Joseph J. Maio

DNA strand reassociation and polyribonucleotide binding in the African green monkey, Cercopithecus aethiops.

Abstract About 20% of the nuclear DNA from tissues and cultured cells of the African green monkey, Cercopithecus aethiops, consists of a homogeneous fraction that is comparable to mouse satellite DNA in the rapidity and precision with which the denatured DNA reassociates. Although this DNA, designated C. aethiops component α, shares several of the properties of mammalian satellite DNAs, it bands in CsCl at the same density (1.699 g/ml.) as the non-reassociating bulk DNA. Component α DNA comprises up to 75% of the DNA from nucleolar preparations from cultured C. aethiops kidney cells (BSC-1 and CV-1). In addition, CV-1 and primary C. aethiops kidney explants contain a heavy satellite (ϱ = 1.711 g/ml.) representing 5 to 7% of the total nucleolar DNA, which was not detected in similar preparations from BSC-1 cells. In alkaline CsCl component α separates into two populations of complementary strands. Neither the duplex molecule nor the separated single strands interact with the homopolyribonucleotides, poly U and poly G, under conditions in which the denatured bulk DNA interacts with both and increases markedly in density. On the other hand, the light strands, which band at a density of 1.757 g/ml. in alkaline CsCl, interact with the copolymer, poly (U,G) as does the bulk DNA. The heavy strands (ϱ = 1.764 g/ml. in alkaline CsCl) do not interact. Alkali-denatured nuclear DNA of C. aethiops cells elutes as three peaks and nucleolar DNA as one peak from methylated albumin kieselguhr chromatography columns with linear salt and pH elution gradients. The elution order could be correlated with the amounts of component α DNA in each fraction as well as the restriction in the ability to bind the polyribonucleotides. The complementary strands of component α could not be separated by this method, however. In the presence of Ag+ or Hg2+, component a bands in Cs2SO4 at a density different from the bulk DNA, although both fractions show the same density in CsCl or in Cs2SO4 in the absence of heavy metals and presumably have the same (G + C) content.

Deoxyribonucleic acid replication in synchronized cultured mammalian cells: I. Time of synthesis of molecules of different average guanine + cytosine content☆

Abstract HeLa and mouse strain L-cells synchronized by release from excess thymidine block were pulse-labeled with [14C]thymidine or 32P at intervals throughout the DNA synthetic period. The nuclear DNA was then isolated and fractionated into molecules of different buoyant densities by preparative CsCl density-gradient centrifugation. Early in the DNA synthetic period, DNA species having high buoyant densities had the highest specific activity. These results indicated that, compared to total nuclear DNA, the DNA replicated early in this period has a higher average buoyant density and hence, higher average G + C content. Late in the period, DNA species having low buoyant densities had the highest specific activities. This pattern was maintained when HeLa cells were synchronized by the selective detachment of mitotic cells. It was also observed when HeLa DNA replicated early in the DNA synthetic period was fractionated in alkaline CsCl and when DNA of the lysed nuclei was analyzed without further purification, thus eliminating the possibility of selective losses of DNA during isolation. By labeling with bromodeoxyuridine, the DNA replicated during a selected interval of the DNA synthetic period could be isolated from the total DNA for base composition analysis. HeLa DNA synthesized in this early period had an average G + C content of 43.6% and that synthesized late in the period had an average G + C content of 38.7%. The average G + C content of pulse-labeled randomly growing cells was 40.1%, similar to the base composition of the total DNA. In thymidine-synchronized mouse L-cells, there was a gradual change in the average G + C content of DNA replicated throughout the synthetic period. The replication of DNA of high G + C content in the early part of the period and the replication of DNA of low G + C content in the late part of the period was similar to the pattern observed in HeLa cells. The specific radioactivities of satellite and main band DNAs were measured after 1-hour pulses with 32P at different times during the DNA synthetic period. The ratio of the specific activity of satellite to main band DNA increased continuously throughout the period. The specific activities of satellite DNA indicated that about 20% of mouse satellite DNA synthesis occurred during the first 2 hours of the period, and about 80% after main band DNA had already reached its maximum rates of synthesis.

Isolated mammalian metaphase chromosomes: I. General characteristics of nucleic acids and proteins

Abstract The metaphase chromosomes of tissue culture cells derived from four different mammalian species have been isolated in relatively large quantities at neutral pH and essentially free of nuclei and microscopically visible cytoplasmic debris. Some characteristics of the nucleic acids and proteins associated with these chromosome preparations are given below. The average chemical composition of the HeLa chromosome preparations is 16% DNA, 12% RNA and 72%, protein. Similar chemical compositions are reported for the chromosomes from Syrian and Chinese hamster cells and mouse L-cells. The DNA and RNA associated with the HeLa chromosomes have G + C contents of 41 and 63%, respectively. The DNA from the isolated metaphase chromosomes of the four cell types studied here exhibits the same density and very similar band profiles in CsCl density-gradient centrifugation as DNA extracted directly from the interphase nuclei of the respective cells. The satellite band of mouse L-cell DNA is present in chromosomal DNA in the same proportion as in DNA from interphase nuclei. More than 80% of the RNA associated with the HeLa chromosome preparations is 28 s and 16 s in the proportion of 2:1. At least 50% of the chromosomal protein is acid-soluble and shows an acrylamide gel electropherogram similar to that of acid-soluble proteins extracted from the nuclei of the respective cells.

Studies on the intranuclear distribution and properties of mouse satellite dna.

Nucleoli isolated from mouse strain L-cells, L-5178Y lymphoblast tumor cells, and mouse liver and kidney cells showed a 3–4-fold enrichment in satellite DNA when compared with DNA from whole nuclei. This amount of satellite DNA associated with the nucleoli accounted for approximately one-third of the total satellite DNA of the cell. Treating the nucleoli with 2 M NaCl removed more main band DNA than satellite, and suggested that satellite DNA was more intimately bound to the nucleoli than main band DNA. However, attempts to demonstrate whether the satellite enrichment could have been due to preferential adsorption to the nucleoli during their isolation were inconclusive. Incubation of native or renatured satellite DNA with Escherichia coli exonuclease I did not produce any change in its buoyant density in CsCl. The base composition of satellite DNA was determined by using three different combinations of enzymes. Incubation with E. coli exonuclease III produced an increase in CsCl buoyant density and converted 45% of the satellite DNA to mononucleotides having the same average base composition as the total DNA. The satellite DNA did not behave differently from the main band DNA towards the deoxyribonucleases used in this study.

Multiple satellite deoxyribonucleic acids in the calf and their relation to the sex chromosomes

Abstract Four distinct nuclear satellite DNAs from calf (Bos taurus) were isolated and the physical properties of native, single-stranded and renatured duplex molecules of each of the four satellite DNAs were studied by buoyant density-gradient centrifugation. These DNAs were localized in the calf nucleus and on calf metaphase chromosomes by in situ hybridization. In all cases, the calf satellite DNAs are preferentially situated at the centromeres of the autosomes, whereas the X and Y sex chromosomes contain little or none of the satellite DNAs. C-banding techniques showed constitutive heterochromatin at the centromeres of all the autosomes, but not on the X and Y chromosomes. Calf satellite 1 DNA (p = 1.716 g/ml) is at the centromeres of all of the autosomes. Although calf satellite II DNA (p = 1.722 g/ml) is the most widely dispersed over the karyotype, two-thirds of the grains were over the autosomal centromeres. Calf satellites III (p = 1.706 g/ml) and IV (p = 1.709 g/ml) are localized at the centromeres of most, but not all, of the autosomes. The four satellite DNAs each showed a strongly clumped distribution in interphase nuclei of both confluent and growing calf kidney cells in vitro.

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.

Isolated mammalian metaphase chromosomes: II. Fractionated chromosomes of mouse and Chinese hamster cells

Abstract Isolated metaphase chromosomes of mouse L-cells, mouse L-5178Y lymphoblast tumor cells and Chinese hamster cells have been separated into several distinct size classes by velocity sedimentation in steep sucrose gradients. Fractions showing relatively homogeneous chromosome size distributions were readily obtained, but none of the fractions was composed entirely of chromosomes of one specific type. Fractions of the smallest chromosomes were the most homogeneous in size, and the heterogeneity of the fractions increased progressively with increasing average chromosome length. Mouse satellite DNA was not restricted to chromosomes of a limited size class or type but was distributed throughout all fractions of the isolated chromosomes in nearly the same proportion of the total DNA. These results are consistent with the interpretation that the amount of satellite DNA in each chromosome is proportional to the total DNA of the chromosome and that it is not limited to a fixed amount per chromosome. Approximately 70% of the total chromosomal DNA remained in the supernatant or sodium chloride-soluble fraction after extracting the isolated L-cell chromosomes twice in 2 m -NaCl and centrifuging after each extraction. The sodium chloride-soluble DNA was main-band DNA, whereas satellite DNA remained associated with the insoluble chromosome pellet fraction. Isolated chromosomes of the Chinese hamster were also separated into several size classes by centrifugation in sucrose gradients. The DNAs from the various fractions showed no significant differences either in densities or band profiles in cesium chloride density-gradient centrifugation. The DNAs from two fractions showing highly restricted chromosome size-distributions also failed to renature after heating and annealing and their behavior in this respect resembled that of the total DNA of mammalian cells.

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.

Subnuclear redistribution of DNA species in confluent and growing mammalian cells

About 20 to 25 percent of the nuclear DNA from cultured cells of the African green monkey, Cercopithecus aethiops, consists of a homogeneous, highly repetitive fraction designated C. aethiops component α DNA. Use of in situ hybridization techniques reveals component α at the centromeres of chromosomes from both diploid and heteroploid African green monkey kidney (AGMK) tissue culture cells. — Component α DNA comprises 47 percent of the nucleolar DNA in actively growing primary AGMK cells, but only 31 percent of the nucleolar DNA in confluent cells which show density-dependent growth inhibition. Further, there is a pronounced shift of both main band and component αDNA from euchromatin to heterochromatin when growing cells attain confluency. Thus, the relative subnuclear distributions of component α and main band DNAs are different in growing and confluent cells. — In situ hybridization techniques indicate that component α sequences aggregate in clumps in nuclei of growing cells and show a diffuse distribution in nuclei of confluent cells. This suggests that centromeric regions of the various chromosomes or groups of chromosomes aggregate and disaggregate reversibly as the culture changes from density-dependent growth inhibition to active cell division. — Hypotonic citrate treatment of primary AGMK cells causes nucleoli of confluent cells to disperse: this dispersion following citrate treatment was not seen in growing AGMK cells or in confluent or growing heteroploid cells. Similarly, this nucleolar dispersion was seen in confluent diploid mouse and human cells but not in growing diploid cells or in confluent or growing heteroploid cells.