Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

Ribosomal RNA cistrons in Allomyces arbuscula

Bulk and nuclear DNA have been fractionated by preparative neutral CsCl equilibrium density gradient centrifugation and each fraction hybridized to labeled rRNA (25 + 18 S). The cistrons coding for rRNA appeared on the light side of the main peak. Hybridization of the nuclear DNA fractionated by preparative Ag+-Cs2SO4 gradients at different pHs showed that the banding profile did not change as compared to the CsCl pattern. In Hg2+-Cs2SO4 gradients, however, the peak of the fRNA-DNA hybrids shifted on the heavier side of the profile. This indicates that the ribosomal RNA cistrons in Allomyces are A-T-rich. Hybridization with homologous rRNA showed that, at saturation, 3.25% of the DNA is complementary to rRNA. With the genome size of 1.7-10(10) daltons, the multiplicity of rRNA cistrons has been found to be close to 270.     

Study of a haploid yeast strain with an unusually high rDNA content. II. Fractionation of the DNA in preparative Ag+-Cs2SO4 density gradients.

Yeast DNA has been fractionated in preparative Ag+-C-S2SO4 density gradients. After complexing with silver ions at a pH of about 7, the rDNA appears in a defined heavy satellite component in the gradient. For our particular strain, the satellite represents about 15% of the total nuclear DNA and has been identified as the gamma-DNA. In alkaline CsCl density gradients, the satellite DNA forms two bands, of which the light component hybridizes with ribosomal RNA.     

Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction.     

Restriction enzyme analysis of satellite DNA components from the bovine genome

A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.     

DNA fragments associated with chromosome scaffolds

Following extensive digestion of HeLa metaphase chromosomes with either Hae III endonuclease or micrococcal nuclease, nonhistone protein scaffolds may be isolated. Scaffolds isolated after Hae III digestion have about 1.5% of the chromosomal DNA attached to them. This DNA is heterogeneous in size, ranging from about 0.2 to 20 kbp. It can be cleaved with either Eco RI or Hae III - Eco RI, producing a series of repeated fragments, of which the most abundant is 1.7 kbp in length. The 1.7-kdp fragment is tandemly repeated and is enriched (about 50-fold) in the scaffold-associated DNA. It is located primarily on human chromosome 1 and is probably a component of human satellites II and III. Scaffolds isolated after micrococcal nuclease digestion have about 0.1% of chromosomal DNA attached. This DNA is present in two size classes - fragments larger than 10 kbp and fragments approximately 0.2 kbp long. Restriction enzyme digestion of this DNA gives no prominent repeated fragments. Its reassociation kinetics are similar to those of total DNA, indicating that it is not enriched in either highly repetitive or middle repetitive sequences.     

THE DNA components of the chicken genome.

The organization of the chicken genome was investigated by centrifuging chicken DNA (Mr = 57 X 10(6) in preparative Cs2SO4/Ag+ and Cs2SO4/BAMD density gradients [BAMD = 3.6-bis(acetato-mercurimethyl)dioxane]. An analysis by CsCl density gradient of the DNA fractions obtained from the preparative experiments revealed that 88% of the genome is made up of four DNA components, characterized by buoyant densities of 1.699, 1.702(5), 1.704(5) and 1.708 g/cm3 and representing 39%, 25%, 15%, and 9%, respectively, of the total DNA. The remaining 12% of the genome is formed by seven minor and/or satellite components. The distribution of the ovalbumin gene in a Cs2CO4/BAMD density gradient, as tested with a cloned cDNA probe, coincides with the distribution of the 1.702(5)-g/cm3 component. This shows that the DNA regions flanking the ovalbumin gene are homogeneous in base composition over along distances and that the gene is located on a DNA segment belonging to the 1.702(5)-g/cm3 component.     

Interspersion of highly repetitive DNA with single copy DNA in the genome of the red crab, Geryon quinquedens.

Kinetic analysis of the reassociation of 420 nucleotide (NT) long fragments has shown that essentially all of the repetitive sequences of the DNA of the red crab Geryon quinquedens are highly repetitive. There are negligible amounts of low and intermediate repetitive DNAs. Though atypical of most eukaryotes, this pattern has been observed in all other brachyurans (true crabs) studied (1,2). The major repetitive component is subdivided into short runs of 300 NT and longer runs of greater than 1200 NT while the minor component has an average sequence length of 400 NT. Both components reassociate at rates commonly observed for satellite DNAs. Unique among eukaryotes the organization of the genome includes single copy DNA contiguous to short runs (approximately 300 NT) of both repetitive components. Although patent satellites are not present, subsets of the repetitive DNA have been isolated by either restriction endonuclease digestion or by centrifugation in Ag+ or Hg2+/Cs2SO4 density gradients.     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Bovine galactosyl transferase. Substrate.managanese complexes and the role of manganese ions in the mechanism.

The dG+dC-rich fractions obtained by density gradient centrifugation of bovine DNA in Cs2SO4/BAMD [J. Cortadas, G. Macaya & G. Bernardi (1977) Eur. J. Biochem. 76, 13--19] were centrifuged in Cs2SO4/Ag+ density gradients. These experiments led to the preparation of the DNA components which had been detected (by analytical centrifugation in CsCl) in the Cs2SO4/BAMD fractions, and also of DNA components which had identical behaviors in Cs2SO4/BAMD gradients and identical buoyant densities in CsCl. A total of eight satellite components and 11 minor components, accounting for 23% and 4% of the bovine genome, respectively, were thus isolated and charcterized in their relative amounts and buoyant densities. The implications of these results on the interpretation of renaturation kinetic data on the bovine genome are discussed.     

Isolation of a DNA fraction highly enriched in transfer RNA genes from Xenopus laevis.

A DNA fraction highly enriched in tRNA genes can be isolated from the Xenopus laevis genome by the use of Ag+/Cs2SO4 density gradients. Ag+ shows a low affinity for some tRNA cistrons, allowing their separation from bulk DNA upon equilibrium centrifugation in a Cs2SO4 density gradient. Contaminating DNA in the resulting tDNA fraction is further removed by two additional CsCl density gradient centrifugations. The final DNA fraction is 60-fold enriched in tRNA genes, compared to the starting DNA material.     

Properties of satellite DNA from Brassica nigra

The DNA components of B. nigra were preparatively separated by equilibrium ultracentrifugation in a CsCl density gradient, the buoyant density of the main component being 1,696 g . cm-3, that of the satellite component--1,704 g . cm-3. The properties of individual DNA fractions were investigated. Four major components could be observed on the differential melting curve of satellite DNA. Using the reassociation kinetics method it was shown that 30% of satellite DNA are presented as a fast reassociating component with a length of a repeated unit of approximately 2,5 . 10(3) nucleotide pairs. The calculated values of Tm and buoyant density suggest that the m5C content in satellite DNA is lower than that in the main component. During equilibrium ultracentrifugation in the density gradients of actinomycin D--CsCl and Hg2+--Cs2SO4 the satellite DNA is split into 4 major components.     

Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.     

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.     

DNA nuclear satellites of the genus Brassica]

Distribution of nuclear DNA of nine species of the genus Brassica in caesium chloride density gradient has been studied. It has been shown that the amount of the satellite DNA component with the density of 1.704 g.cm-3 varies within a wide range. It is completely absent in B. oleracea and its amount reaches 37% in B. nigra. The other species have an intermediate position. The absence of the latent satellite DNA component in B. oleracea has been shown by equilibrium ultracentrifugation in Cs2SO4 density gradient containing Hg++. Denaturation-renaturation properties of the nuclear DNA of B juncea have been studied.     

Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III.

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.     

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.     

The leftward promoter of bacteriophage lambda. Isolation on a small restriction fragment and deletion of adjacent regions

As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.     

A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes.

Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: Exhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde cross-linked into any SDS-resistant nucleoprotein structure nor topologically "locked" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. These findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the "late" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al., 1978).