Fred L. Brown

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: Recurrent periodicities and models for the evolutionary origins of repetitive DNA☆

Abstract A restriction enzyme analysis of the repeat structure of mouse satellite, sheep satellite II, human highly repetitive fractions, calf satellite I, and a repetitive fraction of the rat indicates that those DNAs share repeat periodicites in common with one another and with the highly repetitive component α DNA of the African green monkey. The basic repeat periodicity of component α is 176 ± 4 nucleotide base-pairs: the repeat periodicities of the various highly repetitive fractions described here also seem based on this fundamental unit, but it is disguised by a superimposed, higher order repeat organization in each case. The higher orders of organization are based on integral multiples of the basic unit which may reflect the nucleosome spacing of constitutive heterochromatin. With the exception of component α DNA, which shows a repeat structure based on a monomer of 176 ± 4 nucleotide base-pairs, all of the highly repetitive DNAs examined showed a preference for even-numbered or geometric multiples of the basic unit in their higher order sequence organization. It is suggested that such organization is a relatively recent development in the hierarchical evolution of the sequences. Several models are discussed which may account for the higher order organization and expansion of these highly repetitive DNAs. Either a modified unequal crossover model (Smith, 1973) or a modified replicative loop model (Keyl, 1965 a ) seems consistent with many of the properties of highly repetitive DNAs. The models may have implications for the number, distribution and intranuclear rearrangements of transcribed sequences associated with such DNAs.

Highly repetitive component α and related alphoid DNAs in man and monkeys

The genomes of Old-World, New-World, and prosimian primates contain members of a large class of highly repetitive DNAs that are related to one another and to component α DNA of the African green monkey by their sequence homologies and restriction site periodicities. The members, of this class of highly repetitive DNAs are termed the alphoid DNAs, after the prototypical member, component α of the African green monkey which was the first such DNA to be identified (Maio, 1971) and sequenced (Rosenberg et al., 1978). The alphoid DNAs appear to be uniquely primate sequences. — From the restriction enzyme cleavage patterns and Southern blot hybridizations under different stringency conditions, the alphoid DNAs comprise multiple sequence families exhibiting varying degrees of homology to component α DNA. They also share common elements in their restriction site periodicities (172 · n base-pairs), in the long-range organization of their repeating units, and in their banding behavior in CsCl and Cs2SO4 buoyant density gradients, in which they band within the bulk DNA as cryptic repetitive components. — In the three species from the Family Cercopithecidae examined, the alphoid DNAs represent the most abundant, tandemly repetitive sequence components, comprising about 24% of the African green monkey genome and 8 to 10% of the Rhesus monkey and baboon genomes. In restriction digests, the bulk of the alphoid DNAs among the Cercopithecidae appeared quantitatively reduced to a simple series of arithmetic segments based on a 172 base-pair (bp) repeat. In contrast with these simple restriction patterns, complex patterns were observed when human alphoid DNAs were cleaved with restriction enzymes. Detailed analysis revealed that the human genome contains multiple alphoid sequence families which differ from one another both in their repeat sequence organization and in their degree of homology to the African green monkey component α DNA. — The finding of alphoid sequences in other Old-World primate families, in a New-World monkey, and in a prosimian primate attests to the antiquity of these sequences in primate evolution and to the sequence conservatism of a large class of mammalian highly repetitive DNA. In addition, the relative conservatism exhibited by these sequences may distinguish the alphoid DNAs from more recently evolved highly repetitive components and satellite DNAs which have a more restricted taxonomical distribution.

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

Abstract The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei. Sites for four different restriction enzyme activities, EcoRI, EcoRI∗, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI∗ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI∗ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI∗ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei. EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI∗ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI∗ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI∗ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.

Toward a molecular paleontology of primate genomes

Families of related, but nonidentical repetitive DNA sequences, termed the alphoid DNAs, have been identified and characterized in representative species from seven major primate Families. The sequences appear as old as the primate Order itself: they are found in a prosimian (lemur), in a New World monkey, and in all Old World primates examined, including man. The alphoid DNAs are uniquely primate sequences and they may represent the most abundant repetitive DNAs in the primate genome. — A classification scheme for two major families of alphoid DNAs is proposed that is based upon restriction enzyme analysis and Southern blotting with radioactive probes prepared from component α DNA (Maio, 1971) and from the human EcoRI dimer sequences (Manuelidis, 1976). The family of alphoid DNAs that hybridizes readily with component α is termed the HindIII family of alphoid DNAs. This family shows an almost universal distribution among present-day primates. The family of DNA sequences that hybridizes readily with the human EcoRI dimer probe is termed the EcoRI dimer family of alphoid DNAs. This family may be restricted to the great apes and man. The two probes permitted the discrimination of different, but related alphoid families in present-day primates. Multiple alphoid sequence families are found within the genomes of individual primates and the major primate taxa can be characterized by the representations of the various alphoid DNAs within their genomes. — An Appendix is presented (Brown et al., 1981) indicating that competition hybridization effects may influence the autoradiographic banding patterns, and hence, the interpretations of Southern filter-transfer hybridizations when dealing with related repetitive sequences such as the alphoid DNAs that are present in abundance in eukaryotic genomes.

The repetitive sequence structure of component α DNA and its relationship to the nucleosomes of the African green monkey

Abstract An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for Eco RI, Eco RI ∗ , Hind III and Hae III. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary Eco RI ∗ and Hind III sites, about 59% of the repeat sequences contain secondary Eco RI ∗ sites and about 36% of the repeat sequences contain secondary Hind III sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial Eco RI ∗ or Hind III digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for Eco RI, Eco RI ∗ , Hind III and Hae III have been mapped within the repeat unit. Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 m m -NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion. These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al. , 1977 b ). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al. , 1977).

Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors.

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

Cleavage of mammalian repetitive deoxyribonucleic acids by a mammalian site-specific endodeoxyribonuclease☆

Abstract We probed the structure of mammalian repetitive DNAs with a site-specific mammalian endodeoxyribonuclease, which we recently identified, and which apparently represents a common enzyme activity among the mammals (McKenna et al. , 1981). With several of the DNAs (e.g. mouse satellite, guinea pig β-satellite, variable repeated spacer DNA from mouse ribosomal genes and primate alphoid sequences), the endonuclease activity gave highly specific cleavage patterns when the digestion products were analyzed by gel electrophoresis. These patterns were not always identical to those produced by microbial restriction enzymes. However, in other cases (e.g. bovid and caprid satellites and guinea pig α-satellite) the repetitive DNAs appeared to be degraded randomly. Thus, the mammalian enzyme reveals structural features of the repetitive sequences that are not rendered immediately obvious by microbial restriction enzyme analysis. Evidence from mapping data presented here suggests that the mammalian site-specific endonucleases are not sequence specific but have special affinity for imperfect or hyphenated palindromic sequences in repetitive DNAs and in other eukaryotic DNA sequences.