Methylation of a middle repetitive DNA sequence class during differentiation in Friend erythroleukemia cells

We have examined the methylation patterns within middle repetitive sequences in Friend erythroleukemia cells. Mouse-interspersed-family-1 (MIF-1) and a group characterized by a 1350-bp Eco-Bam fragment cloned into pBR322 as plasmid pFS-13, are both less modified in Friend cell DNA than in normal tissue DNA. The pattern of methylation present in pFS-13 homologous sequences was found to be stable during cell division, i.e., somatically inherited, and stable during differentiation induced by HMBA.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.     

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.     

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted β rabbit and α and β mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted α rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which a 3 kb deletion adjacent to the insertion site was observed.     

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.     

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.     

Rearrangements of microinjected recombinant DNA in the genome of transgenic mice

Summary Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic F_o mouse by the plasmid rescue technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.     

Inhibition of DNA synthesis and cell death

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytotoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Organization of sequences related to U6 RNA in the human genome.

Small nuclear RNAs were isolated from human placenta and fractionated into individual molecular species. They were then iodinated with 125I and used as probes to screen the human genome. Of 2 x 10(4) recombinant phage clones screened, 22 clones hybridized with U6 RNA, suggesting that there were about 200 copies of this sequence family per haploid genome. Southern blots of these cloned DNAs digested with several restriction enzymes gave the following results: 1, each clone had only one fragment that carried the U6 sequence, 2, the lengths of these fragments varied from clone to clone. These observations indicate that U6 sequences exist as dispersed middle repetitive DNA, and that the sequences surrounding these loci vary. Two of the loci and their flanking regions were subcloned into plasmid and sequenced. Both of the loci showed microheterogeneity of mainly A/G and T/C, but had closely related sequences to U6 RNAs of rat or mouse. The divergence of the flanking regions begins immediately outside the loci. The implication on the microheterogeneity of the U6-related sequences is discussed.     

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.     

Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Molecular cloning and characterization of small polydisperse circular DNA from mouse 3T6 cells.

We have isolated, cloned and analyzed small polydisperse circular (spc) DNA from mouse 3T6 cells. The representation of highly repeated mouse genome sequence families in spcDNA has been examined, and the B1 repeat appears overrepresented in spcDNA by two criteria. The majority of spcDNA clones, however, is made out by as yet uncharacterized middle repetitive sequences. We have investigated the increase in the spcDNA population upon cycloheximide treatment of individual sequences, which are found to amplify differentially.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.