Wolfgang Oertel

Electrophysiological pattern of involuntary limb movements in the restless legs syndrome

Patients with restless legs syndrome (RLS) suffer from involuntary limb movements during the day. We studied these leg movements in 18 idiopathic (n = 8) and uremic (n = 10) patients at rest. Electromyographically measured muscle contractions were preceded by sensory discomfort in all patients. The mean duration of the contractions ranged between 0.67 and 5.71 s with a mean frequency of 244 epochs of muscle activity per hour. Seven of 18 patients showed a constant order of recruitment with propagation of muscle activity up or down spinal segments (L3 to S1 and vice versa). No difference in electrophysiologically recorded patterns was observed between patients with idiopathic and uremic RLS. We suggest a brainstem disinhibition phenomenon as the pathological mechanism that activates a spinal generator. The spinal origin of the involuntary limb movements in patients with RLS is confirmed by the long duration of jerks, the recruitment characteristics, and the periodicity of the jerks. No jerk could be elicited by sensory reflexes.

Reflex studies and MRI in the restless legs syndrome

Brainstem and spinal pathways of untreated patients with idiopathic restless legs syndrome (RLS) were examined using magnetic resonance imaging (MRI), blink reflex, first and second exteroceptive suppression (ES1, ES2) of temporalis muscle, and H reflex. MRI of 25 patients elicited no structural lesions beyond age‐related atrophy or white matter lesions on proton density‐ and T2‐weighted coronal and axial images. All patients showed a normal latency of the soleus H reflex (mean·SD latency=31.22·2.81 ms) and the H/M ratio was 48·17%. The duration and onset latency of the direct and indirect blink reflex responses were normal in all patients compared with those of controls (p>0.5). There was no significant difference in ES1 and ES2 latencies or duration between patients and controls (p>0.5). These results suggest that the etiology of RLS symptoms does not involve structural lesions.

Isolation and characterization of the minimal fragment required for autonomous replication (“Basic replicon”) of a copy mutant (pKN102) of the antibiotic resistance factor R1

SummaryThe mini plasmids deriving from pKN102, a copy mutant of the antibiotic resistance factor R1drd-19 of E. coli, share a common DNA sequence of 2.6 kb, which carries the minimal functions for autonomous replication. By cloning of two PstI fragments of this region it could be demonstrated that the “basic replicon” is a DNA segment not larger than 1.8 kb, which carries the origin of replication and the genetic information for at least two proteins. Protein F (MW=11.000 dalton) seems to be synthesed in larger amounts in minicells of E. coli than protein C (20.000 dalton). Plasmids containing this isolated replicon of R1 are completely compatible with the parental plasmid R1drd-19.

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19

SummaryThe recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170–200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possibility that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.

Steele-richardson-olszewski-syndrome: the relation of dopamine D2 receptor binding and subcortical lesions in MRI

Summary. We compared 123I-iodobenzamide single photon emission computed tomography (IBZM-SPECT) for imaging of striatal dopamine D2 receptors in vivo, and MRI in 32 patients with the clinical diagnosis of progressive supranuclear palsy (PSP). We found a significant inter-dependence of reduction of specific striatal IBZM binding indicative of striatal degeneration and of the absence of multiple signal hyperintensities in MRI; age had no influence neither on IBZM binding nor on signal hyperintensities. We conclude that the presence of multiple signal hyperintensities should raise doubt on the correct clinical diagnosis.

Structure and mitotic stability of minichromosomes originating in yeast cells transformed with tandem dimers of CEN11 plasmids

SummaryLarge (10.5–13.5 kbp) circular minichromosomes containing the centromere of chromosome 11 (CEN11) and the MET14 gene of Saccharomyces cerevisiae in the YRp7 vector are considerably more stable during mitosis than smaller ones containing only the 1.6 kbp CEN11 SalI-fragment. Yeast transformants obtained with a tandem dimeric and thus dicentric form derived from this DNA varied in the mitotic stability of the TRP1 marker of the vector. The largest group of transformants contained minichromosomes which carried deletions located quite specifically at one of the two centromeres in the dimer, eliminating its function in mitosis. This group included also some minichromosomes which had been modified by intramolecular tandem amplification of the subunit carrying the deletion without losing the centromere within the unmodified subunit. The second major group carried minichromosomes which had been monomerized. Monomerized minichromosomes showed the relative low degree of mitotic stability typical for the original minichromosomes containing the 1.6 kbp CEN11 SalI-fragment. Increasing numbers of additional subunits carrying the TRP1-ARS1 sequences but lacking additional centromeres improved the mitotic stability considerably.

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

SummaryThe recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.

A new approach to the sequence analysis of DNA

For the analysis of structure and function of DNA it would be of advantage, if short defined segments of the macromolecule could be copied in vitro. DNA polymerase I is able to initiate DNA synthesis at internal sites on a single-stranded DNA template, if oligonucleotides are offered as primers [ 1, 21. Recent experiments indicated that the initiation reaction may be sequence specific, since a high template specificity was found when oligonucleotides were used as primers which were long enough to anneal stably fo a template DNA [3,4]. In the present communication we show that DNA polymerase I can initiate DNA synthesis at a single site on a phage DNA template, if a unique base complementary oligonucleotide is offered as a primer. Fd DNA minus strands were used as template, the polypyrimidine tract C9Tll from the fd plus strand [S] as primer. Pulse-labelled reaction products of various size were characterized by fingerprinting techniques, which allowed to deduce the sequential arrangement of the polypyrimidine tracts in the nucleotide sequence following the 3’ terminus of the primer nucleotide.

Short nascent DNA pieces, accumulating in Saccharomyces cerevisiae after inhibition of DNA chain elongation, hybridize to specific chromosomal sites

SummaryIn temperature-sensitive DNA chain elongation mutants (cdc8ts) of Saccharomyces cerevisiae even at the restrictive temperature a small portion of DNA is synthesized, which can be labeled by radioactivity. Under denaturing conditions this product sediments in alkaline sucrose gradients with about 4S. It is probable that these short nascent DNA pieces are derived predominantly from newly activated origins of replication. An alternative and more direct method of limiting the elongation of DNA chains uses araCMP as an inhibitor in nucleoside monophosphate-incorporating yeast strains. As with the cdc8ts mutants the only radioactive products in labeling experiments with [32P]dTMP are 4S pieces. Potential sites of their formation are small replication “bubbles” and terminal doublestranded loops observed in electron micrographs of the DNA from araCMP-inhibited cells. The pieces hybridize specifically with the replication origin of the 2 μm-plasmid, the chromosome telomeres and a group of chromosomal genes. Other genes and the centromere of chromosome 11 (CEN11) do not react. The 4S pieces hybridize with only three of nine cloned autonomously replicating sequences (ARS). It is concluded that ARS sequences, at least in the presence of araCMP, are not always used as replication origins within their normal environment on the chromosome.