Bovine thymus satellite I DNA sequences, which are highly methylated, are preferentially located in H1-rich chromatin

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5-7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.     

Quantitative evolution of transposable and satellite DNA sequences in Picea species

Clones containing tandemly arranged repeats belonging to two distinct sequence families, (i) PAG004P22F (2F) and PAG004E03C (3C) or (ii) Ty3/gypsy- (8R; PAG004B08R) and Ty1/copia-like sequences (9R; PAG007F19R), were selected from a randomly sheared total genomic DNA library of Picea abies . The inserts were used as probes in dot-blot hybridizations to genomic DNA of P. abies, Picea orientalis , Picea pungens , and Picea pungens var. glauca. All these entities are diploid and share the same chromosome number (2n = 24), but the genome sizes differ largely. The redundancy (copy number per 1C DNA) of sequences related to each probe varied greatly between the genomes. No significant correlation was found between the genome size and the copy number of sequences in any family. The quantitative ratios varied greatly (in each genome) between the two families of satellite DNA, between the sequences that represented copia or gypsy retrotransposons, and between tandemly arranged sequences and retroelements as a whole, suggesting that there is no common factor that controls the quantitative evolution of repeats belonging to different sequence families during speciation in Picea.     

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.     

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

Tissue specificity and organisation of CpG methylation in calf satellite DNA I.

Examination of bovine satellite DNA I methylation within CpG dinucleotides has been made by restriction analysis. It is shown that variations in the methylation patterns occur between different tissues (brain, liver, thymus and sperm) . Some of the 8 Hpa II sites present per repeat are clearly undermethylated in sperm as compared to other tissues. Methylation is considered therefore, as a highly specific event. It is also shown that there is a spatial specificity in the methylation pattern of the 3 Hha I sites in all tissues. These results are discussed in the light of methylation and satellite DNA functions.     

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.     

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue

Summary This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400μg/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB₃ derived from cells transfected with pSV_3neo, and 2RB₅ derived from cells transfected with pSV_3neo, revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.     

Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m⁶A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m⁷GpppX^mpG, m⁷GpppX^mpY^(m)pG, m⁷GpppX^mpY^(m) pNpG and m⁷GpppX^mpY^(m)p(Np)_{> 1}G have distinctive non-random compositions of the 2′-O-methylated constituent X^m; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA². For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m⁷Gppp(m⁶)A^mG, whereas less than 1% are m⁷GpppU^mpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Y^m in the cap II forms of mRNA (8% G^m, 11% A^m, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m⁶A occurs in well-defined sequences of the type: $\text{-N}{}_1\text{-(}\text{G}\text{A}\text{)-m}{}^6\text{A-C-N}{}_2\text{-}$, the 5′ nearest-neighbor of m⁶A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N¹ is a purine about 90% of the time and the nucleotide N² is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m⁶A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed.     

Repetitive DNA sequences in methotrexate- and methasquin-sensitive and -resistant Chinese hamster cell lines

DNA purified from a Chinese hamster cell line of lung fibroblast origin (DC83F) was analyzed by density gradient centrifugation and by gel electrophoresis after restriction endonuclease digestion in order to fractionate discrete repetitive fractions within the total DNA. No obvious satellite DNAs were resolved using the CsCl or Ag-Cs₂SO₄ density gradient conditions described herein. However, analysis of the digestion products of a battery of restriction endonucleases indicated that three of these enzymes, EcoR ₁, HaeIII, and XhoI, yielded discrete fragments which could be visualized with EtBr staining or identified by scintillation counting of [ ³ H] DNA. DNAs from several highly ( hundredfold increased resistance) antifolate-resistant sublines of DC-3F, characterized by a large homogeneously staining region (HSR) in the chromosome complement, were examined with both techniques and compared to the parental, antifolate-sensitive cell line DNA. The density gradient profiles and electrophoretic patterns of restriction endonuclease digests were identical among all the cell lines examined and were indistinguishable from those of the parental DC-3F DNA.This work was supported in part by grants to the Sloan-Kettering Institute and to J.L.B. and P.W.M. by the National Institutes of Health and the Fairchild New Frontiers Fund. Portions of this study were presented at the 18th Annual Meeting of the American Society for Cell Biology held in San Antonio, Texas, November 4–8, 1978.     

Characterization of polyoma viral DNA sequences in polyoma-induced hamster tumor cell lines

The polyoma (PY) viral DNA sequences present in a series of hamster tumor cell lines were evaluated using the blotting technique of Southern ((1975) J. Mol. Biol. 98, 503-517). Remarkably, no cell line contained an intact distal portion of the early gene region which encodes a portion of the large T antigen. All cell lines examined contained the PY DNA Bum I fragment which contains most of the genetic information encoding PY small and middle T antigens, as well as the origin of viral DNA replication. These results provide an explanation for our previous observation that PY virus-induced hamster tumors do not contain the large species of T antigen.     

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.     

The DNA sequences of cloned complex satellite DNAs from Hawaiian Drosophila and their bearing on satellite DNA sequence conservation

A class of restriction endonuclease fragments near 185 bp in length and comprising approximately 20% of the genomes of 3 species of Hawaiian Drosophila has been cloned using bacteriophage M13. The nucleotide sequences of 14 clones have been determined and the variation between clones has been found to be due to deletions and base changes. Analyses of uncloned material show that the cloning system itself does not introduce the variation. The variation of the basic repeat within and between species is high; 15% due to deletions and 10% due to base changes. The Drosophila data are similar in many respects to both the 23 bp calf satellite results (Pech et al., 1979b) and those from sequence analyses of the 170 bp primate restriction fragments (Rubin et al, 1979; Donehower et al., 1980, Wu and Manuelidis, 1980). The intraspecies level of base changes and deletions in the calf satellite approaches 25% as does that in the human/African green monkey/baboon comparisons. The between species variation in the primate group is near 35%. Direct sequencing methods thus reveal a widespread sequence heterogeneity in both invertebrate and mammalian satellite systems of long or short repeat length. This heterogeneity does not support the strict sequence conservation implied by the library hypothesis, which claims a functional role in speciation for the rigid conservation of satellite DNA sequences (Fry and Salser, 1977). Furthermore the Drosophila and primate data reveal that satellite DNAs can change rapidly, though nonrandomly, at the nucleotide sequence level in a relatively closely knit group such as the Hawaiian species, as well as in more distantly related species from amongst the primates. We draw two major conclusions. There is no universal attribute of satellite DNA sequence per se, the only biological variable to date being the amount of satellite DNA and its effects in the germ line. Many aspects of satellite DNA evolution conform to Kimura's (1979) concepts of neutrality.     

Quantitative determination of unchanged cisplatin in rat kidney and liver by high-performance liquid chromatography

A quantitative analytical method for measuring unchanged cisplatin (CDDP) and high- and low-molecular-mass metabolites (fixed and mobile metabolites) in rat kidney and liver was developed. Unchanged CDDP, separated from fixed and mobile metabolites in tissue homogenates by consecutive procedures of fractionation and ultrafiltration, was determined by high-performance liquid chromatography (HPLC) with post-column derivatization. Although unchanged CDDP was found to be partly metabolized to fixed metabolites during the preparation of cytosolic ultrafiltrates, the recovery of unchanged CDDP gave a constant value (about 70%), which was independent of tissue type and CDDP concentration (from 1 to 10 μg/ml). The detection limit for unchanged CDDP in the cytosolic ultrafiltrate was 20 ng/ml, corresponding to a concentration detection limit of 65 ng Pt per g of tissue in the kidney and liver. The concentrations of fixed and mobile metabolites were determined as platinum concentrations in the tissue homogenate and in the cytosolic ultrafiltrate using atomic absorption spectrometry after correcting for transformation of unchanged CDDP to fixed metabolites. The distribution of unchanged CDDP, mobile metabolites and fixed metabolites in rat kidney and liver, after bolus injection of CDDP (5 mg/kg), was determined using this method.     

Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation

Four clonal cell lines subcloned from a clonal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of alpha 1(I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases MspI and HpaII. It was found that the loss in collagen type I expression correlated positively with the degree of methylation of alpha 1(I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen gene regulation.     

The structure of the regulatory region of the rat L1 (L1Rn, long interspersed repeated) DNA family of transposable elements.

Here we report the DNA structure of the left 1.5 kb of two newly isolated full length members of the rat L1 DNA family (L1Rn, long interspersed repeated DNA). In contrast to earlier isolated rat L1 members, both of these contain promoter-like regions that are most likely full length. In addition, the promoter-like region of both members has undergone a partial tandem duplication. A second internal region of the left end of one of the reported members is also tandemly duplicated. The propensity of the left end of rat L1 elements to undergo this form of genetic rearrangement, as well as other structural features revealed by the present work, is discussed in light of the fact that during evolution the otherwise conserved mammalian L1 DNA families have each acquired completely different promoter-like regions. In an accompanying paper [Nur, I., Pascale, E., and Furano, A. V. (1988) Nucleic Acids Res. 16, submitted], we report that one of the rat promoter-like regions can function as a promoter in rat cells when fused to the Escherichia coli chloramphenicol acyltransferase gene.