Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops.

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.

An architectural role of the Escherichia coli chromatin protein FIS in organising DNA.

The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.

Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.

The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.

Muscle fiber conduction velocity at different states of isotonic contraction.

Conduction velocity (CV), relative twitch force (RTF) and contraction time (CT) of single muscle fibers (SF) and small muscle fiber bundles (FB) were measured at different states of isotonic contraction with double impulse stimuli at varying interstimulus intervals (ISI) from 0 to 1000 ms in the biceps brachii muscle in vivo. During an isotonic contraction, muscle fibers conducted the action potential on average 0.18 m/s faster along the muscle fiber membrane than did relaxed muscle fibers. This difference was statistically significant (P < 0.001). There was a significant positive correlation between the degree of isotonic contraction and fiber bundle conduction velocity (FBCV) with a first peak at the maximum RTF at an ISI of 9 ms and a second peak at the maximum CT at an ISI of 100 ms.

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the 'heavy strand' is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have shown that 25 orfs are essential. Of the remaining orfs, functions could be predicted for the products of five of the orfs on the basis of comparison of the deduced amino acid sequence to known proteins. Intergenic regions include most of the 5 PE and the 4 PL promoters. Transcripts are polycistronic. Transcription from the PE promoters is mediated by host RP, whereas recognition of the PL promoters requires an additional unidentified phage-encoded product. Translation of mRNA transcribed from most of the orfs seems to be initiated independently, each from its own ribosomal binding and initiation site, although a few cases of coupled translation have been reported. The organization of SPP1 genes involved in the replication, DNA packaging and phage assembly proteins resembles the organization of genes of equivalent regions of different E. coli double-stranded DNA phages. Absence of aa sequence similarity between analogous proteins of different phages suggested that the conserved gene organization is representative of a primordial bacteriophage.

Isometric muscle contractions after double pulse stimulation. comparison of healthy subjects and patients with myotonic dystrophy.

Isometric contractions of the adductor pollicis muscle were studied in healthy subjects and patients with myotonic dystrophy after single and double stimuli of the ulnar nerve using a wide range of interstimulus intervals (ISI, 0.4-180 ms). In healthy subjects, the force contributed by a second stimulus was greater than the single twitch force being maximal (mean + 140%) at 12-ms ISI. In myotonic dystrophy, the force contributed by the second stimulus was (relative to a reduced twitch amplitude) increased (mean + 204%) with a maximum at 4.8-ms ISI. An abnormal increase of force was only recorded if the single twitch force was clearly reduced. The absolute refractory period of muscle contraction (normal range 1.2-1.6 ms, mean 1.35 ms) was shortened in all patients (mean 1.01 ms) except one (1.2 ms). The ISI showing the maximal force were related to those showing the maximal prolongation of the contraction time in healthy subjects (r = 0.71) but not in patients. The rate of force development contributed by a second stimulus was slower than expected from the summation of two single twitches with short stimulus intervals (3-40 ms) a phenomenon called early depression. In patients, the early depression was reduced or abolished within this range of ISI as has been found in dystrophic mouse muscles. The optimal ISI in patients was shifted towards very short times and together with the other disturbances it is suggested that activation of diseased muscle by motoneurons may be less effective, being an additional factor leading to weakness in myotonic dystrophy.

Nucleotide sequence and complementation studies of the gene 35 region of the Bacillus subtilis bacteriophage SPP1.

Genetic analysis of the Bacillus subtilis bacteriophage SPP1 defective in gene 35 shows that the gene 35 product (G35P) is essential for SPP1 growth. The defect in growth of SPP1tsl17 and SPP1tsl20F at nonpermissive temperature is overcome by wild-type gene 35 expressed from a plasmid. The region where gene 35 maps was cloned and sequenced. Analysis of the nucleotide sequence (5884-bp) around gene 35 revealed 13 open reading frames (orfs). We have assigned the term gene to three of these orfs; gene 35, gene 36, the product of which shares homology with SSB proteins, and gene 38, given the gene order orf 34-orf 34.1-orf 34.2-orf34.3-orf34.4-gene 35-gene 36-orf 36.1-orf 37-orf 37.1-orf 37.2-orf 37.3-gene 38. Gene 35 encodes a protein of 32.0 kDa. By using the T7 promoter-expression system for gene 35 a radioactive band of the expected molecular mass was detected.

Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination.

A recN- (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF, recH and addAB genes, when present in an otherwise Rec+ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline-1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10(2)- to 10(4)-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.

Intramolecular homologous recombination in Bacillus subtilis 168.

Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B. subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene. Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions. These results are consistent with the hypothesis that B. subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Characterization of Bacillus subtilis recombinational pathways.

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.

Characterization of recF suppressors in Bacillus subtilis.

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.

Genetic analysis of rec E activities in Bacillus subtilis.

A recE mutant (recE6) of Bacillus subtilis was constructed by insertion of a selectable marker into the recE coding region. The insertional inactivation of the recE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. The recE6 allele was then introduced into a set of DNA repair-deficient strains of B. subtilis. The removal of DNA damage by the recF, addA addB, recH, recL and recP gene products is strictly dependent on an active recE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in the uvrA42 recE6 and polA5 recE6 strains suggests that such lethal lesions may be removed either by the recE-dependent or by the recE-independent pathway.

A computerized re-evaluation of the collision technique.

A computer program is described for the collision technique by which data acquisition and data interpretation is possible. Paired stimuli with varying interstimulus times evoke a muscle action potential. The amplitude or the rectified area integral as a function of interstimulus times was used to define the scatter of conduction velocities within the ulnar nerve. In normal and slightly distorted EMG signals the amplitude and area produces the same scatter. In distorted potentials the integrated area is more sensitive. The technique was furthermore used to separate two different age groups of normal persons. Significant differences were seen as well as in an age matched group of patients with myotonic dystrophy. The collision technique and the computer program proved to be sensitive and applicable in routine examinations.

Characterization of recombination-deficient mutants of Bacillus subtilis.

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.

Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication. We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology. Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction. Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.

Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage.

Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient.

Habituation of the blink reflex: computer assisted quantitative analysis.

The electrically evoked blink reflex (BR) was elicited in 20 healthy volunteers, varying the stimulus interval by a computer JN 110. Latencies, amplitudes, reflex duration and reflex areas of early and late components of the BR were analysed by a computer program testing the signal-to-noise ratio, the response configuration and frequency distribution. Changes in habituation related to the stimulus interval can be illustrated best by the progressive diminution of R2 areas. Computing the habituation in relation to the stimulation frequency by a standardized program seems to be a quantitative method to assess habituation in various diseases and during treatment studies.