Toyomasa Hatakeyama

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30°C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1×109 cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2×107 transformants/µg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non‐permeating cryoprotectant such as sorbitol. Copyright

A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells

A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10–15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 106 transformants/µg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long‐term for more than 3 months without any significant loss of efficiency. Copyright

Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia.

A region on chromosome 14q32.1 is often involved in chromosomal translocations and inversions with one of the T-cell receptor loci in T-cell lymphoproliferative diseases. The breakpoints of the different rearrangements segregate into two clusters; a cluster due to inversion on the centromeric side and a cluster due to simple balanced translocations on the telomeric side. If the target gene activated by these different types of chromosomal rearrangements is the same, the gene must be localized between the two clusters of breakpoints in a region of around 160 kb. Within this breakpoint cluster region, we isolated two genes; namely, TCL1 and TML1/TCL1b genes. In the course of characterizing the TML1 gene, we further identified a third novel gene, which we named TCL6 (T-cell leukemia/lymphoma 6), from a region 7 kb upstream of the TML1 locus. The TCL6 gene expressed at least 11 isoforms through very complex alternative-splicing, including splicing with the TML1 gene. Those isoforms encode at least five open reading frames (ORFs) with no homology to known sequences. The localization of the proteins corresponding to these ORF was determined by fusing green fluorescence protein at the carboxyl terminal of each ORF. ORF141 and ORF72 were observed in the cytoplasmic region, while ORF105, ORF119, and ORF163 were predominantly localized in the nuclear region. Since the TCL6 gene was expressed in T-cell leukemia carrying a t(14;14)(q11;q32.1) chromosome translocation and was not expressed in normal T-cells (just like the TML1 and TCL1 genes), it is also a candidate gene potentially involved in leukemogenesis.

Cryopreservation of competent intact yeast cells for efficient electroporation

We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long‐term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non‐permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10°C/min and a rapid thawing rate of 200°C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non‐electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at −80°C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long‐term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation. Copyright

Gene transfer and protein release of fission yeast by application of a high voltage electric pulse

A high voltage electric pulse can be applied to induce the uptake of DNA into cells and the release of protein from cells. In transformation procedures, electroporation is widely used since the technique is simple, rapid, reproducible, and highly efficient. In extraction of protein, on the other hand, electroextraction has many advantages over other conventional extractions. We have developed a highly efficient method for the electroporation of fission yeast. In particular, application of a high voltage electric pulse to fission yeast improves the cellular uptake and release of macromolecules controlled by both osmotic conditions and electric field strength.

High osmotic stress improves electro-transformation efficiency of fission yeast

A preincubation of fission yeast cells with hyperosmotic solution improved the electro-transformation efficiency. The efficiency increased approximately five-fold when the cells were preincubated with 2.0 M sorbitol and 1.5 M NaCl at 30 degrees C for 60 min before an applied high electric pulse. Losses in the efficiency of the cells after hyperosmotic stress above 2.5 M sorbitol and 2.0 M NaCl were directly related to the marked reduction of viability. The efficiency at 2.0 M sorbitol gradually increased until 60 min of the preincubation period, but longer exposure resulted in a gradual decrease. On the other hand, when the cells of the osmotic-sensitive mutant were preincubated with isosmotic solution of 0.5 M sorbitol, the efficiency was also dramatically increased by approximately 15-fold. These improvements in efficiency were observed in sublethal conditions of osmotic stress regardless of osmoticums and strains.

Orientation and Characteristic Movement of Yeast Cells Induced by Homogeneous Electric Field

A cylindrical yeast cell ( Schizosaccharomyces pombe ) oriented itself parallel or perpendicular to an alternating homogeneous electric field, depending upon the applied frequency. The turnover frequency was about 65 MHz for the living cells in a non-electrolyte solution but about 1.5 MHz for the dead cells in an electric field. Further, the lower the cell activity due to the refrigeration effect, the lower the turnover frequency was. Similar phenomena were observed for budding yeast cells ( Saccharomyces cerevisiae ). Therefore, these phenomena can be used to investigate the cell activity in the solution. We also found a second turnover frequency of about 15 MHz for the dead cylindrical cells. Moreover, when two living cylindrical cells were placed in contact with each other, they exhibited characteristic movement at applied frequencies near the first turnover frequency.

Orientation and Pearl-Chain Formation of Paramecia Induced by AC Electric Field

Paramecium deciliated with ethanol is able to orient itself in a parallel (positive orientation) or perpendicular direction (negative orientation) to an AC electric field, depending upon the applied frequency. We found that this turnover frequency is between 1 and 10 MHz in a non-electrolyte solution for the cells. The cells also aggregate with one another by the mutual dielectrophoresis in the electric field, provided the distance between the two cells is shorter than about half their length. The two critical field intensities for the orientation and for the aggregation cannot be clearly distinguished. Consequently, when the cell density in the solution is sufficiently high, a positive or negative pearl-chain of the cells is formed, depending upon the applied frequency.

Assignment of the human poly(A) polymerase (PAP) gene to chromosome 14q32.1-q32.2 and isolation of a polymorphic CA repeat sequence

AbstractWe report the chromosomal localization of the gene for human poly(A) polymerase (PAP) and the characterization of a newly isolated CA repeat near the PAP locus. By fluorescence in situ hybridization and polymerase chain reaction (PCR)-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel, this gene was mapped on the q32.1–q32.2 region of chromosome 14. From a genomic clone containing the human PAP locus, we have isolated a polymorphic dinucleotide (CA) sequence. High heterozygosity (0.81) makes this polymorphism a useful marker in the genetic study of disorders localized at the 14q32 region, such as autosomal recessive congenital microphthalmia (CMIC).

ISOLATION AND MAPPING OF A POLYMORPHIC CA REPEAT SEQUENCE AT THE HUMAN VRK1 LOCUS

AbstractVRK1 is a novel human putative serine/threonine kinase, and is located on chromosome 14 at band q32 where an autosomal recessive congenital microphthalmia (CMIC) is mapped. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human VRK1 gene. This polymorphism will be useful in genetic studies of disorders localized at the 14q32 region, such as CMIC.