Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Theoretical Basis of Protocols for Seed Storage III. Optimum Moisture Contents for Pea Seeds Stored at Different Temperatures

In previous work, we demonstrated that there was an optimummoisture level for seed storage at a given temperature (Vertucciand Roos, 1990), and suggested, using thermodynamic considerations,that the optimum moisture content increased as the storage temperaturedecreased (Vertucci and Roos, 1993b). In this paper, we presentdata from a two year study of aging rates in pea (Pisum sativum)seeds supporting the hypothesis that the optimum moisture contentfor storage varies with temperature. Seed viability and vigourwere monitored during storage under dark or lighted conditionsat relative humidities between 1 and 90%, and temperatures between-5 and 65°C. The optimum moisture content varied from 0·015g H₂O g^-1 d.wt at 65°C to 0·101 g H₂O g^-1 d.wt at15°C under dark conditions and from 0·057 at 35°Cto 0·092 g H₂O g^-1 d.wt at -5°C under lighted conditions.Our results suggest that optimum moisture contents cannot beconsidered independently of temperature. This conclusion hasimportant implications for 'ultra-dry' and cryopreservationtechnologies.Copyright 1994, 1999 Academic Press Seed storage, seed aging, seed longevity, water content, temperature, glass, desiccation damage, ultradry, Pisum sativum L., pea, cryopreservation     

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.     

Somatic expansion of the (CAG) n repeat in Huntington disease brains

The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG) _n repeat in the 5 part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG) _n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.     

Identification of cellular prosomatostatin and nonsomatostatin peptides derived from its amino terminus

Rat prosomatostatin was isolated from a somatostatin-producing cell line and was partially microsequenced. This indicated the amino terminal structure of cellular prosomatostatin and implied a 92-amino acid sequence for the somatostatin precursor. Based on the structure for cellular prosomatostatin, a peptide was synthesized and used to develop a radioimmunoassay directed toward the amino terminal portion of prosomatostatin. This assay has revealed two peptides containing the amino-terminal portion of prosomatostatin in a somatostatin-secreting CA-77 rat medullary thyroid carcinoma cell line. These two peptides - MW 4000 and 8000 daltons - lack somatostatin immunoreactivity. Thus, processing of prosomatostatin occurs both at the amino and carboxyl regions. These results open the way for elucidation of the structure, function and metabolism of non-somatostatin peptides derived from the amino terminus of prosomatostatin.     

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.