Normal expression of the viral gene N interferes with growth of bacteriophage lambda in Escherichia coli 15T-.

Summary Growth of and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T^-. Mutants of which overcome this inhibition map in gene N. Some of these hty mutants are temperature sensitive for growth on E. coli K12. Thus plating of on strain 3057 allows one to isolate temperature sensitive N mutants. The hty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus ^- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate can be at least partially reversed by addition of thymidine to the medium and the growth difference between hty and in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by in 3057 at low thymidine concentration is lower than that produced by hty under the same conditions. Only a small percentage of the DNA produced by in 3057 is packaged into viable phage particles. This suggests that not only produces less DNA in 3057 than hty but that an important part of the DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable to grow on E. coli 15T^- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over N ⁺ which confirms the idea that N activity or the activity of a gene under N control interferes with growth in 3057 at low thymidine concentration.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Physiological relationship between the temperate phages lambda and phi80

Summary This work deals with the ability of phage 80 to provide defective mutants of with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of 80 cannot excise prophage . However, 80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between 80 and . Maturation of DNA by 80. The Ter recombinants excised by 80 from tandem dilysogens are packaged into a 80 protein coat. This contrasts with the fact, already mentionned by Dove, that 80 is extremely inefficient for packaging phage superinfecting a -lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of (80hy4 or 80hy41 — see Fig. 1). However, the maturation of the superinfecting is much more efficient if the 80hy used as a helper has the att-N region of (like 80hy1). Conversely a with the att-N region of 80 (hy6 — see Fig. 1) is packaged more efficiently by 80 or 80hy4 than by 80hy1. It is suggested that the maturation of chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by 80. That N mutants are efficiently helped by 80 does not tell that 80 provides the defective with an active N product; the chromosomes are simply packaged into a 80 coat. This shows that 80 is unable to switch on the late genes of . That neither 80 nor any of the 80hy tested can provide an active N product is shown in a more direct way by their complete failure to help N ^-r₁₄; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a N ^- by 80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by 80. This suggests that the products coded by these genes are not interchangeable between 80 and , and that packaging of DNA into 80 coats is possible but inhibited when late proteins are present in the cell. Activation of the Late Genes. Among the im ₈₀ h ⁺ hybrids tested, only 80hy41 is able to switch on the late genes of a N defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of 80 and of the 80hy1 and 80hy4 to activate the late genes of would imply that these phages are unable to provide an Q product active on the chromosome Reciprocally, switches on the late genes of prophage 80hy41, but not of prophages 80hy1 and 80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

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Ce travail a été réalisé dans le cadre du contrat d'association Euratom-U. L. B. 007-61-10 ABIB et avec l'aide du Fonds de la Recherche Fondamentale Collective.     

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Summary Intermediates of DNA replication in the second half of the latent period after phage infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative froms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the genome. Replicating circles with a linear DNA tail longer than one unit length of genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

Host specificity of DNA produced by Escherichia coli. XII. The two restriction and modification systems of strain 15T-

Summary E. coli 15T^- carries two distinct sets of DNA restriction and modification activities. The genetic information for system A is contained in the bacterial chromosome and linked to the thr region. This fact suggests host specificity A to be related to those of strains K and B. The genes controlling system 15 are on a plasmid which is related to phage Pl: it competes with Pl for stable inheritance in the carried state and it genetically recombines with Pl. This recombination may produce plasmid genomes with newly assorted characters (see Table 3). One of them is an active, Pl-like prophage with the 15-specific instead of the parental Pl-specific restriction and modification characters. Superinfection of 15T^- with Pl may also result in curing of the bacteria from the restriction plasmid.Bot A- and 15-specific restrictions and modifications act on bacterial DNA, on the DNA of various sex factors and on the DNA of certain bacteriophages, e.g. of phage . Phage 82 DNA is sensitive only to 15-specific restriction, but not to A-specific restriction.Independently of the A- and 15-specific restrictions, the growth of phage in E. coli 15T^- encounters another limitation of yet unknown nature. No such limitation is observed either with phage 82 or with mutants of occurring at a frequency of about 10^-5.     

Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Role of SSB protein in RecA promoted branch migration reactions

Summary The RecA protein ofEscherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981a). In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.Abbreviations SSB single strand binding protein - ssDNA single stranded DNA - X phage X174 - bp base pairs - ATP[S] adenosine 5-O-(gamma-thiotriphosphate)     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Studies on radiation-sensitive mutants of E. coli. II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda

Summary It has been shown that linear DNA molecules of phage are converted to the twisted circular structure (species I) by covalent closure of the both strands at the cohesive ends after infection to the immune bacteria and that the twisted circular molecules are transformed to the circular form (species II) by a single-strand break in one of the strands of their DNA. This system offers a very sensitive method to study on the strand breaks or their repair. For characterization of the defects of ultraviolet sensitive strains, the structural changes of ultraviolet irradiated DNA in these strains were studied.Ultraviolet irradiation to phage greatly reduced the extent of conversion of the molecules to the species I in the uvrD mutant while the irradiation showed little effect on the conversion in the uvrA, B and C mutants. When infected bacteria carrying species I molecules were irradiated, the species I molecules in the uvrD mutant were disrupted while most of the molecules in the uvrA, B and C mutants kept the structure. These results indicate that in the irradiated DNA strand breaks are rarely introduced or, if introduced, repaired rapidly in the uvrA, B and C mutants and they are introduced in the uvrD mutant leading to the degradation of the DNA. These results provide a firm evidence that the defect of the uvrD mutant is different from other Her^- mutants and in the process of repair synthesis.Ultraviolet irradiation to the uvrD mutants promote the formation of the species I molecules from the infected irradiated -DNA.Such effect was not observed with the uvrA mutant. Since the uvrD mutant has UV reactivation capacity and the uvrA mutant has not, the above phenomenon is probably caused by UV reactivation and may provide a more direct method to study the mechanisms of UV reactivation than the plaque assay.Abbreviations used UV Ultraviolet light - UVr Ultraviolet light reactivation This work was aided in part by a research grant GM 08384 from the United States Public Health Service.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

A mutation in Escherichia coli enhancing the UV-mutability of phage lambda but not of its infectious DNA in a spheroplast assay

Summary A mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage . The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage . However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated -DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.The mutation responsible for the mul phenotype has been mapped by conjugation and transduction to be located between the markers pyrE and ilv and probably marks a new gene. The mul mutation does not increase the UV-sensitivity of the cells nor does it affect their ability to perform host cell reactivation. The presence of a recA allel in a mul mutant abolishes the UV-mutability of phage .Part of this work is from the thesis of the first author in partial fullfilment of the requirements for the doctoral degree of the University of Bochum. Some of the results have already been presented at the 32. Kongreß für Hygiene und Mikrobiologie at Münster, 1969.     

On the mode of antirepressor action in anti-immune cells

Summary The mode of antirepressor action in anti-immune cells was analysed in respect to its two main features, low lysogenization and high cell killing. By means of complementation experiments between and i434 in anti-immune cells, it was shown that the antirepressor no longer channels phages towards lysis in such cells if the genes which are needed for lysogenization are provided in trans by the heteroimmune phage i434. Since complementation could be demonstrated, it was possible to exclude that direct action of the antirepressor over repressor production is responsible for the feature under analysis. It was also shown that both int- and cII-product are required to improve lysogenization and to prevent high levels of killing.Recipient of an EMBO predoctoral fellowship.     

A transducing lambda phage carrying grpE, a bacterial gene necessary for lambda DNA replication, and two ribosomal protein genes, rpsP (S16) and rplS (L19).

Summary A grpE mutation of Escherichia coli K12, which blocks DNA replication of the phage (Saito and Uchida, 1977), was mapped at 56 min on the standard genetic map. A transducing phage, grpE22, carrying the wild type allele of the grpE gene was constructed in vitro. Structures of grpE22 and its viable deletion derivatives were determined by electron microscopic analyses of appropriate heteroduplexes. Proteins coded by the bacterial DNA incorporated into the transducing phages were detected by two-dimensional gel electrophoresis. The results showed that the product of the grpE gene is a weakly acidic protein of molecular weight 24,000. Structural genes for two ribosomal proteins, rplS (L19) and rpsP (S16) were also shown to be carried by grpE22.     

UV-sensitivity and UV-mutability of infectious lambdaDNA: reactivation, protection and mutability in various assay systems

Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca⁺⁺-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr^- spheroplasts and cells, resp., and since survival is also identical in Ca⁺⁺-treated Hcr⁺ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr⁺ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca⁺⁺-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca⁺⁺-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul ^-, could not be induced in DNA when assayed with spheroplasts or Ca⁺⁺-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.