Heinz Schuster

The reaction of tobacco mosaic virus ribonucleic acid with hydroxylamine

The pyrimidine bases of tobacco mosaic virus RNA react with hydroxylamine under rather mild conditions. By using the nucleosides and nucleotides of the corresponding bases the reaction mechanism with hydroxylamine was clarified. Uridine was split into ribosyl-urea and 5-isoxazolone. Cytidine only added hydroxylamine presumably to the C 4 –C 5 double bond, readily converting the C 6 amino-group to the keto-group. In the RNA eytosine at pH 6 reacted with hydroxylamine at least 30 times faster than uracil did, whereas at pH 9 uracil reacted at least 8 times faster than cytosine. Thus the reaction could be controlled to give the rather specific base elimination or alteration without splitting the polynucleotide chain. The applications of the findings for studies with infectious nucleic acid are discussed.

The inactivating and mutagenic action of hydroxylamine on tobacco mosaic virus ribonucleic acid

Abstract Inactivation of tobacco mosaic virus ribonucleic acid by hydroxylamine at pH 6.1 and 9.1 followed first-order kinetics. Treatment at pH 6.1 (alteration of cytosine) led to mutation, whereas treatment at pH 9.1 (elimination of uracil) seemed to have only a lethal effect. The intact viruses of tobacco mosaic virus (TMV) vulgare and TMV strain U2 were resistant to the action of hydroxylamine.

The c4 repressors of bacteriophages P1 and P7 are antisense RNAs.

The c4 repressors of P1 and P7 inhibit antirepressor synthesis and are solely responsible for heteroimmunity of the phages. We show that c4 is a new type of antisense RNA acting on a target, ant mRNA, that is transcribed from the same promoter. Interaction depends on complementarity of two pairs of short sequences encompassing the ribosome binding site involved in ant expression. We demonstrate that heteroimmunity of P1 and P7 is due to just two substitutions in each of the complementary sequences of c4 and ant mRNA. Based on P1-P7 sequence comparison and a mutant analysis, we propose a secondary structure model for c4 RNA, with the complementary regions in loops as important sites for antisense control.

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12.

SummaryA mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage λ is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

SummaryIn E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages λ, P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages λ and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage λ is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.

Replication Determinants of the Broad Host-Range Plasmid RSF1010

RSF1010 is a small (8.7 kb), multicopy plasmid of the incompatibility group IncQ that confers resistances to streptomycin and sulfonamide to its host cells (1). It is very similar or identical with the 2 other representatives of the IncQ group, R300 (2) and R1162 (3). One of the most striking properties of this plasmid is its extraordinary broad host-range among gram-negative bacteria. This feature has made it attractive as a replicon for construction of vectors for gene cloning outside of Escherichia coli species. Both general- and special-purpose cloning vectors, derived from RSF1010, have proved very useful for gene manipulation in different species of soil bacteria (for review, see Ref. 4,5).

Die Basenspezifität bei der Induktion von Mutationen durdh salpetrige Säure im Phagen T2

In phage T2 treated with nitrous acid (HNO2) the deamination rates of the amino-bases G, A and HMC can be shifted relative to each other by altering the pH of treatment. Under various pH conditions the variation of the rates of HNO2-induced r- mutation and inactivation have been measured and compared to the corresponding alteration of the deamination rates. In this way it was shown, that the deamination of the bases A and HMC can lead to mutagenic changes, whereas the deamination of G can only act lethally. Furthermore it was estimated, that the deamination of any one of 370 base pairs of the rII-region give rise to a mutation. Deamination of ∼1/4 of the bases within the total T2-DNA lead to inactivation.

The antirepressor of phage P1 Isolation and interaction with the C1 repressor of P1 and P7

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in‐frame start codon. Another open reading frame, icd, 5′ upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene‐carrying plasmids we have overproduced and purified Ant½ in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N‐terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N‐terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co‐precipitate with the Ant½ complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1‐inactivating function by a direct protein—protein interaction.

The fate of newly made DNA in Escherichia coli mutants thermosensitive in DNA synthesis

SummaryE. coli mutants exist in which DNA synthesis is thermosensitive. In one class of these mutants DNA synthesis stops immediately if a critical temperature (42°C) is reached. When DNA replication in such mutants is followed by 3H thymidine incorporation at 33°C, it is found that 1. only the newly made DNA is degraded at 42°C, 2. the discontinuously replicated DNA is lost predominantly at 42°C, 3. 1–3% of the chromosomal DNA is rendered acid soluble at 42°C without concomitant loss of viability of the cells at 33°C.Replication of phage λ DNA is inhibited in the same mutant at 42°C. However, when DNA synthesis is followed in λ infected cells at 33°C it is found that 1. no degradation of λ specific DNA seems to occur at 42°C in the early phase of infection, 2. replicating λ DNA molecules in the late phase of infection are completed at 42°C before DNA synthesis comes to a halt.

Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor.

The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis. The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene. We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22. Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced. On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.