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We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBCsbcB-host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, XPpA10, and in a 2. (7,8). The stability of palindromic structures, artificially constructed in plasmid (9) and X DNA (10), has also been studied. It has been found that propagation of these sequences is permitted only in bacterial hosts lacking both exonucleases I and V, the products of the sbcB and recBC genes, respectively (for reviews, see refs. 11 and 12). It has been shown by electron microscopy (13, 14) that in singlestranded Physarum DNA, "foldback" structures can be observed after intramolecular reassociation; these structures are regularly spaced throughout the genome. Here we present evidence that palindromic structures in Physarum DNA may cause low plating efficiencies of a genomic Physarum library in the X replacement vector EMBL3 on Rec' E. coli hosts and that these structures also may cause instability of actinrelated sequences obtained from this library that are cloned either in phage X or the plasmid pBR322. MATERIALS AND METHODSConstruction of the Genomic Library. DNA was isolated from nuclei of axenically grown Physarum amoebae, strain Cld-Axe (15). The nuclei were lysed in 50 mM Tris Cl, pH 8/0.1 M EDTA/proteinase K (100 gg/ml)/0.5% N-lauroylsarcosine for 1 hr at room temperature. High molecular weight DNA was obtained by phenol extraction, RNase treatment, and extensive dialysis against 10 rnM Tris Cl, pH 8/1 mM EDTA (16). Electrophoresis in a 0.4% agarose gel revealed a major DNA band at -100 kilobases (kb) and a minor band at 60 kb, which probably represents the intact linear, extrachromosomal ribosomal DNA of Physarum. The DNA was partially digested with restriction endonuclease Mbo I, and 15-to 20-kb fragments were isolated from a sucrose gradient (1). DNA from bacteriophage EMBL3 was digested with BamHI and EcoRI, and the small linker fragment was removed by isopropanol-precipitation (17). The Physarum DNA fragments were then ligated to the BamHI sites of the phage arms in a 1:4 molar ratio. The ligated DNA was packaged in vitro according to Mullins et al. (18) or with an in vitro packaging kit (Amersham).Isolation and Characterization of Bacteriophage and Plasmid DNA. Bacteriophages were prepared from cultures of infected E. coli strains LE392 and CES200 in L-broth, and DNA was isolated as described (19,20 mapping by multiple digestion were done as described (23,24). For DNA blotting and hybridization we followed the procedure of Southern (25) with modifications (3...

The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ard, ardC, ardO) are duplicated laVy, corresponding to the first 10% of the genome. The fourth locus (ard4) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various tinmes during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.It has been clearly demonstrated, both by cytological methods and density-labeling, that certain segments -of the eukaryotic genome replicate at defined time intervals in S phase of the cell cycle (1, 2) in lower eukaryotes like Physarum (3) as well as in human cells (4). Heterochromatin is generally, found to replicate late in S phase (5-7), whereas certain potentially active genes (8, 9) replicate early. Employing electron microscopic spreads of chromatin, we have recently visualized actively transcribing genes in early-replicated DNA of Physarum. Typically, both coding strands of a newly replicated locus are transcribed in early S phase (10). A relationship between the onset of DNA replication in S phase and transcription has been documented for yeast (11), Drosophila qmbryos (17), and tissue culture cells (13,14) (16,18), the evidence for the temporal order of DNA synthesis (3), and tfhe preferential transcription of newly replicated DNA in early S phase (10, 15), prompted us to determine the chronology of replication of the four actin loci.In this communication we demonstrate that three of the four actin loci are replicated at the time when only 5-10% of the genome has replicated. The synchrony of DNA replication at the gene level is such that two allelic DNA sequences of one actin locus are coordinately replicated. The fourth actin locus is replicated when DNA synthesis is 75% completed. Moreover, this temporal order of replication remains invariant, when monitored either through two consecutive S phases in a single macroplasmodium or over a period of at least 400 generation times in suspension culture. The significance of this fixed order of replication is discussed in light of the replication-transcription-coupling hypothesis. MATERIALS AND METHODSCultures. The strain Tu 291, provided by F. Haugli (Tromso University, Norway), was used throughput this investigation. Obtained by crossing stains RSD4 by'RSD8 amoebae, it is 'a diploid derivative of the Wis 1 natural isolate (19). The synchronous cultures (macroplasm...

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.