral cavity is one of the most complex human body environments. Indeed, the continuous variation of this habitat conditions reflects the high dynamism of the resident microbial community. Two key actors in the oral diseases are the bacteria Streptococcus mutans and Fusobacterium nucleatum, both implicated in the formation of oral biofilms and consequently in the generation of common pathologies such as caries and various gingival and soft tissue inflammation diseases. In addition, F. nucleatum is also implicated in the halitosis phenomenon, thanks to its demonstrated ability to produce as second metabolite the hydrogen sulphide (H2S), one of the volatile sulphur compounds (VSCs) that, with methyl mercaptan (CH3SH) and the dimethyl sulphide (CH3SCH3)24, is produced by periodontopathic anaerobic bacteria and causes the awkward bad breath in halitosis patients.MethodsIn this study, the oral preparation Vea® Oris constituted only by vitamin E and capric/caprylic acid was evaluated as a potential treatment of caries and periodontal diseases; the effect of the product at different concentrations on the growth and the ability of both strains to form biofilm was investigated. Regarding to F. nucleatum also the influence of Vea® Oris on the production of H2S was evaluated. ResultsOur in vitro results suggested that the Vea® Oris treatment could considerably reduce the growth and biofilm formation of both S. mutans and F. nucleatum. For F. nucleatum an appreciable reduction of the H2S production can be also obtained. ConclusionsOverall, this study highlighted the potential of Vea® Oris as a more “natural” adjuvant to prevent the biofilm and plaque formation and to reduce the smelly odour of halitosis.

We present a survey of the introduction and evolution of microscopy techniques in Spain, and the concepts and lines of research developed around this instrument, particularly in the field of Biomedical research. We cover in our article the long period from the XVII Century to the arrival of the great figure of Santiago Ramon y Cajal (1853-1934). We particularly want to mention many of the previously neglected pioneers who certainly paved the route for his discoveries and, we believe that without them, he would never have arrived to his important position in the annals of Biology and Medicine. The historical, scientific and social framework of that period also helped the approach to important biological concepts such as the cell and tissue, which are previous and essential ideas for a correct understanding of Development.

5 páginas -- PAGS nros. 293-297, Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions

Neurobiological experiments on 4 animal species (Xenopus laevis, Pleurodeles waltl, Drosophila melanogaster, Acheta domesticus) were performed to study effects of microgravity on development and aging of neuronal, sensory and motor systems. Animal models were selected according to their suitability to answer questions concerning μg-effects on neuroanatomy, neuronal activity, and behaviour. The studies were performed on the Soyuz Taxi flights Andromede, Cervantes, Eneide and LDM-TMA8/TMA7. Observations from these flights include: (1) In tadpoles and cricket larvae, morphological features of sensory cells and neurons are rarely affected by microgravity. (2) In crickets, in-flight fertilization was successful; after landing, flight larvae hatched earlier than ground reared siblings. (3) In crickets, proliferation of peptidergic neurons and their projection patterns within the nervous system were not affected by microgravity. (4) During aging, the impact of microgravity on peptidergic neurons of male Drosophila was limited to the size of cell body. (5) In Xenopus, neurophysiological features of the spinal motor system during fictive swimming were partially modified. (6) In Xenopus tadpoles, the vestibuloocular reflex was affected in an age-related manner. Modifications were also related to the occurrence of a tail lordosis induced by microgravity. It is concluded that adaptation to microgravity during development and aging is mainly based on physiological mechanisms within the central nervous system while structural modifications of the sensory and neuronal system contribute less.

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms

In Drosophila melanogaster two high molecular weight tropomyosin isoforms, historically named heavy troponins (TnH-33 and TnH-34), are encoded by the Tm1 tropomyosin gene. They are specifically expressed in the indirect flight muscles (IFM). Their N-termini are conventional and complete tropomyosin sequences, but their C-termini consist of different IFM-specific domains that are rich in proline, alanine, glycine and glutamate. The evidence indicates that in Diptera these IFM-specific isoforms are conserved and are not troponins, but heavy tropomyosins (TmH). We report here that they are post-translationally modified by several phosphorylations in their C-termini in mature flies, but not in recently emerged flies that are incapable of flight. From stoichiometric measurements of thin filament proteins and interactions of the TmH isoforms with the standard Drosophila IFM tropomyosin isoform (protein 129), we propose that the TmH N-termini are integrated into the thin filament structural unit as tropomyosin dimers. The phosphorylated C-termini remain unlocated and may be important in IFM stretch-activation. Comparison of the Tm1 and Tm2 gene sequences shows a complete conservation of gene organisation in other Drosophilidae, such as Drosophila pseudoobscura, while in Anopheles gambiae only one exon encodes a single C-terminal domain, though overall gene organization is maintained. Interestingly, in Apis mellifera (hymenopteran), while most of the Tm1 and Tm2 gene features are conserved, the gene lacks any C-terminal exons. Instead these sequences are found at the 3' end of the troponin I gene. In this insect order, as in Lethocerus (hemipteran), the original designation of troponin H (TnH) should be retained. We discuss whether the insertion of the IFM-specific pro-ala-gly-glu-rich domain into the tropomyosin or troponin I genes in different insect orders may be related to proposals that the IFM stretch activation mechanism has evolved independently several times in higher insects., This research was supported by grants from the Spanish Government PB96-0069, ESP1999-0379-C02, ESP-2001-4521-PE, ESP2002-11913-E and ESP2003-09475-C02-01.

The proteolytic activites of Artemia salina immediately after hatching are found masked in a precursor of high molecular weight (∼ 100000). The molecular weight of this precursor decreases slightly as development proceeds. However, its kinetic and chromatographic properties vary greatly. Shortly after hatching, the activities are highly inhibited, can be activated severalfold by the chaotropic agent potassium iodide and are not proportional to the amount of enzyme-containing extract added. Later after hatching, its kinetics become normal. From these observations we have concluded that (a) the majority, if not all, of the proteinase activities found later in development are already present immediately after hatching, although in an inhibited state that is gradually activated; (b) the proteinases are not found free in the cytoplasm but in a complex which may allow regulation of their activity., This work has been supported by grants of the J. March Foundation and Fondo Nacional para la Investigación Científica R. G. is a fellow of the Consejo Superior de Investigaciones Científicas.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at −18 or +4°C.

To define the transcriptional mechanisms contributing to stage- and tissue-specific expression of muscle genes, we performed transgenic analysis of Drosophila paramyosin gene regulation. This gene has two promoters, one for paramyosin and one for miniparamyosin, which are active in partially overlapping domains. Regions between -0.9 and -1.7 kilobases upstream of each initiation site contribute to the temporal and spatial expression patterns. By comparing the Drosophila melanogaster and Drosophila virilis promoters, conserved binding sites were found for known myogenic factors, including one MEF2 site and three E boxes. In contrast with previous data, our experiments with the paramyosin promoter indicate that the MEF2 site is essential but not sufficient for proper paramyosin gene transcription. Mutations in the three E boxes, on the other hand, do not produce any effect in embryonic/larval muscles. Thus MEF2 site- and E box-binding proteins can play different roles in the regulation of different muscle-specific genes. For the miniparamyosin promoters, several conserved sequences were shown to correspond to functionally important regions. Our data further show that the two promoters work independently. Even when both promoters are active in the same muscle fiber, the transcription driven by one of the promoters is not affected by transcription driven by the other.

The complete genomic organization of the Drosophila troponin T (TnT) gene shows many interesting features, including the presence of a microexon of only 3 nucleotides conserved among Drosophilidae. It is the smallestbona fide exon so far described, placing a new lower limit on the nucleotide number required for correct splicing. Four muscle-type specific transcripts are generated by developmentally regulated alternative splicing. Exons 3, 4, and 5 are absent in the transcript present in jump and flight muscles. A total of 11 exons are present in the adult hypodermic muscles transcript, whereas the microexon is absent in the larval hypodermic musculature. The two isoforms differ in a lysine residue. Post-translational regulation of the flight muscles/tergal depressor of the trochanter-specific isoform is involved in flight and/or jump function. The interaction domains of TnT in the tropomyosin-troponin complex are strongly conserved in the known vertebrate and invertebrate TnT sequences, whereas the terminal regions show an important variability. The COOH-terminal region shows important phylogenetic variations, whereas the NH2-terminal domain is associated with specific muscle types in a particular organism, a finding that discloses a selective value for these domains in the functionality of distinct muscles in different organisms.

Two sets of muscle polypeptides showing calcium-binding capacity and intense labelling in vivo with 32P were purified and characterized from Drosophila melanogaster adult extracts. The polypeptides exhibit crossed immunoreactivity and share similar biochemical properties such as those involved in purification. They have been identified as isoforms of troponin-T (TnT) by sequence analysis of a cDNA clone isolated from an embryonic library. The two sets of TnT polypeptides correspond to the fibrillar and non-fibrillar muscle isoforms, respectively. The non-fibrillar muscle isoforms separate into two bands which are differentially expressed during development. Analysis of TnT isoforms in bee thoraces indicates that the expression of the fibrillar muscle isoform correlates with the acquisition of functional flight capability. In vivo labelling experiments reveal that the two TnT sets are readily phosphorylated. The Drosophila TnTs show calcium-binding properties by three different types of assays. Our results suggest that this property could be specific to insect TnTs and may be related to the long, extremely acidic polyglutamic carboxy-terminus present in these polypeptides, which does not occur in non-arthropod TnTs.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

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.

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.

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.

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.

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).

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.

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.

Several molecules of a protein specified by gene 3 of M13 comprise a minor fraction of the phage coat and have been assigned a role in adsorption to the bacterial cell. We find that the gene-3 protein molecules of the virion are fully conserved in phage that have attached irreversibly to the host cell, and they form a complex with the phage DNA when it has been converted to a duplex replicative form. In cells infected at a restrictive temperature with a thermosensitive mutant in gene 3, there is no conversion of the phage DNA to the replicative form. Both the adsorbed phage and the complex of replicative form DNA with the gene-3 protein were isolated with the inner membrane fraction of the cell. We suggest that the gene-3 protein may perform an essential function in the synthesis of replicative form by linking the phage DNA to a cellular replicative system in or on the inner cell membrane.

Certain kinetic properties of the pyruvate kinase activity in different tissues of the rat have been studied in fresh extracts with particular attention to their possible value for the regulation of gluconeogenesis and glycolysis. The pyruvate kinase activity in fresh extracts of the gluconeogenic tissues liver and kidney cortex has allosteric properties very marked in near physiological conditions, as follows: (a) co-operativity in the kinetics with respect to the concentration of phosphoenolpyruvate; (b) strong allosteric inhibition by alanine and ATP, each of which raises the [S]0.5 value and increases the sigmodicity respect to the concentration of phosphoenolpyruvate; and (c) very strong activation by fructose diphosphate, which greatly lowers the [S]0.5 value and shifts the kinetics from markedly sigmoid to hyperbolic, and can fully counteract the inhibitory effects of alanine and ATP. Keeping the extracts of liver and kidney at low temperature (0–2°) leads to desensitization of the pyruvate kinase to the homotropic cooperativity of the phosphoenolpyruvate substrate and to the allosteric inhibition by alanine and ATP and the activation by fructose diphosphate. This cold desensitization is reversible. These facts and their time dependence make it possible to understand previous difficuties to obtain reproducible allosteric effects with the pyruvate kinase of rat liver. The pyruvate kinase of other tissues examined, including heart and adipose tissue, did not exhibit any of the allosteric properties above described for the enzyme of the gluconeogenic tissues. The μmolar range of fructose diphosphate concentrations required for activation of liver pyruvate kinase in the presence of the two physiological inhibitors and within the physiological range of concentrations of phosphoenolpyruvate fits well in order of magnitude with the calculated range of concentrations of free fructose diphosphate prevailing in liver. These results strongly support the hypothesis that an isoenzyme of pyruvate kinase present in liver and kidney has evolved with built-in specific regulatory mechanisms in order to prevent the diversion of phosphoenolpyruvate from its way to glucose in glucoenogenic situations. An efficient regulation in the reversible switch over from glycolysis to gluconeogenesis seems to be feasible by the interplay of two feedback inhibitors, alanine and ATP, and a forward activator, fructose diphosphate.

Biological development, i.e., the process by which a highly complicated multi-tissular organism develops from an aparently uniform homogeneous cell, the fertilized oocyte, remains as mysterious today as the first time that it was observed by the curious eye of a man. Even though, especially in recent times by the joint effort of the genetical, cell biological and molecular approaches (Sambroek, 1985, Wilkins, 1986, Davidson, 1987), our knowledge of the details of embryonic development has been greatly extended, we still lack a true understanding of how this process occurs. In fact a fascinating story of complex tempo-spatial patterns of gene activity is currently being described (for instance, Lawrence et al, 1987), but its mechanistically linkage to the phenoytpe produced, the normal larva and adult, remains basically untackled.

A partial purification scheme applied to the Triton X-100 insoluble pellet of adult flies homogenates yielded a fraction which upon polymerization reproducibly produces filaments which in the electron microscope have all the typical properties of intermediate filaments. This is the first report of the presence of protein components with such properties in Drosophila. Thus, it is highly possible that insects and arthropodes, like other lower invertebrates, may contain the third extremely insoluble element of the cellular cytoskeleton.

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.

Interaction between the filamentous phage M13 and the Escherichia coli host progresses through stages of binding, eclipse, and penetration, defined as follows: binding of phage to cell receptor may be reversible or irreversible, eclipse of phage makes it noninfective and susceptible to DNase, and penetration entails uptake of phage components in a manner tightly coupled to replication. Binding (or adsorption) was specific for F+ cells, was relatively slow, did not follow simple bimolecular kinetics, and attained a value of about one phage per cell. It was blocked by low temperature, cyanide, or starvation and was unaffected by rifampicin. Binding capacity of phages was removed by subtilisin treatment which also released the gene 3 (adsorption) protein from the viral particle. Rifampicin which blocks replication prevents penetration of an irreversibly attached and altered (“eclipsed”) phage. Thus, decapsidation and penetration of the phage are coupled to replication of the viral DNA. Binding or eclipse triggered an alteration of the gene 3 protein to a lower molecular-weight form. The coat proteins (gene 3 and gene 8) were taken up by the cells to the same extent as the DNA. Cells disrupted by lysozyme-detergent treatment, as judged by release of β-galactosidase, retained their capacity to bind and eclipse the phage particle; binding to disrupted cells remained cyanide sensitive. After this treatment F− cells were as active in the binding of M13 as F+ cells. Such preparations from both F+ and F− cells had the capacity of replicating the DNA in intact phage particles to the duplex replicative form. In a discussion of these findings, current models for the key role of the F pilus (of F+ cells) in phage adsorption are discussed and a larger role for the phage adsorption protein is proposed.

To extend to the crustacean class the information concerning the genomic organization of the mitochondrial DNA (mtDNA) a double strategy has been used: Southern blot analysis with cloned Drosophila mtDNA probes and sequence comparison to the Drosophila mtDNA of the sequenced termini of different subclones along the Artemia mitochondrial genome, probably the smallest mtDNA studied at this level to date. These approaches have allowed us to localize the 16S rRNA gene, two tRNA genes and eleven protein genes. The genome organization is surprisingly similar to the Drosophila mtDNA, with the 16S rRNA and the protein genes located in the same positions and orientations as their Drosophila counterparts. The only changes detected are at the level of tRNA genes, although the position and orientation of some of these are also conserved. These results contrast with the important rearrangements detected among other invertebrates mtDNAs and suggest that the genome organization of the mitochondrial DNA may be more conserved in the arthropods than in other invertebrate phyla.

The yolk granules of Artemia behave as unstable structures during isolation, especially after hatching. A stringent dechorionization of the cysts is required for an easy homogenization and good extraction of the yolk granules. The use of Ficoll and the avoidance of high dilutions in the homogenization process allows the isolation in an intact state of a pure yolk granules fraction without nuclear contamination. Previously described methods for the isolation of yolk granules lead to low recoveries of these structures in preparations with nuclear contamination. Lipovitellin, the major yolk protein, can be used as a biochemical marker to monitor the recovery of yolk granules and the possible yolk contamination of other subcellular fractions. The isolation of nuclei is also improved, their double membrane preserved in an enriched fraction with less yolk contamination than usually obtained with previously described methods. The other major storage structure, the lipoid bodies, can be isolated intact in these conditions, whereas they are also disrupted in common homogenization media., This work was supported by grants of the Rodriguez Pascual Foundation and the Fondo Nacional para la Investigación Científica. R.P. has a fellowship from Caja de Ahorros y M.P. de Madrid. R.G. was a fellow of the Consejo Superior de Investigaciones Científicas.

A systematic examination of the properties of the clot assay, a highly sensitive and simple method to detect DNase activities, is presented. It is shown that it is both quantitative and specific for monitoring the double-strand cleavage of DNA produced by specific and nonspecific endonucleases. Its simplicity and ease of quantification, which exceeds that of other methods of similar sensitivity, indicates that the clot assay can be advantageous in monitoring the activity of both types of endonucleases, especially if carried out in parallel with other more informative assays on the mechanism of action of the enzymes. As an example of its use, it is shown that pancreatic DNase I is capable, in the absence of external metal addition, of a limited attack on DNA containing bound divalent cations.

Previous work by members of our group [1,2,3] suggested that in Artemia early development a considerable proportion of the mitochondria were stored in an inactive state in the yolk granules from which they were released during the developmental process, becoming active as power generators in the nauplii. In order to test conclusively this idea, we turned to the mitochondrial DNA as we thought that it could provide the best marker for the presence and quantitation of these organelles, independent of their functional status. Very little was known about this interesting informational molecule in Artemia [4], but in other systems as distant as sea urchin, amphibians and mammals [5,6,7] it is known that it is basically synthesized during oogenesis, the mature oocyte containing all the mitochondria necessary for early embryogenesis.