JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 4, 275-277. 1991

NOTES

Autonomously Replicating Fragments of Mitochondrial DNA from Penicillium urticae in Saccharomyces cerevisiae

HIROKI Y A M A M O T O , YOUKO Y O S H I D A , YASUNORI K I T A M U R A , AND JU N ICH I SEKIGUCHI*

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386, Japan

Received 13 November 1990/Accepted 5 January 1991

Eight fragments which cover the whole range o f the mitochondrial genome o f Penicillium urticae were subcloned into the yeast integration vector YIp5. Transformat ion o f Saccharomyces cerevisiae with the con- structed plasmids by the alkali cat ion method indicated that six plasmids are able to replicate in yeast. Both closed and open circular forms o f the plasmids ~ere detected in the D N A extracts from transformants . Distr ibution o f the a u t o n o m o u s l y replicating sequence in the mitochondrial genome was similar to that in P. chrysogenum except for one small region.

Penicillium is an industrial ly impor tant genus which pro- duces commercial antibiotics, secondary metaboli tes and enzymes (1). However , there has been little research on its mi tochondr ia l DNA (rot DNA). We have recently cloned mt DNA fragments from P. urticae and characterized the genome structure (2). Two mt DNA fragments which cover 20%o of the P. urticae mt D N A genome have been analyzed for the function of the au tonomous ly replicating sequence (ARS) in S. cerevisiae (2). In this report , we describe ARS activity for mt DNA fragments which cover the total range o f the mt genome.

Since a physical map of the P. urticae mt DNA has been constructed (2), the 7.0 kb SalI-EcoRI fragment which covers the large subunit of r ibosomal RNA gene was cloned into the EcoRI and SalI sites of pUC19, yield- ing plasmid pME2. The 7.0 kb Sall-EcoRI insert was hybrid- ized to the appropr ia te fragments of mt DNA digested with EcoRI, HindIII or EcoRI+HindIII by Southern hy- br idizat ion (data not shown).

Mi tochondr ia l D N A fragments cloned in the plasmid pME2 and the previous five pUC19 derivatives (2) were subcloned into the yeast integrat ion vector YIp5 (Ap r Tet' URA3) which has one restriction site each for EcoRI, HindIII, BamHI and SalI (3). A 5.0 kb BamHI-SalI frag- ment of pMB1, a 7.0 kb SalI-EcoRI fragment of pME2, 1.4 kb, 4.8 kb and 0.9 kb HindIII fragments of pMH7, pMH2 and pMP3, respectively, and a 3.0 kb EcoRI frag- ment of pME4 were subcloned into the corresponding site(s) of YIp5, yielding plasmids YIp5BI, YIp5E2, YIp5H7, YIp5H2, YIp5H8 and YIp5E4, respectively. Inserted mt DNA fragments of these derivatives, and YIp5E5 and YIp5EI2 described previously (2), are shown in Fig. 1.

Trans format ion with these plasmids of S. cerevisiae SHY3 (a ste-VC9 ura3-52 trpl-289 leu2-3 leu2-112 his3- Zl adel-lOl canl-lO0) (5) was done to assay ARS activity of the mt D N A fragments. Transformat ion with six plasmids (YIp5B1, YIp5E2, YIp5H8, YIp5E5, YIp5E4 and YIp5E12) yielded 52-95 t ransformants per /zg DNA, but two plasmids (YIp5H7 and YIp5H2) did not yield an)"

* Corresponding author.

colonies (Table 1). Al though the t ransformat ion frequen- cies with the six plasmids were slightly low, the frequencies were at least 500 times higher than that of the control YIp5 (Table 1). Ura segregants were frequently obtained when the t ransformants were cultured in YEPD medium. These results indicate that the six mt DNA fragments contained ARSs.

To confirm the presence of plasmids in the t ransfor- mants, total DNAs extracted by the rapid alkaline method (7) were subjected to agarose gel electrophoresis (Fig. 2A), fol lowed by Southern hybridizat ion with YIp5 as a probe (Fig. 2B). Lanes 1 to 7 in Fig. 2A show common bands which correspond to closed circular (cc) and open circular (oc) forms of 2-/~m DNA. Plasmids YIp5H8, YIp5E5, YIp5E4 and YIp5E12 were detected as both cc and oc forms (lanes 3 to 6 in Fig. 2A and 2B). For YIp5BI and YIp5E2, both forms were visible in Southern hybridizat ion (Fig. 2B lanes 1 and 2, respectively). These results support the finding that the six mt DNA fragments contained ARSs. Judging from a compar ison with the density of

TABLE 1. Transformation frequencies of S. cerevisiae SHY-3 with chimera plasmids"

Plasmid Size of inserted Transformants per pg DNA mt DNA (kb) Exp. 1 Exp. 2

YIp5 0 YIp5BI 5.0 YIp5E2 7.0 YIp5H7 1.4 YIp5H2 4.7 YIp5H8 0.9 YIp5E5 2.0 YIp5E4 3.0 YIp5E12 3.4

• 0.1 68.2 67.8 0.1 0.1

52.0 58.0

94.8

• 0.1

75.0 79.0

275

A 0.5 ml cell suspension of S. cerevisiae SHY3 (2 × l0 s cells/ml) and 10/zg plasmid DNA of a YIp5 derivative were used for transfor- mation (6). Ura- transformants were selected on SD agar medium [6.7 g yeast nitrogen base w/o amino acids (Difco), 20 g glucose and 15 g agar per liter (pH 5.3)] supplemented with histidine (35 Ftg/ml), leucine (30/tg/ml), tryptophan (100 ~g/ml) and adenine (30 ltg/ml).

276 YAMAMOTO ET AL. J. FERMENt. BIOENG.,

S l Y I p 5 t l 1 ¥ 11 ,51 t8

S l

F-- Y I p 5 E 2 _; 'i Y,.s,,2 ;i.,,,sE. i

B S1 X H N H E A tt P tt l'tN I L I II11 1 I II I _

0 L-rRNA

HE H I1 t I I

S-rRNA

iiiiiii iiJ ;!iiiii i i ! i i i i i i i i i i ! ! iiiiiii!iiiii iiiiiiiiiii! iii!iiiiiiiii i; iiJi iiii!ii ii iiiiii

15

P H Ell is P E E I I II [ I I ]

B

t 27.2kb

I!iiiii!iJS••;ii!iii!iiiiii•!JiJ•ii•iiiiiiiiiiiiiii!!!;ii[;iiiiiiiii!•iiiiiiiiiii•i•ii!•iii!i!•!!!i!!i!i!iiii•

II E H

B S l lil 11 tt E H l l P H H E P E E t B

0 27. l_kb

]II s,,, 'i B E A tl H N HE tl PH tiE P EH E

I I l l [ I I I II 1 [ I II I I 1 I

I 0 L-r RNA S-rRNA 25.8kb

FIG. 1. Restriction maps and comparison of ARS regions of P. urticae and P. chrysogenum nat DNAs. Clone designation and the inserted fragments of P. urticae 2159A mt DNA are shown above restriction map I. I to Ill are mt DNAs of P. urticae 2159A (2), P. chrysogenum BC2 (4) and NRRL 1951 (2), respectively. Dotted and open boxes represent ARS and non-ARS regions, respectively. L-rRNA, large subunit of rRNA; S- rRNA, small subunit of rRNA. A HindIII site closer to the Ncol site in YIp5E2 was recently found and added to the restriction map of P. urticae mt DNA (2). The Ncol site(s) is undetermined for the mt DNA of P. chrysogenum BC2. A, Aval; B, BamH1; E, EcoRI; H, HindIII; N, Ncol; P, Pstl; S, SacI; Sl, Sail; X, XhoI.

b a n d s o f the h igh copy n u m b e r 2-/tm p lasmid , the smal ler p la smids (YIp5H8, YIp5E5 and YIp5E4) seem to exist as r a the r h i g h - c o p y n u m b e r p l a smids in S. cerevisiae (lanes 3 to 5).

Loca t i ons o f the ARSs in the P. urticae mt D N A g e n o m e are s h o w n in Fig. 1I. The two f r agmen t s which con ta in the S - r R N A region and f lanking region did not con- ta in A R S activi ty, A R S activity for the f r a g m e n t con ta in -

oh, mt P

2~m oc P

21]m cc --~

FIG. 2.

A B

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

Southern hybridization analysis of plasmids from transformants of S. cerevisiae SHY-3. A, 1% agarose gel electrophoresis; B, Southern hybridization of panel A. YIp5 DNA was labeled with a nick translation kit (Nippon gene) containing [n-32p]dATP (HAS) and used as a probe. DNA which had been separated by agarose gel electrophoresis was transferred to a nylon membrane (magnagraph nylon: Micron Separa- tion inc.) by the method of Southern (8). Hybridization was done basically as described by Maniatis et al. (9). Lane 0, HindIIl-digested 2 phage DNA; lanes 1-6, DNAs prepared from transformants with YIp5BI, YIp5E2, YIp5H8, YIp5E5, YIp5E4 and YIp5E12, respectively; lane 7, DNA preparation from S. cerevisiae SHY-3. Closed and open triangles in lanes 1 to 6 represent positions of closed and open circular DNAs of YIp5B 1, Ylp5E2, YIp5HS, YIp5E5, YIp5E4 and YIp5E 12, respectively, ch, Chromosomal DNA; mr, mitochondrial genomic DNA; 2/tin oc and 2 Ima cc, open and closed circular forms of 2-/~m DNA, respectively.

VOL. 71, 1991 NOTES 277

ing the L - r R N A gene o f P. chrysogenum has not been repor ted (4). H o w e v e r , when P. urticae and P. chry- sogenum are c o m p a r e d , the A R S d i s t r ibu t ion in P. ur- ticae is seen to be very similar to that in P. chrysogenum ex- cept for the r ight f lanking reg ion o f S - r R N A (Fig. 1).

H e t e r o l o g o u s mt D N A f ragments with A R S act ivi ty in S. cerevisiae have been repor t ed for several yeasts and fungi (4, 10-12). H o w e v e r , in the genus Penicillium, they have h i ther to been repor ted only for P. chrysogenum. M o r e o v e r , repor ts o f A R S tests which covered the whole range o f the mt g e n o m e are very rare. The re fo re , our results give basic i n f o r m a t i o n abou t the d i s t r ibu t ion and a p p r o x i m a t e n u m b e r o f A R S s in the mt g e n o m e of Penicillium. Our current research is directed t oward the d e v e l o p m e n t o f a t r a n s f o r m a t i o n system and func t iona l analysis o f the A R S s in P. urticae.

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