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J Kur

Publications and source records attributed to J Kur.

62 records · Page 4Linked to original sources
PubMed1990

The role of the direct repeat in qut-controlled antitermination in phage lambda.

For antitermination of transcription from the late p'R promoter of phage lambda, a cis-acting qut sequence, which overlaps with p'R, is required, together with the product of lambda gene Q. Using our BspMI-mediated multicycle technique for generation of precise deletions, we have confirmed that deletions removing DNA downstream of +18 bp (counted from the p'R-controlled transcriptional start point s'R = +1) do not affect the efficiency of qut antitermination; at the same time we found that deleting one more bp (shifting the right-hand boundary to bp +17) reduces antitermination by only 20%. Deleting another 5 or 6 bp (+11 or +12 bp right-hand qut boundary), decreases antitermination by about 80%. These deletions reduce the 9/10-bp-direct repeat (5'-TGGGT(A or T)AATT)2 in qut to only the five italicized bp. Similar strong reduction in antitermination (by about 68%) was obtained with +16 bp qut boundaries, in constructs which also contained 2- or 3-bp insertions between bp +11 and +12 or between +12 and +13. Since the latter deletions retain only 5/10 bp of the direct repeat, it appears that antitermination is dependent on the length and intactness of the direct repeat.

Bacteriophage lambda↗
PubMed1989

An MboII/FokI trimming plasmid allowing consecutive cycles of precise 1- to 12-base-pair deletions in cloned DNA.

A novel trimming plasmid has been designed which allows, in a preprogrammed fashion, the precise deletion of up to 12 bp per cleavage cycle, from one end of a cloned fragment. The plasmid, which carries the dhfr gene, contains unique recognition sites for two class-IIS restriction enzymes, MboII and FokI, which are arranged in the form of a cassette, so that consecutive cleavages with these endonucleases, followed by blunting with mung bean nuclease (MB), will precisely delete 12 bp of adjacent cloned DNA. When either MboII or FokI is used alone (followed by MB), 1 or 4 bp are removed, respectively. The final step in the trimming cycle is religation of the plasmid with T4 ligase. After required number of cycles, plasmids were transformed into Escherichia coli C600, and transformants selected by resistance to trimethoprim. Since the MboII/FokI cassette remains intact during these operations, one can repeat the cycle, consisting of cleaving, MB blunting and religation, several times, each time removing up to 12 bp from the cloned target DNA. Examples are provided of one-, two- and three-cycle trimmings.

Base Sequence↗
PubMed1989

Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.

Bacterial Proteins↗
PubMed1989

Repression of transcription from the b2-att region of coliphage lambda by integration host factor.

The central b2-att region of coliphage lambda is known to be transcriptionally active in vitro, but silent in vivo in lambda lysogens. To explain such in vivo repression of transcription originating in the b2-att region, we explored the effect of the Escherichia coli integration host factor (IHF), the product of E. coli genes himA and himD, especially since the att region contains several IHF-binding sites. Using various lambda DNA templates, we mapped the transcripts which are initiated in vitro in the attP region by the RNA polymerase and found that there are three rightward (RI, RII, and RIII) and one leftward (LI) transcripts. All four of them are repressed by a factor of about 10 by 10 micrograms IHF/ml. Moreover, in in vivo experiments we found that plasmids carrying the attP fragment cannot be established and maintained in IHF-hosts. These results indicate that IHF may play a significant auxiliary role in repressing transcription in the prophage state.

Attachment Sites, Microbiological↗
PubMed1987

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda.

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.

Bacterial Proteins↗
PubMed1987

Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme.

The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector. The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level. Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.

Bacteriophage lambda↗
PubMed1981

ADP-ribosylation of proteins in non-infected Escherichia coli cells.

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-(3)H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radioactive group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them.

Adenosine Diphosphate Ribose↗