The structure and function of the replication initiator protein (Rep) of pSC101: an analysis based on a novel positive-selection system for the replication-deficient mutants.

Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.

Open strands adjacent to iterons promote the binding of the replication initiator protein (Rep) of pSC101 to the unit sequence of the iterons in vitro.

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.

A single-stranded region located downstream of the repeated sequence in the ori region of pSC101 is required for binding of the Rep protein in vitro.

Purified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.

Exonuclease III promotes in vitro binding of the replication initiator protein of plasmid pSC101 to the repeated sequences in the ori region.

Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S. et al. (1990) J. Biochem. 107, 369-376). We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli. In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep. On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor. We purified extensively the factor and identified it as exonuclease III (exo III). Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA. This binding of Rep to duplex DNA treated with exo III is DR-sequence specific. Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.

Acute antihypertensive effect of nifedipine by sublingual route in cases with clinically severe systolic hypertension. A study up to 4 h after administration.

In a multi-center study, nifedipine (Bay a 1040, Adalat) (10 mg capsule) was administered, in liquid form and via sublingual route, to 22 cases who were diagnosed to have clinically severe systolic hypertension, and the depressor effect of the treatment was studied over a period of 4 h. In patients of "emergent" admission (cerebral hemorrhage n = 8, cerebral thrombosis n = 4, subarachnoid hemorrhage n = 1, renal failure n = 6, essential hypertension n = 3), 3 bouts of blood pressure measurement at intervals of 10-15 min during the control period were carried out. In case of systolic blood pressure higher than 200 mmHg, at least one time, nifedipine at a dose of 10 mg was sublingually administered. Thereafter, blood pressure and pulse rate were recorded at intervals of 15 min up to the end of the first one hour, then at intervals of 30 min up to the end of 4 h. Results were as follows. In terms of the average values for all cases, blood pressure fell to near lowest levels by the end of about 30 min after the administration and it stayed at near lowest levels up to the end of 120-240 min. During this period of time, pulse rate remained substantially unchanged. In terms of the pattern of the blood pressure fall, all cases could be classified generally into two types, namely (a) the "dip" group (7 cases) in which blood pressure fell to the lowest level to form a "dip" and remained below the control level, although it showed a trend to return to control level for 4 h and (b) the "flat" group (15 cases) in which blood pressure declined gradually for about 1 h and, then, remained low or below the control level, although it showed a trend to return to control level throughout the rest of the observation period of 4 h.

Origin of sodium ions appearing in the venous blood plasma during acute venous congestion and hemorrhagic hypotension: a study on kidneys and skeletal muscle of dogs.

We studied the origin of sodium ions which appeared in a higher concentration in the venous blood plasma obtained from the renal or femoral vein, than in the arterial blood plasma during acute renal venous congestion, acute venous congestion of hindlimbs and acute hemorrhagic hypotension. We measured the sodium concentration in the blood plasma as well as in hemolyzed blood obtained with sonication. In addition, sodium contents of the kidneys and skeletal muscle were determined. All experiments were performed on anesthetized mongrel dogs. Sodium contents of the kidneys and skeletal muscle of hindlimbs, in terms of milliequivalent per gram dry tissue weight, was decreased significantly 30-40 min after exposure of the kidneys and hindlimbs to acute venous congestion, or after exposure of hindlimbs to hemorrhagic hypotension, indicating a release of sodium ion into the blood stream. The sodium ion concentration became slightly higher in the venous blood than in the arterial blood, in terms of both blood plasma and hemolyzed blood, during 15-30 min of exposure of hindlimbs to local venous congestion or hemorrhagic hypotension, again indicating a release of sodium ion into the blood stream. However, acute renal vein congestion caused the sodium ion concentration to become slightly higher in the venous blood than in the arterial blood, only in terms of blood plasma, and not in terms of hemolyzed blood, indicating that red blood cell sodium is the origin of sodium ions which appeared in the renal venous plasma during acute renal vein congestion.