Sequence analysis of bovine satellite I DNA (1.715 gm/cm3).

The 1402 bp Eco RI repeating unit of bovine satellite I DNA (rho CsCl = 1.715 gm/cm3) has been cloned in pBR322. The sequence of this cloned repeat has been determined and is greater than 97% homologous to the sequence reported for another clone of satellite I (48) and for uncloned satellite I DNA (49). The internal sequence structure of the Eco RI repeat contains imperfect direct and inverted repeats of a variety of lengths and frequencies. The most outstanding repeat structures center on the hexanucleotide CTCGAG which, at a stringency of greater than 80% sequence homology, occurs at 26 locations within the RI repeat. Two of these 6 bp units are found within the 31 bp consensus sequence of a repeating structure which spans the entire length of the 1402 bp repeat (49). The 31 bp consensus sequence contains an internal dodecanucleotide repeat, as do the consensus sequences of the repeat units determined for 3 other bovine satellite DNAs (rho CsCl = 1.706, 1.711a, 1.720 gm/cm3). Based on this evidence, we present a model for the evolutionary relationship between satellite I and the other bovine satellites.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.     

Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture.

Over the finite proliferative life span of cultured bovine adrenocortical cells, satellite I DNA shows a progressive and extensive loss of methylation at CCGG sites. This was shown by Southern blotting after digestion with the methylation-sensitive enzyme HpaII alone, which provides a sensitive indicator of methylation loss, or digestion with the combination of EcoRI and HpaII, which provides a quantitative indication of loss of methylation. Bovine tissues, including adrenal cortex, all showed a much higher level of satellite methylation than cultured adrenocortical cells. After adrenocortical cells are placed in culture, some demethylation of satellite I is seen as early as 10 population doublings. By 80 population doublings, loss of satellite DNA methylation is extensive. The loss does not appear to prevent continued cell division, since an extended life span clone of bovine adrenocortical cells transfected with SV40 T antigen showed a similar pattern of extensive demethylation. Satellite demethylation has been reported in aging in vivo and the present cell culture system may provide an in vitro model for this form of genetic instability.     

Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5–8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.     

New satellite DNA in Lacerta s. str. lizards (Sauria: Lacertidae): evolutionary pathways and phylogenetic impact

A new tandemly repeated (satellite) DNA family namely Agi160, from Lacerta agilis and Lacerta strigata (Lacerta sensu stricto (s. str.), Linnaeus 1758) have been cloned and sequenced. Agi160 is found in the above two species, as well as two other representatives of the same genus, L. viridis and L. media. DNA hybridization did not reveal it in Darevskia, Podarcis, Zootoca, Eremias, Ophisops, and Gallotia - the other genera of the family Lacertidae. The results suggest that Agi160 is a Lacerta s. str. specific family of tandem DNA repeats. However, a comparison between sequences of Agi160 and CLsat repeat units revealed 60 bp regions 62-74% identical. The latter is a satellite DNA family typical for Darevskia (syn. "L. saxicola complex") (Grechko et al., Molecular-genetic classification and phylogenetic relatedness of some species of Lacertidae lizards by taxonoprint data. Mol Biol 32:172-183, 1988.). Both Agi160 and CLsat tandem repeats share several common features (e.g., the same AT content and distribution of multiple short A-T runs, internal structure of repeated units, the presence of conservative regions). These data are indicative of their common origin and a possibly strong selective pressure upon conserving both satellites. A comparative analysis of structure, organization, and abundance of these two families of satDNA reveals evolutionary pathways that led to their formation and divergence. The data are consistent with the hypotheses of the concerted evolution of satellite DNA families. The possibility of use of Agi160 as a phylogenetic tool, defining relationships within Lacerta s. str., as well as within the whole family of Lacertidae is discussed.     

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.     

Nucleotide sequence of satellite I and II DNA from alpaca (Lama pacos) genome

Agarose and polyacrylamide gel electrophoresis of a total alpaca (Lama pacos) DNA, digested with several restriction enzymes, revealed the presence of two tandemly organized repetitive DNA sequences, named Satellite I and Satellite II. Three Xhol-monomers from Satellite I DNA and two BspRI-monomers from Satellite II DNA were sequenced. As determined by dot hybridization analysis, the total alpaca DNA consists of 3.4% and 1.3% Satellites I and II, respectively. Computer search revealed no homology to any primate, rodent or mammalian sequences published in the Gen Bank Release 48.0 (February 1987) and the EMBL Bank Release 10 (December 1986).     

Adjacent satellite DNA segments in Drosophila: Structure of junctions

The structure of eight satellite DNA molecules containing a junction between tandem arrays of different repeated sequences is described. In one class of junctions there was an abrupt switch with the juxtaposition of two satellite arrays. These arrays were closely related and the periodicity of repeats was maintained in phase across the junction. These arrays usually showed extreme homogeneity in their repeating sequences. A second class of junctions was more complex, and in two cases may have arisen by the insertion of a mobile element into a satellite array. A novel mechanism of satellite formation is proposed to explain the precision of junctions and sequence similarities of neighboring satellite arrays. Homogeneous satellite arrays would be generated enzymatically by synthesis of a repeat using the preceding repeat as template. Occasional errors in copying of the template, either single base changes or misreading the length of the repeat unit, would lead to abrupt switches in the repeating sequence.     

Cloning of calf thymus satellite I DNA in Escherichia coli

Summary The 1400 base pair repeat produced by digestion of calf satellite I DNA (=1.714 g/cm³) with EcoRI, was cloned in E. coli. The hybrid plasmid (pGM 214) which contains the ColE1-Ap vector (pSF 2124) and the 1400 base pair fragment replicates stably in E. coli and can be amplified by chloramphenicol treatment.No clone was found in which more than one repeat unit of the satellite I DNA was present in the chimaera plasmid.Digestion of the original satellite I and the plasmid pGM 214 with R · SmaI shows that the satellite DNA replicated in E. coli is cleaved by the restriction endonuclease SmaI whereas the original satellite I DNA from calf thymus is not, suggesting that the satellite I contains a large amount of modified cytosine or guanosine, probably 5-methyl-cytosine.R · EcoRI^* produces a number of fragments with the satellite I in the range of 300 base pairs to 1400 base pairs.A physical map of pGM 214 (and pSF 2124) with R · EcoRI, R · HincII, HindIII, R · SmaI, R · BamI and R · EclI was constructed.The 1400 base pair repeat unit in the pGM 214 is efficiently transcribed in vitro by purified RNA polymerase, starting from a pSF 2124 promoter.The restriction enzyme EclI produces a 350 base pair repeat with calf satellite II (=1,722 g/cm³), whereas the satellite I is not cut by this enzyme.     

Polypyrimidine segments in drosophila melanogaster DNA: I. Detection of a cryptic satellite containing polypyrimidine/polypurine DNA

Very long runs of pyrimidine nucleotides (polypyrimidines), previously detected in DNA from Drosophila melanogaster, have now been localized to a “cryptic” satellite. These polypyrimidines have an average length of 750 nucleotides and account for about 3% of the thymine residues in total DNA. The buoyant density of the DNA component which contains the polypyrimidines was detected by centrifuging native DNA to equilibrium in a CsCI gradient, and then assaying each fraction for its content of polypyrimidines. A peak was detected at a density of about 1.707 gm/cm³, distinctly heavier than the main band of DNA (1.702 gm/cm³). The buoyant density of polypyrimidine-containing molecules was little affected by differences in the molecular weight of the starting DNA in the range 10⁵-10⁷ daltons (single-stranded). Thus polypyrimidines (and their complementary polypurines) appear to form all or part of a “cryptic” satellite.Polypyrimidines have been isolated and characterized with respect to composition and buoyant density. Direct nucleoside analysis of unlabeled material indicated 34.5% deoxycytidine, 65.5% thymidine. Their banding position in neutral and alkaline CsCI gradients was consistent with a single-stranded DNA polymer of this composition.     

Sequence and evolution of related bovine and caprine satellite DNAs: Identification of a short DNA sequence potentially involved in satellite DNA amplification

The satellite II DNAs of the domestic ox Bos taurus and sheep Ovis aries have been sequenced, and that of the domestic goat Capra hircus partially sequenced. All three are related, and consist of repeat units of about 700 base-pairs. There is no evidence of internal repetition within these repeat units. When matched for maximum homology, the goat and sheep sequences show 83% homology, whereas the ox and sheep sequences share only 70% homology. Factors contributing to the uncertainty of the exact homology between these sequences are discussed, but the results are nevertheless consistent with their progenitor sequence being present in the common ancestor of cattle and sheep. Goat satellite II DNA is shown to contain another, unrelated, tandemly repeated sequence, which is composed of 22 base-pair repeat units. Both this sequence and a region of ox satellite II share good homology with the 11 base-pair progenitor sequence of ox 1.706 g/cm³ satellite DNA. It is suggested that this shared sequence could play a role in bovine satellite DNA amplification.     

DNA damage recognition and repair by 3-methyladenine DNA glycosylase I (TAG)

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3-methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix-hairpin-helix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA.     

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.     

Ag staining of the nucleolus organizer (NO) and its relationship to satellite association

Summary The frequency of involvement in satellite association and the frequency of selective staining of the secondary constrictions with silver solutions have been studied in five phenotypically normal individuals, all carriers of morphological variants of the nucleolus organizing region (NOR). The results show the preferential involvement of some morphological markers in satellite association, and also their preferential staining with Ag-I. It has also been shown that acrocentric chromosomes involved in satellite association are always stained by silver.     

GeneFizz: A web tool to compare genetic (coding/non-coding) and physical (helix/coil) segmentations of DNA sequences. Gene discovery and evolutionary perspectives

The GeneFizz (http://pbga.pasteur.fr/GeneFizz) web tool permits the direct comparison between two types of segmentations for DNA sequences (possibly annotated): the coding/non-coding segmentation associated with genomic annotations (simple genes or exons in split genes) and the physics-based structural segmentation between helix and coil domains (as provided by the classical helix-coil model). There appears to be a varying degree of coincidence for different genomes between the two types of segmentations, from almost perfect to non-relevant. Following these two extremes, GeneFizz can be used for two purposes: ab initio physics-based identification of new genes (as recently shown for Plasmodium falciparum) or the exploration of possible evolutionary signals revealed by the discrepancies observed between the two types of information.     

Caprine pregnancy-associated glycoproteins (PAG): their cloning, expression, and evolutionary relationship to other PAG

Pregnancy-associated glycoproteins (PAG) are structurally related to aspartic proteinases and belong to an extensive, rapidly evolving family of recently duplicated genes expressed in the placentas of artiodactyl species. The aim of the present study was to clone PAG from the goat, study their temporal and cell-specific expression, and determine their phylogenetic relationship to PAG from other species. RT-PCR was used to generate PAG cDNA from pooled placental RNA obtained between days 45 and 115 of pregnancy. A total of 11 cDNA, which differed by > 5% from each other, were selected for complete bidirectional sequencing from 60 clones analyzed. A group of nine (caPAG1, caPAG3-7(var), caPAG9-11), which displayed > 80% sequence identity with each other, were expressed after day 45 of pregnancy and were localized to trophoblast binucleate cells. These PAG demonstrated an unusually high ratio of nonsynonymous (amino acid changing) to synonymous nucleotide differences. CaPAG2, by contrast, was detectable only in early pregnancy (days 18 and 19) and expressed throughout trophectoderm. It was of more ancient origin than the PAG1 group, but more recent than caPAG8. The latter was expressed at all stages examined (days 18 to 115). The data confirm that many PAG genes, with different patterns of temporal and spatial expression, are transcribed in the placenta of the goat. The data also suggest that the recently duplicated PAG genes are being selected for rapid diversification of function.     

Hoechst 33258, distamycin A,and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.by E.R. Schmidt