First Round Of DNA Replication Research Articles

Asymmetric parental genome engineering by Cas9 during mouse meiotic exit.

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.

DNA replication is an integral part of the mouse oocyte's reprogramming machinery.

Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24–36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

BackgroundPrevious work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Metfl/fl;Alb-Cre+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.Methodology/Principal FindingThe priming events appeared to be intact in Metfl/fl;Alb-Cre+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Metfl/fl;Alb-Cre+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.Conclusion/SignificanceIn conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.

Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

MyoD is a transcriptional factor that is required for the differentiation of muscle stem cells (satellite cells). In this study, we describe a previously unknown function for MyoD in regulating a gene (Cdc6) that is vital to endowing chromatin with the capability of replicating DNA. In C2C12 and primary mouse myoblasts, we show that MyoD can occupy an E-box within the promoter of Cdc6 and that this association, along with E2F3a, is required for its activity. MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6. Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase. Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.

MicroDNAs and Transcriptional Regulation

AbstractSynthetic "microDNAs (MIDs)"is a new class of ~ 20-25 nucleotide-long DNAs capable of repressing the activity of the target gene at the level of transcription by mechanisms that have not been clarified yet. However they are designed to target non-coding regions of the cancer causing genes, thus interfering with transcription. The inhibition might be possible through the direct binding of MIDs to cis-regulatory sites and/or to some Transcription Factors (TF) that normally activate transcription. Synthetic MIDs in some ways are similar to the newly discovered microRNAs a mechanism by which cell regulates its genetic activities at post-transcriptional level. Synthetic MIDs can provide a powerful tool to prevent massive production of mRNA by undesired gene activities. Therefore drugs are not required to interact with overwhelming number of mRNA and microRNA copies that may present unwanted side effects. In vitro studies suggest that the inhibition of the target gene starts after the first round of DNA replication, usually 24 hours after treatment depending on cell doubling time. Cell growth suppression maximizes at day 6 or 7 and the inhibition effect is sustained for weeks. We have shown that blocking of both bcl-2 and k-ras transcription by their specific microDNA Inhibitors induced apoptosis in HL60 leukemia cells and B-cell lymphomas.

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.

DNA Mismatch Repair-dependent Response to Fluoropyrimidine-generated Damage

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts

In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.

Involvement of chromatin structure in the regulation of mouse zygotic gene activation

ABSTRACTWe investigated the contribution of the global changes in chromatin structure to the mechanism regulating zygotic gene activation in mouse embryos by in vitro transcription assay and in situ DNase I sensitivity assay. Changes in the DNase I sensitivity and transcriptional activity were similar from the late 1‐cell to the late 2‐cell stage, but were not in the early 1‐cell stage. The DNase I sensitivity decreased at the late 1‐cell stage when compared with the sensitivity at the early 1‐cell stage. It increased at the early 2‐cell stage and then decreased at the late 2‐cell stage. Similarly, the transcriptional activity increased at the early 2‐cell stage when compared with the activity at the late 1‐cell stage, and then decreased at the late 2‐cell stage. The DNase I sensitivity was not decreased in embryos which were treated with aphidicolin, where the first round of DNA replication was inhibited. A similar result was observed in embryos when the second DNA replication was inhibited. These results suggested that the chromatin structure is one of the important factors regulating gene expression from the late 1‐cell stage and that DNA replication is an event responsible for the establishment of the repressive state in the chromatin structure. The inhibition of protein synthesis did not affect the DNase I sensitivity in the 1‐cell embryos, while it is known that protein synthesis is indispensable for gene activation. This result suggests that the protein synthesized after fertilization, but not the chromatin structure, is responsible for the initiation of the gene activation.

Reprogramming of gene expression during preimplantation development.

A fundamental problem in early mammalian development is the transformation of the highly differentiated oocyte into the totipotent blastomeres by the 2-cell stage. This remarkable transformation probably entails reprogramming the pattern of gene expression. The maternal-to-zygotic transition occurs during the 1-cell stage, i.e., 1-cell embryo is transcriptionally active. DNA replication is a likely locus of regulation, since DNA replication, with consequent nucleosome displacement, would serve to facilitate the access of maternally derived transcription factors to their cis-acting DNA-binding sequences. In fact, the first round of DNA replication is essential for the expression of two genes, eIF-1A and the transcription-requiring complex (TRC), as well as global transcription. A growing body of evidence suggests that a transcriptionally repressive state develops during the 2-cell stage. Interestingly, inhibiting the second round of DNA replication inhibits the decrease in expression of both the TRC and eIF-1A, as well as total endogenous gene expression. This repression may be linked to a change in chromatin structure, since treatment of 2-cell embryos with histone deacetylase inhibitors prevents the decrease in expression of the TRC and eIF-1A, and also inhibits the development of the transcriptionally repressive state for global gene expression. The findings that histone deacetylases can be targeted to chromatin provide a mechanism to link histone deacetylation with repression of gene expression. J. Exp. Zool. (Mol. Dev. Evol.) 285:276-282, 1999.

Effect of stimulating maize germination on cell cycle proteins

The germination process can be accelerated if seeds are stimulated either by adding cytokinins or by osmopriming. Under these conditions, cells in maize (Zea mays) embryo axes shorten the time at which the first round of DNA replication and mitosis takes place, thus advancing the cell cycle. Using heterologous antibodies against different cell cycle proteins, we have followed the behaviour of several markers for G1 phase (cyclin D, E2F and p53) and a marker of G2 phase (cyclin B) under either control or “accelerated” germination conditions. The results showed two classes of behaviour: either there was no variation in the amount of the protein present under control or accelerated germination conditions, represented by cyclin Band E2F‐type proteins, or the amount of the proteins was drastically reduced, more rapidly under accelerated germination, as was the case for cyclin D‐ and p53‐type proteins. Although the cyclin D‐type protein was synthesized de novo during germination, the balance was towards degradation so that there was no cyclin D detected 15 h after germination in benzyladenine‐treated and osmoprimed seeds. A Cdk4‐type protein seemed to be present in cyclin D immunoprecipitates and its kinase activity paralleled the fluctuations of the cyclin amount during germination. These data are discussed in the context of early seed germination.

Cyclin D2 ArrestsXenopusEarly Embryonic Cell Cycles

Xenopuscyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. WhenXenopuscyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However,Xenopuscyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced byXenopuscyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. RadiolabeledXenopuscyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest thatXenopuscyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.

Role of the first round of DNA replication in reprogramming gene expression in the preimplantation mouse embryo.

The first round of DNA replication has been proposed to provide a window of opportunity for maternally-derived transcription factors to gain access to their cognate cis-binding DNA sequences and thereby reprogram the pattern of gene expression that occurs during the 2-cell stage. Using high-resolution, two-dimensional electrophoresis of metabolically radio-labeled polypeptides, we report the expression of a group of several polypeptides whose synthesis in the 2-cell embryo is due to transcription (i.e., alpha-amanitin-sensitive) and depends on the first round of DNA replication (i.e., aphidicolin-sensitive). We also describe the synthesis of another subset of alpha-amanitin-sensitive polypeptides whose expression does not require the first round of DNA replication (i.e., aphidicolin-insensitive). These results are consistent with the proposed role of the first round of DNA replication in reprogramming the pattern of gene expression.

Regulation of Transcriptional Activity during the First and Second Cell Cycles in the Preimplantation Mouse Embryo

Transcription of endogenous genes in preimplantation 1- and 2-cell mouse embryos was determined by monitoring the incorporation of BrUTP by plasma membrane-permeabilized embryos. Incorporation is observed starting by mid-S phase in the 1-cell embryo and increases progressively; the amount of incorporation by the 1-cell embryo in G2 is about 20% that of the 2-cell embryo in G2. Incorporation by the male pronucleus is always about four to five times greater than that of the female pronucleus. Nevertheless, the amount of incorporation by the female pronucleus present in parthogenetically activated eggs is similar to the total amount of incorporation in inseminated eggs, i.e., the transcriptional capacity of the female pronucleus is not inherently less than that of the male pronucleus. Inhibiting the first round of DNA replication does not prevent the initiation of transcription in the 1-cell embryo, but does inhibit the extent of BrUTP incorporation by 35%. The transcriptional machinery of the 1-cell embryo appears to be rate-limiting, since the total amount of BrUTP incorporation by parthenogenetically activated and dispermic eggs is similar to that in monospermic eggs; trispermic eggs incorporate BrUTP to only about 60% the level of monospermic eggs. A transcriptionally repressive state may start to develop in the 2-cell embryo, since inhibiting the second round of DNA replication results in an 50% increase in BrUTP incorporation. Trapoxin treatment, which induces histone hyperacetylation, enhances incorporation by 2-cell embryos 1.8-fold and suggests that histone hyperacetylation can relieve this repression.

Transient Expression of Translation Initiation Factor eIF-4C during the 2-Cell Stage of the Preimplantation Mouse Embryo: Identification by mRNA Differential Display and the Role of DNA Replication in Zygotic Gene Activation

Zygotic gene activation (ZGA) definitely occurs by the 2-cell stage in the mouse embryo. Analysis of protein synthesis by two-dimensional gel electrophoresis reveals a class of genes whose expression transiently increases in the 2-cell embryo. Although the paucity of biological material has prevented a systematic identification of these genes, the mRNA differential display method circumvents this problem. Using this approach we find a transient increase in the mRNA abundance of the translation initiation factor eIF-4C that is inhibited by α-amanitin and correlated with a transient increase in the relative rate of protein synthesis for eIF-4C. We confirm the transient increase in eIF-4C mRNA abundance by a reverse transcription–PCR-based assay using eIF-4C-specific primers. The first round of DNA replication seems critical for eIF-4C expression, since addition of aphidicolin prior to S phase in the 1-cell embryo inhibits the magnitude of the increase in eIF-4C expression. Aphidicolin treatment also inhibits the synthesis of an accepted marker for ZGA, the transcription-requiring complex (TRC), which is also transiently expressed during the 2-cell stage. Incubating late 1-cell/early 2-cell embryos in medium containing aphidicolin reveals that the second round of DNA replication is not required for the increase in eIF-4C expression but DNA replication is required for the decrease in both eIF-4C expression and TRC synthesis. The decrease in eIF-4C expression, however, does not require cytokinesis or mitosis, since it occurs when 2-cell embryos are cultured in the presence of cytochalasin D or nocodazole, respectively. Changes in chromatin structure may be involved in the decrease in both eIF-4C and TRC expression, since neither decrease occurs when 2-cell embryos are cultured in trapoxin, which is a specific and irreversible inhibitor of histone deacetylase. Results of these experiments suggest that the first round of DNA replication is permissive with respect to ZGA and that the second round is repressive.

Requirements for DNA transcription and replication at the beginning of mouse development

In mice, the first round of DNA replication occurs in fertilized eggs (1-cell embryos), while the onset of zygotic gene transcription begins approximately 20 hours after fertilization, a time that normally coincides with formation of a 2-cell embryo. One approach to investigating the mechanisms that control these developmentally regulated events has been to microinject plasmid DNA into the nuclei of mouse oocytes and embryos in order to determine the requirements for unique DNA sequences that regulate transcription and replication. The results from these and other studies have revealed two important mechanisms that regulate the beginning of animal development. The first is a time dependent "zygotic clock" of unknown detail that delays the onset of transcription, regardless of whether or not a 2-cell embryo is formed. The second is a mechanism that represses the activity of promoters and origins of replication specifically in maternal pronuclei of oocytes and 1-cell embryos, and in all nuclei of 2-cell embryos, regardless of their parental origin or ploidy. This repression is linked to chromatin, but the striking ability to relieve this repression with specific embryo-responsive enhancers first appears with formation of a 2-cell embryo. The need for a TATA-box to mediate enhancer stimulation of promoter activity appears even later when cell differentiation becomes evident. Thus, a biological clock delays transcription until both paternal and maternal genomes are replicated and remodeled from a post-meiotic state to one in which transcription is repressed by chromatin structure in a manner that can be relieved by cell-specific enhancers at appropriate times during development.

The genetics of the Luria-Latarjet effect in bacteriophage T4: evidence for the involvement of multiple DNA repair pathways.

The Luria-Latarjet effect is an increase in resistance of a virus to DNA damage during infection of a host. It has often been assumed to involve recombinational repair, but this has never been demonstrated experimentally. Using nine bacteriophage (phage) T4 mutants, I present evidence indicating that, for phage T4, the Luria-Latarjet effect is due to three repair pathways-excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR) (a second form of recombinational repair). The results also show that the Luria-Latarjet effect develops in two stages. The first stage starts soon after infection. Damage which occurs during the first stage can be repaired by excision repair or PRRR. The second stage appears to start after the first round of DNA replication is complete. DNA damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways. The results of this study support the hypothesis that recombinational repair has been selected to ensure that the progeny phage genomes which are packaged have minimum DNA damage. Since other viruses which infect bacterial, animal and plant cells show a Luria-Latarjet effect similar to that in phage T4, the conclusions from this study may have wide applicability.

Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis

A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990).

Site-directed mutagenesis of virtually any plasmid by eliminating a unique site

We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.