Organization of African green monkey DNA at junctions between alpha-satellite and other DNA sequences

Segments of African green monkey DNA containing sequences of the highly reiterated cryptic satellite DNA called α-satellite were selected from a library in λ bacteriophage. This λ library was constructed to enrich for monkey segments that contain (1) irregular regions of α-satellite and (2) α-satellite linked to other monkey sequences. At least 11 of 15 cloned monkey segments between 13 × 10³ and 16 × 10³ base-pairs in length, selected by hybridization to α-satellite, also include other monkey sequences.In general, α-satellite sequences close to the junctions with non-α-satellite DNA contain an abundance of divergent forms compared to the average frequency of such forms within total α-satellite. Many of the cloned segments are missing some of the HinIII sites that occur once in most monomer units of α-satellite, and likewise several of the cloned segments contain restriction sites that rarely occur in α-satellite as a whole. In some segments HinIII sites occur that are spaced at distances other than the basic multiple of 172 base-pairs. At least one of the cloned segments, however, is composed mainly of typical 172 base-pair long α-satellite monomer units.Several of these cloned DNAs have been mapped by restriction endonuclease digestion and Southern blot analysis and the arrangements of α-satellite and non-α-satellite sequences have been determined. In addition to segments that contain a boundary where satellite meets other types of sequence, some contain two such boundaries and thus satellite flanks a non-α-satellite segment. Further, two different types of non-α-satellite sequence appear to be common to more than one phage, perhaps indicating some recurring organization at boundaries.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

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.

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, EcoRI¹, 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-RI¹ 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-RI¹ 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, EcoRI¹ 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 EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ 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.     

Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

Cartographie de Restriction Comparée du Satellite DNA chez l'Homme et le Chimpanzé

Highly repeated DNA satellite α sequences from man and chimpanzee (Pan troglodytes) have been compared, using restriction endonucleases. The two species share a 340 base pairs tandemly represented DNA, that is cut once by EcoRt. Pan troglodytes differ from man by loss of the two MboI and EcoRI star sites and by the gain of an Hae III site in the repeated sequence.     

Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.     

Deca-satellite: A highly polymorphic satellite that joins α-satellite in the African green monkey genome

Three different cloned segments of African green monkey DNA that contain α-satellite sequences linked to a previously undescribed, distinct monkey satellite (called deca-satellite) are described here. The cloned segments were derived from a monkey DNA library in λCharon4A that was constructed to select for junctions between α-satellite and other DNA sequences.The structure of the deca-satellite and of a junction between deca-satellite and α-satellite were studied by subcloning appropriate fragments of the original cloned segments and by sequence analysis. Deca-satellite has a ten base-pair repeat unit: the consensus sequence of the repeat units is 5′ A-A-A-C-C-G-G-N-T-C. Sequences homologous to the deca-satellite are in the middle repeated class of genomic DNA. Analysis of the organization of deca-satellite sequences by digestion of total DNA with various restriction endonucleases and hybridization with a cloned deca-satellite probe revealed extensive polymorphism in the genomes of different individual monkeys but not among the tissues of one organism. These observations indicate that the arrangement of deca-satellite sequences is continually changing.An unusual α-satellite repeat unit occurs at a junction between the α-satellite and deca-satellite. It resembles the major baboon α-satellite more closely than it does monkey α-satellite and thereby provides evidence in favor of the “library” hypothesis for satellite evolution.     

Comparaison des séquences de fragments de restriction répétés spécifiques du chromosome Y humain chez le chimpanzé et le gorille

Analysis of the evolutionary conservation and change of the Y human specific Hae III 2.4 kb repeated sequence were studied between man and anthropoïd species after restriction with EcoRI endonuclease. In chimpanzee, blocks of these repeats are absent, the remaining copies being interspersed with other sequences. A close similarity of the restriction patterns of these sequences is found between gorilla and man.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

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

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 EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary $\text{Eco}\text{RI}{}^1$ and HindIII sites, about 59% of the repeat sequences contain secondary $\text{Eco}\text{RI}{}^1$ sites and about 36% of the repeat sequences contain secondary HindIII 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 $\text{Eco}\text{RI}{}^1$ or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-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., 1977b). 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).     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Circular Permutation of the Genome of a Temperate Bacteriophage from Streptococcus cremoris BK5

The temperate bacteriophage BK5-T was isolated from Streptococcus cremoris BK5 by induction with mitomycin C. Electron microscopy revealed that BK5-T DNA consists of linear molecules, ranging in size from 39.7 to 46 kilobase pairs. Restriction analysis of self-ligated BK5-T DNA showed that the ends of the DNA were not cohesive. The EcoRI restriction fragments of the phage genome were cloned into pACYC184. Restriction enzyme analysis of both the phage DNA and the cloned EcoRI fragments with EcoRI, BstEII, PstI, ClaI, and XbaI yielded a 37.6-kilobase-pair-long circular restriction map for the phage genome. It was concluded that the BK5-T DNA molecules in the population differ in their sequence by a circular permutation and that individual DNA molecules are terminally redundant. The map location of the sites at which packaging of BK5-T DNA into phage heads is initiated (pac) and at which the phage integrates into the bacterial chromosome (att) were established.     

Different regions of a complex statellite DNA vary in size and sequence of the repeating unit.

The 1.688 g/cm³ satellite DNA of Drosophila melanogaster is composed primarily of 359 base-pair units repeated in tandem. Most of these units contain a single cleavage site for both HaeIII and HinfI restriction endonucleases; however, some units lack one or both sites. Previously we had shown that the distribution of HaeIII and HinfI endonuclease sites varies widely between different regions of 1.688 g/cm³ satellite DNA; for example, some regions contain HaeIII sites in every unit and other regions (>10,000 base-pairs) contain no HaeIII sites (Carlson &; Brutlag, 1977). We have now cloned molecules of 1.688 g/cm³ satellite DNA which lack HaeIII sites and have shown that the absence of sites is caused by sequence variation rather than base modification. This result indicates that regions of 1.688 g/cm³ satellite DNA with different distributions of restriction sites differ in the sequence of their repeating units. We also show that a large fraction of the satellite DNA which is not cleaved by HaeIII endonuclease still contains HinfI endonuclease sites (and AluI sites) spaced about 359 base-pairs apart. However, one cloned segment lacking HaeIII sites was found to contain 33 tandem copies of a novel 254 base-pair unit. Sequence analysis showed that this 254 base-pair unit is homologous to the 359 repeat except for a 98 base-pair deletion. These data suggest that both units have evolved from a common ancestor and that each has subsequently become amplified into separate tandem arrays.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.     

Analysis of LINE-1 family sequences on a single monkey chromosome.

The structure of LINE-1 (L1Ca) family members present on African green monkey chromosome CAE-19 is compared with that of the entire set of L1Ca sequences present in the monkey genome. The analysis involved annealing of cloned subsegments of monkey L1 family members to DNA-blots containing restriction endonuclease digests of either total monkey liver DNA or DNA isolated from a monkey/mouse somatic cell hybrid carrying the single monkey chromosome. In addition, L1Ca segments cloned from hybrid cell DNA were characterized by restriction endonuclease mapping and hybridization. The data indicate that, taken as a whole, the set of L1Ca sequences on CAE-19 tends to differ in characteristic ways from the set present in the total monkey genome.