5.1 Evaluation of the mechanism of 5FC resistance in C. tropicalis
The mechanisms of 5FC resistance of the clinical isolate YM060607 and the derivatives of seven clinical isolates (YM020438, YM020671, YM060075, YM060097, YM060210, YM060369 and YM060616) require further investigation.
Based on the nucleotide sequence analysis, the 5FC resistant clinical isolate, YM060607, has a heterozygous SNP (C/T) at the position 431 of the FUR1 gene, and a heterozygous SNP (A/G) at the position 775 of the URA3 gene. Both single nucleotide variations (C431T in FUR1 and G775A in URA3) lead to single amino acid changes (T144I in UPRT and A259T in ODCase), which might influence the functions of proteins involved in the 5FC resistance mechanisms. The contribution of these nucleotide changes to the 5FC resistance can be further investigated by constructing the point mutation strains. Genes, other than URA3, encoding proteins that are involved in the de novo pathway may play a role in 5FC resistance. Therefore, these genes can be examined if there are correlations between particular polymorphic nucleotides in the coding region and the resistance to 5FC. Also, alternations on gene expression, which may be due to changes in the 5’ or 3’UTR region, or the interference of regulators of genes associated with 5FC resistance, could result in 5FC resistance. Accordingly, the mRNA level of these genes should be investigated. In addition, as mentioned in the discussion 4.3, the FCY2-like gene, CTRG_00460, might involve in 5FC toxicity and, therefore, the disruption of this gene is likely to influence the 5FC resistance in C. tropicalis. However, the contribution of this homolog gene to the 5FC resistance needs to be examined.
5.2 Examination of the features of the boundaries that might be related to recombination event.
At least three known factors might be associated with recombination and therefore relate to the LOH boundaries (i.e., the GC content, the SNP frequency and the distance to tRNA). Previous research has implied that high GC contents at local
scales might stimulate recombination rate (Marsolier-Kergoat and Yeramian, 2009).
Nevertheless, a gene is often characterized by having a higher GC content relative to the background GC content for the entire genome and the length of a gene is directly proportional to a higher GC content (Pozzoli et al., 2008). Therefore, the GC content of the boundaries with a comparable length scale could compare to either the background GC content or the GC content of the coding regions. Furthermore, the frequency of SNPs also plays a role in the recombination, which is considerable higher in hot spots relative to the genomic average (Brandstrom et al., 2008). SNP frequency of one of the LOH boundaries, located in a coding region, is higher (one SNP per 67 bases) than the average rate of polymorphism in C. tropicalis (one SNP per 576 bases) (Pozzoli et al., 2008); however, whether the SNP frequency around this boundary is actually higher than other regions of supercontig 2 needs to be determined by additional static analyses. Apart from the location of the LOH boundary and the SNP frequency, several researches have proposed the association of tRNA with gross chromosomal rearrangements (Diogo et al., 2009; Dunham et al., 2002; Dunn et al., 2005). Here, two tRNA genes are found near the boundary of YM060800 (the closest distance of which was 3 kb), but their associations with the boundary should be determined by additional static analyses. Last but not the least, the sequence of each boundary can be compared and analyzed to search for hot spots of the recombination.
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吳佳真 (2009) 熱帶念珠菌失去異質合子性導致 5-Flucytosine 抗藥性及鑑別 CaNDT80 活化區重要胺基酸之研究 Loss of heterozygosity contributes to the resistance to 5-flucytosine in Candida tropicalis and the study of identifying the crucial amino acids in the activation domain of CaNDT80
Strain Parental
strain FCY2 genotypea Source
YM020291 wild-type, flucytosine susceptible TSARYb
YM020291-1 YM020291 FCY2R/ FCY2R Hui-Ching Ko
YM020291-2 YM020291 FCY2R/ FCY2R Hui-Ching Ko
YLO417 YM020291 FCY2R/ FCY2R :: SAT1-FLIP Chia-Chen Wu YLO418 YM020291 FCY2R/ FCY2R :: SAT1-FLIP Chia-Chen Wu YLO415 YM020291 FCY2S/ FCY2S :: SAT1-FLIP Chia-Chen Wu YLO454 YM020291 FCY2R/ fcy2S(G486T) :: SAT1-FLIP this work YLO455 YM020291 FCY2R/ fcy2S(G486T) :: SAT1-FLIP this work YLO456 YM020291 FCY2S/ fcy2R(T486G) :: SAT1-FLIP this work YLO457 YM020291 FCY2S/ fcy2R(T486G) :: SAT1-FLIP this work YLO466 YM020291 FCY2R/ fcy2S(G145T) :: SAT1-FLIP this work YLO467 YM020291 FCY2R/ fcy2S(G145T) :: SAT1-FLIP this work YLO468 YM020291 FCY2S/ fcy2R(T145G) :: SAT1-FLIP this work YLO469 YM020291 FCY2S/ fcy2R(T145G) :: SAT1-FLIP this work YLO470 YM020291 FCY2R/ fcy2S(C1731T) :: SAT1-FLIP this work YLO471 YM020291 FCY2R/ fcy2S(C1731T) :: SAT1-FLIP this work YLO472 YM020291 FCY2S/ fcy2R(T1731C) :: SAT1-FLIP this work YLO473 YM020291 FCY2S/ fcy2R(T1731C) :: SAT1-FLIP this work YM060800 wild-type, flucytosine susceptible TSARYb
YM060800-1 YM060800 FCY2R/ FCY2R Hui-Ching Ko
YM060800-2 YM060800 FCY2R/ FCY2R Hui-Ching Ko
YLO440 YM060800 FCY2R/ FCY2R :: SAT1-FLIP Chia-Chen Wu YLO441 YM060800 FCY2R/ FCY2R :: SAT1-FLIP Chia-Chen Wu YLO447 YM060800 FCY2S/ FCY2S :: SAT1-FLIP Chia-Chen Wu YLO448 YM060800 FCY2S/ FCY2S :: SAT1-FLIP Chia-Chen Wu
a FCY2R, 5-FC resistant allele; FCY2S, 5-FC susceptible allele; SAT1-FLIP, the SAT1 flipper cassette
b TSARY, Taiwan Surveillance of Antimicrobial Resistance of Yeasts Table 1. Yeast strains used in this study
Lab No. Backbone description source LOB319 pSFS2A pSFS2A-derived plasmid containing
an insertion of FCY2S allele and its flanking sequences, which was amplified from YM020291.
Chia-Chen Wu
LOB320 pSFS2A pSFS2A-derived plasmid containing an insertion of FCY2R allele and its flanking sequences, which was amplified from YM020291.
Chia-Chen Wu
LOB381 LOB319 Mutation of LOB319, introducing a point mutation G273T (G486T) into FCY2S allele.
this work
LOB382 LOB320 Mutation of LOB320, introducing a point mutation T273G (T486G) into FCY2R allele.
this work
LOB383 LOB319 Mutation of LOB319, introducing a point mutation G-69T (G145T) into FCY2S allele.
this work
LOB384 LOB320 Mutation of LOB320, introducing a point mutation T-69G (T145G) into FCY2R allele.
this work
LOB385 LOB319 Insertion of the HindIII-HindIII fragment from LOB320
this work LOB386 LOB320 Insertion of the HindIII-HindIII
fragment from LOB319
this work Table 2. Plasmids used in this study
(Continued)
a The position of this primer is at the downstream of the stop codon of the FCY2 gene.
b Lower case shows restriction sites for KpnI.
c Lower case shows restriction sites for XhoI.
d Primers that were used to introduce single mutation at position 486 by fusion PCR.
The single mutation is indicated by the underlined bold letters.
e Primers that were used to introduce single mutation at position 145 by fusion PCR.
The single mutation is indicated by the underlined bold letters.
Name Sequences (5' to 3') Position
HJL814 CTCAACATGGAACGATCTAGC CaSAT1: 465 bp to FRT
HJL1207 TGCCCATAAATTAAATGCAGAA CtFCY2: +126~+147
HJL1208 GGAAGCAACAAACCCAAAAA CtFCY2: +863~+844
HJL1210 GTGAAAACGAGCCAATCCAT CtFCY2: +1550~+1530
HJL1420 ggtaccTCAACTCAACCCCAAAGT b CtFCY2: -287~-270
HJL1422 CGGATTCAATGTAGCCAG CtFCY2: +1485~+1502
HJL1423 GTCATTCCATGTCGTGGT CtFCY2: +1971~+1954
HJL1424 ctcgagGTCATTCCATGTCGTGGT c CtFCY2: +1971~+1954 HJL1477 CTGTTGCTCCAGGTGAATCA CtFCY2: +2065~+2084 a HJL1740 GTGCAGTTAATATCATGTCTTCCGCTC d CtFCY2: +470~+496 HJL1741 GAGCGGAAGACATGATATTAACTGCAC d CtFCY2: +496~+470 HJL1742 GTGCAGTTAATATCATTTCTTCCGCTC d CtFCY2: +470~+496 HJL1743 GAGCGGAAGAAATGATATTAACTGCAC d CtFCY2: +496~+470 HJL2100 CATAAATTAAATGCAGAAACTAAAGGTATTG e CtFCY2: +130~+160 HJL2101 CAATACCTTTAGTTTCTGCATTTAATTTATG e CtFCY2: +160~+130 HJL2102 CATAAATTAAATGCATAAACTAAAGGTATTG e CtFCY2: +130~+160 HJL2103 CAATACCTTTAGTTTATGCATTTAATTTATG e CtFCY2: +160~+130 HJL2104 CTTCTCCTTAACTACCTTTTCCTCC CtFCY2: -243~-219 HJL2117 TCATCTCATTTCATTTGGTACAAAGG CtFCY2: +1255~+1280
HJL2124 CAGACTTTGTTGGTGGTAGAAC CtFCY2: +692~+713
HJL1937 TCCGACTTCTCCAAAGTTGA CtEFB1: +10~+29
HJL1938 TTGGACGTGGTCTTCATCTTC CtEFB1: +819~+799
HJL2299 GTAATGCTGCTACTTTAGCC CtFCY2: +1139~+1158
HJL2300 TTCACCGATTTGTCTACCA CtFCY2: +1437~+1419
HJL2301 ATCACCATTGGTAACGAAAG CtACT1:+637~+656
HJL2302 TGGAAAGTAGACAAGGAAGC CtACT1:+953~+934
Table 3. Primers used in this study.
Name Sequences (5' to 3') Position HJL1494 AAGAAACAGAATACGATGAGTGATG CTRG_01076: +58~+82 HJL1495 AGGTTGGTACTGGGTAACATGA CTRG_01076: +1071~+1050
HJL1496 ATGCCAAAGAAAACTCAGGG CTRG_01434: +1~+20
HJL1497 GGGTACATTTAGAAAGACTAGCCG CTRG_01434: +1063~+1040 HJL1498 TGTCATCATCTTCATCTAATCAAGC CTRG_01816: +2~+26 HJL1499 CCATCTCCACCAAGATAACCAA CTRG_01816: +1052~+1031 HJL1500 ATTGATTAGCGACCCAACTGC CTRG_02123: +111~+131 HJL1501 CCCATTGTTCCAAACCCTTT CTRG_02123: +1054~+1035 HJL1621 TCGTTACTGGAGCAACAGGATTC CTRG_01886: +23~+45
HJL1622 TAAGGTAAGGAATCGCCAAGG CTRG_01886: +1037~+1017
HJL1623 TTTCATCTACTGCTGCTCCTGG CTRG_01926: +3968~+3989
HJL1737 TGCTGGAGATGATGAGGATGAAG CTRG_01926: +4974~+4952 HJL1627 AACACCTGAAGATACCAAACTGTC CTRG_02012: +12~+35 HJL1628 AGTGGAAGTAGCAGTAGTAGCAGTG CTRG_02012: +1530~+1506 HJL1629 AGGGATTCAGCAAGGTGACTTTG CTRG_02080: +192~+214 HJL1630 TCTTTCTCCTCATCTTTCTGTTCG CTRG_02080: +1145~+1122
HJL1631 TTGGGCTGATGATGCTGTTG CTRG_02099: +609~+628
HJL1632 TTTCACCCGCTCTCCATCTC CTRG_02099: +1468~+1449 HJL1717 AAACGCCATTAAGAGATTCAGTCC CTRG_01843: +39~+62 HJL1718 TTCAATGCTAGTGACAGTGTTGGTG CTRG_01843: +1077~+1053 HJL1719 TGCCGTAAAGATATTGGGATTCAG CTRG_01858: +48~+71 HJL1720 TGGGTGTGACTTATGGTTGATGAC CTRG_01858: +1080~+1057 HJL1721 AAGGTTACAGGATGCCGTGGAG CTRG_02083: +96~+117
HJL1722 TGAGATCCCAGACGCAAATCAG CTRG_02083: +1238~+1217 HJL1723 TGTATGCCGATAAGACAGTACCTGG CTRG_02089: +2~+26
HJL1724 ATCCAAACAATCAGATAAGCCACC CTRG_02089: +1227~+1204
HJL1725 CCATTCAAGGCAGACATAGATGA CTRG_02092: +3001~+3023 HJL1726 AATCTCCTGCCTTGTAGAAATGG CTRG_02092: +3799~+3777
HJL1727 ATGACAACGACAGTGCCATTG CTRG_02095: +1~+21
HJL1728 TTACCAGCAGCACCTTCATTAAC CTRG_02095: +938~+916 HJL1729 TTCGCACACACACACACTCTCTC CTRG_02103: -459~-437 Table 3. Continued
Name Sequences (5' to 3') Position
HJL1730 GATAAGCCACCAGTTGAAGAAGATG CTRG_02103: +884~+860
HJL1758 TGTTGTTGTTGTTTTCTTGCTC CTRG_02108: -44~-23
HJL1732 TCGGCATAGCGTTGGCATAC CTRG_02108: +1200~+1181
HJL1832 TATCGTTGGTCTAGTGTTGTGAATC CTRG_02109: -274~-250 HJL1833 ATCCATATTCTCCATTCCTTCATC CTRG_02109: +834~+811 HJL1733 AAAGAAGAGAGGTTTGTTTGGTTCC CTRG_02114: -231~-207
HJL1734 ACTTGATGGTGGAGTAATCGGC CTRG_02114: +821~+800
HJL1735 AAAGAATGTGATACGAGCCAAGC CTRG_02117: -411~-389 HJL1736 AGGGCTCTAGTCAACTCAACATCAC CTRG_02117: +786~+762 HJL1759 CATCAACAACAACAAGAACATCG CTRG_01823: +97~+119 HJL1760 ACCTGGGTGTTGAATGTACTGTC CTRG_01823: +965~+943 HJL1769 CAAAGTTGTGGTGAAGACCTCATC CTRG_01837: +192~+215 HJL1770 ACTGTGTCTCATCATCATTGGCAC CTRG_01837: +991~+968 HJL1824 GCTTCTTGGATATGAACACTAACC CTRG_01944: +11~+34
HJL1825 AAATAGTTGGGTGTTGACCTTG CTRG_01944: +964~+943
HJL1826 TCCTTCCTACATACATTGCCTCG CTRG_01991: -403~-381
HJL1827 TGGAGCAGGAACAGGAGCAG CTRG_01991: +735~+716
HJL1828 AGCATCATTACAGCCTACCACTC CTRG_02034: +327~+349 HJL1829 AGAAGAATCAGATGTCACCTCAGC CTRG_02034: +1641~+1618 HJL1830 CTAAGATGTTGTTCAAGAACCGTG CTRG_02082: +1175~+1198 HJL1831 GACACTCTTCAACTATGTGCTTACC CTRG_02082: +2236~+2260 HJL1834 GTCACACAGCCAAGAGATAATAGTC CTRG_02119: +3~+27 HJL1835 ATGACCTGCCTGAAGTAATATCAC CTRG_02119: +927~+904 HJL1836 AAATGGGATGAATGGGTAGG CTRG_01820: +223~+242
HJL1837 CAGTTGTAGATACACCATCCATTG CTRG_01820: +901~+878
HJL2113 CATACGAGGCTAGAACGATTGG CTRG_01817: -374~-353
HJL2114 TGGAAATATCCCGAATCTCTACAAG CTRG_01817: +963~+939
HJL2115 GGTCAACAAGATGCCATTCTG CTRG_01993: +352~+372
HJL2116 AACCTTCAGGAGTTTCACCAAC CTRG_01993: +1345~+1324 HJL2129 ATACCTCTTTGTAGAGTGTGAAACG CTRG_02120: -472~-448
HJL2130 TTCCATCATACTGACCCTTTG CTRG_02120: +767~+787
Table 3. Continued
Name Sequences (5' to 3') Position
HJL2131 GTATGGCTGCTTGTTTGGTAAC CTRG_02122: -2~+20
HJL2132 GGCAACCAACCTCTGAACAT CTRG_02122: 712~731
HJL2179 GAAAGAGAAAGATGGCTCGTTCATC CTRG_01999: +14~+38 HJL2180 CATTTGTCCTCATTGTGCTGAATG CTRG_01999: +927~+950 HJL2183 AAGATCCCCTGTTTGTTCCCTCAAC CTRG_02008: -40~-16
HJL2184 CAATGAACTTCTTTCCCACCAGC CTRG_02008: +1075~+1097 HJL2189 ACCAGACATCCCGATTTATAGTTC CTRG_02080: +161~+138
HJL2190 AGCAACACTGTGATGTAAGTTGTTC CTRG_02080: -977~-953
HJL2191 CACTGTCAAACAGCATTTCCAG CTRG_02081: +1224~+1203
HJL2192 GAGATCCAAGTTGTCCATCATTC CTRG_02081: +39~+61 HJL2193 TGTCTTTCACCAAGGGAGAAGTTTG CTRG_02081: +233~+209 HJL2194 GAATGTCAAAGCTGTTGCCAAGA CTRG_02082: +2886~+2908 HJL2195 AAGGGATCTTTGCGTTGCTTC CTRG_02082: +3192~+3172
HJL2196 GAAGTTGTTACCAGCAGGTGATAG CTRG_02082: +2079~+2102 HJL2197 CGAAAGATTAGCCACTCAATATG CTRG_02009: +1113~+1135
HJL2198 TGCTGAAACTCCTCCAAGTC CTRG_02009: +2211~+2192
HJL2199 TGAGTGAGGGAGTATGTTACCAAG CTRG_02010: -447~-424 HJL2200 CCTGAAGGATGATGTCTTGTTG CTRG_02010: +1265~+1244 HJL2201 GATGGTGATGGTGAAGGTGATA CTRG_02011: -431~-424 HJL2202 ATTCTTCGGAAATAACGGTAGC CTRG_02011: +529~+508 HJL2203 TAATGACGATCCCACTTATTGGAAG CTRG_01138: -2~+23 HJL2204 TCACCTGAAACCAATGAACAACAC CTRG_01138: +1090~+1067 HJL2205 CATTCGTCCTTCAAGTTGGTTTG CTRG_01190: +102~+124 HJL2206 TTCTTATGAGGTTTCTCACTTCCCA CTRG_01190: +698~+674 HJL2207 ATACCCACAGGACTGTTCCATTG CTRG_01239: +540~+562 HJL2208 CGCACCTGGATCTTTGTAGTCAG CTRG_01239: +1635~+1613
HJL2209 TGGTTTCAGTTACTTTGGGAAC CTRG_01337: -46~-25
HJL2210 CAGCGTCAGTATATCCCATTATG CTRG_01337: +886~+864
HJL2211 CCGATTTCACTATTGATTCACATGG CTRG_01490: +140~+164 HJL2212 CCCAAAACAGCTCTTTCACTAACAA CTRG_01490: +1163~+1139 HJL2213 ACCACAATCATCCAGAAAAGGGAAG CTRG_01532: +39~+63 Table 3. Continued
Name Sequences (5' to 3') Position
HJL2214 GCAACTGGAACTCTTGATTCAACCT CTRG_01532: +1187~+1163 HJL2215 GTCGCAAGAAACGATGTTGAACATG CTRG_01598: +463~+487 HJL2216 CCTTCTTTTGACCCAATCTATGTGC CTRG_01598: +1555~+1531 HJL2217 TCCTAGAAGTTTGAAAAGCGAATCC CTRG_01630: +21~+45 HJL2218 TGAATCTGCTCTTCTTGGCGATATT CTRG_01630: +1074~+1050 HJL2219 TCTGCTAGAACCTCCTTGAAGAACG CTRG_01722: +7~+31
HJL2220 CCAGCATCTTTCAAAGCCTTCTTA CTRG_01722: +1055~+1032 HJL2221 ACCACCCTTGAAAAGAGTTCTATC CTRG_02010: +39~+62
HJL2222 AAACAACCATCGCAAGACAAGG CTRG_02084: +333~+354
HJL2222 AAACAACCATCGCAAGACAAGG CTRG_02084: +333~+354