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H Schmieger

Publications and source records attributed to H Schmieger.

At least 37 records · Page 2Linked to original sources
PubMed1990

Isolation of fragments with pac function for phage P22 from phage LP7 DNA and comparison of packaging gene 3 sequences.

Three PstI DNA fragments of the P22-related Salmonella phage, LP7, have been cloned. They contain sequences recognized as pac signals by the packaging apparatus of P22. One of these fragments corresponds to the P22 DNA fragment carrying gene 3 which comprises the pac signal of phage P22. The product of gene 3, Gp3, is involved in the recognition of pac and the packaging process. Gene 3 of LP7 and most of the adjacent gene 2 have been sequenced. The pac analogous segments of the other two PstI fragments have been narrowed down by subcloning and by transduction of the resulting hybrid plasmids under recombination-defective conditions.

Amino Acid Sequence↗
PubMed1990

A molecular analysis of terminase cuts in headful packaging of Salmonella phage P22.

Fragments of DNA molecules of Salmonella phage 22 which represent the molecular termini created by the terminase reaction have been cloned and sequenced. The terminase cleavage separates a headful-sized piece of DNA from the concatemeric precursor; by successful cloning strategy it was shown that the terminase produces blunt ends. The termini of 20 different phage DNA molecules fall into a region located between about 600 and 4000 bp from the pac signal and show a Gaussian distribution. The average terminal redundancy was calculated to be about 2230 bp (= 5.3%) and is therefore higher than was previously reported. A comparison of the nucleotides flanking the terminal bases of 20 different end clones does not support the suggestion that the terminase recognizes some specific sequence and/or structural information in determining the actual cleavage site.

Base Sequence↗
PubMed1990

Cloning and expression of packaging gene 3 of Salmonella phage P22.

Packaging genes 3 of Salmonella phage P22 wild type and two mutants with altered packaging properties (HT12/4 and NT1/1) have been cloned in an expression vector. By plasmid transduction, it has been shown that the amino terminus of gene 3 is not functional in DNA packaging when fused with the Escherichia coli lacZ gene. The reconstituted genes 3, however, express functional gp3. The transduction experiments also have shown that the pac signal, which is part of gene 3, is intact in all three phages. Expression of gene product gp3 has been demonstrated in the minicell system.

Base Sequence↗
PubMed1987

In vitro assay of packaging protein gp3 of Salmonella phage P22.

The packaging proteins, gp2 and gp3, of Salmonella phage P22 were separated by DEAE-sephacel column chromatography. The activity of both proteins was measured by complementation in an in vitro phage packaging assay. For the first time gp3 activity was demonstrated in vitro. The majority of the gp3 activity could be separated from gp2. While gp2 was stable during chromatography and storage, gp3 was very unstable. However, it was stable when stored as a crude extract. The ratio and interaction of these two proteins critical for phage maturation are still uncertain.

Chromatography↗
PubMed1986

Spot-transformation with plasmids.

A method is described which allows transformation of bacterial cells on the surface of agar plates. It is suitable for transferring large numbers of different plasmids, such as gene libraries, into a new genetic background. One nanogram of plasmid DNA is sufficient for transformation.

Escherichia coli↗
PubMed1986

Selection of bacterial pac sites recognized by Salmonella phage P22.

A gene library of chromosomal PstI fragments from Salmonella typhimurium strain DB5575 has been established. By means of phage P22 mediated transduction, ten different clones which contained inserts that promoted plasmid transduction were selected out of a total of about 7,000 clones. Seven of these clones carried inserts that stimulated transduction independently of general and int-promoted recombination and were interpreted as carrying pac analogous signals. The remaining three clones carried inserts that promoted transduction under recombination proficient conditions, whereas transduction occurred at reduced rate in the absence of recombination. These were believed to have short regions of homology with P22 DNA.

Cloning, Molecular↗
PubMed1985

In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro.

In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length.

DNA Transposable Elements↗
PubMed1984

pac sites are indispensable for in vivo packaging of DNA by phage P22.

F' pro+ plasmids were selected and used as donors to prepare P22 transducing phages. Two types of result were observed. pro+ from type I donors cannot be packaged by wild-type P22 to yield transducing particles unless a prophage pac site is introduced into the plasmid. Transposon Tn10 also allows initiation of packaging. pro+ from type II plasmids can be transduced with the same efficiency as pro+ DNA on the chromosome, indicating that a chromosomal pac site was included when the F' pro+ was excised from the Hfr strain. The usefulness of type I plasmids as a test substrate for pac signals is discussed.

Chromosome Deletion↗
PubMed1984

Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22.

Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).

Cloning, Molecular↗
PubMed1984

In vitro packaging of mature phage DNA by Salmonella phage P22.

Mature, headful-sized DNA extracted from the Salmonella phages P22 and L, and P22/L-hybrid phages can be encapsulated in vitro by means of a packaging system for exogenous DNA. The probability of packaging reaches about 10(-3) per headful-sized molecule. The absence of in vitro recombination was demonstrated, to eliminate the possibility that such a process had created concatemers. The endonucleolytic cut at the pac site, which initiates sequential packaging in vivo, does not occur with the mature DNA substrate in vitro. The position of pac on the molecule is not important but the pac-recognizing phage protein gp3 is indispensable for in vitro encapsulation.

DNA, Viral↗
PubMed1982

The packaging initiation site of phage P22. Analysis of packaging events by transduction.

P22 lysates were grown on Salmonella strains carrying P22 prophages deleted to various extents. Transducing bacterial markers at both sides of the prophage insertion site it could be shown that: (i) transduction of markers can be enhanced by the prophage pac site; (ii) the recognition signal pac is in the area of gene 3 on the phage genome and thus close to the cutting site(s); (iii) transposon Tn10 may also act as a signal for packaging initiation; (iv) (at least) Tn10 initiates packaging sequences in both directions.

Genes, Viral↗
PubMed1982

Packaging signals for phage P22 on the chromosome of Salmonella typhimurium.

Numbers of transducing particles for a set of 28 different chromosomal markers were determined in a lysate of Salmonella phage P22. The results were plotted with regard to the map positions of the transduced loci. Maxima of the resulting curve are interpreted as positions of start signals ("pac-sites") for packaging series on the Salmonella chromosome. 5-6 pac-sites could be found as a minimum estimate.

Chromosome Mapping↗
PubMed1981

The influence of host DNA replication on the formation of infectious and transducing Mu-particles.

We have investigated the influence of bacterial DNA replication on the formation of infectious and transducing Mu-particles. The data obtained agree with the previous findings that growth of phage Mu is independent of the host dnaA gene product (Toussaint and Faelen 1974), but required bacterial replication forks (Fitts and Taylor 1980). The replication of transducing DNA during phage development (Teifel and Schmieger 1979) is controlled by the host and is not a precondition for its packaging. Packaging of transducing DNA does not require a a nearby Mu genome.

Bacteriophage mu↗
PubMed1980

Growth of Salmonella bacteriophage P22 in Escherichia coli dna(Ts) mutants.

Salmonella bacteriophage P22 grows in two deoxyribonucleic acid initiation mutants of Escherichia coli under nonpermissive conditions, dnaA and dnaC. Functional products of genes dnaE, dnaZ, lig, dnaK, and dnaG are indispensable for deoxyribonucleic acid replication of P22. In 11 E. coli dnaB mutants belonging to all phenotypic groups, phage were produced at 42 degrees C.

Bacterial Proteins↗
PubMed1979

The origin of the DNA in transducing particles of bacteriophage Mu. Density gradient analyses of intact phages.

The origin of DNA in transducing particles of bacteriophage Mu was investigated by density labelling techniques. Unlabelled plaque-forming and leu+-transducing particles were of about the same density. Preinfection labelling of DNA with 5-bromodeoxyuridine increased the density of the transducing particles, but not that of the infective ones. Postinfection labelling increased the density of the infective particles twice as much as that of the transducing particles. We conclude that half of the transducing DNA is synthesized before infection and half is synthesized after infection, similar to the results obtained with P1kc transducing phages (Ikeda and Tomizawa, 1965).

Bacteriophage mu↗
PubMed1979

Replication and maturation of phage P22 in a mutant of Salmonella typhimurium temperature sensitive in initiation of DNA replication.

TB37 is a dna A-mutant of Salmonella typhimurium in which the initiation of DNA replication at the origin is stopped at 42 degrees C. DNA synthesis in uninfected cells of this strain and in cells infected by phage P22 was followed by the pulse labelling technique. DNA replication ceases completely after about 50 minutes at the high temperature. After lytic infection with P22 (c2) at this time, DNA synthesis starts immediately and increases at a rate well comparable to the permissive control. Obviously the temperature sensitive function of the dnaA-product is dispensable for P22 DNA replication, especially for its initiation. This result is confirmed by the normal yield of phage particles under these conditions, provided that a late step in P22 maturation which naturally is temperature sensitive can proceed at low temperature. If TB37 is infected at 42 degrees C with P22 wild type, an unexpected high rate of phage controlled DNA synthesis is observed. Preliminary results seem to indicate that the process of integration is a prerequisite for part of this synthesis.

DNA Replication↗