Biomedical subjects

R Garesse

Publications and source records attributed to R Garesse.

At least 37 records · Page 2Linked to original sources

PubMed1998

[Human mitochondrial genetic system].

The mitochondria are subcellular organelles devoted to energy production in form of ATP that contain their own genetic system. Mitochondrial DNA codify a small, but extremely important, number of polypeptides of the respiratory chain. The other mitochondrial proteins are encoded in the nucleus. Therefore, mitochondrial biogenesis require the coordinated expression of nuclear and mitochondrial genetic systems. The gene arrangement in mitochondrial DNA is extremely compact with the tRNA genes interspersed with the rRNA and protein-coding genes. This organization has its precise counterpart in the mode of expression and distinctive structural features of the RNAs. Both mitochondrial DNA strands are transcribed as a whole in the form of three polycistronic molecules that are later cut by specific enzymes that recognize the 5' and 3' end of the tRNA sequences, to produced the mature rRNA, mRNA and tRNA. The mitochondrial coded mRNAs are translated into proteins by a mitochondrial specific protein-synthesizing machinery. The genetics of the mitochondrial DNA differs from that of the nuclear DNA in several features. In particular, the mitochondrial genome is inherited from the mother that transmit their mitochondrial DNA to all her offsprings. Another characteristic of this genome is its tendency to mutate more frequently than the nuclear DNA. This provides a powerful tool for studying the evolution of man.

DNA, Mitochondrial↗

PubMed1998

[Studies of pathogenicity and characterization of molecular phenotype caused by mutations in human mitochondrial DNA].

Mitochondrial DNA evolves and accumulates mutations more rapidly than nuclear DNA. These nucleotide variation may produce neutral polymorphisms or affect to functional conserved positions being very deleterious. On the other hand the relation between the type of mutation (genotype) and the observed clinical symptoms (phenotype) is nowadays practically unknown. Therefore it is very important to demonstrate clearly that the new described mutations are pathogenic and understanding the molecular mechanisms responsible for the energetic metabolism dysfunction produced by these mutations at cellular level. In the last years several procedures have been developed, including in situ hybridization, single-fiber PCR and the use of patient myoblast, fibroblast and lymphoblast cell culture lines. Specially relevant is the cybrid technology that allow repopulate a cell line depleted of mtDNA with mitochondria obtained from patient fibroblasts, producing transmitochondrial cell lines. The use of these methodology in the last few years has been very important to understand the pathogenic mechanism of some of the classical mutations associated to mitochondrial pathology.

Bone Neoplasms↗

PubMed1997

Artemia mitochondrial genome: molecular biology and evolutive considerations.

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.

Animals↗

PubMed1996

Preservation of viable biological samples for experiments in space laboratories.

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.

Animals↗

PubMed1996

Microgravity effects on Drosophila melanogaster behavior and aging. Implications of the IML-2 experiment.

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

Aging↗

PubMed1996

Mitochondrial differentiation during the early development of the brine shrimp Artemia franciscana.

During the early development of Artemia there is an increase in mitochondrial enzyme activities of about one order of magnitude, whereas the activities of two cytoplasmic enzymes tested as controls remain unaltered. The mitochondrial enzyme activation correlates with (i) large changes in mitochondrial morphology, (ii) a 5-fold increase in the amount of the H+-ATP synthase beta-subunit and (iii) a dramatic increase in the steady-state level of mitochondrial mRNAs, whereas mitochondrial rRNA concentrations remain mostly unchanged. In contrast, the level of mitochondrial DNA does not change significantly during the first 20 h after resumption of development. After hatching, the mitochondrial DNA content increases twice in parallel with one round of cellular division, thus indicating that mitochondrial and nuclear replication are coupled in Artemia postgastrular development. The data presented strongly suggest that mitochondrial maturation in the absence of significant mitochondrial proliferation is responsible for the dramatic increase in mitochondrial function that takes place after resumption of development in Artemia.

Adenosine Triphosphatases↗

PubMed1995

Analysis of the mitochondrial ATP synthase beta-subunit gene in Drosophilidae: structure, transcriptional regulatory features and developmental pattern of expression in Drosophila melanogaster.

We have cloned and determined the structure of the gene encoding the H(+)-ATP synthase beta subunit in two distantly related Drosophila species, D. melanogaster and D. virilis. The gene contains three exons that are extremely well conserved at the amino acid level, not only in the region encoding the mature protein but also in that encoding the leader peptide. Primer extension analysis indicates that the 5' untranslated region is extremely short, and reveals the presence of multiple initiation sites of transcription in both Drosophila species. The promoters of D. melanogaster and D. virilis H(+)-ATP synthase beta-subunit genes contain a conserved region surrounding the initiation transcription sites. Nucleotide sequence analysis has revealed the absence of canonical TATA and CCAAT boxes and the presence of several putative regulatory elements in both promoter regions, including GAGA, GATA and Ets binding sites. We have analysed the pattern of gene expression during D. melanogaster development. The mRNA is stored in oocytes, and activation of transcription takes place after 10 h of development. The expression of the nuclear-encoded H(+)-ATP synthase beta subunit is strictly coordinated with the expression of subunits 6 and 8 of the same complex that are encoded in the mitochondrial genome.

Amino Acid Sequence↗

PubMed1995

Thyroid hormone-regulated brain mitochondrial genes revealed by differential cDNA cloning.

Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain.

Animals↗

PubMed1994

A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.

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.

Animals↗

PubMed1994

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

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.

Animals↗

PubMed1994

Speciation in the Artemia genus: mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps.

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<==>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.

Amino Acid Sequence↗

PubMed1994

Arthropod model systems for studying complex biological processes in the space environment.

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 exobiology 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.

Aging↗

PubMed1993

The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies.

The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced. The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins. We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts. This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla.

Amino Acid Sequence↗

PubMed1993

Mitochondrial DNA remains intact during Drosophila aging, but the levels of mitochondrial transcripts are significantly reduced.

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.

Aging↗

PubMed1993

Genomic organization and developmental pattern of expression of the engrailed gene from the brine shrimp Artemia.

We report the isolation and characterization of an engrailed gene in the crustacean Artemia franciscana. The Artemia gene spans a genomic region of 15 kilobases and the coding sequence is interrupted by two introns. It appears to be the only gene of the engrailed family present in the Artemia genome. The predicted engrailed-like protein is 349 amino acids long and contains several domains including the homeodomain, well conserved when compared to other proteins of the engrailed family. Based on sequence comparisons we have detected, in the Artemia engrailed protein, several features which are in common with the Drosophila and Bombyx engrailed proteins. It also has some features specific for invected proteins. Therefore, this gene appears to have diverged from an ancestral gene common to both the engrailed and invected insect genes. Whole-mount in situ hybridization experiments show that the expression of this gene in postembryonic development of Artemia is restricted to the posterior part of at least the thoracic and maxillary segments. The pattern is generated sequentially from a growth zone organized in columns of cells close to the caudal region of the larvae. Cell proliferation in the growth zone follows an interspersed pattern without evidence of early lineage restrictions. The engrailed expression is detected in the growth zone before any segmentation is visible and continues to be expressed in a posterior location in the segments that are morphologically defined. Initially expressed in isolated cells, it spreads into rows broadening to two-three cells as segments mature. The evidence presented here is compatible with the hypothesis that intercellular signaling mechanisms are in part responsible of the early activation of selector genes.

Amino Acid Sequence↗

PubMed1992

Drosophila melanogaster paramyosin: developmental pattern, mapping and properties deduced from its complete coding sequence.

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.

Amino Acid Sequence↗

PubMed1992

Microgravity effects on Drosophila melanogaster development and aging: comparative analysis of the results of the Fly experiment in the Biokosmos 9 biosatellite flight.

The results are presented of the exposure of Drosophila melanogaster to microgravity conditions during a 15-day biosatellite flight, Biokosmos 9, in a joint ESA-URSS project. The experimental containers were loaded before launch with a set of Drosophila melanogaster Oregon R larvae so that imagoes were due to emerge half-way through the flight. A large number of normally developed larvae were recovered from the space-flown containers. These larvae were able to develop into normal adults confirming earlier results that Drosophila melanogaster of a wild-type constitution can develop normally in the absence of gravity. However, microgravity exposure clearly enhances the number of growing embryos laid by the flies and possibly slows down the developmental pace of the microgravity-exposed animals. Due to some problems in the experimental set-up, this slowing down needs to be verified in future experiments. No live adult that had been exposed to microgravity was recovered from the experiment, so that no life span studies could be carried out, but adult males emerged from the recovered embyros showed a slight shortening in life span and a lower performance in other experimental tests of aging. This agrees with the results of previous experiments performed by our groups.

Aging↗

PubMed1988

Genome organization of Artemia mitochondrial DNA.

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.

Amino Acid Sequence↗