Title Page
ACKNOWLEDGEMENT
contents
ABBREVIATION 12
ABSTRACT 13
Ⅰ. Introduction 14
Ⅱ. Materials and Methods 16
Ⅱ.1. Yeast Strains, Genotypes and Culture Methods 16
Ⅱ.2. Plasmid Constructs and Mutagenesis 16
Ⅱ.3. Isolation of RNA and RT-PCR 17
Ⅱ.4. Western Blot Analysis 17
Ⅱ.5. Determination of Sodium Tolerance and Temperature-Sensitivity 18
Ⅲ. Results 19
Ⅲ.1. Mgl-1 is a Mouse Homologue and Contains the WD-40 Repeat Sequence 19
Ⅲ.2. Expression of Rgl-1 in Yeasts 21
Ⅲ.3. Complementation of a Cold-Sensitive Yeast Mutant by Rgl-1 24
Ⅲ.4. Five Point Mutant Constructs of Mgl-1 are Generated by Site-Directed Mutagenesis 26
Ⅲ.5. All Five Point Mutants of Mgl-1 are Expressed at the DNA and Protein Levels 27
Ⅲ.6. W D-40 Repeat Motif Mgl-1 Protein is Required for Cation Homeostasis and Restrictive Temperature 31
Ⅳ. Discussion 33
Ⅴ. Conclusions 36
References 37
Summary in Korean 41
Table 1. Specific primers used for RT-PCR.These sequences shows specific primers for sop 1, Rgl-1, Mgl-1 and ACT-1. 30
Figure 1. Comparison of sequence for the WD-40 repeat motif in L(2)gl family members. Alignment of WD-40 repeat sequences for L (2) gl (fly), Mgl-1(mouse), Rgl-1(rat), Bgl-1(bovine) and Hugl (human) using MegAlign software (cluster method) from DNASTAR. 20
Figure 2. Expression of Rgl-1in the Saccharomyces cerevisiae. The transcription was confirmed by RT-PCR analysis using specific primers for Rgl-1. The specific primers forsop1 and ACT1 were used as controls.A, sop1expression;B, Rgl-1expression;C, ACT1expression. Lane 1:DNA molecular marker;2,W303 wild-type strain + pYX212;3, sop1△sop2△ mutant strain;4, sop1△sop2△ mutant strain + pYX212;5, sop1△sop2△ mutantstrain+pYX212-Rgl-1. 22
Figure 3. An immunoblot showing the expression ofRgl-1 proteins in the sop1△sop2△ double mutant carrying pYX212-Rgl-1-HA. The molecular weight of Rgl-1 is approximately 112 kDa. 23
Figure 4. Complementation of a cold-sensitive growth forthe sop1△sop2△ double mutant. The sop1△ sop2△ mutant strain transformed with Rgl-1 cDNA inserted in the multicopy pYX212 plasmid was analyzed. The W303 wild-typeand the sop1△ sop2△ mutant strains were used as controls. Cell were streaked on YEPD plate and incubated for 3 days at 30℃ and 20℃ prior to photography. 25
Figure 5. (A) Mgl-1 transcripts are confirmed by RT-PCR analysis using specific primers for Mgl-1(Table 1). The specific primers for sop1 and ACT1 were used as a control (Table1). Cells were routinely cultured at 30°C in either rich medium (YEPD) containing 120 μl/mladenine or syntheti minimal medium (SD) containing glucose, necessary amino acids and nucleotides. 28
Figure 5. (B) Mgl-1 and its mutant proteins expressed in sop1△sop2△ double mutants were also confirmed by immunoblotting analysis. The order of lane is the same arrangement for RT-PCR analysis 29
Figure 6. Complementation of salt tolerance and temperature-sensitivity of sop1△sop2△ double mutants. The sop1△sop2△ mutants were transformed with either the Mgl-1cDNA oreach of Mgl-1 mutant cDNAs inserted in the multicopy pYX212 plasmid. Cells were grown overnight in YEPD medium adjusted to OD600 1.0 and serial10-fold dilutions were spotted on YEPD plates containing 0 M and 0.5 M NaCl. They were also spotted on YEPD plates and then incubated at 20°C and 30°C for 3-5 days. 32