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R H Plasterk

Publications and source records attributed to R H Plasterk.

103 records · Page 6Linked to original sources
PubMed1985

The invertible P-DNA segment in the chromosome of Escherichia coli.

In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region. This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences. The element e14 contains a gene which kills the host cell. This can be repressed by a function also coded by e14. The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element. The invertible segment and pin gene were sequenced. The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment. Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly). The complete DNA sequences of both invertible segments were screened for homology. No resemblance was found. The P segment is flanked by inverted repeat sequences of 16 bp. Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region. This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.

Bacterial Proteins↗
PubMed1984

Analysis of the methylation-regulated Mu mom transcript.

The Mu mom gene is transcribed only when DNA is methylated by Dam; this methylation dependence is determined by a region upstream of the gene containing three Dam-methylation sites. The mom operon contains two open reading frames, one of which codes for Mom. Experiments were done to find a function for the other reading frame; it is shown that it does not affect phage immunity or the regulation of mom expression. Deletion mapping and S1 nuclease mapping experiments identify the +1 of the mom mRNA: it is 45 bp downstream of the three Dam-methylation sites. Possible models for the short-distance transcription regulation by methylation are discussed. The mom promoter is surrounded by sequences that are shown to be homologous to the oriC region of E. coli. This suggests a relation between replication and methylation-dependent mom expression. It is shown however, that replication is not essential for mom expression.

Bacteriophage mu↗
PubMed1984

A genetic switch in vitro: DNA inversion by Gin protein of phage Mu.

Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.

Bacteriophage mu↗
PubMed1983

Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator.

The product of the bacteriophage Mu gene mom modifies adenine residues of DNA within the consensus sequence CGAGCNPy, providing protection against various restriction endonucleases (ref. 1 and D. Kamp, personal communication cited in ref. 2). The mom gene is only expressed during lytic development of the phage. It is known that mom is nonfunctional in Escherichia coli host mutants in a gene (dam) which itself encodes an adenine methylation system. We show here that the E. coli dam gene is essential for transcription initiation of the mom gene, and that this dependence on dam seems to lie in a short segment preceding the mom coding region, which also contains the mom promoter. The sequence of this segment reveals the presence of dam methylation sites (GATC), and suggests a model for the regulation of mom gene expression based on DNA secondary structure, which may explain why mom is only expressed during phage lytic development. We also show that expression of phage-coded proteins (A, B and C) is needed for transactivation of mom transcription.

Bacteriophage mu↗
PubMed1983

Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions.

A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the PL-promotor of phage lambda. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of E. coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus inversions can be detected or selected as a switch from Lac- to Lac+. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.

Bacterial Proteins↗
PubMed1983

DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems.

The gene product of bacteriophage Mu gin catalyzes a 3,000-base-pair inversion in the DNA of the phage, thus changing its host range. In some strains of Escherichia coli there is a function that can complement Mu gin mutations. This function (pin) was cloned and shown to catalyze an inversion of 1,800 base pairs in the adjacent E. coli DNA (P region). pin- derivatives carry the P region frozen in the (+) or (-) orientation. The function of the switch is not yet clear. The sequences of gin and pin were determined; they exhibit 70% homology. The sequences around the recombination sites of Gin and Pin are also largely homologous; a consensus sequence is derived for the recombination sites of Gin and Pin, and of Hin in Salmonella typhimurium. The amino acid sequences of Gin, Pin, Hin, and TnpR are compared, and the evolutionary relationship between these prokaryotic site-specific recombination systems is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Transposition of structural genes to an expression sequence on a linear plasmid causes antigenic variation in the bacterium Borrelia hermsii.

In Borrelia hermsii, a spirochaete that causes relapsing fever, the switch between expression of two frequent variable major protein (VMP) types (7 and 21) is associated with a DNA rearrangement. Both cell types 7 and 21 contain untranscribed 7 and 21 VMP genes on linear plasmids. The serotype 7 cells contain an additional copy of the 7 VMP gene fused to an expression sequence on another linear plasmid. Switching to the 21 serotype involves removal of the transcribed 7 VMP gene and fusion of a copy of the 21 VMP gene to this same expression sequence. Thus recombination between linear plasmids can activate different VMP genes.

Bacterial Proteins↗