Replication signals in prokaryotic DNA.
Explore the source record for details and available documents.
Biomedical subjects
Publications and source records attributed to G Hobom.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The DNA sequences for the origins of replication of the lambdoid bacteriophages phi80, 434, phi21, and lambdaimm21 (identical to phi21) have been determined and compared to the lambda structure. Two presumptive elaborate binding sites for two initiator proteins have been identified in their outer sections, while a replicational primer start site seems to be located in their centres.
In a pBR313-lambda dv hybrid plasmid system, stepwise deletion and serial cloning procedures have led to a functional dissection of the DNA replication region of lambdoid bacteriophages lambda, 434 and 21. A simple system for initiation of DNA replication has been detected within lambdoid replicator DNAs, which is active in the absence of several normal replication elements, including the origin of replication (ori) and product of gene O. This "minimal" (or "mini") initiation system depends on the p0 or substitute leftward promoter in conjunction with the newly discovered "inceptor" (ice) element, which is located within the cII gene. Even the fragments containing ori are unable to initiate replication in these hybrid plasmids as long as fragments containing ice are missing. The base sequence of ice resembles transcriptional terminators and it appears to control both termination of primer RNA and inception of daughter strand DNA synthesis. Initiation in the p0-ice mini system of lambda or 21 phages requires the gene P product. Hwever, mini replication of 434 DNA hybrid plasmids required neither O nor P proteins, although there are only two single-base changes in the 434 inceptor sequence. The mini system is repressed by the elements of the maximal lambda replication system, as described in the accompanying publication.
In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter). The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication. In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication.
In our hybrid-plasmid reconstruction analysis of lambda (lambdoid) DNA signal structures involved in phage DNA replication, we have detected a dual system alternatingly able to initiate a first primer-RNA synthesis. Both of them--the major, primase-dependent ori system and the minor and usually suppressed, RNA-polymerase-dependent oop system--act in conjunction with a common signal structure for inception of DNA synthesis. It appears that in situations such as this, where one has to deal with the existence of regular as well as backup systems serving the same function, straightforward conclusions are no longer possible in their genetic analysis. For example, even though the oop-DNA segment can be deleted entirely from bacteriophage lambda DNA without disturbing its ability to replicate, it may not be valid to conclude that the oop system has no function in DNA replication. Dual systems of this type or organization in general have also been observed previously for some other replicons such as the R-factors R6-5 and R6K (Timmis et al. 1978; Crosa et al., this volume) or the F factor (Helinski et al., this volume), and they may be more common than presently expected.
A nucleotide sequence comprising 960 base pairs of bacteriophage lambda DNA has been determined. The sequence includes the entire genes of the regulatory proteins cro and cII, and part of the O gene, together with control elements for their transcription and translation. The right-hand boundaries of the lambdaimm434 and lambdaimm21 substitutions and the cy42 mutation have been located.
The sequence of a 123 base pair HpaII restriction fragment of bacteriophage lamdba DNA has been determined by the dimethylsulphate-hydrazine technique. Of this fragment 65 nucleotide pairs are transcribed into the 5' proximal part of the oop RNA. The remaining 58 nucleotide pairs preceding the start point of transcription show homologies to the lamdbaL and lamdbapR promoter regions, and are concluded to contain the lamdbapo promoter sequence.
New techniques for cleaving DNA molecules at precisely defined points, and for successful DNA-transformation of E. coli have allowed genetic engineers to ligate DNA fragments of all possible origins and to convert them into replicating molecules sustained within bacterial cells. For this purpose, typically, segments of eukaryotic DNA are linked to bacterial "vectors", in particular plasmids carring their own replication machinery. This procedure allows to study the genetic organization of eukaryotic DNA and to detect whether or not the eukaryotic genes will function in a foreign environment. Work has just been initiated aiming at the complementary procedure, i.e., at introducing prokaryotic or foreign eukaryotic genes into eukaryotic cells which may carry hereditary defects.
Restriction nucleases from Escherichia coli carrying the resistance transfer factor RI, from Hemophilus influenzae, Hemophilus parainfluenzae, and Bacillus subtilis were used for the generation of specific DNA fragments from lambdadv plasmids. Cleavage maps were established for various plasmids containing different but overlapping parts of bacteriophage lambdaDNA by analysis of digestion patterns obtained in polyacrylamide gel electrophoresis. The correlation between the plasmid cleavage maps and the genetic map of lambda was based upon the location of the EcoRI cleavage site at 81.3% of lambda fractional length and the position of promoters PL and PR on the plasmid DNA fragments.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Spontaneous mutants of the bacteriophage fd and mutants resulting from fd-infected cultures of E. coli grown in the presence of 2.7-diaminofluoren and proflavin were isolated by means of free flow electrophoresis.The amino acid analyses of the mutant coat proteins (B-proteins) show significant differences in comparison with the amino acid analyses of wild type coat protein.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.