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582.282.23+575.113 ARO4 ̲ вƲ CANDIDA FAMATA (CANDIDA FLARERI) HANSENULA POLYMORPHA (PICHIA ANGUSTA) . . ...

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ARO4 ̲ вƲ...

582.282.23+575.113

ARO4 ̲

вƲ CANDIDA FAMATA (CANDIDA FLARERI)

HANSENULA POLYMORPHA (PICHIA ANGUSTA)

. .

㳿 , . , 1416, 79005,

e-mail: dmytruk@cellbiol.lviv.ua ARO4 3--D--7- (), , . ARO4m, -, Candida famata Hansenula polymorpha, DL-4-. 50 / .

: , , Candida famata, Hansenula polymorpha.

ᒺ. , , ( , , ) [17].

Candida famata (Candida flareri Debaryomyces subglobosus) ᒺ , . , , ( 2) . [17]. C. famata , , 䳿 / Studia Biologica 2010 4/2 . 512 6 .. , , 2 [12, 18, 16].

H. polymorpha . ֳ ᒺ , , , [11]. H. polymorpha . H. polymorpha , . Saccharomyces cerevisiae, H. polymorpha , , .

- - . . , Candida albicans, Cryptococcus neoformans Aspergillus fumigates, , . -, , [10, 1]. , . .

, , [2].

, DL-4- C. famata H. polymorpha.

в ˲

C. famata L20105 leu2 [19], H. polymorpha CBS4732 leu2-2 [14] Debaryomyces hansenii CBS767. YPD (0,5% , 1% , 2% ) YNB (0,67% YNB, 2% ) 30. 40 / .

C. famata H. polymorpha 0,8 / DL-4- 2,5 0,5 /, .

E. coli DH5a (80dlacZM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK-, mK+), supE44, relA1, deoR, (lacZYA-argF)U169) 37 䳿 / Studia Biologica 2010 4/2 . 512

ARO4 ̲ вƲ...

LB (0,5% , 1% , 1% NaCl).

100 /.

- [15].

Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). (Fermentas, Vilnius, Lithuania). E. coli Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). () GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA), Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) . C. famata H. olymorpha [19, 5].

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ARO4m ARO4 Saccharomyces cerevisiae DL-4- () [8]. . , - , , , [9]. ARO4m , [7]. ARO4m 496 (), -166 (Q166K) [6]. ARO4m [3]. C. famata H. polymorpha.

C. famata . CUG, , C. famata [4]. ARO4 Debaryomyces hansenii C. famata . D. hansenii Genolevures (http://www.genolevures.org/deha.html#). 㳺 ARO4 (YBR249C) S. cerevisiae D. hansenii (DEHA2A04774g), 368 68% 81% . , - , (. 1).

ARO4 (DEHA2A04774g) D. hansenii - - . , Ko153 (CTA TTA TTA TAC ATT AAG CAA AGA GCT CAT GAG TAA

   

. 1. -, . . dz , (Q157K). : Sc S. cerevisiae; Cg Candida glabrata; Pg Pichia guilliermondii; Dh D. hansenii; Hp H. olymorpha Fig. 1. Alignment of DAHP synthases site sequences responsible for feedback inhibition by tyrosine from several yeasts. Conserved sequences are on black background. Asterisk indicates amino acid was changed (Q157K). Abbreviations of yeasts species decoded as follows: Sc S. cerevisiae; Cg Candida glabrata; Pg Pichia guilliermondii; Dh D. hansenii ; Hp H. olymorpha

AAC ACC AAT CCC) / Ko154 (CAA AAA GTC TGA GAA GTA TTT TGG AGA AAT GGT

ATC CAA C) Ko155 (GTT GGA TAC CAT TTC TCC AAA ATA CTT CTC AGA CTT TTT G) / Ko156 (CGC GGA TCC GAT AAG AAA ATG AAA CGA GAT CC) D. hansenii CBS767 469 , Q157K ( , ). ARO4m TEF1 C. famata, a [4, 13].

TEF1 C. famata (683 ..) ARO4m (1328 ..) Ko153 / Ko156 Ko151 (CCG GAA TTC TAA CGA ACA GCT CAT CAG) / Ko152 (GGG ATT GGT GTT TTA CTC ATG AGC TCT TTG CTT AAT GTA TAA TAA TAG). Ko151 Ko156 , ARO4m D. hansenii TEF1 C. famata. 1964 .. EcoRI BamHI EcoRI/BamHI- pUC57. pTDhARO4m (. 2). ³ .




140 .


C. famata H. olymorpha. , , (0,1; 0,25; 0,5; 0,75; 1; 1,25; 1,5; 1,75;

2; 2,25; 2,5; 2,75; 3; 4; 5 /). , C. famata H. olymorpha 2,5 0,5 /, .

EcoRI- pTDhARO4m ﳺ C. famata H. olymorpha. ϳ 䳿 / Studia Biologica 2010 4/2 . 512

ARO4 ̲ вƲ...

   

. 2. ˳ pTDhARO4m (4,7 ...). TEF1 C. famata ARO4m D. hansenii , . ( ). , ble, , lacZ E. coli . : H HindIII;

BI BamHI; Sc SacI; RI EcoRI Fig. 2. Linear scheme of plasmid pTDhARO4m (4.7 kb). Promoter TEF1 C. famata gene and ORF ARO4m D. hansenii gene with own terminator are designed with white and grey lines, respectively. Primers are designed with dashed narrows (explanation in the text). Bacterial origin of replication, gene ble for selection of bacterial transformants on the ampicillin containing medium and fragment of gene lacZ E. coli are designed with narrows. Restriction sites: H HindIII; BI BamHI; Sc SacI; RI EcoRI , , .

, , 5 20 50 1 C. famata H. olymorpha, . pTDhARO4m Ko151 / Ko156 (. 3, , ).

   

. 3. - C. famata () H. olymorpha () pTDhARO4m. 25 1114 C. famata H. olymorpha, .

7 9 ( pTDhARO4m), 6 10 ( C. famata H. olymorpha) . 1 8 ( ...) Fig. 3. Electrophoresis of the PCR assay of C. famata () and H. olymorpha () transformants with the plasmid pTDhARO4m. Lines 24 and 1114 are transformants of C. famata and H. olymorpha, respectively. Lines 7 and 9 represent positive (plasmid pTDhARO4m), while 6 and 10 negative (chromosomal DNA of C. famata and H. olymorpha) controls. 1 and 8 are a marker of the molecular mass of the fragments (the fragment values are expressed in kb) 䳿 / Studia Biologica 2010 4/2 . 512 10 .. , ARO4m D. hansenii, C. famata H. polymorpha. , .

, ARO4m D. hansenii, C. famata H. olymorpha 20 50 1 , .

. . . . , . . . .

1. Adam A.C., Gonzlez-Blasco G., Rubio-Texeira M., Polaina J. Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates. Appl. Environ. Microbiol, 1999, 65(12):53035306.

2. Akada R. Genetically modified industrial yeast ready for application. J. Biosci. Bioeng, 2002, 94(6):536544.



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