Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.     

Structure and genomic organization of proretrovirus-like elements partially eliminated from the somatic genome of Ascaris lumbricoides.

A clone containing a middle repetitive element next to satellite DNA has been isolated from a germ line genomic library of the chromatin eliminating nematode Ascaris lumbricoides var. suum. The structure of this element has been elucidated by comparison of several clones containing the element in different environments. It is flanked by 256-bp-long terminal repeats (LTRs) and has an internal region of approximately 7 kb. The nucleotide sequences of both the 5' and the 3' LTRs have been determined. The element has a strong structural similarity with retroviral proviruses and related mobile elements. It was therefore named 'Tas', for transposon-like element of Ascaris. Approximately 50 Tas copies are dispersed over approximately 20 different chromosomal sites. Their genomic distribution varies between individuals, indicating that Tas elements are mobile in the Ascaris genome. Two variant forms, Tas-1 and Tas-2, present in a ratio of approximately 2 to 1 in the germ line genome, have been characterized. They differ not only in their restriction pattern, but also in their elimination behaviour. While only about one-fourth of the Tas-1 elements are expelled from the somatic cell lineage, all Tas-2 copies are specifically eliminated and are thus confined to the germ line cells. We have demonstrated that a cloned representative of Tas-1 elements is expelled concomitantly with its flanking DNA sequences during the chromatin elimination process.     

Isolation of a variant family of mouse minor satellite DNA that hybridizes preferentially to chromosome 4

Two cosmids (HRS-1 and HRS-2) containing mouse minor satellite DNA sequences have been isolated from a mouse genomic library. In situ hybridization under moderate stringency conditions to metaphase chromosomes from RCS-5, a tumor cell line derived from the SJL strain, mapped both HRS-1 and HRS-2 to the centromeric region of chromosome 4. Sequence data indicate that these cloned minor satellite DNA sequences have a basic higher order repeat of 180 bp, composed of three diverged 60-bp monomers. Digestion of mouse genomic DNA with several restriction enzymes produces a ladder of minor satellite fragments based on a 120-bp repeat. The restriction enzyme NlaIII (CATG) digests all the minor satellite DNA into three prominent bands of 120, 240, and 360 bp and a weak band of 180 bp. Thus, the majority of minor satellite sequences in the genome are arranged in repeats based on a 120-bp dimer, while the family of minor satellite sequences described here represents a rare variant of these sequences. Our results raise the possibility that there may be other variant families of minor satellites analogous to those of alphoid DNA present in humans.     

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33258 — CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 × 10⁵ copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a theme and variation pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA.

A recombinant DNA plasmid library was constructed from HeLa cell extrachromosomal circular DNA and the sequence organization of one family of clones, which contain sequences enriched in HeLa small polydisperse circular (spc) DNA, was studied by restriction mapping and base sequence analysis. Restriction mapping revealed each clone to be composed solely of imperfect tandem repeats of ca. 170 bp. The entire DNA sequence of one clone was determined and found to be alphoid satellite with a variant monomeric construction.     

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.     

Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver.

The location of nucleosomes on the nucleotide sequence of rat satellite I DNA was investigated using micrococcal nuclease, exonuclease III, and restriction nucleases as tools. Hae III cleaved the satellite DNA containing chromatin very preferentially in the linker region. Nucleosomes were found predominantly in three defined positions on the 370 bp satellite I monomer unit. This type of arrangement occurs on not more than half of the satellite DNA containing chromatin while the rest of this chromatin is arranged differently. The arrangement of nucleosomes with high probability in preferred frames and with low probability in less preferred frames may be a general phenomenon which can be discussed as a possible mechanism to modulate sequence recognition.     

Nucleotide sequence of a highly repetitive component of rat DNA.

A highly repetitive component of rat DNA which could not yet be enriched by density gradient centrifugation was isolated with the help of the restriction nuclease Sau3AI. This nuclease converted the bulk of the DNA to small fragments and left a repetitive DNA component as large fragments which were subsequently purified by gel filtration and electrophoresis. This DNA component which was termed rat satellite DNA I is composed of tandemly repeated 370 bp blocks. According to sequence analysis the 370 bp repeats consist of alternating 92 and 93 bp units with homologous but not identical sequences. Methylation of CpG residues was correlated to the rate of cleavage by restriction nucleases. Significant homologies exist between the sequences of rat satellite DNA I and satellite DNAs of several other organisms. The divergence of the sequence of rat satellite DNA I was discussed with respect to evolutionary considerations.     

Characterization of a complex satellite DNA in the mollusc Donax trunculus: Analysis of sequence variations and divergence

A highly repetitive sequence in the genomic DNA of the bivalve mollusc Donax trunculus (Dt) has been identified upon restriction with EcoRV. During the time-course of DNA digestion, genomic fragments resolved electrophoretically into a ladder-like banding pattern revealing a tandem arrangement of the repeated elements, thus representing satellite DNA sequences. Cloning and sequence analysis unraveled the presence of two groups of monomer units which can be considered distinctive satellite subfamilies. Each subclass is distinguishable by the presence of 17 evenly spread diagnostic nucleotides (nt). The respective consensus sequences are 155 bp in length and differ by 11%, while relevant internal substructures were not observed. The two satellite subfamilies constitute 0.23 and 0.09% of the Dt genome, corresponding to 20 000 and 7600 copies per haploid complement, respectively. Sequence mutations often appear to be shared between two or more monomer variants, indicating a high degree of homogenization as opposed to that of random mutational events. Shared mutations among variants appear either as single changes or in long stretches. This pattern may arise from gene conversion mechanisms acting at different levels, such as the spread of nt sequences of a similar length to the monomer repeat itself, and the diffusion of short tracts a few bp long. Subfamilies might have evolved from the occasional amplification and spreading of a monomer variant effected by gene conversion events     

Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements

The complete nucleotide sequence of L1Md-A13, a 6372 base-pair (bp) member of the L1Md repetitive family isolated from a BALB/c mouse genomic DNA library, is reported. The nucleotide sequence of 4331 bp from the 5' end of L1Md-9, which is located in the beta-globin complex of the C57BL/10 mouse, is also reported. Parsimony analysis of these sequences plus two previously reported L1Md sequences allows the determination of an ancestral L1Md sequence. Analysis of the L1Md population indicates that this ancestral sequence is likely to represent a functional L1 sequence. This ancestral sequence confirms that the length (1137 bp and 3900 bp) and relationship (14 bp overlap) of the two large open reading frames previously reported are conserved features of the L1Md family. It also allows the determination of an ancestral amino acid sequence for these two open reading frames. Full-length L1Md elements have one of two sequences tandemly repeated at the 5' end. These two monomers are called A-type and F-type. Our data define the 5' end of A-type full-length L1Md elements. L1Md elements of the A-type have varying numbers of tandemly repeated 208 bp monomers, but each element ends about 78 bp from the 5' end of the terminal 208 bp monomer.     

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.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Analysis of highly purified satellite DNA containing chromatin from the mouse.

A purification scheme for satellite DNA containing chromatin from mouse liver has been developed. It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases. Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions. Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively. In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA. This material has the same histone composition as bulk chromatin. No significant differences were detected in an analysis of minor histone variants. Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.     

Patchwork structure of a bovine satellite DNA

According to a previous restriction nuclease analysis, bovine 1.706 satellite DNA (density 1.706 g/cm3 in CsCl) is organized in an unusual structure of superimposed long- and short-range repeats (Streeck and Zachau, 1978). We have now determined the nucleotide sequence of this satellite DNA in both cloned fragments and fragments from the total satellite DNA. Each long-range repeat unit (about 2350 bp) is divided into four segments. Each segment consists of different variants of a basic 23 bp sequence which is itself composed of a dodecanucleotide and a related undecanucleotide. A total of 2400 nucleotides have been sequenced. Detailed analysis of the sequence divergence reveals that both the overall extent of divergence and the frequency of base changes at individual positions of the 23 bp repeats are characteristically different in the various segments. Preferentially methylated sites and a high incidence of symmetry elements are found. In two of the four segments, 22 of 23 bp of the prototype sequence are included in six overlapping elements of dyad symmetry and in a palindrome. A scheme for the evolution of the satellite DNA from a basic dodecanucleotide is proposed which is based on the different degrees of divergence for the various repeats superimposed in this satellite DNA.