Genomic dynamics of a low-copy-number satellite DNA family in Leptestheria dahalacensis (Crustacea, Branchiopoda, Conchostraca)

The LEP150 satellite DNA (satDNA) family found in Leptestheria dahalacensis (Ruppel, 1837) (Conchostraca) is a low-copy-number satellite with a canonical monomer of 150 bp. Nucleotide variation analyses suggest a 14-bp palindromic region as a possible protein binding site with constraints acting on the whole sequence but a 25-bp variable box. Besides the head-to-tail arrangement of 150 bp monomers, multimers analyses evidenced incomplete monomers, one duplication event, and three inversions. Both observed rearrangements and the higher values of sequence variability scored suggest that rearranged monomers reside in regions with a lower degree of homogenisation efficiency. Sixty-seven percent of the breakpoints occurs at kinkable dinucleotides, thus supporting their role in rearrangements as documented in alphoid satDNA recombination events. Monomers of different lengths may result from crossing over between repeats misaligned through the direct and inverted subrepeats of LEP150 monomers. ANOVA results indicate that the same range of sequence diversity is experienced at the individual and population ranks; therefore, the evolution of the L. dahalacensis satDNA is concerted.     

DNA sequence dependence of guanine-O6 alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5 and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5 adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Stability of Tandem Repeats in the Drosophila Melanogaster HSR-Omega Nuclear RNA

The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing >5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is ~280 bp. Sequences of 191/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than ~5 kb nor more than ~16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA.     

Sobo, a recently amplified satellite repeat of potato, and its implications for the origin of tandemly repeated sequences

Highly repetitive satellite DNA sequences are main components of heterochromatin in higher eukaryotic genomes. It is well known that satellite repeats can expand and contract dramatically, which may result in significant genome size variation among genetically related species. The origin of satellite repeats, however, is elusive. Here we report a satellite repeat, Sobo, from a diploid potato species, Solanum bulbocastanum. The Sobo repeat is mapped to a single location in the pericentromeric region of chromosome 7. This single Sobo locus spans approximately 360 kb of a 4.7-kb monomer. Sequence analysis revealed that the major part of the Sobo monomer shares significant sequence similarity with the long terminal repeats (LTRs) of a retrotransposon. The Sobo repeat was not detected in other Solanum species and is absent in some S. bulbocastanum accessions. Sobo monomers are highly homogenized and share >99% sequence identity. These results suggest that the Sobo repeat is a recently emerged satellite and possibly originated by a sudden amplification of a genomic region including the LTR of a retrotransposon and its flanking genomic sequences.     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization

The complete nucleotide sequence of hepatitis B virus (HBV) DNA from Dane particles of subtype adr was determined. The 3215-bp sequence showed the presence of genes for the surface antigen (226 amino acids) and core antigen (183 amino acids), in addition to two (long and small) open reading frames (ORFs) capable of coding the 843 and 154 amino acids. These ORFs differed from those of the other adr clones so far reported [Ono et al., Nucl. Acids Res. 11 (1983) 1747–1757; Fujiyama et al., Nucl. Acids Res. 11 (1983) 4601–4610]. The gene organization of HBV DNA was found to be well conserved irrespective of subtype. The direct repeat of the undecanucleotide sequence near the 5′ ends of the short (S) and long (L) strands of HBV DNA and the two small direct repeats between both 5′ ends were found to be characteristic structures.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Determination of the polymorphic composition of smooth pea starch

A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.     

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.     

Hymenoic acid, a novel specific inhibitor of human DNA polymerase lambda from a fungus of Hymenochaetaceae sp

Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1′E,5′S)-5′-carboxy-1′-methyl-1′-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase λ (pol λ) in vitro, and 50% inhibition was observed at a concentration of 91.7 μM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols , γ, δ, ε, η, ι, and κ), but also showed no effect even on the activity of pol β, which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol λ. This compound had no inhibitory activities against two N-terminal truncated pol λ, del-1 pol λ (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133–575]), and del-2 pol λ (lacking NLS, BRCT, domain and proline-rich region [residues 245–575]). The compound 1-induced inhibition of intact pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.     

Structure Analysis of Two <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="BoldItalic">Neospora caninum</Emphasis> Satellite DNA Families and Evolution of Their Common Monomeric Sequence

A family of repetitive DNA elements of approximately 350 bp—Sat350—that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1) an arrangement of AB1CB2 350-bp repeats and (2) an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database ( ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.     

Molecular analysis of a deletion polymorphism in alpha satellite of human chromosome 17: evidence for homologous unequal crossing-over and subsequent fixation.

The human alpha satellite DNA family is organized into chromosome-specific subsets characterized by distinct higher-order repeats based on a approximately 171 basepair monomer unit. On human chromosome 17, the predominant form of alpha satellite is a 16-monomer (16-mer) higher-order repeat present in 500-1000 copies per chromosome 17. In addition, less abundant 15-monomer and 14-monomer repeats are also found constitutively on chromosome 17. Polymorphisms in the form of different higher-order repeat lengths have been described for this subset, the most prominent polymorphism being a 13-monomer (13-mer) higher-order repeat present on approximately 35% of all chromosomes 17. To investigate the nature of this polymorphism, we have cloned, sequenced and compared the relevant regions of the 13-mer to the previously characterized 16-mer repeat. The results show that the repeats are virtually identical, with the principal difference being the exclusion of three monomers from the 13-mer repeat. We propose that the 13-mer is the product of an isolated homologous recombination event between two monomers of the 16-mer repeat. Sequence comparisons reveal the approximate site of recombination and flanking regions of homology. This recombination site corresponds to a position within the alphoid monomer which has been previously implicated in an independent homologous recombination event, suggesting that there may exist a preferred register for recombination in alphoid DNA. We suggest that these events are representative of an ongoing process capable of reorganizing the satellite subset of a given chromosome, thereby contributing to the establishment of chromosome-specific alpha satellite subsets.     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.     

Molecular and structural evolution of Citrus satellite DNA

Highly repeated satellite DNA (stDNA) of citric plants was characterized by cloning and sequencing 10–14 repeats of each plant (Citrus limon, C. sinensis, C. ichangensis, Poncirus trifoliata). The monomers are mostly 181 bp in length with a GC-content between 60% and 68% (significantly higher than the average GC-content of the citrus group genomes). Similarity among the repeats indicates that they belong to a satellite family that underwent species-specific modifications, which are reflected in the phylogenetic relationships. Curvature provoked by dA-stretches of the repeats analyzed by gel shifts revealed structural conservation, even though the nucleotide sequences vary among species, thereby probably supporting the heterochromatic structure of stDNA. We show that the species-specific modification of the satellite consensus involves changes in the position and number of dA tracts. The molecule shapes of satellite oligomeres predicted by computer modelling indicate a superhelical structure of the tandem repeats which is in a good agreement with the satellite sequence dendrogram. The contribution of DNA bending elements to the evolution of plant satellite repeats is discussed. Received: 27 November 2000 / Accepted: 12 January 2001     

Neolignans from Ocotea catharinensis

Bark, wood and leaves of Ocotea catharinensis contain respectively 10 (average yield 0.7%.), 15 (average yield 0.004%.) and one (yield 0.4%.) neolignans of the bicyclo[3.2.1]octanoid and the hydrobenzofuranoid structural types, including the new rel-(7S,8R,1′R,4′S,5′R,6′R)-Δ^8′-4′,6′-dihydroxy-5′-methoxy-3,4-methylenedioxy-3′-oxo-8.1′,7.5′-neolignan, (7S,8S)-Δ^{1′,3′,5′,8′}-5,3′,5′-trimethoxy-3,4-methylenedioxy-8.1′,7.O.6′,4.O.7′-neolignan, (7R,8S,1′R,3′R)-Δ^5′,8′-3,4,3′,5′-tetramethoxy-4′-oxo-8.1′,7.O.6′-neolignan and rel-(7R,8S,1′R,2′S)-Δ^4′,8′-2′-hydroxy-3,4-dimethoxy-3′-oxo-8.1′,7.O.2′-neolignan.