Satellite DNA sequences in the red kangaroo (Macropus rufus)

There is a complex pattern of satellite DNA sequences in M. rufus which are revealed by addition of Ag+ or dye (Hoechst 33258) to the DNA ink Cs2SO4 or CsCl equilibrium density gradients. Six satellite DNA fractions have been isolated; these have buoyant densities in neutral CsCl of 1.692, 1.704, 1.705, 1.707 (two), 1.710 and 1.712 g/ml compared with 1.696 g/ml for the main band DNA. Each satellite accounts for 1-3% of the DNA of the genome. The satellites are located in the centromeric heterochromatin of the chromosomes, in the nucleolar organizer region and in interstitial bands on some of the autosomes, each satellite having a unique distribution. Nucleic acid hybridization showed that six of the satellite sequences are also present in the genomes of the wallaroo and the red-necked wallaby, with sequence divergences of only 1-2% relative to the sequences in the red kangaroo.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

Genetic variability in a family of satellite DNAs from tilapia (Pisces: Cichlidae).

We have cloned and sequenced members of a family of satellite DNAs from three genera of the tilapiine tribe of fishes: Oreochromis, Sarotherodon, and Tilapia. The satellite DNAs, visualized as intensely staining bands following electrophoretic separation of EcoRI-digested genomic DNA, consist of three size variants differentially distributed in the various tilapiine species. The sizes of the monomers are approximately 237 bp (type I), 230 bp (type II), and 209 bp (type III). Several cloned monomers were sequenced from Oreochromis niloticus (type III), Oreochromis placidus (types I and II), Sarotherodon galilaeus (type I), Tilapia zillii (type I), and Tilapia rendalli (type I). Comparison of derived consensus sequences for the monomer units of the satellite DNAs revealed sequence identities within and between species that ranged from 89 to 96%. The type II and type III size variants appear to have arisen by deletions of 9 and 29 bp, respectively, within different regions of the type I satellite. Hybridization of a cloned monomer satellite from O. niloticus (type III) to PalI digests of genomic DNA from all three genera detected polymorphic, high molecular weight restriction fragments that produced fingerprint-like patterns. The complexity of these DNA fingerprints varied from one species to another, suggesting a markedly different genomic organization for these polymorphic satellite DNAs.     

Characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver

11 Beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11 beta-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11 beta-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11 beta-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K(m) and V(max)) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11 beta-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K(m) 1959+/-797 nM, V(max) 766+/-88 pmol/mg*min; reductase: K(m) 778+/-136 nM, V(max) 112+/-4 pmol/mg*min). 11 beta-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K(m) 184+/-24 nM, V(max) 74+/-3 pmol/mg*min). In the liver, only 11 beta-HSD1 is detected exerting reductase activity (K(m) 10462 nM, V(max) 840 pmol/mg*min). Sequence analysis of conserved parts of 11 beta-HSD1 and 11 beta-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.     

Sex chromosome associated satellite DNA: Evolution and conservation

Satellites visible in female but not in male DNA were isolated from the snakesElaphe radiata (satellite IV, p = 1.708 g · cm^–3) andBungarus fasciatus (BK¹ minor, p=1.709 g · cm^–3). The satellites cross hybridize. Hybridization of³H labelled nick translated BK minor satellite DNA with the total male and female DNA and/or chromosomes in situ of different species of snakes revealed that its sequences are conserved throughout the snake group and are mainly concentrated on the W chromosome. Snakes lacking sex chromosomes do possess related sequences but there is no sex difference and visible related satellites are absent. The following conclusions have been reached on the basis of these results. 1. The W chromosome associated satellite DNA is related to similar sequences scattered in the genome. 2. The origin and increment in the number of the W satellite DNA sequence on the W chromosome is associated with the heterochromatinization of the W. 3. Satellite sequences have become distributed along the length of the W and resulted in morphological differentiation of sex chromosomes. 4. Evolutionary conservation of W satellite DNA strongly suggests that functional constraints may have limited sequence divergence.     

A highly repetitive DNA component common to all Cervidae: its organization and chromosomal distribution during evolution

In recent work we have isolated and characterized a highly repetitive DNA (MMV satellite IA) from Muntiacus muntjak vaginalis, the species with the most reduced karyotype in the Cervidae family. We have now analysed the genomes of nine related species for the presence of MMV satellite IA components, and have determined their organization and chromosomal distribution. Repetitive satellite IA type DNA is present in all species of the Cervidae, and also in the bovine, but not in a species of the Tragulidae suggesting that these sequences were generated after the phylogenetic separation of Bovidae and Tragulidae. Studies on the organization of the satellite IA DNA in the various species revealed three main repeat lengths: 1400, 1000 and 807 bp. The relative proportion of satellite IA sequences present in any one of the three registers is strikingly different within the various species and can be correlated with the phylogeny of the Cervidae. The chromosomal locations of the satellite IA sequences were determined in seven species by in situ hybridization. It turned out that the chromosomal rearrangements leading to the reduction in the number of chromosomes during karyotype evolution have led to the elimination of satellite I DNA at most locations. In all tandem fusions, the satellite IA sequences located at the centromeres of the ancestral acrocentric chromosomes are lost. In contrast, during the centric fusion that generates the M. m. vaginalis X chromosome satellite IA sequences are amplified. Sequence motifs, which are known to be involved in recombinational events are present in the satellite IA and might have contributed to the unique karyotype variation in the Cervidae.     

Isolation and identification of a novel satellite DNA family highly conserved in several Cervidae species

In an attempt to amplify cervid satellite II DNA from the genomes of Indian muntjac and Chinese muntjac, a pair of primers derived from the white tailed deer satellite II DNA clone (OvDII) yielded a prominent approximately 1 kb polymerase chain reaction (PCR) product (in addition to the expected 0.7 kb satellite II DNA fragments) in both species. The approximately 1 kb products were cloned, sequenced, and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). This revealed that the approximately 1 kb cloned sequences indeed represent a previously unknown cervid satellite DNA family, which is now designated as cervid satellite IV DNA. Approximately 1 kb PCR clones were also obtained from the genomes of the black tailed deer and Canadian woodland caribou with similar primer pairs. Extremely high sequence conservation (over 90% homology) was observed among the clones generated from all four deer species and PCR-Southern hybridization experiments further verified the co-amplification of two kinds of satellite DNA sequences with the same pair of primers. This satellite DNA was found to co-localize with centromeric proteins at the kinetochore by a simultaneous FISH and immunofluorescence study. Due to its high sequence conservation and close association with kinetochores, the newly identified satellite DNA may have a functional centromeric role.     

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae)

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.     

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.     

Synthesis of a carboxamide linked T*T dimer and its incorporation in oligonucleotides.

The condensation of 5'-O-protected 3'-O-(2-aminoethyl)thymidine with 1,2-dideoxy-1-thyminyl-beta-D-erythro-pentofuranuronic acid gives a T*T dimer with * representing a 3'-OCH2CH2NHC(O)-4' linkage connecting the two pentofuranosyl moieties. The incorporation of this dimer in oligonucleotide sequences show only moderately lowered Tm values when hybridized with a complementary DNA relative to the unmodified DNA duplex. Consistently, no looped-out or bubble-type structure could be detected in DNA duplexes with an internal T*T module. Moreover, the 5-atom carboxamide linker causes complete stop on DNA polymerization and on exonuclease III degradation.     

A satellite DNA evolutionary analysis in the North American endemic dioecious plant Rumex hastatulus (Polygonaceae)

We studied the evolution of RAE180 satellite DNA family in the North American endemic dioecious plant Rumex hastatulus. In this species, the Texas race is characterized by a single XX/XY sex chromosome system, whereas the North Carolina race has evolved a derived complex XX/XY(1)Y(2) sex chromosome system. RAE180 repeats were autosomic and poorly represented (2 × 10(-4)% of the genome) with no differences between individuals of different genders or different races of R. hastatulus. In fact, the sex chromosomes of the North Carolina race are still euchromatic, and they have not accumulated satellite DNA sequences, which contrasts with that occurring in the rest of dioecious XX/XY(1)Y(2) Rumex species. In R. hastatulus, we detected the existence of three RAE180 subfamilies. Notwithstanding, while in the Texas race the TX1/NC1 subfamily is the most frequent, the TX2/NC2 subfamily is the most abundant in the North Carolina race. Additionally, the third, less represented subfamily (TX3/NC3) appears currently as relict sequences in both genomes. A common feature of RAE180 satellite is the sudden replacement of one sequence variant by another in different species (or populations as in R. hastatulus races). Thus, the phylogenetic analysis of RAE180 repeats from six dioecious Rumex species supports the "library" hypothesis. According to this hypothesis, we assume that a set of divergent RAE180 variants were present in the ancestral genome of dioecious Rumex species, from which novel tandem arrays originated by the amplification of different variants in different lineages. Differential levels of RAE180 satellite DNA amplification in each lineage, at different evolutionary times, and in different chromosomal positions gave rise to differential patterns of sequence evolution.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.     

The organisation of repetitive sequences in the pericentromeric region of human chromosome 10.

Three satellite DNA families are present in the pericentromeric region of chromosome 10; the alpha satellite and two 5 bp satellite families defined here as satellites 2 and 3. Pulsed field gel electrophoresis (PFGE) demonstrates that these sequences are organised into five discrete arrays which are linked within a region of approximately 5.3 Megabases (Mb) of DNA. The alpha satellite is largely confined to a 2.2 Mb array which is flanked on its p arm side by two 100-150 kb satellite 3 arrays and on its q arm side by a 900 kb satellite 2 array and a further 320 kb satellite 3 array. This linear order is corroborated by fluorescent in situ hybridisation analyses. In total, these arrays account for 3.6 Mb of DNA in the pericentromeric region of chromosome 10. These data provide both physical information on sequences which may be involved in centromere function and a map across the centromere which has the potential to link yeast artificial chromosome (YAC) contigs currently being developed on both arms of this chromosome.     

Isolation and characterization of two families of satellite DNA with repetitive units of 135 bp and 2.5 kb in the ant Monomorium subopacum (Hymenoptera, Formicidae)

Analyzing the satellite DNA in the ant species Monomorium subopacum we found two unrelated families of satellite DNA. Because these satellite DNA families were isolated using the two enzymes HaeIII and EcoRI we called the two families HaeIII and EcoRI family, respectively. The HaeIII family proved to be organized in a 135-bp basic unit repeat, the EcoRI family in a 2.5-kb basic unit repeat. The latter represents perhaps the longest satellite DNA isolated up to now in insects. The HaeIII family apparently comprises about 10% of the total genomic DNA whereas the EcoRI family represents only about 1-2%. A comparative analysis of the two satellite DNA sequences showed no homology between the two families although both sequences possessed long A and T stretches. Eight of the 34 chromosomes showed hybridization with the HaeIII family and hybridization signals are visible in six chromosomes with the EcoRI family. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that the HaeIII family is only slightly curved. However, the unit of the EcoRI satellite DNA family has curvature, especially the first 1000 bp of the monomeric repeat, in which this DNA is AT rich and has numerous A and T stretches. There are also internal inverted subrepeats in each family. The sequences of satellite DNA families found in Monomorium subopacum are different from the sequences of other satellite DNAs cloned in insects, including other species of ants.