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Biomedical subjects

S Molin

Publications and source records attributed to S Molin.

At least 109 records · Page 6Linked to original sources
PubMed1985

Stable inheritance of plasmid R1 requires two different loci.

The largest EcoRI fragment from plasmid R1 mediates a stability phenotype which is required to ensure the stable inheritance of this low-copy-number plasmid. When covalently linked to small, unstable R1 derivatives, this fragment makes the plasmids as stable as the wild-type R1 plasmid. A genetic analysis showed that two independently acting stabilization functions are encoded by this EcoRI fragment, both of which have the potential of partial stabilization of mini-R1 plasmids. The two loci are located at opposite ends of the fragment. Stabilization was also obtained by inserting these regions in unrelated, unstable plasmids from the p15 group. One of the two functions was very efficient in stabilizing such foreign replicons. Besides the stability phenotype, these genes exert incompatibility in an allele-specific manner. The stability functions do not seem to interfere seriously with the copy number of the plasmid.

Cloning, Molecular↗
PubMed1985

Two functions of the E protein are key elements in the plasmid F replication control system.

By using a plasmid carrying a translational fusion between the E gene of the IncFI plasmid F and the lacZ gene, we located the operator of the autogenously regulated E gene to an inverted repeat overlapping the E-gene promoter and showing perfect homology to part of the sequence found in all the direct repeats of two regions exerting an inhibitory effect on F replication, incB and incC. Excess E protein provided in trans to an F plasmid increased the replication frequency of the F plasmid. This stimulatory effect was counteracted by increased dosages of incB or incC. A model is proposed for the replication control system of F in which the key elements are autoregulation of E-gene expression and titration of E protein by incB and incC.

Bacterial Proteins↗
PubMed1984

Regulated expression of a gene important for replication of plasmid F in E. coli.

Fusions between the gene encoding the E protein of the IncFI plasmid F and the lac genes were constructed. Analysis of the expression of beta-galactosidase from these fusions shows that the promoter for the E protein gene is located between the incB region and the structural gene for the E protein. Near this promoter is a regulatory site on which a negative control element acts. Most likely the E protein itself acts as a repressor of E gene expression and thus autoregulates its own expression. No other gene products seem to affect the expression of the E protein gene.

Bacterial Proteins↗
PubMed1984

Copy mutants of plasmid R1: effects of base pair substitutions in the copA gene on the replication control system.

Five different copA copy mutants of plasmid R1 have been identified by nucleotide sequencing. Independent measurements of the activities of the mutant inhibitor RNA and of the mutant target properties were carried out using several different methods. Correlation of these measurements with the location of th nucleotide substitutions resulted in the following conclusions: (1) The copy number of plasmid R1 is controlled primarily by interaction between the CopA RNA molecule and its target, the RepA mRNA. (2) The binding of th inhibitor to its target is based on nucleotide interactions within two complementary sequences of ten nucleotides and dependent on the secondary structure of the active site. (3) The secondary structure of both the CopA target and the CopA RNA is a stem-loop structure. Mutations in the loop region interfere with binding affinity between inhibitor and target, whereas mutations in the upper stem mainly interfere with secondary structure. Mutations in the latter region create temperature-dependent copy number phenotypes.

Base Sequence↗
PubMed1984

How the R1 replication control system responds to copy number deviations.

The copy number of R1 and of a repA-lacZ gene fusion was increased above normal by coupling to a plasmid which is present at a fivefold higher copy number at 30 degrees C. This carrier plasmid is deficient in replication at 42 degrees C, and it was thus possible after a temperature shift to analyze the response to the increased plasmid concentration of the R1 replication control system. Both the frequency of replication per plasmid molecule and the rate of repA expression per gene copy were reduced under these conditions, and the data strongly suggest that there is an inverse proportionality between the specific rate of plasmid replication viz repA expression and the copy number/gene dosage of the plasmid.

DNA Replication↗
PubMed1984

Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system.

Plasmids are autonomously replicating DNA molecules that are present in defined copy numbers in bacteria. This number may for some plasmids be very low (2-5 per average cell). In order to be stably inherited, replication and partitioning of the plasmid have to be strictly controlled. Plasmids carry genetic information for both processes. In the present paper we summarize what is known about the replication control system of one low-copy-number plasmid, R1, belonging to the FII incompatibility group. We do so because the FII group seems to be one of the best understood examples with respect to genetics, molecular biology, and physiology of the replication control system. The paper is not a classical review, but rather an essay in which we discuss the aspects of replication control that we regard as being important.

Bacterial Proteins↗
PubMed1984

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter.

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.

DNA Replication↗
PubMed1983

Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule.

Fusions between the repA gene of plasmid R1 (required for autonomous plasmid replication) and the lac genes have been the basis for in vivo studies of regulation of repA expression. Two gene products--the CopA RNA and the CopB protein--act as inhibitors of repA expression. Comparison of the effects of addition in trans of the two Cop functions on transcription and translation of repA-lac gene fusions show that the CopB protein represses transcription of the repA gene, whereas the CopA RNA interferes with the RepA mRNA in such a way that effective translation is inhibited. The CopA RNA does not seem to have a direct effect on the transcription of the repA gene but, as a consequence of the posttranscriptional regulation, transcriptional polarity within the repA gene is observed. It is also shown that the CopA RNA interacts with its target (CopT) only when the region is transcribed to form RepA mRNA.

Artificial Gene Fusion↗
PubMed1982

Convergent transcription interferes with expression of the copy number control gene, copA, from plasmid R1.

The copy number control gene, copA, of plasmid R1 codes for an 80-nucleotide untranslatable RNA. In Escherichia coli minicells, some copA hybrid plasmids and R1 miniplasmids also express a transcript of 200 nucleotides. Only the small RNA mediates the CopA phenotype. The switch between 80- and 200-nucleotide RNA synthesis is shown to be caused by convergent transcription; if transcription proceeds in both directions the inactive larger RNA is synthesised; the active small RNA is formed when copA transcription is not opposed by transcription from the other direction. The data presented indicate that convergent transcription interferes with copA expression by abolishing or reducing normal copA transcription termination.

Journal Article↗
PubMed1982

The sites of action of the two copy number control functions of plasmid R1.

Two negatively acting functions - the CopA-RNA and the CopB protein - are involved in the control of replication of plasmid R1. They both act as inhibitors of expression of a gene, repA, which seems to be positively required for autonomous plasmid replication. Here we show that the two control functions act separately and independently. The CopB protein represses initiation of transcription of the repA gene, and its target site lies within a 60 base pair region containing the repA promoter. The CopA-RNA acts downstream of the repA promoter in the leader sequence containing the copA gene itself, preceding the repA structural gene. Measurements of RepA-beta-galactosidase expression from wild-type and a copA mutant fusion hybrid in the presence of extra copies of the respective copA genes show that a point mutation affecting the activity of the CopA-RNA can also affect CopA target properties. It is therefore concluded that the target site for the CopA-RNA resides within the copA gene in a small region encoding the loop of a stem-loop structure in the CopA-RNA. In addition, the data indicate a direct nucleic acid-nucleic acid interaction as the basis for the CopA inhibitor activity.

Bacterial Proteins↗
PubMed1982

Expression of a copy number control gene (copB) of plasmid R1 is constitutive and growth rate dependent.

The copy number control gene copB from plasmid R1 was fused to the lacZ gene in vitro, resulting in expression of a fused polypeptide consisting of the first 53 amino acids of the CopB polypeptide and the beta-galactosidase polypeptide minus its first 8 amino acids. Based on measurements of specific activities of this fused protein under various conditions, it was concluded that expression of copB is gene dosage dependent, unregulated by plasmid-coded functions, and proportional to growth rate between 0.4 and 2.0 doublings per h. The rate of expression of the copB gene is surprisingly high compared with other known cases of regulatory proteins.

Bacterial Proteins↗
PubMed1982

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.

Cloning, Molecular↗
PubMed1981

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19.

The region of plasmid R 1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wild-type R 1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.

Base Sequence↗
PubMed1981

Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control.

Site-specific deletions and insertions in the replication region of plasmid R1 have generated a new class of copy mutants that are present in the cell with 10-15-fold increased copy number. All mutations described inactivate a copy number control gene which is distinct from another cop inc gene that was identified previously (Molin and Nordström 1980). Insertion of the lac operon lacking the normal lac promoter has been used to determine the direction of transcription of this cop gene. The mutants may all be complemented by wild-type plasmid derivatives and are thus recessive. In incompatibility tests with wild-type R1 plasmids, these mutants are indistinguishable from the wild-type plasmid. It therefore seems that this cop function does not play an important role for the incompatibility function. A polypeptide, molecular weight 11,000, has been identified as being the product of this cop gene.

Bacterial Proteins↗
PubMed1981

RNAs involved in copy-number control and incompatibility of plasmid R1.

Replication of plasmid R1 is controlled by the products of two genes, copA and copB, that act as inhibitors of replication. Here it is shown that one small RNA synthesized from the copA gene acts as replication inhibitor. This RNA molecule was identified from analyses of RNAs synthesized in EScherichia coli minicells carrying R1 miniplasmids or chimeric plasmids containing the copA gene. In minicells, This RNA was found to be unstable with a half-life of less than a few minutes. Two mutant hybrid plasmids lacking the inhibitor function did not express the RNA normally made from plasmids carrying the wild-type copA allele. Nucleotide sequence analysis of one of the copA mutants showed that a base substitution had occurred within the promoter sequence in front of the copA gene. DNA sequence analysis of the other mutant showed that a putative transcription-termination sequence was affected. The DNA sequence analysis also showed that the RNA molecule synthesized from the copA gene is untranslatable but has the potential for a high degree of secondary structure.

Base Sequence↗
PubMed1980

Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region.

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol., 118, 1123-1131).

Cell Division↗