Abstract: An object of the present invention is to provide a method for amplifying a nucleic acid using a PCR reaction solution with a volume as large as 30 mL or more, and an apparatus for amplifying a nucleic acid using a PCR reaction solution with a volume as large as 30 mL or more. The present invention provides a method for amplifying a nucleic acid, comprising performing polymerase chain reaction (PCR) by controlling a temperature of one or more reaction container(s) housing a reaction solution containing a DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), a template DNA, a forward primer, a reverse primer, and a buffer solution, wherein the reaction solution is in an amount of 30 mL or more, and the polymerase chain reaction is performed while the reaction solution is stirred.

Abstract: In order to provide a culture medium eliminating variation by lot of component concentrations, the present invention provides a culture medium, and preferably a chemical-synthetic culture medium, imparting a proliferation ability to yeast that is equivalent to or greater than that of a YPD culture medium. A culture medium is provided which includes sugars as a carbon source capable of being assimilated by yeast; amino acids as a nitrogen source; vitamins; inositol; zinc ion (Zn2+); potassium ion (K+), and magnesium ion (Mg2+), and in which inositol concentration is between 50 and 10,000 mg/L.

Abstract: Plasmid vectors have been widely used as a carrier of a DNA sequence capable of expressing a target RNA in cells. However, construction of these plasmid vectors requires technical skill and time. Thus, a quicker and easier method is required therefor. To solve this problem, a method using a linear DNA that has been amplified by the PCR method is examined. However, this method is disadvantageous in that RNA expression in cells is extremely low. Under these circumstances, the present inventors attempted to develop an RNA expression method using a linear DNA which can be produced mainly by using the PCR method alone and which enables a high level of RNA expression. As the results of intensive studies on terminator sequences to be used in a linear DNA, the present inventors found a smallest unit of a terminator sequence enabling linear DNA expression equivalent to that when using a plasmid vector.

Abstract: The promoter of the present invention causes a desired gene to be highly expressed, especially in thermotolerant yeast. The promoter is located upstream of the PIR1 gene or the CTR1 gene on the Kluyveromyces marxianus chromosome and comprises a region controlling expression of the PIR1 gene or the CTR1 gene.

Abstract: An object to be solved by the present invention is to provide, for example, a method for producing a Kluyveromyces marxianus transformant by a method for conveniently and efficiently connecting the ends of DNA fragments without using specific restriction enzymes or their recognition sequences, and a method for producing a useful substance using the transformant.

Abstract: A yeast of the genus Kluyveromyces is modified to improve the ethanol yield from xylose by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.

Abstract: In order to provide a culture medium eliminating variation by lot of component concentrations, the present invention provides a culture medium, and preferably a chemical-synthetic culture medium, imparting a proliferation ability to yeast that is equivalent to or greater than that of a YPD culture medium. A culture medium is provided which includes sugars as a carbon source capable of being assimilated by yeast; amino acids as a nitrogen source; vitamins; inositol; zinc ion (Zn2+); potassium ion (K+), and magnesium ion (Mg2+), and in which inositol concentration is between 50 and 10,000 mg/L.

Abstract: The promoter of the present invention causes a desired gene to be highly expressed, especially in thermotolerant yeast. The promoter is located upstream of the PIR1 gene or the CTR1 gene on the Kluyveromyces marxianus chromosome and comprises a region controlling expression of the PIR1 gene or the CTR1 gene.

Abstract: Provided is a novel high-expression promoter, namely a GAL1 promoter, derived from Kluyveromyces marxianus. Also provided are the following, characterized by the use of the provided high-expression promoter; a recombinant polynucleotide containing said high-expression promoter; a vector containing said recombinant polynucleotide; a transformant obtained by introducing said recombinant polynucleotide or vector into yeast; a method using said transformant for high expression of a target gene; and a method using said transformant to manufacture the gene product of a target gene.

Abstract: Plasmid vectors have been widely used as a carrier of a DNA sequence capable of expressing a target RNA in cells. However, construction of these plasmid vectors requires technical skill and time. Thus, a quicker and easier method is required therefor. To solve this problem, a method using a linear DNA that has been amplified by the PCR method is examined. However, this method is disadvantageous in that RNA expression in cells is extremely low. Under these circumstances, the present inventors attempted to develop an RNA expression method using a linear DNA which can be produced mainly by using the PCR method alone and which enables a high level of RNA expression. As the results of intensive studies on terminator sequences to be used in a linear DNA, the present inventors found a smallest unit of a terminator sequence enabling linear DNA expression equivalent to that when using a plasmid vector.

Abstract: A yeast of the genus Kluyveromyces is modified to improve the ethanol yield from xylose by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.

Abstract: Provided is an efficiency improving agent for gene transfer to mammalian cells, a method for improving efficiency of gene transfer to mammalian cells, and a method for transforming mammalian cells. The method is characterized in that tRNA is used in combination with a lipofection reagent. Preferably, the agent may be used so that the tRNA concentration in a lipofection solution falls within the range of 3 to 50 ?g/mL, and the concentration in a culture is approximately 1/10. More preferably, tRNA and PEG may be used in combination with a lipofection reagent. According to the present invention, gene transfer to mammalian cells with high efficiency can be achieved.

Abstract: Provided is a novel high-expression promoter, namely a GAL1 promoter, derived from Kluyveromyces marxianus. Also provided are the following, characterized by the use of the provided high-expression promoter; a recombinant polynucleotide containing said high-expression promoter; a vector containing said recombinant polynucleotide; a transformant obtained by introducing said recombinant polynucleotide or vector into yeast; a method using said transformant for high expression of a target gene; and a method using said transformant to manufacture the gene product of a target gene.

Abstract: An object to be solved by the present invention is to provide, for example, a method for producing a Kluyveromyces marxianus transformant by a method for conveniently and efficiently connecting the ends of DNA fragments without using specific restriction enzymes or their recognition sequences, and a method for producing a useful substance using the transformant.

Abstract: Provided is an efficiency improving agent for gene transfer to mammalian cells, a method for improving efficiency of gene transfer to mammalian cells, and a method for transforming mammalian cells. The method is characterized in that tRNA is used in combination with a lipofection reagent. Preferably, the agent may be used so that the tRNA concentration in a lipofection solution falls within the range of 3 to 50 ?g/mL, and the concentration in a culture is approximately 1/10. More preferably, tRNA and PEG may be used in combination with a lipofection reagent. According to the present invention, gene transfer to mammalian cells with high efficiency can be achieved.

Abstract: It is to provide a novel Kluyveromyces marxianus transformant having thermotolerance and flocculation property, suitable for the industrial production of bioethanol, by introducing a foreign flocculation gene into Kluyveromyces marxianus, and an efficient method for producing the transformant. The present inventors focused on the flocculation gene FLO of Saccharomyces cerevisiae as a foreign gene to confer flocculation property to Kluyveromyces marxianus and produced a linear DNA fragment comprising a known expression promoter sequence and a FLO gene sequence derived from Saccharomyces cerevisiae. As a result of introducing this linear DNA fragment into Kluyveromyces marxianus, the present inventors have confirmed that Kluyveromyces marxianus transformant can be obtained efficiently, and that the flocculation property of the above transformant is unexpectedly and significantly enhanced. The present invention has been thus completed.

Abstract: It is to provide a novel Kluyveromyces marxianus transformant having thermotolerance and flocculation property, suitable for the industrial production of bioethanol, by introducing a foreign flocculation gene into Kluyveromyces marxianus, and an efficient method for producing the transformant. The present inventors focused on the flocculation gene FLO of Saccharomyces cerevisiae as a foreign gene to confer flocculation property to Kluyveromyces marxianus and produced a linear DNA fragment comprising a known expression promoter sequence and a FLO gene sequence derived from Saccharomyces cerevisiae. As a result of introducing this linear DNA fragment into Kluyveromyces marxianus, the present inventors have confirmed that Kluyveromyces marxianus transformant can be obtained efficiently, and that the flocculation property of the above transformant is unexpectedly and significantly enhanced. The present invention has been thus completed.