Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.     

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.     

Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis

Cloned DNA sequences were introduced into the cytoplasm of unfertilized sea urchin eggs by a simple microinjection technique. Sperm was then added, and development allowed to proceed. If linearized plasmids are injected they form random concatenates, and during the early development of the embryos replicate repeatedly. Eukaryotic sequences are not required for replication of the exogenous DNA. Injected supercoiled DNAs neither ligate nor replicate. Both forms of exogenous DNA persist in the embryo through pluteus stage.     

Replication of injected DNA templates in Xenopus embryos

We have analysed the replication of both exogenous frog DNAs and heterologous DNAs during development from the first cleavage through the blastula stage, by their microinjection into fertilized eggs of Xenopus laevis. The data show that various plasmids increase to different extents and that the differences cannot be attributed to size alone. Plasmids containing the Xenopus ribosomal gene repeat unit do not replicate efficiently, and they also inhibit the replication of co-injected DNA templates. This inhibitory effect may be due to DNA sequences contained in the intergenic ribosomal gene spacers.     

Initiation of SV40 DNA replication after microinjection into Xenopus eggs

We have examined the capacity of Xenopus laevis eggs to support replication of microinjected SV40 DNA. As previously reported, microinjected DNA undergoes semi-conservative replication. Unlabeled SV40 DNA was microinjected with [³H]dTTP and, after a 3 h incubation period, the DNA was recovered and adsorbed to BND-cellulose. Elution with an NaCl gradient removes molecules that are entirely double-stranded but not those with single-stranded regions. The latter DNA population is eluted with caffeine. The radioactive DNA that eluted with NaCl was comprised mostly of supercoiled and open circular SV40 DNAs. The radioactive DNA eluted with caffeine was comprised mainly of endogenous DNA but also contained replicative forms of SV40 DNA. Analysis of SV40 DNA replication intermediates by electron microscopy revealed mainly Cairn's forms of varying degrees of maturation. Digestion with BamH1, which cleaves SV40 DNA almost opposite the normal viral replication origin, indicated that SV40 DNA microinjected into frog eggs does not initiate DNA synthesis at its normal initiation site nor at any other obvious preferred site. Rather, it appears that when this template is injected into activated Xenopus eggs, replication may initiate at random.     

Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast

We examined the controversial question concerning DNA sequences required for replication in Xenopus eggs. First we used yeast to isolate ARS elements from the Xenopus genome. They show a striking sequence homology with the yeast ARS consensus sequence. The cloning vector and the ARS-containing plasmids replicate equally after injection into Xenopus eggs. Second, we compared a wide range of DNA templates from procaryotes and eucaryotes. All DNA molecules tested replicate as monomeric molecules, and the efficiency is proportional to their size for templates between 4 and 12 kb. Third, we re-examined two reports of replication origins from the Xenopus genome. In both cases, the vector and the recombinant molecules replicate equally under all conditions tested. The apparent lack of sequence specificity for replication in Xenopus eggs does not prevent the injected molecule from being under cellular temporal control of replication. These results are compared with those from yeast.     

E.coli galk和gpt基因重组质粒(pIDB103)导入金鱼受精卵内的研究

用显微注射法把含有E.coli galk和gpt基因的环状和线状重组DNApIDB103分别导入金鱼受精卵的细胞质内。这些注射过的卵子一般都能正常发育。从各不同发育时期的胚胎分离DNA与~(32)P标记的pIDB103探针杂交表明,导入的环状外源重组DNA在胚胎发育的早期,绝大部分以各种环状构型存在。从原肠胚晚期开始,它们逐渐形成串联状高分子量DNA。在尾芽期仍能检测到它们的序列。但尚未证明,它们是否与受体的染色体DNA发生整合。我们从囊胚期的胚胎中回收到了能转化大肠杆菌的环状重组DNA,它的酶切图谱和pIDB103极其相似。导入金鱼受精卵内的线状重组质粒pIDB103,除少量DNA与金鱼的染色体DNA可能发生整合外,其余绝大部分也形成高分子量DNA。     

The telomeres of Tetrahymena ribosomal DNA are not sufficient for stabilizing linear DNA in Xenopus oocytes

Restriction fragments that include the telomeres of ribosomal DNA from Tetrahymena thermophila (TtrDNA) were ligated to the ends of linearized simian virus 40 (SV40) DNA. The linear SV40 DNA with TtrDNA ends, circular SV40 DNA, linear SV40 DNA, and intact TtrDNA were injected into the nuclei of Xenopus laevis oocytes and assayed for stability. The intact linear 21-kb TtrDNA and circular SV40 DNA were maintained stably for at least 72 h after injection while the linearized SV40 DNA, either with or without telomeric ends, was degraded rapidly. Limited digestion with micrococcal nuclease revealed that neither the intact TtrDNA nor the SV40 DNA with telomeric ends reconstituted into chromatin containing regularly spaced nucleosomes. Another linearized plasmid DNA (pBamC), 14 kb in length, also was not stable in Xenopus oocytes with or without the addition of TtrDNA telomeres. Therefore, TtrDNA telomeres by themselves are not sufficient for stabilization of linear DNA in Xenopus oocytes. Rather, linear TtrDNA is maintained stably because of additional sequence or structural information encoded within the molecule.     

Replication of plasmid DNA in fertilized Xenopus eggs is sensitive to both the topology and size of the injected template

The behavior of various plasmid templates was examined following their microinjection into fertilized eggs of the frog Xenopus laevis using an assay that permits the examination of both replicated and unreplicated plasmids in single eggs. Our results show that both the size and the topology of the template drastically affect the fate of the injected plasmid. Only a small proportion of injected monomeric supercoiled plasmids underwent replication during 6 h of incubation, although not all injected cells supported replication. Nicked circles were less stable than supercoiled molecules, and we could not detect their replication. Linear monomeric molecules polymerized into large, randomly oriented multimers which were extensively, but not entirely, replicated. Similar results were obtained when linear templates were ligated into polymeric forms in vitro prior to injection. Thus large molecules or molecules which, due to their topology, could be converted into high molecular weight forms following injection were preferred templates for replication. On rare occasions tandemly repeated, high molecular weight DNA was generated following the injection of supercoiled plasmid monomers. This large DNA was shown to be almost entirely replicated.     

Fate of amplified nucleoli in Xenopus laevis embryos

We have followed the fate of two components of extrachromosomal nucleoli, amplified ribosomal DNA (rDNA) and 7.5 kb precursor rRNA, during early embryogenesis of Xenopus laevis. Other workers have shown that the amount of amplified rDNA accumulated during oogenesis remains unchanged through the 16-cell stage of embryogenesis. Here we show that as embryonic cleavage continues, the amount of amplified rDNA decreases until it is no longer detectable in the early gastrula embryo. In contrast, the amount of 7.5 kb precursor rRNA in eggs, early cleavage stage embryos, or blastula stage embryos is the same as in oocyte nuclei. Since no rRNA synthesis occurs during these early stages, we conclude that the precursor rRNA sequences synthesized in the oocyte are neither processed nor degraded during early development. The amplified rDNA is not replicated in the early embryo even though the chromosomal DNA of the embryo replicates every 30 min during the first 7.5 hr of embryogenesis. When amplified rDNA is purified and then injected into cleaving embryos, however, we find that it is replicated. This finding suggests that some factor(s) prevents the endogenous amplified rDNA from responding to the cellular replication signals. We show that methylation of cytosine in the rDNA is not related to the DNA's capacity for replication in this system since amplified (unmethylated) and chromosomal (methylated) rDNA are both replicated when injected into embryos. The methylation pattern of these rDNAs appears to be maintained after replication in the embryo.     

Conformation-selective methylation of geminivirus DNA

Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially.     

In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was transformed with restriction endonuclease-digested (linear) DNAs containing the replication origin of the yeast 2 microns plasmid and selectable markers with efficiencies of 10(3) to 10(4), 10(3), and 10(2) to 10(3) transformants per microgram of DNA in the cases of transformations with linear DNAs containing the same cohesive ends, flush ends, and non-complementary cohesive ends, respectively. The results of a restriction analysis of the circular plasmids recovered from transformed cells suggested that the linear DNA molecules were ligated to produce circular forms in the recipient protoplasts.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.