Comparison of theoretical denaturation maps of phiX174 and SV40 with their gene maps.

When the theoretical denaturation maps of phiX174 and SV40 are compared with their gene maps, it is observed that the beginning and the end of each gene in these two DNA's fall in a region of lower melting temperatures. Local (A+T)-contents evaluated from the known sequences at these regions support the above implication that the beginnings and the ends of nearly all the genes in phiX174 and SV40 are relatively rich in (A+T)-content.     

Denaturation maps of DNA: experimental and theoretical maps of phiX174 DNA.

A formaldehyde denaturation map of the replicative form of phiX174 DNA is obtained. The RFI DNA was converted into a linear state by restriction endonuclease pst I which introduces into this DNA a single double-stranded break. The map has four clear-cut peaks. Their positions excellently correlate with the peak positions on the map of equilibrium denaturation theoretically obtained earlier from the known nucleotide sequence of phiX174 DNA. The sequence is also used for a calculation of the maps of smoothed AT-content. The maxima on these maps correlate well with the peaks on the denaturation maps. To reveal the causes of a good correlation between the experimental formaldehyde and theoretical equilibrium denaturation maps, the theoretical formaldehyde denaturation maps are calculated for different conditions (temperature, formaldehyde concentration) using the detailed theory of DNA interaction with formaldehyde developed earlier.     

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.     

A graphical representation of genetic and physical maps: the Marey map.

A novel, simultaneous, visual representation of sex-specific genetic maps and physical maps is introduced. Such maps, called Marey maps, provide direct comparisons of multiple genetic maps and elucidate the relationship of recombination frequency to physical distance.     

Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Bacteriophage phiX174 growth in an Escherichia coli dnaIts mutant, KS810.

A bacteriophage phiX174-sensitive Escherichia coli dnaIts mutant, KS810, was constructed and growth of phiX174 in the cells was investigated. phiX174 and phiX174am3trD could grow normally at 43 degrees C as well as 27 degrees C, therefore we conclude that the growth of bacteriophage phiX174 is not dependent upon the host dnaI gene product.     

Dimeric circular duplex DNA of bacteriophage phiX174 and recombination

Summary Bacteriophage X174 replicative from DNA (RF DNA) was formed in the presence of chloramphenicol at a concentration of 40 g per ml and isolated at 12 and at 55 min. after infection. The component I RF DNA (double stranded covalently closed and twisted form) was separated and divided into a monomer and multimer (dimer) fraction.The frequency of recombinants found after phage formation in the chloramphenicol treated cells and that found after spheroplast infection with the monomer molecules both increase with the time of RF formation. However, the frequency of recombinant molecules among the dimers remained constant. This finding is explained by the hypothesis that two separate mechanisms act in X174 recombination, one of which is restricted to the formation of dimers.Irradiation with UV of phage prior to infection showed that the frequency of recombinants in monomers increased, as the recombination frequency of phage after (a single) growth (step) did, but that neither the frequency of recombinant molecules in dimers is raised, nor the frequency of dimers. Using a recombination negative host the frequency of recombinant dimer molecules was three to fourfold decreased, whereas the frequency of dimers was only slightly lower (relative to the normal host). These results support the hypothesis mentioned above and moreover lend support to the view that the greater part of the dimers is not formed by recombination events.     

Deletions in bacteriophage P2. Circularity of the genetic map and its orientation relative to the DNA denaturation map

Several types of viable chromosomal deletions of bacteriophage P2 were isolated. One type gives the immunity insensitive phenotype and may extend to the genes for the immunity repressor (C) and for integrative recombination (int). Two other types delete genes (old and fun) known to be active in the lysogenic state. For such deletion mutants the relationship between particle density and DNA length was established. The deletions were located in respect to previously mapped genes and the results were compared with electron microscopical studies (by Inman and collaborators) of the P2 chromosome. It is concluded that the best representation of the genetic map of P2 is circular. The cohesive ends of the linear P2 DNA molecule are most likely formed between genes old and Q. Except for the neighborhood of gene old, the previously published, linear genetic map of P2 (Lindahl) is colinear with the melting map of the P2 chromosome (Inman). Preliminary evidence for some specific recombination event often accompanying integrative recombination between phage chromosomes is presented.     

New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map

An F1 mapping population was bred by crossing an accession of wild cardoon with a single Argentinian globe artichoke plant of the variety Estrella del Sur FCA with a view to generating new Cynara cardunculus linkage maps. Genotyping was conducting using a set of 553 SRAP, SSR, AFLP and SNP markers. The 1,465.5 cM map based on the segregation of alleles present in the wild cardoon parent comprised 214 loci distributed across 16 linkage groups (LGs), while the 910.1 cM globe artichoke-based map featured 141 loci falling into 12 LGs covering the total length. Three of the morphological traits (head spininess, leaf spininess and head color) for which the parents contrasted were inherited monogenically, and the genes conditioning them were mapped. A set of 48 co-dominant loci was used to align the LGs with those derived from a reference SSR-based consensus map of the species.     

DNA heteroduplex analysis of the relation between bacteriophages phiX174 and S.13

When observed in the electron microscope using the isodenaturing methods of Davis &; Hyman (1971), only one small segment (4.7 ± 1.9%) of the DNA of phage φX174 is highly homologous with phage S.13 DNA; the rest is partially homologous with an over-all average 36% base mismatch. The two phage DNA molecules appear to be identical in length and have no regions of complete base non-homology. The phage-coded proteins were compared by electrophoresis on slab polyacrylamide gels and only one of the S.13 coded proteins migrated identically with its φX174 counterpart. The other eight S.13 coded proteins varied in size from their φX174 counterparts by +4.6% to ?6.0% (± ten amino acid residues). The relevance of these data to the complementation and recombination between these two phages is discussed.     

Excision repair of uracil during replication of phiX174 DNA in vitro.

Each of the stages in the replication of ØX174 DNA $\text{in vitro}$, e. g., conversion of circular single stranded parental DNA to the duplex replicative form (SS → RF), replication of the closed circular duplex form (RF → RF), and synthesis of circular single stranded progeny DNA (RF →SS), may be affected by a reduced level of dUTPase. Thus, in enzyme preparations from mutant strains defective in dUTPase ($\text{dut}$^?), the complementary strand synthesized in the SS → RF reaction is abnormally short (7–8S vs. 14S), and the extent of RF replication is decreased 10-fold. Preferential removal of dUTPase during fractionation of enzyme preparations from wild type ($\text{dut}$⁺) cells may produce comparable effects. In particular, the single stranded circular DNA synthesized in the RF → SS reaction by a set of highly purified enzymes is rapidly degraded upon incubation with the less pure enzymes required for its conversion to RF. All of these effects are plausibly accounted for by the incorporation into DNA of uracil from dUTP, possibly present as a contaminant in one or more components of the reaction, followed by excision of the uracil and phosphodiester bond cleavage at the resulting apyrimidinic site.     

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.