pll9t-4'paxU Etub2l3 .. :> .. .. > > u u . .. - _ . .. . - - - -- - - . 23.1 1.& - 0.3 4.4 - 2.3- 2.0-
Figure 3.8 PCR screen ing and Southe rn a n alysis of CY2 transfonnants containing pSS1 6 (paxGMC)
(A) PCR screen i ng for pSS 1 6 (paxGMC) in CY2 backg roun d . Each transformant was screened with one primer set at either end of the construct (pI199-3/pax1 22 and p1l99- 4/pax64). The transformants (labelled black) that gave a PCR product for both sets of p ri m e rs a n d a l s o CY2 a n d C Y 2 . V . 2 ( n ega tive co ntrol a n d vecto r o n l y co n t ro l, respectively) were screened further by Southern analysis as shown in (B). See Figure 3 . 7 for primer positions. tub2 was used as a control (amplified with primer set Etub2/3).
( B ) Southern analysis of CY2/paxGMC transformants. Hindl l l-digested genomic DNA
from CY2/paxGMC tra nsformants hybrid ised with the [32P ]-labelled Hind I I I frag ment from the p S S 8 construct . T h e n u m be rs o n the left corre s p o n d to the sizes of ADNNHindl l 1 fragments and those on the right correspond to the expected sizes of the restriction fragments that hybridised to the probe.
All fragment sizes are shown in kb. P 1 6=pSS 1 6; V=p1 l99 vector.
Standard PCR components (Section 2 . 1 4.2) were used with the followi ng thermocycle conditions: 1 cycle of 94°C for 2 min; 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for either 2 min (for p1l99-3/pax1 22 and p I l99-4/pax64) or 1 min (for Etub2/3); 1
cycle of 72°C for 5 min.
The TLC analysis ( Sectio n 2 . 2 1 ) of extracts of CY2/pSS 1 6 tra nsformants conta i ni ng the pSS 1 6 i nsert showed no detectable i ndole-d iterpenes (Fig u re 3 . 9A) . This was fu rther confirmed by H P LC ana lysis (Section 2 . 2 1 ) (Fig u re 3 . 98). These res ults confirmed that the introduction of pSS 1 6 i nto C Y2 was not sufficient to restore the ability to synthesise paspa li ne.
3.1 .5 Preparation of pSS20
Since the i ntrod ucti on of p SS 1 6 i nto CY2 fa iled to restore the abi lity to synthesise paspaline the con struct pSS20 containing paxGAMC was prepared . To p repare this construct, a paxA-paxM fragment containing 479 bp of the seq uence 5' of the paxM ATG , all 1 559 bp of the paxM gene seq uence, all 752 bp of the seq uence between paxM and paxA, all 1 1 3 1 bp of the paxA gene seq uence and 22 bp of the seq uence fla nking the 3' end of paxA was PC R amplified from wild-type genomic D NA (Section 2 . 1 4 .4) using the pri mers SS9 and paxMSpe F 1 , contai ning native and introduced Spel sites, respectively (Fig u re 3 . 1 0) . The paxA-paxM PC R prod u ct was subcloned into pGEM®-T Easy vector (Promeg a) to generate pSS 1 9 and subseq uently seq uenced. A clone witho ut any PC R-i ntrod u ced error was then used to obtain the Spel fragment, contai ning paxA-paxM, wh ich was then l igated i nto the Spel site of pSS8 to generate pSS20.
3.1 .6 pSS20 transformation of pax deletion mutant CY2
Protoplasts of CY2 were transformed with 5 I-Ig of ci rcu lar pl l99 or p SS20 and genetici n-resista nt tra nsforma nts selected on RG med i u m supplemented with 1 50 !-Ig/ml of geneticin (Secti on 2 . 1 3 . 2) . Ten a rbitrarily selected genetici n resistant CY2/pSS20 tra n sforma nts were screened for i ntegration of the pSS20 insert by PCR ( Section 2 . 1 4 .2) usi ng primer sets designed to amp l ify each end of the insert (Figure 3 . 1 1 A) . Eight out of ten transformants showed both produ cts . The absence of PCR prod ucts i n two transformants was n ot due to template q ual ity as confi rmed by the amp lification of tub2 prod uct in these tran sforma nts . The positive transformants were fu rther a nalysed by Southern blotting a nd hybridisation (Section 2 . 1 0) using a
e
2P]-label led Hind l l l restriction frag ment from pSS8 as the p robe (Figure 3 . 1 1 B). I n these transformants theA
13-dp paspaline paxilline N :> N N >- >- 0 0B
CY2.P16·9 CY2.P16-8 CY2.P1 6·7 CY2.P16-8 CY2.P16-5 CY2.P16 .... CY2.P1 6-3 CY2.P1 6·2 CY2.P16·1 CY2.V.2 CY2 wlld-type ... � <? .,. o<t et � or fit tI co co co co co co co co co�
... ... ... ... ... ... ... ... ... � � � � � � c., � Il. N N N N N N N N N :2 >- >- >- >- >- >- >- >- >- 'i 0 0 0 0 0 0 0 0 0 paspaline o 5 10 1 5 20 25 30Retention time (minI
Figure 3.9 NP-TLC and RP-HPLC analysis of CY2/pSS1 6 transformants
CY2 transformants containing the construct pSS 1 6 (paxGMC) were analysed by (A) N P-TLC a n d
(8)
R P- H P LC for the p rese n ce of indole-d iterpen es (Section 2 . 2 1 ) . Mycelium was g rown for 7 days in COVE, supplemented with trace element m ix (Section 2.3.2 ), at 28°C with shaking at 200 rpm, freeze d ried a n d e xt racted in 2 : 1 chlorofo rm-metha n ol mixtu re. V=p l l 99 vecto r; P 1 6=pSS 1 6 ; 1 3-dp= 1 3-desoxypaxilline.SS9 paxMSpeF1
paxM �
paxA
pax122 Spel Spel* pax64
�
pax G SpeJ paxA paxM pax B Spel paxC
Hind 11 I Hind 11 I
Hind 11 I 0.5 kb
pll99 nptlf
5249 bp
Fig ure 3 . 1 0 pSS20 (paxGAMC) construct
The pSS20 construct was made by ligating a peR generated copy of paxA-paxM
( a m p l ified with TripleMaster@ p e R System using the prim e r pair S S 9 a n d paxMSpe F 1 ) into the Spel-digested pSSB.
Restriction sites used for cloning and positions of primers used for peR a n d
screening transformants are shown i n green and pink, respectively. * ind icates