A cloned tryptophan-synthesis gene from the Ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans

79

Gene, 42 ( 1986) 79-88 Elsevier GENE

1548

A cloned tryptophan-synthesis gene from the Ascomycete Escherichia coli, yeast and Aspergillus nidulans (Recombinant

DNA;

bacteriophage

plant

pathogen;

fungal

genetics;

C~e~Zi~~~las~eterostpuphus functions in

heterologous

complementation;

shuttle

vector;

IL)

B. Gillian Turgeon, W. Donald MacRae,

Robert C. Garber, G.R. Fink * and O.C. Yoder **

Department qf Plant Pathology, Cornell Univ., Ithaca, NY 14853, Tel. (607)255-3243: and * Whitehead Institute. 9 Cambridge Center, Cambridge, h&i 02142 [U.S.A.) Tel. j617)2.%-5215 (Received

October 23rd.

(Revision

received

1985)

and accepted

December

18th, 1985)

SUMMARY

A gene (TRPI ) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene also complemented an E. co& trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trpl mutant missing PRAI activity and an Aspergillus nidulans trpC mutant. It functioned in E. coli and A. niduluns without apparent rearrangement but in yeast only after the 5’ end of the gene was deleted. The gene was subcloned on a 4.65kb DNA fragment and the PRAI domain was localized to a 2.9-kb region. It showed homology to the A. nidulans trpC and Neurospora crassa #p-l genes. There was one predominant transcript of C. heterostrophus TRPI, the same size (2.6kb) as one of the two functional transcripts produced by A. niduIans trpC. The constitutive activity of the C. heterostrophus TRPl gene was high whereas that of the A. nidulans trpC gene was low.

iNTRODUCTiON

The tryptophan pathways in E. coli, yeast, and N. crussa are well understood (Schechtman and Yanofsky, 1983). There are five steps from choris.____ ** To

whom

correspondence

and reprint

requests

should

be

addressed. Abbreviations: serum

Ap, ampicillin;

albumin;

GAT.

illdoleglycerolphosphate

EDTA;

0378-I

isomerase;

sulfate;

0.015 M Na,

Sm,

BSA, bovine

amidotransferase;

synthetase;

phoribosylanthraniiat~ dodecyl

bp, base pair(s);

glutamine

IGPS,

kb, 1000 bp; PRAI, R. resistance;

streptomycin;

SSC,

SDS,

phossodium

0.15 M NaCi,

‘citrate, pH 7-8; TE, IO mM Tris (pH 7.5) 10 mM

[ 1,designates

I lY~86:$0?.50

0

plasmid-carrier

1986 Elsevier

state.

Science

Publishers

B.V.

mate to t~ptophan, catalyzed by seven enzymatic activities; the steps are the same in bacteria, yeast, and lilamentous fungi (Schechtman and Yanofsky, 1983). The seven enzymatic functions of E. cofi are encoded in a single operon and designated tlpA through trpG. In the filamentous fungi N. crassu and A. nidulu~s four unlinked genes encode four polypeptides, of which two are monofunctional, one is bifunctional, and one is trifunctional. The activities of the trifunctional gene (tip-I in N. crassa, trpC in A. nidulans) correspond to the trpG, trpC, and trpF functions of E. co& which encode CAT, IGPS, and PRAI, respectively (Schechtman and Yanofsky, 1983; Yelton et al., 1983). We isolated a gene encoding PRAI from the fila-

~Biomedi~~ Division)

mentous

maize

pathogen

gene is potentially construction

This

(b) Isolation and manipulation

of DNA

useful as a selectable marker in the

0ftr~sforInation

transformation study

C. heterostrophus.

technology

the molecular

vectors. We are using (Turgeon

were prepared

lysis (Birnboim

from E. cob’ by either the and

Daly,

boiling

(Holmes

C. heterostrophus. The strategy employed to clone the

nomic

DNAs

C. heterostrophus PRAI gene, complementation

A, niduluns and yeast were isolated

corresponding

has been

of pathogenicity

Plasmids alkaline

in

E. colt’ trpF mutant,

genetics

et al., 1985) to

used

gene from yeast (Struhl

N. crassa (Keesey and Yanofsky,

and DeMoss,

to clone

of an the

et al., 1979)

1982; Schechtman

1983) and A. nidufans (Yelton

et al.,

1983).

described

and Quigley, from

previously

1979) or the

1981) method.

C. heterostrophus, (Garber

Yelton et al., 1984; Sherman

Ge-

N. crassu,

and purified

and

Yoder,

as

1983;

et al., 1981). Restric-

tion enzyme digestions, hybridization analyses, and nick translations were performed according to standard

procedures

(Maniatis

et al.,

1982).

Filter

hybridizations were in 50 mM Tris buffer pH 7.5. containing 1 M NaCl. 1 mM EDTA, 10 x Denhardt’s and 10 iig salmon sperm DNA/ml at 65 -C; for lower stringency the temperature was SO’C. MATERIALS

AND METHODS

(c) Construction

of C. herewstroprkus genomic lib-

(a) Strains and media

raries

E. coli K-12 strain DHl [recA 1, e&A 1, gyrA96, thi-1, hsdR 17 (r, - , mk ), supE44] was used for

Genomic DNA was partially digested with Suu3A, size fractionated, and ligated into the BavrzHI site of either the yeast/E. cofi shuttle vector YEp24 (Botstein et al., 1979) or the i replacement vector EMBL4 (Frischauf et al., 1983). The plasmid library contained approx. 10J clones, 80”/, of which had inserts averaging 11 kb; the probability of finding any given sequence in the library was 0.98

library construction. Strain JA300 (thr, leuB6, thi, thyA, trpClI17, hsd~, hsdR) was used to isolate the C. heterostrophus TRPl gene and, atong with strain HBlOl [hsdS20(r,-,m,-), recA13,ara-14, pruA2, lacY1, galK2, rpsL20, Sm R, ~$5, mtl- 1, supE44] to propagate plasmids. Mutants of strain W3 1IO, each carrying a different mutation in the trp operon (Table I) were from Dr. C. Yanofsky. Since strain W3110 is hsdR +, all plasmids used in attempts to complement the various trp - mutations were first methylated by passage through strain DB6656 (pyrF: : Mu, rrp,,,,, IacZ, ,,,, hsdR _ hsdM * ). S. cere~~~iue strain F762 (trpl-A 1, ura3-52)

(Clarke and Carbon, 1976). The i, library consisted of 4 x 10’ clones with inserts of about 1%kb. (d) Transformation E. colt’ strains

and

A. nidulans strain UCDl (argB2, yA2, puhuA 1, metG, trpC801, biA1) were used to study the expression of the C. heterostrophus TRPl gene in heterologous fungal hosts. C. heterostrophus strain C3 (MA T-2. toxl ; ATCC 48330), described earlier (Leach et al., 1982) was the source of DNA and RNA used for all studies reported here. All microbial strains were stored at -70°C in glycerol (50’,!, for bacteria, 25 y0 for fungi) and recovered fresh for each experiment. Standard complete and minimal media for E. coli (Maniatis et al., 1982), yeast (Sherman et al., 1981), C. heterostrophus (Leach et al., 1982) and A. nidulans (Kafer, 1977; Barratt et al., 1965) were used where appropriate.

were transformed

by either

the

CaCl, (M~iatis et al., 1982) or the hexamine cobalt chloride (Hanahan, 1983) method, yeast by the lithium acetate protocol (Ito et al., 1983), and A. niduluns by the procedure of Yelton et al. (1984).

RESULTS

(a) Isolation of the C. heterostrophus from the plasmid library

TRPl

gene

Competent ceils of E. coii JA300 were transformed with 0.5 /lg C. heterostrophus plasmid library in YEp24 and plated on supplemented M9 medium

81

without

tryptophan.

After 2 days at 37 “C, 25 col-

onies were visible; 24 of these were Ap resistant. of the 24 grew vigorously lacking

tryptophan,

which transformed

Six

when returned

to medium

and five contained

a plasmid

strain

JA300

to Trp’

at high

frequency. One plasmid, designated pChTRP24, was labeled with [r-‘2P]dGTP and used to probe C. heterostrophus

genomic

DNA

and

ize

to

C. heterostrophus

lane a4) or to those fragments

sites in pChTRP24 rostrophus

of pChTRP24

gene

which (Fig. 1, enzyme

carried

by pChTRP24

is called

TRPl.

pChTRP24,

restriction

enzymes.

from the 3, library

hybridizing

genomic

at least

co-migrated

one

of the C. heterostrophus

TRPI

gene

with a

fragment from the cloned DNA (e.g. Fig. 1, lanes b3 and b4), identifying the isolated sequence as C. heterostrophus DNA. [ 32P]YEp24 did not hybrid-

To determine

if selection

for Trp’

in E. coli

caused rearrangement of the C. heterostrophus TRPl gene, approx. 7000 recombinant clones from a C. heterostrophus genomic library in the 1. vector EMBL4 were probed with pChTRP24. Seven plaques hybridized strongly and three of these were analyzed further. All three overlapped one end of the pChTRP24 insert and one overlapped and extended beyond the other end. Comparison of fragments from restriction digests of the EMBL4 clones with

2341234

1

(Fig. 1,

is shown in Fig. 2. The C. hete-

(b) Isolation

In every digest

DNA

contained only C. heterostrophus DNA lane a3 vs. lane b3). A map of restriction

both of which had been digested with each of several fragment

genomic

9.4.

6.6

0.6. b

a Fig. 1. Autoradiogram to fragments

presenting

of genomic

hybridization

C. heterosfrophus

sample was digested

with HindIlI,

in an 0.6”,, agarose

gel in Tris-acetate

tate,

0.002 M EDTA),

(Schleicher (a)YEp24

& Schuell, or

the fragments to

BA 85) and

probed

nitrocellulose

Lane 1, yeast

with

C. heterostrophus genomic

DNA

fragments (compare faint bands fragments. digestion.

of pChTRP24

DNA acepaper

“P-labeled

genomic

(2 icg): lane 2, YEp24 (50 ng); lane 3, pChTRP24 to C. hererostrophus genomic

Each

were separated

buffer (0.04 M Tris

transferred

(b)pChTRP24.

of pChTRP24

DNA.

DNA

(50 ng); lane 4,

( 10 pg). pChTRP24

hybridized

DNA (lane b4) and to restriction

which

did not hybridize

lanes a3, b3). The film was overexposed in lane b4, which represent

to YEp24 to show the

the vector-insert

border

Faint bands in other lanes are the result of incomplete Fragment

sizes (in kb) are specified on the left margin.

Fig. 2. Map ofpChTRP24. 8.2-kb

C. heferostrophus

portion tion;

Dashed insert.

of the yeast 2~ plasmid

containing

gene; 2~ is a

its origin of replica-

amp is the ApR gene from pBR322. TRPl lies between 3.5

and 8.2 kb. Clockwise

numbers

kb coordinates.

The shaded

cates a deletion

that occurred

yeast,

line is YEp24; solid line is the

URA3 is a yeast

within

of the transcript

was reverted

(Fig. 4) and a Trp’

the direction

of transcription;

have not been precisely

the

6.5 and 9.7 kb indi-

when pChTRP24

giving rise to pChTRP24d2

type. The arrow indicates

the circle represent

area between

mapped.

in

phenothe ends

82

AChTRPl-1

A A

I

A

.I.

AChTRPl-2

b

I

A

.I.

J

lI*

.

A

*I.

.

AChTRPl-3

Fig. 3. Comparison 1 library

I

A A

pChTRP24 insert

of the restriction

with [32P]pChTRP24. in the portions

map of the pChTRP24

extends

end. Vertical bars areEcoR1

of the 1 clones that overlapped

I

I

I

I

I

I

I

insert with those of three clones recovered

One of the clones (IChTRPl-1)

/z clones extend beyond the right-hand sites mapped

.

beyond

the left-hand

sites, dots are Sal1 sites, and triangles

pChTRP24

aligned

areHind

(c) Complementation

(d) Deletion mapping of pChTRP24

To determine how many E. coli trp functions could be complemented by the C. heterostrophus clone, trp - cells were transformed with pChTRP24 and selected for Ap resistance. Each plate oftransformed colonies was then printed to minimal medium to test

The recombinant

(Fig. 2) was

I of E. coli trp _ mutants

Complementation

Cells of each strain were transformed minimal

plasmid pChTRP24

digested with MndIII, EcoRI, BumHI, Sal1 or XhoI, and fragments from each digestion were self-ligated and used to transform E. coli strain JA300 to Ap resistance. The plasmids recovered were analyzed by restriction enzyme digestion and fractionation on agarose gels. Those with deletions in the

for tryptophan-independent growth. The C. heterostrophus sequence complemented E. coli trpF-

TABLE

all three

not trpD or trpE to test directly for because that enzyme E. coli (Schechtman

functional complementation of E. cofi had not undergone major rearrangement. These results are diagrammed in Fig. 3. of E. coli trp - mutants

insert;

sites. All restriction

with sites in pChTRP24.

(PRAI) and trpC (IGPS) but (Table I). It was not possible trpG + (CAT) activity in E. cofi is not essential for growth of and Yanofsky, 1983).

fragments of pChTRP24 digested with the same enzymes indicated that the sequence isolated by

a C. heterosrrophu.v

by probing

end of the pChTRP24

medium

with or without

Strain

by pChTRP24

with pChTRP24

and plated

on L agar containing

Ap. After one day, colonies

were printed

to

tryptophan. Relevant

Growth

on minimal

medium:

genotype With tryptophan

Without

JA300 rrpC1117

rrpF

+

+

W3110 drrpClO-16

trpC _, trpF _

+

+

W3 110 wpC782

trpC

+

W3 110 AtrpES

trpE

+

+ _

w3110 AtrpLD102

rrpE -. , trpD _ a

+

I’ Lack of trpD activity

was determined

indole but not on medium

supplemented

by showing

that W3 IlOAfrpLD 102[pChTRP24]

with anthranilate.

tryptophan

grew on minimal

medium

supplemented

with

83

t rpC-F+

t rpC+F-

t rpC-F‘

8 pChTRP24B

. . . . . . . . . . .‘B

pChTRP24H

.............

pChTRP24E

. . . . . . . . . . . . . . . . . E

pChTRP24X

B

x

m

.

H ............

.”

E . . . . . . . . . . . .

x . . . . . . . . . . . . . . -

B

s s . . . . . . . . . . .

B

pChTRP24S

B

B pChTRP24A2

’

. . . . . . . . . .

pChTRP24A2B.

Fig. 4. Deletion by digesting digestion represent

.’ -

.........

mapping

the plasmid

products, deletions.

tryptophan-independent

of the Trp functions with BarnHI

and recircularizing To assess

trpC _ F‘ , trpC_ Fm are mutants

C. heterostrophus DNA function in various tip

in pChTRP24.

Deletions

were introduced

either by reversion

(B), Hind111 (H), EcoRI (E), XhoI (X), or Sal1 (S), recovering it by ligation. Thick lines are C. heterosmphus sequences,

Trp function,

growth.

B

..........

+ indicates

E. coli cells were transformed normal growth;

- indicates

of E. coli (see Table I). kb markers

insert were tested for Trp mutants of E. coli.

The region of pChTRP24 necessary for trpC+ (IGPS) and trpF+ (PRAI) functions in E. cofi was localized between 3.6 and 8.3 kb on the map (Figs. 2 and 4). Both functions were retained when the remainder of the C. heterostrophus sequence, from 0 to 3.6 kb, was deleted (Fig. 4; pChTRP24B). If the C. heterostrophus sequence between 3.6 and 5.5 kb was deleted (Fig. 4, pChTRP24 H, E, and X), both IGPS and PRAI functions were lost. If all or part of the sequence between 5.3 and 8.3 kb was deleted (Fig. 4, pChTRP24 S, 42, and A2B), IGPS function was lost but PRAI function was retained. These observations determine the relative locations of the IGPS and PRAI domains on the clone and indicate that the function of the IGPS domain depends on an intact PRAI domain, but not vice versa.

with each

no growth;

are included

plasmid

of pChTRP24

the largest

fragment

in yeast or among

the

thin lines are YEp24, and dotted lines and transformants

42, plasmid isolated by reversion

were tested

for

in yeast. trpC + F-,

for pChTRP24.

(e) Homology to N. crassa and A. nidulans trp genes To test for homology

among

C. heterostrophus

TRPl, A. nidulans trpC and N. crassa trp-1, plasmids

containing the cloned genes from each organism were digested with enzymes which cut all or part of the insert out of the vector. The digestions were fractionated in agarose gels, Southern-blotted, and with 32P-labeled plasmids probed at 50°C pChTRP24,

pNC2 (N. crassa), or pHY201

(A. nidu-

lam).

Probing PvuII-digested pChTRP24 with pNC2, which carries N. crassa trp-I or with pHY201, which carries A. nidulans trpC, indicated that the three genes are homologous. For example, pNC2 and pHY201 hybridized to the 3.8-kb PvuII fragment of pChTRP24 (Fig. 5, arrowhead). This fragment contains only C. heterostrophus DNA and includes

84

123456

pNC2, and selected recovered

were

rate of one/l0

kig pChTRP24

6//lg of pNC2.

wild-

growth rates and morphologies

com-

plete from pChTRP24

genomes

which

had

received

and probed with

with [“P]pBR322 firmed

transformants ofA.

con-

that (not

their This homology that the

C. heterostrophus

TRPI

gene is functionally There was no homology

between DNA. (g) Expression

Fig. 5. Autoradiograms

representing

hybridization

of C. het~~

trophus TKPI DNA to A. niduluns rrpC and N. UXSSNrrp-I DNAs. details. Lane 1. i, DNA digested

See Fig. 1 for experimental

HindIII; digested digested and

lane 2, pNC2 with

EcoRI + XbaI;

with PvuII. Lanes

/1 DNA

[72P]pHY201; indicates

digested

digested

lane 6 probed

the 32%kb PvuII

C. heterosirophus

domains

and hybridizes

Psrl;

lane4,

with

pHY2OI

lanes 3, 5. and 6. pChTRP24

1, 2 and 3 probed

with

entirely

with

HirldIII;

with [‘*P]pNC?

lanes4,

5 probed

with [“P]pChTRP24. fragment

DNA,

of pChTRP24

spans

the IGPS

to probes containing

hith

Arrowhead which and

is

PRAI

N. UO.WI II~)-I and

A. niduluns trpC. Other bands in lanes 3 and 5 which hybridized to pChTRP24

contained

both vector

and insert sequences.

the IGPS and PRAI domains (Fig. 4). The heterologous hybridizations were less intense than the homologous hybridization, suggesting that there may be substantial sequence differences between C. heterostrophus TRPI and the other two genes. (f) Expression

of

C. heterostrophus

TRPI

A. nidulans Protoplasts of A. nidzduns transformed with

UCDl

in

TRPI in yeast

When of yeast F762 transformed with ‘, colonies arose at the of about 2OOO/LlgDNA. ’ colonies were Trp on medium lacking tryptophan. To obtain Trp’ colonies, the Ura+ Trp transformants were grown for 2 days on minimal agar medium plus tryptophan, then printed to the same medium without tryptophan. After 4 days about 50 colonies appeared, several of which were purified by streaking on minimal medium. Mitotic co-segregation of the yeast URA3 and C. heterostrophus TRPl genes was demonstrated by growing the cells to stationary phase in complete liquid medium, then plating on complete agar medium followed by printing to minimal medium supplemented with either tryptophan or uracil. About 10”; of the cells lost both URA3 and TRPI activities while the rest retained both genes, demonstrating that the TRPl activity was plasmid-encoded. Cells of E. coli JA300 (trpF ) were transformed with plasmid DNA isolated from twelve yeast revertants, selected for Ap resistance, and printed to supplemented M9 medium lacking tryptophan. All transformants grew vigorously, indicating that each plasmid retained PRAI activity. Restriction enzyme digests of plasmid DNA isolated from one E. co/i transformant (chosen at random) from each of the twelve E. coli transformations showed that two of the twelve clones tested had deletions of about 3 kb (Fig. 6). These two apparently identical clones, designated pChTRP24d 1 and pChTRP24d2, trans-

85

the construction

which had the single Hind111 on the

PvuII

fragment

nearest

tation

1) produced

the SP6 promoter

a transcript

(orien-

which hybridized

to

poly(A)+ RNA; the transcript from the opposite construction (orientation 2) did not hybridize. When C. heterostrophus poly(A)’ runoff transcript band

and

several

smaller

(Fig. 7, lanes 3,4).

of pChTRP24

that function

from twelve Trp + yeast colonies to transform

E.

coli

with EcoRI,

stained

with

details.

Lane 1, pChTRP24;

from

different

HirrdIII.

found to be Trp(pChTRP24dl about

fractionated bromide.

E. co/i clones;

All plasmids

pChTRP24

to Ap resistance.

digested

ethidium

carrying

Plasmid

were used were

gel, and

See Fig. 1 for experimental

lanes 2-13,

plasmids

lane 14. i. DNA except

DNAs

DNAs

on a 0.6’,,, agarose

were identical

in yeast,

in yeast.

recovered

digested

to pChTRP24 for those

and 42), each of which

in lanes 3 and IO

sustained

3 kb and was Trp + when transformed

with

and were

a deletion

of

back into yeast.

formed cells of yeast strain F762 to Trp’ at high frequency whereas the other ten (which were indistinguishable from pChTRP24) did not. Thus, although the native C. heterostrophus TRPl gene did not function in yeast, occasional mutations of the recombinant plasmid permitted expression in yeast. Failure to recover Trp + plasmids from some of the Trp + yeast colonies probably reflects differential segregation of pChTRP24 and its rearranged derivatives either in yeast or in E. coli cells. To determine if the deletion caused loss of trpC activity, cells of E. coli strains JA300 (trpF ), W3110trpC782 (trpC_), and W3110dtrpClO-16 (trpC trpF -) were transformed to Ap resistance with pChTRP24d2 and printed to supplemented medium without tryptophan. Transformed JA300 cells were Trp + , confirming trpF activity, whereas cells of trpC782 and AtrpClO-16 remained Trpeven though they were Ap resistant, indicating that pChTRP24d2 had lost trpC activity. (h) Transcriptional

analysis

The PvuII fragment between 3.5 and 7.75kb on the map of C. heterostrophus TRPl (Fig. 2) was inserted in both orientations into vector pSP64. Only

The

1 a single strong

weak bands strongest

slightly more intense

in RNA isolated medium

on

minimal indicating

substantial

hybridized

band

grown medium, Fig. 6. Revertants

RNA was probed with a

from orientation

than

was only

from fungus on

complete

constitutive

tran-

scription of TRPl. When A. nidulans RNA from cells grown on minimal medium was probed with a transcript derived from the A. nidulans trpC gene, a weak band was seen that corresponded in size to the C. heterostrophus main band, along with a stronger band about 200-bp shorter and several other small minor bands (Fig. 7, lane 2). No hybridizing bands were seen in RNA isolated from A. nidufans cells grown in complete medium, confirming that transcription of the trpC gene is very low when this fungus grows in a rich medium (Yelton et al., 1983). Sequence analysis of the A. nidulans trpC gene has shown that the two largest transcripts are authentic mRNAs which are translated; their sizes are 2.6 and 2.4-kb (Mullaney et al., 1985). The most abundant C. heterostrophus transcript must therefore be 2.6-kb since it comigrated with the largest A. nidulans transcript (Fig. 7). Markers included in the gel, however, sized the largest transcript at approx. 2.4-kb; this apparent discrepancy probably reflects the variability sometimes script lengths 1983). (i) Organization

encountered

(Mullaney

in determining

tran-

et al., 1985; Yelton

et al.,

of TRPZ

Because the runoff transcript from orientation 1 and not orientation 2 of the pSP64 constructions containing the C. heterostrophus TRPl gene hybridized to poly(A)+ RNA, we were able to determine the direction of transcription. The transcript which hybridized to C. heterostrophus poly(A) + RNA was by definition produced from the DNA coding strand. The transcript which failed to hybridize to C. heterostrophus poly(A) + RNA had the same sense as the native mRNA. We therefore concluded that the direction of transcription of the C. heterostrophus

86

buffer

pH 7.0. Electrophoresis

10 mM Na’ phosphate

was

buffer,

from the gels to nitrocellulose

paper

identified using RNA probes generated (Riboprobe,

Promega

C. heterostrophus TRPI,

219-

between

agarose

gels in

was transferred

by blotting

in 20 x SSC.

of C. heterostrophus TRPI and A. nidulans trpC were

Transcripts system

in 1.2%

pH 7.0. RNA

the

with an SP6 transcription

Biotec.

Madison,

PvuII fragment

WI).

For

of pChTRP24

3.5 and 7.75 kb on the map (Fig. 2), was electroeluted

from a gel and ligated

into the HincII site of the Riboprobe

plasmid

For A. nidulans trpC, a 3.3-kb BarnHI-

vector

SmaI fragment

pSP64.

of pKBY2 that spanned

from a gel and ligated pSP64 and pSP65. the protocol 1.25-2.5 Na

RNA probes

provided

RNA.

pH 6.5,50”,

Filters

0.05””

Ficoll,

25Opg

salmon

sperm

RNA/ml.

Filters

Na phosphate

were washed

sites of

according

to

Biotec. Specific activities were were probed

formamide,

TA, 0.05 “, BSA, denatured

restriction

were synthesized

by Promega

x 10s cpm/‘pg

phosphate

the trpC gene was eluted

into the appropriate

0.05”,

polyvinylpyrrolidone,

DNA/ml 3-5 times

pH 6.5, 50 mM NaCl,

in 50 mM

0.8 M NaCl, 1 mM EDand

5OOpg yeast

at 75°C

in 20 mM

1 mM EDTA

and 0.15,

SDS. of C. heterostrophus TRPI and A. nidulans

Fig. I. Transcripts trpC. Poly(A)‘RNA

from each fungus

glyoxal gel (McMaster cellulose

paper,

and Carmichael,

and probed

1977) blotted

with 32P-labeled

1 and 2, A. nidulans RNA

Lanes

was fractionated

on a to nitro-

(25 pg/lane)

probed

with

a

from A. nidulans trpC; lanes 3 and 4, C. heterostrophus

transcript

RNA (25 pg/lane)

probed

from C. heterostro-

with a transcript

phus TRPl. RNA in lanes 1 and 3 was from fungi grown in complete medium; indicate

lanes 2 and 4 from minimal

sizes (in kb) of cucumber

RNA was isolated

following

the method

(1969) with some modifications. was seeded

with conidia

with shaking was ground

(2

(250 rev./mm) in a mortar

One volume

of TE-saturated

for 48 h at 20°C. liquid

homogenized

in a Waring

The aqueous

then extracted

twice with chloroform

Nucleic acid was precipitated with 2.5 vol. of ethanol, RNA from DNA,

dissolved

14” triisopropyl

pellet,

with glass

alcohol

(24

of 0.15 M Na and

: 1). ace-

suspended

in

et al., 1982). To to a final concen-

held at 4’C contained

after

phenol - cresol,

(Maniatis

LiCl was added which

: 1) was added

blender

- isoamyl

centrifuged, H,O

of 2 M, then the solution The

50 mM EGTA,

with

in the presence

diethyl-pyrocarbonate-treated

centrifuged.

to

layer was removed

x g). reextracted

overnight

total

in 20 mM Tris buffer pH 7.5 containing

RNA,

and was

500 mM NaCl,

1mM EDTA and 0.1% SDS. Poly(A)‘RNA

was

from total RNA by passage

column (type 3,

Collaborative mendations. denatured presence

Research) For

through following

separation

for 1 h at 50°C

an oligo(dT)

with

of 50”,, dimethylsulfoxide

separated

the manufacturer’s

in gels,

RNA

I5?0 deionized

(j) Conclusions

grown

The mycelium

phenol - cresol (9

(17000

separate

medium

acid, at the rate of 1 g fresh mycelium/ml.

for 60 s at 4°C.

tration

Fungal

and Leaver

N, and transferred

acid, and

centrifugation

tate,

Markers

or complete

pH 8.5 containing

sulfonic

and the mixture

of Lovett

Minimal

6:~ p-aminosalycilic

naphthalene

beads

medium. virus RNAs.

104/ml) and the culture

x

under

cold 200 mM Tris buffer 250 mM NaCl,

mosaic

gene is the same as that of the pSP64 C. heterostroTRPl construction (orientation 2) which gave rise to a non-hybridizing transcript (see preceding section and Fig. 2). The order of functional domains on the C. heterostrophus sequence is thus 5’-IGPSPRAI-3 ’ and the direction of transcription (arrow in Fig. 2) is counterclockwise.

phus

runoff transcripts.

recoin

samples

were

glyoxal

in the

and 10 mM Na

phosphate

The C. heterostrophus TRPI gene, isolated by complementation of an E. coli trpF mutant, is functional in both prokaryotic and eukaryotic cells. This observation supports previous evidence that the activities encoded by the gene are highly conserved (Schechtman and Yanofsky, 1983; Yelton et al., 1983). The N. crassa trp-I gene is known to be trifunctional because it complements three different N. crassa trp mutants, each lacking IGPS, PRAI, or GAT activity (Schechtman and Yanofsky, 1983). A. mdulans trpC is also apparently trifunctional since its primary sequence has extensive homology with N. crassa trp-1 (Mullaney et al., 1985; Schechtman and Yanofsky, 1983). We have found that C. heterostrophus TRPl is similar to the corresponding N. crassa and A. nidulans genes. It clearly encodes IGPS and PRAI because it complements E. coli trpC and trpF mutants. It probably carries a GAT domain as well since the size of the C. heterostrophus

81

TRPl transcript

is the same as those of the N. crassa

trp-1 and A. nidulans trpC genes

1985; Schechtman

and Yanofsky,

There are also differences

(Mullaney

et al.,

1983).

among the three fungal

genes. First, the A. nidulans trpC gene is highly regulated whereas

C. heterostrophus TRPl is not. We ob-

Agriculture

Competitive

and Pioneer

Hi-Bred

was supported

Research

Grants

International.

Office,

W.D. MacRae

by a postdoctoral

fellowship

from

NSERC of Canada. The N. crassa plasmid pNC2 was from M. Schechtman, A. nidulans plasmids pHY201

served only a slight difference in the amount of TRPl

thank

transcript when C. heterostrophus was grown in minimal medium compared with the amount produced in

Barbara

and pKBY2 were from W. Timberlake. Peter

Mullin

Mosher

for technical

for preparation

We

assistance

and

of the manuscript.

complete medium, but no detectable trpC transcript when A. nidulans was grown in complete medium. Second, the fungal genes may differ in size. Sequencing of N. crassa trp-1 and A. nidulans trpC has revealed that all three functional domains are encoded by about 2300 bp and transcripts correspond to that length. Deletion subcloning indicates that up to 3 kb of the C. heterostrophus TRPl sequence may be required for both IGPS and PRAI activity in E. coli (Fig. 4) although the TRPl transcript is the same size as that of A. nidulans trpC. C. heterostrophus TRPl complements both trpC and trpF mutants of E. coli while cloned N. crassa trp-I complements only trpF, even though a trpC domain is present on the clone. The failure of N. crassa trp-1 to complement trpC in E. coli may be caused by lack of a binding site upstream of trpC that E. coli ribosomes can recognize (Schechtman and Yanofsky, 1983). Fusion of a bacterial ribosomeThird,

binding site to trp-1 enables it to complement both trpF and trpC mutations in E. cob (Schechtman and Yanofsky, 1983). PRAI activity of C. heterostrophus TRPl in E. coliis apparently initiated from within the coding region and not from the C. heterostrophus promoter, since the 5’ end of the gene can be eliminated and PRAI activity retained. It is likely that the C. heterostrophus TRPl promoter does not function in yeast, since unrearranged pChTRP24 did not confer a Trp’ phenotype on yeast cells that carried it, while an altered version of the plasmid gave rise to a Trp’ phenotype. The functional version appeared to be pChTRP24 with a 3-kb deletion at the 5’ end of the TRPl coding region.

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