Biomedical subjects

D Ugarković

Publications and source records attributed to D Ugarković.

At least 19 recordsLinked to original sources

PubMed2006

Wide distribution of related satellite DNA families within the genus Pimelia (Tenebrionidae).

Major satellites of species in the genus Pimelia comprise large portions of their genomes and belong to seven major satellite families which all originate from a common ancestral sequence. Here we present the results of comprehensive screening of 26 Pimelia species belonging to three distinct geographic groups (Ibero-Balearic, African and Canary Islands) for the presence of different Pimelia satellite families in their genomes. Dot-blot hybridization experiments suggest that together with one dominant, highly abundant satellite family, other families are also present in genomes of the majority of examined Pimelia species, but as low-copy number repeats. The estimated abundance of these underrepresented repeats is about 4,000 copies per haploid genome. Signals of highly abundant satellite family from P. scabrosa (PSCA) in examined congeneric species, obtained after PCR amplification and Southern hybridization under high stringency conditions, corroborate sequence preservation of low-copy representatives of satellite families. PRINS localized low-copy repeats within the pericentromeric regions of all chromosomes. These results point to the existence of an extensive library of repetitive DNAs that was already present in the genome of the common ancestor of extant Pimelia taxa, and shifts the period of diversification of Pimelia satellites far in the history of this genus.

Animals↗

PubMed2000

A novel interspersed type of organization of satellite DNAs in Tribolium madens heterochromatin.

Analysis of arrangement of satellite DNA sequences in Tribolium madens (Insecta, Coleoptera) by Southern analysis of pulsed-field blots and two colour FISH on extended chromosomes and DNA fibres revealed a novel type of heterochromatin organization. Two satellite DNAs, distributed over the whole pericentromeric heterochromatin of all chromosomes form clusters, ranging in size from 150 kb up to several Mb. Within the clusters, both satellites are in the form of highly interspersed, short homogeneous arrays which vary in size with a lowest length limit of only few kb. The longest arrays composed of a single satellite are relatively short, up to 70 kb for satellite I, and up to 45 kb for satellite II. Only a small fraction of about 15% of satellite II is organized in long tandem repeats, while the rest is in the form of only a few repeats intermingled with satellite I. The results indicate that large clusters composed of interspersed arrays of both satellites represent a major component of T. madens heterochromatin, which is mostly devoid of long regions of other sequences. The same organizational pattern probably also includes a region of the functional centromere. We propose that such an organizational pattern of DNA sequences in heterochromatin might be common in genomes characterized by a high rate of interchromosomal exchange. This pattern of organization is different from that in other animal as well as plant species analysed up to now, in which every satellite in heterochromatin is organized in a small number of large separate domains.

Animals↗

PubMed2000

Comparative study of satellite sequences and phylogeny of five species from the genus Palorus (Insecta, Coleoptera).

Major satellite sequences are analysed in the three tenebrionid beetles Palorus cerylonoides, P. genalis, and P. ficicola, and compared with the ones from P. ratzeburgii and P. subdepressus reported elsewhere. All of them are A+T rich, pericentromerically located, and with lengths of about 150 bp, either in the form of monomers or formed by more complex repeating units. A preliminary phylogenetic analysis of Palorus species using the 3' end of the mitochondrial Cytochrome Oxidase I gene shows that the five Palorus species have been diverging for a considerable amount of evolutionary time, with the pair P. ratzeburgii and P. genalis being the most closely related. Only these two taxa showed some similarity between their respective high-copy-number satellite sequences, while other satellites are mutually unrelated and might have originated independently. However, all the satellites have in common tertiary structure induced by intrinsic DNA curvature, a characteristic which is conserved within the genus. Palorus major satellites were previously detected in the genomes of congeneric species as low-copy-number clusters (Mestrović et al., Mol. Biol. Evol. 15: 1062-1068. 1998). Given the divergences between the analysed species, the substitution rate deduced from high- and low-copy-number repeats is unexpectedly low. The presence of sequence-induced DNA curvature in all Palorus satellites and similar satellite DNAs in the species pair P. ratzeburgii and P. genalis suggest (i) that constraints are at the tertiary structure; and (ii) that the satellite DNA evolutionary turnover can be dependent on the history of the taxa under study, resulting in retention of similar satellites in related taxa.

Animals↗

PubMed1998

Evolution of satellite DNAs from the genus Palorus--experimental evidence for the "library" hypothesis.

Satellite DNA profiles have been characterized in the congeneric species Palorus ratzeburgii, Palorus subdepressus, Palorus genalis, and Palorus ficicola (Coleoptera, Insecta), each of which contains a single, A + T-rich satellite DNA comprising a considerable portion of the genome (20%-40%). These satellites exhibit insignificant mutual sequence similarity. Using PCR assay, it has been shown that all four sequences are present in each of the tested Palorus species: one of them is amplified into a high copy number or a major satellite, while the three others are in the form of low-copy-number repeats estimated to make up approximately 0.05% of the genome. Each of the four satellites is interspecifically high conserved concerning the sequence, monomer length, and tandem repeat organization. Major, as well as low-copy-number, satellites are colocalized in the regions of pericentromeric heterochromatin on all chromosomes of the complement. The low-copy-number satellites are dispersed between the large arrays of the major satellite over the whole heterochromatic block. Our results explain satellite DNA evolution, confirming the hypothesis that related species share a "library" of conserved satellite sequences, some of which could be amplified into a major satellite. Due to the evolutionary dynamics of satellite DNAs, the content of the "library" is variable; the elimination of some sequences parallels the creation of the new ones. Quantitative changes in satellite DNAs, induced by occasional amplification of satellite repeat from the "library", could possibly occur in the course of the speciation process, thus forming a species-specific profile of satellite DNAs.

Animals↗

PubMed1997

Conservation of satellite DNA in species of the genus Pimelia (Tenebrionidae, Coleoptera).

Satellite DNA has been characterized in six allopatric species from the genus Pimelia: P. interjecta, P. integra, P. variolosa and P. baetica, inhabiting Iberian Peninsula, and P. elevata and P. criba, endemic to Balearic Islands Ibiza and Mallorca, respectively. All species show the presence of a single satellite DNA of a basic monomer length of 357 bp and A+T content of 69%, comprising a considerable amount of the genome (39%-45%, corresponding to about 4.5 x 10(5) copies per haploid genome). The sequence analysis of 22 cloned repeats reveals very high intra- and interspecific sequence similarity. Phylogenetic analysis separates the satellite sequences into two clusters, each comprising clones from three species exclusively. Within the clusters, satellite clones are not grouped species-specifically, except those of P. integra where species-diagnostic nt substitutions are detected with a pattern that could be produced by gene conversion. Such high sequence conservation could be related to preservation of satellite DNA curvature, resulting in a higher order helical structure, proposed to act as a specific protein binding domain.

Animals↗

PubMed1996

Identification of serotonin transporter mRNA in rat platelets.

Total RNA isolated from rat platelets by guanidinium-acid-phenol extraction, and mRNA for the serotonin (5-hydroxytryptamine) transporter (5HTt) was identified. From a typical starting sample of 20 mL of rat blood (approximately 9 x 10(9) platelets), 14 to 17 micrograms of total platelet RNA was obtained. Northern blot analysis, using 32P-labeled 5HTt cDNA as a probe, identified approximately 3.3 kb long 5HTt mRNA. After rehybridization with cyclophilin cDNA, approximately 1 kb long mRNA for cyclophilin, which could serve as a reference for 5HTt mRNA quantification, was also identified. Densitometric analysis demonstrated clearly measurable signals for both mRNAs. The possibility of quantification of rat platelet 5HTt mRNA should enable parallel studies on 5HTt gene expression in platelets and brain of the same animal, and the evaluation of the platelet model at the molecular genetic level.

Animals↗

PubMed1996

Satellite DNA of the red flour beetle Tribolium castaneum--comparative study of satellites from the genus Tribolium.

A highly abundant satellite DNA comprising 17% of the Tribolium castaneum (Insecta, Coleoptera) genome was cloned and sequenced. The satellite monomer is 360 bp long, has a high A+T content of 73%, and lacks significant internal substructures. The sequence variability is 3.6%, essentially due to random distribution of single-point mutations. The satellite is evenly distributed in the regions of centromeric heterochromatin of all 20 chromosomes, as shown by fluorescent in situ hybridization. Comparison of T. castaneum satellite with those from three different but congeneric species reveals the highest sequence similarity of 47.1% with the satellite from the sibling species Tribolium freemani. The phylogenetic relationships among Tribolium species deduced from satellite sequence agree with those based on karyological, chemotaxonomic, and hybridization data. This indicates a parallel in the divergence of satellites and some genetic and cytogenetic characters. Despite low mutual sequence similarity, which makes them species-specific, Tribolium satellites have a common structural characteristic: a block of about 95% A+T content, 20 to 42 bp long, flanked at one side by an inverted repeat which can potentially form a thermodynamically stable dyad structure. Since similar structural features are found in centromeric DNA of Saccharomyces cerevisiae and Chironomus pallidivittatus, their possible importance in centromere function may be inferred.

Animals↗

PubMed1996

Evolution of Tribolium madens (Insecta, Coleoptera) satellite DNA through DNA inversion and insertion.

Two different satellite DNAs from tenebrionid species Tribolium madens (Insecta, Coleoptera) have been detected, cloned, and sequenced. Satellite I comprises 30% of the genome; it has a monomer size of 225 bp and a high A + T content of 74%. Satellite II, with a monomer size of 711 bp and A + T content of 70%, is less abundant, making 4% of the total DNA. Sequence variability of the monomers relative to consensus sequence is 4.1% and 1.2% for satellite I and II, respectively. Both satellites are localized in the heterochromatic regions of all chromosomes. A search for internal motifs showed that both satellites contain a related subsequences, about 100 bp long. The creation of satellite I monomer is explained by duplication of the basic subunit, followed by subsequent divergence by single point mutations, deletions, and gene conversion. Inversion of the subsequence in addition to its duplication has occurred in satellite II. The result of this inversion is possible formation of a long, stable dyad structure. The 408-bp sequence, inserted within satellite II monomer, shares no similarity with a basic subunit. Frequent direct repeats found within the inserted sequence point to its evolution by duplication of shorter motifs. It is proposed that both satellites have been derived from a common ancestral sequence whose duplication played a major role in the formation of satellite I monomer, while insertion of a new sequence together with inversion of an ancestral one induced the occurrence of satellite II.

Animals↗

PubMed1995

Uniform distribution of satellite DNA variants on the chromosomes of tenebrionid species Alphitobius diaperinus and Tenebrio molitor.

The chromosomes of tenebrionid species Alphitobius diaperinus contain large blocks of pericentromerically located constitutive heterochromatin, as revealed by C-banding procedure. As previously reported, satellite DNA of this species is composed of two related monomeric units organized in three satellite subfamilies. In order to analyze the chromosomal location of the satellite DNA and the distribution of monomeric variants within it, and compare it with the distribution of monomer variants in Tenebrio molitor satellite DNA, the methods of in situ hybridization and restriction enzyme/nick translation were performed. Fluorescent in situ hybridization with the entire satellite DNA reveals the pericentromerically located signals on all chromosomes of the complement, coinciding with heterochromatic blocks. Results of fluorescent in situ hybridization with particular monomeric variants and of in situ restriction enzyme/nick translation show that monomeric variants are homogeneously dispersed within the entire satellite DNA. The spreading of satellite monomeric variants of the related species T. molitor within the pericentromeric heterochromatin of the entire complement, is demonstrated using the method of in situ restriction enzyme/nick translation. Although the complexity of organization of satellite DNAs is quite distinct in these two species, obtained results suggest similar efficiency of mechanisms of spreading and homogenization resulting in random chromosomal distribution of their satellite variants.

Animals↗

PubMed1994

Tandemly repeated pentanucleotides in DNA sequences of eucaryotes.

Genetic sequence data banks were scanned in order to retrieve tandemly repeated pentanucleotides (pnts). It was found that among 102 (=(1024-4)/2/5) possible distinct pnts roughly each fourth is involved in tandem repeats. It is shown that tandemly repeated pnts are composed of frequently occurring di- and trinucleotides and that those pnts which occur frequently in the form of mono- or di-pnts form also tandem repeats either in the form of satellites or in the form of shorter tandem repeats. Human satellite III is taken as a specific example. It is shown that the first guanine within GG-AAT pnt exhibits the highest mutability. Sequential distribution of base changes gives evidence that the mutations do not occur at random positions but in a correlated fashion so that long stretches of original pnts remain intact. It is found that pnts related to the satellite III are present in introns and flanking regions of some structural genes, but are not preserved between orthologous genes of related species. The results corroborate the most plausible mechanism of their evolution--rapid amplification followed by successive divergence of repeat units by various mutational processes.

Animals↗

PubMed1994

Characterization of two abundant satellite DNAs from the mealworm Tenebrio obscurus.

Two highly abundant satellite DNAs comprise 36% of the Tenebrio obscurus (Tenebrionidae, Coleoptera) genome. They are designated as satellite I and satellite II with the monomer length of 344 and 142 base pairs (bp), respectively. Both satellites differ in their nucleotide (nt) sequences, but the frequency of point mutations, well-conserved length of monomer variants, stretches of shared mutations characteristic for the process of gene conversion, and distribution of both satellites in regions of centromeric heterochromatin of all chromosomes indicate that the same evolutionary processes act on both of them with the same, or similar, rate. While satellite I shares no sequence similarity with any other known nt sequence, satellite II is 79.7% homologous with the highly abundant satellite from closely related Tenebrio molitor. Difference in the frequency of point mutations and absence of shared mutations indicating gene conversion strongly suggest that in these two closely related species mutational processes affecting satellite DNAs seem to be changed. Retarded electrophoretic mobility, due to sequence-induced curvature of DNA helix axis, was observed for T. obscurus satellite II, but not for satellite I. Although evolutionary processes act with different rates in T. obscurus and T. molitor satellites the monomer length and sequence-induced curvature are well preserved in both 142-bp satellites, as well as in, at the nt sequence level completely divergent, Palorus ratzeburgii (Tenebrionidae) satellite, indicating potential importance of these parameters in their evolution.

Animals↗

PubMed1994

Analysis of divergence of Alphitobius diaperinus satellite DNA--roles of recombination, replication slippage and gene conversion.

Satellite DNA is highly abundant in Alphitobius diaperinus (Tenebrionidae, Coleoptera), comprising 25% of the total genomic DNA. Sequence analysis reveals an average GC content of 50.8% and the presence of three different groups of satellite monomer variants, tH1, tH2 and tH3 with corresponding lengths of 123, 128 and 126 bp. Their mutual homologies range between 65 and 81%. Sequence comparison shows that the monomer variant tH2 has been formed by a recombination process between tH1 and tH3, which have a low average homology of only 65.15%. The longest stretch of 100% homology between the recombining units is 17 bp and is located 3' to the predicted recombination site. There is also an indication from sequence analysis that replication slippage and gene conversion play a part in the formation of satellite units and contribute to their divergence. The tH1, tH2 and tH3 monomer variants are organized in higher order repeating structures: a dimer, composed of tH1 and tH3, and a trimer containing tH1, tH2 and tH3 in series. The dimeric and trimeric repeat units furthermore create three higher order satellite subfamilies. Two of them contain either tandemly arranged dimers or trimers, while the third one is composed of both types of repeats, mutually interspersed.

Animals↗

PubMed1994

Tenebrio obscurus satellite DNA is resistant to cleavage by restriction endonucleases in situ.

Satellite DNA from the mealworm beetle, Tenebrio obscurus, is composed of 344 bp long monomers of high AT content (68%), and represents 15% of the total DNA. In situ hybridization reveals the positions of the satellite on the pericentromeric heterochromatin of all T. obscurus chromosomes. To compare restriction enzyme (RE) effects with those on naked DNA, fixed chromosomes were digested with REs having recognition sites in most of the satellite monomers, and also with enzymes having target sites present only partially, or very rarely in the satellite units. All enzymes produce similar C-like banding patterns showing heterochromatin resistance to digestion regardless of the enzyme used. In situ nick translation suggests the inability of REs to cleave satellite DNA rather than the inefficient extraction of DNA fragments. DNA in heterochromatin was only extensively digested when the chromosomes were preincubated with proteinase K, indicating that accessibility of REs to DNA is increased by the removal of chromosomal proteins. This is in contrast to recently obtained results in Tenebrio molitor, where cleavage of satellite DNA is equally efficient in both fixed chromosomes and in naked DNA. The satellite DNAs of the two congeneric species differ in their AT content, and their primary and higher order structure, which could influence both heterochromatin structure and the accessibility of REs to satellite DNA.

Animals↗

PubMed1993

Satellite DNA and heterochromatin of the flour beetle Tribolium confusum.

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40-45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 x 10(5) per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.

Animals↗

PubMed1992

Evidence for random distribution of sequence variants in Tenebrio molitor satellite DNA.

Tenebrio molitor satellite DNA has been analysed in order to study sequential organization of tandemly repeated monomers, i.e. to see whether different monomer variants are distributed randomly over the whole satellite, or clustered locally. Analysed sequence variants are products of single base substitutions in a consensus satellite sequence, producing additional restriction sites. The ladder of satellite multimers obtained after digestion with restriction enzymes was compared with theoretical calculations and revealed the distribution pattern of particular monomer variants within the satellite. A defined higher order repeating structure, indicating the existence of satellite subfamilies, could not be observed. Our results show that some sequence variants are very abundant, being present in nearly 50% of the monomers, while others are very rare (0-1% of monomers). However, the distribution of either very frequent, or very rare sequence variants in T. molitor satellite DNA is always random. Monomer variants are randomly distributed in the total satellite DNA and thus spread across all chromosomes, indicating a relatively high rate of sequence homogenization among different chromosomes. Such a distribution of monomer variants represents a transient stage in the process of sequence homogenization, indicating the high rate of spreading in comparison with the rate of sequence variant amplification.

Animals↗

PubMed1992

Expression of P-glycoprotein gene in marine sponges. Identification and characterization of the 125 kDa drug-binding glycoprotein.

In the present paper it is shown that the marine sponges Geodia cydonium and Verongia aerophoba contain the gene coding for P-glycoprotein P170, also known as a multidrug-resistance gene. Western blot studies revealed that polyclonal antibodies raised against hamster P170 cross-react with the sponge polypeptide of Mr 125,000. After endoglycosidase F treatment, the sponge P125 is converted to a polypeptide of Mr 105,000. Northern blot studies, using the human P170 cDNA probe, revealed a size of 4.2 kb for the sponge P125 transcript. The level of this transcript does not change in response to incubation with the aggregation factor. Confocal laser scanning microscopy showed that P125 is a cell membrane bound protein. In addition, sponge membrane vesicles possess a potential to bind in vitro 2-acetylamino-fluorene, vincristine and daunomycin. This process is Verapamil-sensitive, a characteristic known also for the mammalian vesicle associated P170. The data reported demonstrate that the classical multidrug resistance mechanism, described in drug-resistant tumor cell lines, functions also in sponges and may explain the relative resistance of these animals to pollution.

2-Acetylaminofluorene↗

PubMed1990

cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges.

Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8). It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process. In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I. The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide. Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids. The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+. On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges.

Amino Acid Sequence↗

PubMed1990

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium.

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.

Amino Acid Sequence↗