[Is the replicon model applicable to higher eukaryotes?]. / Le modèle du réplicon est-il applicable aux eucaryotes supérieurs?

Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication. In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication. This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms. The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes. In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication. Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle. This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought. In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes. In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins. Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs. Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.

Inter-species DNA polymerase delta chimeras are functional in Saccharomyces cerevisiae.

The catalytic subunits of DNA polymerase delta of Schizosaccharomyces pombe and Saccharomyces cerevisiae share over 50% identity. The capability of S. pombe DNA polymerase delta to complement two thermosensitive mutants of S. cerevisiae was studied in vivo and it was determined that complementation was allele dependent. However, DNA polymerase delta from S. pombe did not restore growth of a S. cerevisiae strain containing a disrupted chromosomal copy of the POL3 gene that encodes DNA polymerase delta. To identify the regions of DNA polymerase delta responsible for species-specific interactions, we constructed different chimeras with S. cerevisiae and S. pombe DNA polymerase delta genes. The growth of a S. cerevisiae strain with a disrupted chromosomal POL3 gene was studied after transformation with plasmids expressing different chimeras. A 1254-bp region located in the 3' region of the S. cerevisiae POL3 gene is responsible for species-specific functions.

DNA polymerase delta is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe.

DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome. The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control. Deletion of the chk1/rad27 gene abolishes the radiation but not the replication feedback control. Thermosensitive mutations in the DNA polymerase delta, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle. We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27. We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1+/rad27+ pathway, which monitors directly DNA for signs of damage.

Peduncular 'rubral' tremor and dopaminergic denervation: a PET study.

Lesions causing so-called rubral tremors frequently involve the substantia nigra or the nigrostriatal fibers, suggesting dopaminergic denervation as possibly contributory. We examined this hypothesis using PET and [18F]-fluorodopa in six patients with a contralateral tremor following a peduncular lesion. The denervation revealed by PET was even more marked than in severe parkinsonian patients. All patients showed partial to complete improvement with levodopa therapy. PET evaluation of D2-receptors with [76Br]bromolisuride showed no asymmetry of the D2 binding despite the important asymmetry of 18F-fluorodopa uptake. Our results indicate an important involvement of the nigral dopaminergic system in peduncular tremors that appears to be independent of postsynaptic dopamine receptors.

Brain metabolism after recurrent insulin induced hypoglycaemic episodes: a PET study.

Neuropsychological testing was carried out and the rate of oxygen metabolism in the brain was measured by PET in 15 highly selected patients with type 1 diabetes. The aim was to investigate the impact on the brain of hypoglycaemic comas resulting from insulin treatment. No significant difference was found between nine patients with a history of more than 10 hypoglycaemic comas and six others who denied any history of such events. These data suggest that intensified insulin treatment, although increasing the frequency of hypoglycaemic coma, may not always be harmful for the brain. This may be explained by the limited duration of hypoglycaemic coma induced by conventional insulin treatment.

In vivo phosphorylation, mitotic behavior, and nuclear binding of the catalytic subunit of DNA polymerase alpha in fission yeast.

We studied the phosphorylation of fission yeast p170 (the catalytic subunit of DNA polymerase alpha) and its relationship to the cell cycle. In exponentially growing cells, p170 was phosphorylated at serine residues. Its phosphorylation level did not quantitatively change when cell strains carrying conditional cell division cycle (cdc) mutations arrested at different stages of the cell cycle, under restrictive growth conditions. Especially, phosphorylation did not significantly vary when cells carrying the temperature-sensitive cdc2-33 mutation were shifted to the restrictive temperature, which indicates a minor role, if any, of p34cdc2 in this process. Also, the extent of p170 phosphorylation did not remarkably change during mitosis, a situation which differs from that reported for human DNA polymerase alpha. We used immunofluorescence microscopy and cell fractionation to study the intracellular distribution of p170. We here provide evidence that the protein remains tenaciously associated with nuclear structures throughout the cell cycle and is not redistributed into the cytoplasm at mitosis, as it is in human cells. A possible correlation between phosphorylation, nuclear binding, and mitotic behavior of DNA polymerase alpha catalytic subunits in eukaryotes is therefore conceivable.

[Positron-emission tomographic study of the dopaminergic system in a case of secondary unilateral tremor after mesencephalic hematoma]. / Etude du système dopaminergique en tomographie par émission de positons dans un cas de tremblement unilatéral secondaire à un hématome mésencéphalique.

A 30 year-old woman developed a postural and rest tremor of the left hand following a right peduncular post-traumatic hematoma. Two years later, positron emission tomography showed a marked decrease in [18F] fluorodopa uptake contrasting with a normal [76Br] bromolisuride uptake in the right striatum. This suggests that: 1) chronic unilateral dopaminergic striatal denervation may occur without persistent D2 dopaminergic receptor upregulation in humans; and 2) symptomatic mesencephalic tremor may be, at least in part, related to dopaminergic striatal denervation.

DNA polymerase alpha in the fission yeast Schizosaccharomyces pombe: identification and tracing of the catalytic subunit during the cell cycle.

A recombinant protein was obtained in Escherichia coli by subcloning part of the Schizosaccharomyces pombe POL1 gene at the 3'-end of lacZ. Antibodies raised against this protein were used to identify the POL1 gene product in extracts of exponentially growing S. pombe cells. A major 170-kDa protein, whose structure and properties were typical of the catalytic subunit of eukaryotic DNA polymerases alpha (pol alpha), was detected. The same antibodies were used to trace pol alpha and to quantify its level during the S. pombe cell cycle. We found that pol alpha was present at all stages of the cycle and that its cellular pool was subject to limited (three-fold) increase in G1 and S phases, with a decline to the initial level soon after. In addition, we found that a second form of pol alpha with slightly lower molecular weight (165 kDa) existed only during late G1 and S phases. Moreover, absence of initiation or perturbations in the course of DNA replication induced overproduction of the 165-kDa form.

Expression of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe.

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe. Okadaic acid treatment of S. pombe spheroplasts in amounts known to inhibit phosphatases 1 and 2A resulted in decreased proteolysis of both pol alpha and pol delta. Computer analysis of pol alpha and pol delta sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy of S. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol alpha produced a clear increase of the nuclear signal, no increase was detectable in cells transformed with pol delta. This observation suggests the existence of a mechanism limiting the cell concentration of pol delta in the cell. Constitutive expression of S. pombe pol delta in E. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol delta on E. coli growth.

Characterization of the POL3 gene product from Schizosaccharomyces pombe indicates inter-species conservation of the catalytic subunit of DNA polymerase delta.

The Schizosaccharomyces pombe POL3 gene was isolated by sequence homology with a region of the Saccharomyces cerevisiae POL3 gene, the only gene sequenced to date encoding the catalytic subunit of eukaryotic DNA polymerase delta. The fission yeast POL3 gene contains a 52 base-pair (bp) intron and encodes a 3600 bp transcript the 5'-end of which is located 32 bp upstream from the initiation codon. The polypeptides predicted from budding and fission yeast POL3 genes share 52% of conserved amino acid residues and have a 60% identical central region. This structural conservation of the catalytic subunit of DNA polymerases delta is probably related to functional constraints. A portion of the most conserved region was used to raise antibodies against an S. pombe polymerase delta/beta-galactosidase fusion protein expressed in Escherichia coli. The purified antibodies recognized a 123,000 Da protein in S. pombe wild-type cell extracts and inhibited an aphidicolin-sensitive DNA polymerase activity that was distinct from DNA polymerase alpha. The antibodies also detected a 140,000 Da protein in extracts from different proliferating mammalian cells, indicating that the catalytic subunits of DNA polymerase delta are highly conserved between yeast and higher eukaryotes.

The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha.

The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 bp intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160,000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.

Identification of a gene encoding the predicted ribosomal protein L7b divergently transcribed from POL1 in fission yeast Schizosaccharomyces pombe.

A 0.85 Kb RNA molecule is transcribed in the region upstream from the 5'-end of the S. pombe POL1 gene encoding the catalytic subunit of DNA polymerase alpha. The nucleotide sequence of the DNA region hybridizing with the 0.85 Kb transcript allowed us to identify an open reading frame coding for a predicted peptide which shows 50% identity with the rat ribosomal protein L7 and which is transcribed divergently from POL1. We have named this gene RPL7b because of the existence in S. pombe of a different sequence, named RPL7, which also codes for a putative protein showing homology with the rat ribosomal protein L7. The RPL7b gene includes a 291 bp-long intron containing the sequences necessary for intron excision and RNA splicing in S. pombe. The precise location of the intron was established by amplification and sequencing of a partial cDNA copy of the mRNA, whereas the initiation site of transcription was determined by reverse transcription of the 5' region of the mRNA. The 320 bp separating the starting methionine codons of RPL7b and POL1 genes should contain the signals necessary for their divergent transcription and regulation. The sequence 5'-AAGACAGTCACA-3', whose primary structure is homologous to a conserved block present in the 5'-untranscribed regions of other S. pombe genes of ribosomal proteins, is located about 50 bp upstream the transcription initiation site of RPL7b.

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction.

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.