• No results found

The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression.

N/A
N/A
Protected

Academic year: 2020

Share "The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression."

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

Copyright 0 1990 by the Genetics Society of America

The CCRI Gene From

Saccharomyces cerevisiae

Is

Required for Both

Nonfermentative and sfit-Mediated Gene Expression

Clyde

L.

Denis

and Thomas

Malvar

Department of Biochemistry, University of New Hampshire, Durham, New Hampshire 03824

Manuscript received April 1 1, 1989 Accepted for publication November 3, 1989

ABSTRACT

Mutations in the yeast CCR4 gene inhibit expression of the glucose-repressible alcohol dehydrogen-

ase ( A D H P ) , as well as other nonfermentative genes, and suppress increased A D H 2 expression caused by the crel and ere2 alleles. Both the ere1 and ccr4 alleles were shown to affect ADH I1 enzyme activity

by altering the levels of ADHZ mRNA. Mutations in either C R E l or C R E 2 bypassed the inhibition of A D H 2 expression caused by delta insertions at the A D H 2 promoter which displace the A D H 2 activation

sequences 336 bp upstream of the T A T A element. These crel and ere2 effects were suppressible by the ccr4 allele. T h e crel and ccr4 mutations also affected A D H 2 expression when all the A D H 2

regulatory sequences upstream of the T A T A element were deleted. The relationship of the C R E

genes to the SPT genes, which when mutated are capable of bypassing the inhibition of H I S 4 expression caused by a delta promoter insertion (his4-912delta allele), was examined. Both the crel and cre2

mutations allowed his4-912delta expression. ccr4 mutations were able to suppress the ability of the cre alleles to increase hid-912delta expression. C R E 2 was shown to be allelic to the SPT6 gene, and C R E l was found to be allelic to SPTlO. We suggest that the CRE genes comprise a general transcriptional

control system in yeast that requires the function of the CCR4 gene.

T

H E glucose-repressible alcohol dehydrogenase (ADH 11: encoded by the ADH2 gene) from Saccharomyces cerevisiae functions in the use of ethanol as an energy and carbon source (CIRIACY 1975). ADH2 is controlled by two separate regulatory pathways (CIRIACY 1975; DENIS 1984). One pathway is com- prised of the activator CCR4 an d its apparent negative effectors CREl and CRE2 (DENIS 1984). T h e second pathway is composed of the transcriptional activator A D R l (CIRIACY 1975), which acts through a region of dyad symmetry upstream of the ADH2 T A T A element (SHUSTER et al. 1986; EISEN et al. 1988). A D R l is regulated by the CAMP-dependent protein kinase phosphorylating system (CHERRY et al. 1989; DENIS a n d GALLO, 1986). Genetic evidence suggests that the CCR4 pathway is independent of the ADRl pathway, though both are required for full ADH2 expression (DENIS 1984).

T h e CRE genes were initially identified by muta- tions which allowed ADH2 expression under glucose repressed growth conditions (DENIS 1984). Mutations in both genes were also observed to affect cell mor- phology, indicating that they were pleiotropic (DENIS

1984). T h e o t h e r processes affected by the CRE genes were not identified. Mutations in th e CCR4 gene were observed to suppress the effect of th e cre alleles at ADH2. In addition, ccr4 mutations inhibited the

l ' h e publication coqt\ o f this article were partly defrayed by the payment

oflx~gr clxu-ges. T h i s article must therefore be hereby marked ''advertisement" ill accordance with 18 U.S.C. 41734 solely to indicite this fact.

(knctica 1 2 4 283-291 (February, 1990)

expression of several other nonfermentative genes

(DENIS 1984). The effect of ccr4 mutations on ADH2

expression under glucose growth conditions was

shown to be specific to cre-induced ADH2 transcrip- tion . T he ccr4 mutation did not suppress the increased ADH2 transcription caused by an ADR1-5' allele (DENIS 1984), high ADRl dosage, or the presence of a T y element in the ADH2 promoter (our unpublished observations). T h e observation that the cre alleles allow ADH I1 enzyme activity when glucose is t he carbon source, conditions when no ADH2 RNA has been observed in wild-type strains (DENIS, CIRIACY and YOUNG 1 98 1 ; WILLIAMSON et al. 1983), suggest that the CRE genes affect ADH2 at the transcriptional level. Similarly, t he ability of ccr4 mutations to affect cre-induced ADH2 expression but not that of ADRl- 5' also suggests CCR4 acts to control the transcription

of ADH2 mRNA (DENIS 1984).

T o further characterize the function of the CCR4I CRE genes, we have undertaken studies to define their roles in controlling ADH2 expression. Both ccr4 a nd

crel mutations were shown to affect ADH2 R NA

levels. In addition, we report that CCR4 a n d t he CRE genes, unlike ADRl, do not require sequences up-

(2)

284 C. L. Denis and T. Malvar

TABLE 1

Yeast strains

Strain Cenotvpe

5324a

195-6c 553-4b

1 72-6;1 258-1 Od

147-6cl

258-&I

356"

392"

Rfi49.1-4A R649.1-4A-2

121-4a

2 16-2c

291-la

553-4b-10

665-2a

553-4h FW235 402-8d 397-4h 348-1 Sa

396-2b 402-4a

L22 1

237-lb

CCI 2

500-16-14

408" 403-4d 39"

4 14-2-2d

4 14-2-3~

678-4c

678- 1 b

677-7h

L323-1

FW226-1

FM'7 12-1 755-7c

756-2h

759-7h

MATa adhl-11 adh2-60ldelta adh3 ade2 trp2 d\.IATaadhI-I1 A D R I - 5 ' t r p l u r a l

,VIATaadhl-lI adh3 crel-1 t r p l ural leu2 MATa adhl-11 adh3 ccr4-I0 ural leu2 M9Ta adhl-11 adh2-60ldelta adh3 crel-1 ade2

M A T a a d h l - 1 1 a d h 3 rre2-1 hi54 t r p l ural leu2 ,VlATa adhl-11 adh2-60Idelta adh3 trpl trp2

MATa adhl-11 adh3 rre2-I rcr4-IO hi54 t r p l

.\lATa adhl-1 I adh2-601 delta adh3 cre2-1 his4

MATa adhl-1 I ADH.2-8' adh3 ura I

Isogenic to strain R649-1-4A except adh2-

M A T a a d h l - 1 I adh3 crel-I ural leu2 .CIATa adhl-11 adhZ-MI adh3 hi54 u r a l hi54

MATa adhl-11 adh2-MI adh3 crel-1 his4 trpl

Isogenic to strain 553-4b except ccr4-I0 MATa adhl-11 adh3 crel-I ccr4-I0 adh2-

MATa adhl-I1 adh3 crel-1 ural trpI leu2 M A T a his4-912delta leu2-3

MATa adhl-11 ccr4-I0 his4-912delta .\.IATa adhl-1 I crel-1 ural leu2 his4-912delta MATa adhl-11 ccr4-I0 ura3 trpl

M A T a a d h l - 1 I leu2 his4-912delta

d.IATa adhl-11 ccr4-I0 his4-912delta ural trpl MATa his4-917 lys2-1288 urajl-52 sptI0-HL27 MATa adhl-11 adh3 ura3 hi53 trpl leu2 MATa his4-912 lys2-901 spt6-I40

MATa adhl-11 adh3 adrl-I cre2-I his4 trpl

M A T a a d h l - 1 I crel-1 his4-912delta leu2 M A T a a d h l - 1 I ccr4-I0 his4-912delta u r a l M A T a a d h l - 1 1 his4-912delta cre2-1 u r a l t r p l

M A T a a d h l - l I c c r 4 - I 0 his4-912delta cre2-1

MATa adhl-11 ccr4-10 his4-912delta cre2-1

M A T a a d h I - I I cre2-1 his4-912 delta ural leu2

MATa adhl-11 cre2-I his4-912delta u r a l trpl

M A T a a d h l - 1 I ccr4-35 his4-912delta leu2 trpl M A T a a d h l - I::URA3spt4-3his4-9I2deltaura3-

,MATa adh I - I :URA3 spt5-194 his4-91 Zdelta

M A T n a d h l - I : : U R A 3 his4-912deIta ura3-52 MATaadhI- I::URA3ccr4-l0 his3/his4-912delta

MATn adhl- 1::URA3 crel-1 his3/his4-912delta

,\IATa adhl- I::URA3 crel-I ccr4-I0 his3lhis.l- ura I

leu2

ura I

trpl ural leu2

40Idelta

trp I

ural leu2

6OIdelta trpl u r a l

ura I

1 Q U 2

u r a l trpl

u r a l trpl leu2

t r p l

leu2

5 2

ura3-52

leu2 ura3-52

trpl leu2 ura3-52

912delta ura3-52

A R C

FIGURE I .-(;ell morphology o l ~ s l l - a i n s carryillg rhc r w l - I allele. I'hotographs are of cells grown on )'El' medium supplemented w i t h

X% glucose. A, strain 553-4b ( r r e l - I ) : B, strain R234 (ADRI-5'): C , strain 3.53-4h-10 (rrel-I wr4-IO).

whose upstream activation sequences have been de-

leted or displaced (CLARK-ADAMS and WINSTON 1987;

NEICERORN, CELENZA and CARLSON 1987). CREI was also found to be allelic to SPTlO (FASSLER and WIN-

STON 1988). Both crel- and cre2-induced his4-912delta

expression can be suppressed by ccr4 mutations. We suggest that the CRE genes comprise a general tran- scriptional control system in yeast requiring the func- tion of the transcriptional factor CCR4.

MATERIALS AND METHODS

Yeast strains: Yeast strains are listed in Table 1. In crosses involving ccr4 and those involving crel and ccr4, comple- mentation tests were used to identify all segregants carrying the ccr4 and/or crel alleles.

Growth conditions and enzyme assays: Conditions for growth of cultures on YEP medium (2% Bactopeptone, 1 %

yeast extract, 20 mg/liter adenine and uracil) have been described (DENIS and YOUNG 1983). CAA trp- medium consisted of 0.5% casamino acids (CAA), 6.7 g/liter yeast nitrogen base without amino acids, and 20 mg/liter each of uracil, adenine and tyrosine. ADH I1 enzyme activities were assayed as previously described (DENIS and YOUNG 1983). Yeast transformations were conducted by the LiAc proce- dure (ITO et al. 1983).

Strain and plasmid constructions: The adh2-8Oldelta allele was derived from strain R649.1-4A, which contains a T y element at bp -1 25 in the ADH2 sequence (WILLIAMSON et al. 1983), by selection on allyl alcohol-containing medium as previously described (CIRIACY and WILLIAMSON 1981). Presence of a solo delta sequence at the adh2-80ldelta locus was determined by Southern analysis of chromosomal DNA prepared from allyl alcohol resistant colonies (CIRIACY and WILLIAMSON 198 1 ; DENS 1987). The plasmid CC 14 carries the SPT6 gene on the YEp24 vector (CLARK-ADAMS and WINSTON 1987). Plasmids 943 and 943.7, were gifts of V.

WILLIAMSON. Plasmid 943 contains all the ADH2 sequences present on plasmid YRp7-ADH2-Bsb (WILLIAMSON, YOUNG and CIRIACY 1981) placed in a pBR322-TRPl-CEN3 plas- mid vector. Plasmid 943.2 is the same as 943 except it contains a 2.3-kb segment of maize DNA inserted between the SslI site (-653 bp) and the SphI site (-176 bp) of the ADH2 upstream region present on plasmid 943. The 943- like plasmids containing random yeast DNA segments were obtained by cutting yeast chromosomal D N A with SstI and SphI and ligating the mixture into plasmid 943.7, cut with SphI and S s t l .

Mutant isolation: Mutagenesis of strain 408-6b was con- ducted as previously described using the mutagen ethyl methyl sulfonate (CIRIACY 1975). Selection of mutants lack- ing ADH I 1 activity was carried out by plating cells on YEP plates (YEP medium plus 2% agar and 2% glucose) supple- mented with 10 m M allyl alcohol (CIRIACY 1975).

(3)

ccr4 and @-Mediated Expression 285

Northern analysis was conducted as previously described (DENIS, FERGUSON and YOUNG 1983). The 2.4-kb BamHI

fragment from plasmid 943 containingADH2 sequences was radiolabeled using a random priming kit (Boehringer-Mann- heim). Densitometric analysis was conducted using an E-C- 610 densitometer. The ADH2 mRNA levels were normal- ized to the level of rRNA loaded onto the gel as described (DENIS, FERGUSON and YOUNG 1983).

RESULTS

The

CCR4

and the CRE genes affect cell mor-

phology and growth at elevated temperatures:

Strains carrying the recessive cre2-1 allele were found to be temperature sensitive for growth (data not shown), suggesting that CRE2 is a gene essential for cellular growth. T h e temperature sensitive phenotype segregated 2:2 and cosegregated with the ability of the cre2-1 allele to cause increased ADH2 expression under glucose growth conditions. The temperature sensitive phenotype conferred by cre2-1 was not ob- served to be suppressed by the ccr4-I0 or other ccr4 mutations, even though ccr4 mutations suppress the effect of cre2-1 on ADH2 expression. None of four crel alleles conferred temperature sensitivity under either fermentative or nonfermentative growth con- ditions. Four of seven previously characterized ccr4 alleles including ccr4-I0 displayed a temperature sen- sitive phenotype for growth on nonfermentative car- bon sources such as ethanol and glycerol (data not shown).

Strains carrying a crel allele show an elongated cell morphology under glucose growth conditions (Figure

1 A). This phenotype is not a result of increased ADH I1 expression since strains carrying an ADRI-5" allele and expressing a comparable amount of ADH I1 ac- tivity do not display an elongated cell morphology (Figure 1B). T h e elongated cell shape cosegregates with the crel allele and becomes more pronounced in homozygous crellcrel diploids (data not shown). Mu- tations in the CCR4 locus, which suppress the glucose insensitive ADH I1 phenotype caused by a crel muta- tion (DENIS 1984), also suppress the crel-induced aberrant cell shape (Figure 1C). These results suggest that CREl acts through CCR4 in the control of proc- esses in addition to that of ADH2 expression. CRE2, on the other hand, does not appear to act through CCR4 in its effect on cell growth.

Mutations in the CRE genes bypass the inhibition

of ADH2 expression caused by delta promoter inser- tions: In studying the effect of delta insertions on ADH2 expression, it was observed that the crel-I mutation allowed ADH2 expression. A solo delta in- sertion occurs at ADH2 following the transposition of a T y element into and its recombination out of the

ADHB promoter (CIRIACY and WILLIAMSON 1981;

WILLIAMSON et al. 1983; WILLIAMSON, YOUNG and

CIRIACY 1981). The orientation of the T y elements

TABLE 2

Effect of ccr4/cre mutations on ADHZ-delta insertions

Other relevant

ADH 11 activity

(mU/mg)

ADH allele genotype Glucose" Ethanolb

adh2-60ldelta' 4 34

adh2-601deltad ADRI-5' 5 110

adh2-60ldelta' crel-1 94 54

adh2-60ldeltaf crel-1 ccr4-I0 12 18

adh2-601deltag cre2-1 132 N D ~

adh2-60ldelta' cre2-1 ccr4-10 18 N D ~

adh2-80ldelta' 2 5

adh2-801deltak crel-1 45 90

ADH2' 8 2500

ADH2' crel-1 175 2200

ADH2' crel-1 ccr4-I0 23 500

ADH2' cre2-1 77 2900

ADH2' cre2-1 ccr4-I0 6 110

ADH2' ADR 1-5' 280 6600

ADH2" ccr4-I0 5 400

a Yeast were grown on YEP medium supplemented with 8%

Yeast were grown on YEP medium supplemented with 3%

' Average of eleven segregants from crosses: 532-6a X 195-6c glucose as described previously (DENIS 1984).

ethanol as described previously (DENIS 1984).

and 553-4b X 532-6a.

Average of four segregants from cross: 532-6a X 195-6c. e Average of nine segregants from cross: 553-4b X 532-6a and

f Average of four segregants from cross: 172-6a X 258-10d. Average of five segregants from cross: 147-6d X 258-6a. ND indicates that these values were not determined.

Average of four assays of R649.1-4A-2.

Average of three segregants from cross: 12 1-4a X R649.1-4A-2. ADH 11 values taken from Ref. 1 1.

In strains carrying the ccr4-10 allele, ADH I1 enzyme levels under ethanol growth conditions can range from 100 to 1000 mU/ mg depending on the genetic background.

ADH I1 enzyme activities for individual segregants differed at most by 30% from the average value given in the Table except as indicated above. When one strain was assayed repeatedly, ADH 11

enzyme activities differed by less than 20% from the average.

in the ADH2 promoter are such that the direction of transcription is opposite to that of ADH2 transcription (WILLIAMSON et al. 1983). Strains carrying delta inser- tions (336 bp) upstream, at, or downstream of the ADH2 TATA sequence are incapable of derepressing ADH2 (CIRIACY and WILLIAMSON 198 1 ; Table 2). This phenotype has been suggested to be the result of displacement of the ADRI-responsive upstream acti- vating sequence (UASl) normally located at -27 1 bp (SHUSTER et al. 1986), 336 bp farther upstream (CIR- IACY and WILLIAMSON 1981). Transcription from the delta when an intact T y is present at ADH2 does not affect the transcription initiation site at ADH2 (WIL-

As shown in Table

2,

the crel-1 allele still allowed glucose insensitive ADH2 expression when a delta was present at the ADHB TATA sequence located at

-

16 1 bp (adh2-60Idelta allele, Figure 2). N o significant derepression was observed due to the displacement

172-6a X 258-10d.

' Average of six segregants from cross: 356-8d X 392-3d.

(4)

286 C . L. Denis and T. Malvar

B VAS TATA ADH2

.

I I

b

. I x ) I -271 -161

B UAS 6 TATA

.

I h\,\\\\\\\* b

-161

B UAS TATA 6 ADH2

.

4 l - 1

-125 w

B UAS TATA

.

I

B maize DNA 2.3 kb TATA

-

ADH2

-53 17b

ADH2

odh2-M) I6

odh2-8016

Plasmid 943

Plosmld 943 2

FIGURE ~.-Deletions;n~d insertions i n the ;\Dff2promoter. The . W H 2 promo~er sequences :Ire not dr;twn to scale. The major

fi.;~r~trrs ;Ind points of insertion or cleletion are identified by base pair nun~brrs. Kcstriction sites are designated a s B, R a m H I . “8”

refers t o the dc11;t insertion. Direction of transcription from the

delta would be opposite to t11at of . W H 2 tI-;nlscription.

upstream of the ADRI-UAS1 element. T h e crel-1 allele also allowed ADH2 expression under glucose repressed conditions when a delta sequence was pres- ent 3’ to the TATA element at bp -1 25 (allele adh2-

80ldeZta; Table 2 and Figure 2). In this case a TATA

sequence in the delta is thought to be used for pro- motion of ADH2 transcription when an intact T y is located at that site (WILLIAMSON et aZ. 1983). T h e

cre2-1 allele also allowed ADH2 expression under glu-

cose growth conditions in the presence of the adh2- 60ldelta allele (Table 2). In contrast to the crel and cre2 effects on adh2-60ldeZta expression, the presence of the delta at the ADH2 TATA prevented the ADRI-

5‘ allele from allowing increased expression of ADH2 under glucose repressed conditions (Table 2). T h e inability of ADRI-5‘ to affect adh2-60ldeZta expres- sion is consistent with previous data that demonstrated movement of the ADRl-responsive UAS upstream of

the TATA by about 300 bp blocked ADRI-5c-me-

diated expression (BEIER and YOUNG 1982).

The ability of the ccr4-I0 mutation to suppress the effects of crel and cre2 on delta insertions in the ADH2

promoter was tested. Strains carrying an adhZ-

60ldelta allele together with the crel-1 and ccr4-I0 alleles expressed eightfold less ADH I1 enzyme activity under glucose growth conditions than did strains car- rying only the crel-1 and adh2-60IdeZta alleles (Table 2). Consistent with this finding, strains carrying the ccr4-I0 allele were antimycin A sensitive, indicative of low ADH2 expression (CIRIACY 1979). A ccr4-I0 allele also suppressed the increased ADH2 expression caused by the combination of the cre2-1 allele and the adh2- 60ldelta allele (Table 2).

Allelism of CRE genes to SPT and SSN genes: T h e temperature-sensitive phenotype of cre2-1 and its abil- ity to bypass delta insertions at ADHZ led us to test whether CRE2 was allelic to SPTG (WINSTON et aZ.

1984). T h e spt6 mutation displays a temperature sen- sitive phenotype and overcomes the effect of delta insertions at the his4-912deZta locus (WINSTON et aZ.

1984; CLARK-ADAMS and WINSTON 1987). HIS4 en-

codes a required enzyme for histidine biosynthesis. A

cre2-l/spt6-140 diploid was constructed and was found

to be unable to grow at 37”C, indicating that the cre2 allele did not complement an spt6 mutation. After sporulation of the diploid 15 tetrads were analyzed for segregation of the temperature sensitive pheno- type. All segregants were temperature sensitive, indi- cating that CRE2 and SPTG are indeed allelic. SPT6 is

also allelic to SSN20 (CLARK-ADAMS and WINSTON

1987; NEIGEBORN, CELENZA and CARLSON 1987), mu-

tations in which bypass defects in the transcriptional activator s n f 2 and allow SUC2 (invertase) derepression

(NEIGERORN, RUBIN and CARLSON 1984).

Inactivating SPT6, as well as increasing the dosage of SPT6 in the cell, allows his4-912deZta expression (CLARK-ADAMS and WINSTON 1987). In a similar man- ner, increasing or decreasing SSN20 dosage allowed

SUC2 expression in a snf2 background (NEIGEBORN,

CELENZA and CARLSON 1987). T o test whether an increase in CRE2 dosage could bypass ADH2 glucose repression in a manner similar to that of the cre2-1

allele, plasmid CC14 containing the SPTG gene

(CLARK-ADAMS and WINSTON 1987) was transformed

into strain 237-1b. Transformants carrying this mul- ticopy plasmid, however, did not allow a glucose- insensitive ADH2 phenotype (data not shown). These results indicate that overexpression of CRE2 affects SUC2 and his4-912deZta expression but does not affect ADH2 expression.

T h e allelism of CRE2 to SPTG suggested that the CRE genes and CCR4 are not specific to the regulation of carbon metabolism and function as general effec- tors of transcriptional processes. T o test whether ccr4 and crel also affect genes not related to carbon me- tabolism, their potential effects on his4-912deZta expression were analyzed. T h e crel-1 mutation, like cre2-I, was capable of overcoming the block in his4-

912deZta expression caused by the delta insertion

(Table 3). Because crel behaves similar to spt alleles in affecting his4-912delta expression, allelism of ere1 to the other spt alleles was tested. Of 15 spt genes (FASSLER and WINSTON 1988; WINSTON et aZ. 1984) tested, only sptlO/crel diploids allowed his4-912deZta expression (F. WINSTON, personal communication), indicating that sptlO and crel failed to complement each other. Our subsequent analysis showed that

sptlO/creI diploids grew slower on medium containing

(5)

ccr4 and spt-Mediated Expression 287

TABLE 3

Effect of ccr4/cre mutations on his4-912delta and ADHZ expression

Relevant his4-YZ2delta Antimycin A genotype expression resistance

Wild-type" - -

ccr4-IOb -

c r e l - I '

+

+

cre2-I"

+

+

ccr4-I0 crel-I' -

c c r 4 - I O c r e 2 - I d

*

-

sptIO-HL27'

+

+

ccr4-35 cre2-lg

-

-

ccr4-35 creI-Ih - -

spt4-3'

+

-

spts- 19 4'

+

-

-

-

~ p t 6 - 1 4 0 '

+

+

Growth was scored either on minimal plates lacking histidine at 30" or on antimycin A plates (YEP medium supplemented with 2% agar, 2% glucose, and 0.1 mg/ml antimycin A). For cells to grow on medium containing antimycin A , they generally must contain at least 20 mU/mg ADH I1 activity. f indicates that some segregants from cross 403-4d X 395-5c grew while others did not on plates lacking histidine. At least eight segregants of each genotype were assayed.

a Segregants from cross 147-6d X FW235.

Segregants from cross 402-8d X 397-4b and 348-13a X 396-

Segregants from cross 402-8d X 397-4b and 402-4a X 397-4b. Segregants from crosses 403-4d X 395-5c, 414-2-2d X 678-4c, and 414-2-3c X 678-1b.

Segregants from cross CC12 X 500-16-14 were assayed for antimycin A resistance. T h e his4-912delta phenotype is taken from WINSTON et al. (1 984).

f Segregants from cross L221 X 237-1b were assayed for anti- mycin A resistance. T h e his4-912delta phenotype is taken from FASSLER and WINSTON (1 988).

2b.

Segregants from cross 677-7b X 678-1b.

*

Strain 408-6d-35 which is allelic to 408-6d except for the ccr4-

' Strain L323-1.

35 allele.

Strain FW226-1.

are typical of crel haploid and homozygous crel dip- loids (DENIS 1984; see Figure 1) and indicate that crel

and sptl0 are members of the same complementation

group. The cloned SPTlO gene was also able to com- plement by transformation the crel-1 mutation in vivo

(C. DOLLARD and F. WINSTON, personal communica-

tions). Tetrad analysis of sporulated sptlO/crel dip- loids, however, was confounded by poor spore viabil- ity. When the ability to allow his4-912delta expression was scored, we found that

2

of 46 surviving meiotic segregants displayed a His- phenotype, suggestive of a low frequency of recombination events between crel and spt10. In other tetrad analysis involving a cross between a strain carrying an SPTlO allele marked with the URA3 gene and a strain carrying the crel-1 allele, no recombination events between crel-1 and SPTlO were observed in more than 50 tetrads dissected (C.

DOLLARD and F. WINSTON, personal communication).

We conclude from the complementation and tetrad data that CREl is most likely the same gene as SPTIO.

The effect of ccr4-IO on crel- and cre2-induced his4-

TABLE 4

Number of ccr4 mutations with crel phenotypes

crel phenotypes Antimycin A

resistance G l y ~ e r o l - ~ ~ T s expresslon ccr4 alleles

his4-912delta No. of

-

+

+

3

+

9

+

5

26

- -

-

-

- -

-

For antimycin A resistance and his4-912delta expression growth was scored as described in Table 4. G l y ~ e r o l - ~ ~ T s growth (indicated by a "+") was determined by incubating glycerol plates (YEP me- dium supplemented with 2% agar and 3% glycerol) at 37". The

ccr4 alleles are derived from strain 408-6d ( c r e l h i s 4 - 9 1 2 d e l t a ) .

912delta expression was also examined. As shown in Table 3 the ccr4-I0 allele was capable of suppressing crel effects at his4-912delta. This result indicates that CCR4 is not solely involved in controlling nonfermen- tative gene expression. T h e ccr4-I0 allele was also able to suppress the effect of cre2-1 on his4-912delta. The ability of ccr4-I0 to suppress the effect of cre2-1 on his4-912delta was found, however, to vary with genetic background (Table 3).

A

similar analysis of the effects of ccr4-35 on cre2-1 was also conducted. In this case ccr4-35 was fully capable of suppressing the effects of cre2-1 on his4-912delta (Table 3).

Two other spt alleles were tested for their ability to allow glucose-insensitive ADH2 expression. T h e spt4 and spt5 mutations cause cell lethality when combined with an sptb mutation, suggesting that the SPT4, -5

and -6 proteins may be functionally related (WINSTON et al. 1984). We observed, however, that neither spt4 nor the spt5 mutation allowed increasedADH2 expres- sion (Table 3), indicating that not all spt mutations can affect ADH2.

Phenotypic differences of ccr4 alleles at ADH2 and his4-912delta: In order to further study the re- lation between ADH2 and his4-912delta gene expres- sion, additional suppressors of crel-1 effects at ADH2 were sought. Strain 408-6d (crel-I his4-912delta) was mutagenized and mutants displaying little or no ADH I1 enzyme activity were identified on plates containing allyl alcohol. Those mutants which were also sensitive to the inhibitor antimycin A (indicative of low ADH

11 activity) were selected for further analysis.

(6)

288

a b c d e

ADH2.

C . L. Denis and T. Malvar

I‘IGCRE J . ” A D H P mRS:\ l c . \ t , l \ i n stwi11\ ca1-1.1 ing err4 and c r e l

mutations. Three n1icrogr;tms ot’total y e a s t K S A were lo;detl into lanes :I ; ~ n d I, ant1 1 1 pg w r e loaded into lanes c. d and e , and Northern analysis was contluctetl a s previously described (DENIS. FERCVSON and YOL’NG 1983). A 2.4-kb fr;lgtnent of D N A contain- ing the entire ;\Dlf2 coding sequences ;Ind 1.2 k b of upstrean1 sccluence~ \ v a s used a s the radioactive probe. Undcr glucose growth conditions. strail1 FM’712-I espressed 5 mU/mg ,4DH I 1 (lane c). strain ’756-21) rspresserl lti0 InU/mg ADH I 1 (lane (1). and strain /:)9-7bespressetl 15 mU/nlg ADH I 1 (lane e ) . 1.mea. R N A isolated frotll strain FIV7 12-1 (CCR4 C R K I ) under ethanol growth condi- tions: Ianc I), R N A iso1;ltc.d from strain 7.5.5”ic ( c c r 4 - l O ) under r.th;lnol gro\\.th conditions: I;lne c, same a s lane ;I escept glucose growth conditions; lane tl. R N A isolated fronl strain 5.56-21, ( c r r l )

under glucose growth conditions: lane e. R N A isolated from strain

559-7b ( c w l ccr4) under glucose growth conditions. A duplicate gel

stained w i t h ethidium bromide is displayed i n the bottom panel and shows the anlount of rRNA (25s rop: 1 XS bottom) present i n each lane. The r R N A levels i n lanes ;I and b were found to be equivalent following densitometric analysis of the rRNA bands. The r R N A levels i n lanes c. d and e were also found to be equivalent. ”

induced his4-912delta expression or cause a nonfer- mentative defect at high temperatures suggests that the CCR4 protein is capable of protein-specific con- tacts at different promoters.

crel and ccr4 mutations affect ADH2 mRNA lev-

els: Previous genetic analysis suggested that the crel and ccr4 mutations affected ADH2 at the transcrip- tional level. In order to formally show that the crel

and ccr4 alleles affect ADH2 RNA levels, Northern

analysis was used to detect and quantitate ADH2 mRNA. Under ethanol growth conditions, strains car- rying the ccr4-I0 allele displayed reduced levels of ADH2 RNA relative to that found in strains carrying the CCR4 gene (Figure 3, lane a, CCR4, and lane b,

ccr4-IO). T h e sevenfold reduction in ADH2 RNA

levels that was observed with the ccr4-I0 allele agreed with the sixfold reduction in ADH I1 enzyme activity that was measured (5500 mU/mg ADH I1 in the strain carrying the CCR4 gene vs. 900 mU/mg for the strain carrying ccr4-IO). T h e crel-1 allele was also found to affect ADH2 expression at the RNA level in that strains carrying the crel-1 allele expressed ADH2 RNA

under glucose growth conditions. N o ADH2 RNA was detected for strains carrying the C R E l gene grown under glucose growth conditions even after long au- toradiographic exposures (Figure 3, lane d, crel-I, and lane c, C R E I ) . In addition, the ccr4-I0 allele

suppressed the increase in ADH2 mRNA levels under

glucose growth conditions caused by the crel-1 allele (Figure 3, lane e, crel-1 ccr4-IO). These results con- firm that the crel and ccr4 alleles affect ADH I1 enzyme levels by affecting the level of ADH2 RNA present in the cell.

The crel and ccr4 mutations affect ADH2 expres- sion when its upstream activation sequences have been deleted: T h e above observations that crel and

ccr4 mutations continue to affect ADH2 expression

when the upstream activation sequences have been diplaced by a delta promoter insertion suggests that the CREl and CCR4 factors do not require the up- stream activation sequences as their site of action. T o investigate whether CCR4 requires upstream activa- tion sequences to affect ADH2 expression, we studied the effect of the ccr4-I0 and cre-1 alleles on a promoter deletion of the ADH2 upstream sequences. The dele- tion, represented on plasmid 943.2 (Figure 2), consists of the ADH2 gene in which bp -176 to -653 have been replaced with a 2.3-kb segment of maize DNA. Plasmid 943.2 lacks, therefore, all ADH2 sequences upstream of the TATA that have been shown previ- ously to be required for ADH2 regulation (from bp

-176 to -479) (BEIER, SLEDZIEWSKI and YOUNG

1985). T h e maize DNA was used as a filler between bp -1 76 and -653 to prevent overstimulation of

ADHB transcription by pBR322 sequences brought

too close to the ADH2 TATA element (BEIER and YOUNG 1982). Plasmid 943.2 and the control plasmid 943, containing the complete ADHB upstream se- quence (Figure 2), were used to transform three yeast strains: 216-2c (CREI CCR4 adh2), 291-la (crel-I

CCR4 adh2), and 665-2a (crel-1 ccr4-I0 adh2). Trans-

formants were identified and their ADH I1 enzyme activities were determined (Table 5).

(7)

ccr4 and @-Mediated Expression 289

TABLE 5

Promoter deletion of ADHZ in combination with ccr4 and ere1 mutations

ADH I1 activity (mU/mg) 2 16-2c 291-la 665-2a

CRE C C R 4 c r e l - 1 CCR4 crel-l ccr4-10 a d h 2

a d h 2 a d h 2

Plasnlid G" Eb G E G E

None 5 5 30 10 0 20

943 80 6200 850 7500 170 5200

9 43. 2 260 280 1000 1100 79 70

943.Y-4.0' 110 130 3800 1600 47 79

943.Y-2.0' 180 ND 950 ND 19 ND

943.Y-0.4' 390 N D 4100 ND 300 ND

a Refers to glucose growth conditions.

Refers to ethanol growth conditions.

' These plasmids are the same as 943.2 except a random segment of yeast DNA has been substituted for the 2.3-kb maize DNA. The size of the yeast DNA insert is 4.0 kb, 2.0 kb, or 0.4 kb, respectively. Growth conditions were as described in Table 2 except cells containing plasmids were grown on CAA-trp- medium. Use of other strains with genotypes similar to the strains given above gave similar ADH I1 activities when transformed with the 943 and 943.2 plasmids.

Not done.

of ADH2 (Table 2). However, defects in the A D R l gene have also been observed to have less effect on

ADH2 derepression when ADHZ sequences were pres-

ent on a plasmid (BEIER and YOUNG 1982). Plasmid-

borne ADH2 sequences respond under glucose re-

pressed conditions to both ADRI (BEIER and YOUNG 1982; BEIER, SLEDZIEWSKI and YOUNG 1985) and to the CCR4 and CRE genes (Table 5).

The above analysis was duplicated using plasmid

943.2 which lacks the upstream ADHB regulatory sequences. Strain 291-la ( c r e l ) when transformed with the 943.2 plasmid expressed increased levels of

ADHZ under glucose growth conditions as compared

to strain 216-2c ( C R E l ) transformed with 943.2

(Table 5 ) . In both cases no significant derepression of

ADH2 occurred since the ADR1-responsive UAS had

been deleted. T h e crel-induced increase in ADH2 expression caused by the 943.2 plasmid was still ca- pable of being completely suppressed by the ccr4-10 allele under both repressed and derepressed growth conditions (Table 5). These results together with those obtained using the 943 plasmid indicate that the crel and ccr4 alleles are able to affect ADH2 expression when the ADH2 upstream regulatory sequences have been deleted.

In order to determine whether the responsiveness of the 943.2 plasmid to the crel and ccr4 mutations was a result of sequences present in the maize insert,

three random fragments of yeast chromosomal DNA

were substituted for the maize insert in plasmid 943.2.

These yeast sequences ranged in size from 0.4 kb (comparable to the size of the original ADH2 se- quences that were deleted) to 4 kb. Table 5 shows

that regardless of which DNA segment was used be- tween bp -176 and -653 in plasmid 943, the crel mutation resulted in elevated ADH2 expression (5-

30-fold) and the ccr4 allele was capable of suppressing this increase in activity. T h e different yeast DNAs

were not totally neutral to the crel and ccr4 effects, however, since differences in absolute and relative

ADH2 expression were observed between the differ-

ent plasmids. We conclude that the ADH2 upstream sequences are not required for the crellccr4 alleles to affect ADHP, although sequences in that region may play some role in the way the CRElICCR4 genes affect transcription.

DISCUSSION

T h e crel and cre2 mutations were originally identi- fied by their ability to release ADH2 from glucose repression (DENIS 1984). Our identification of the CRE genes as members of the SPT gene family impli- cate them in the control of processes other than re- lated directly to carbon metabolism. Mutations in

CRE2 (SPT6 and SSN20) increase SUC2, his4-912delta,

and ADH2 transcription (NEIGEBORN, CELENZA and

CARLSON 1987; CLARK-ADAMS and WINSTON 1987; DENIS 1984) and mutations in C R E l ( S P T l O ) affect

his4-912delta and ADH2 expression (FASSLER and

WINSTON 1988; DENIS 1984; Table 3). In addition to these effects on the regulation of known genes, SPT6

(SSN20) has been shown to be an essential yeast gene

(NEIGEBORN, CELENZA and CARLSON; CLARK-ADAMS

and WINSTON 1987) and mutations in C R E l result in altered cell morphology (DENIS 1984). These pheno- types suggest that the CRE genes play a major role in regulating the expression of a variety of genes. T h e CRE genes may, therefore, encode essential compo- nents of the transcriptional machinery or general tran- scriptional regulatory elements.

T h e CCR4 gene was identified by mutations which suppressed the ability of crel to cause increased ADH2

mRNA levels (DENIS 1984; also Figure 3). We have now provided evidence in this report that ccr4 muta- tions are capable also of suppressing the effects of crel

and cre2 at his4-912delta (Table 3). Furthermore,

when additional suppressors of crel were sought only additional ccr4 mutations were obtained (Table 4).

ccrl mutations also suppressed the aberrant cell mor-

phology caused by the crel allele (Figure 1). These results suggest that the CRE genes function through CCR4 in exerting their negative effects on transcrip- tion.

(8)

290 C. L. Denis and T. Malvar

able to suppress the high temperature lethality caused by the cre2-1 mutation. On the other hand, it is possible that some of the CRE2 effects on cellular processes are not mediated by CCR4. This could be imagined if CCR4 were an important factor for cre2- induced transcription but was not the agent through which CRE2 exerted its effects on gene expression,

Further evidence supporting the role of the CRE and CCR4 genes as modulators of general transcrip- tion derives from their proposed sites of action. Mu-

tation of SSN20 has been shown to affect SUC2 even when all the sequences upstream of the SUC2 TATA

element have been removed (NEIGEBORN, CELENZA

and CARLSON 1987). Similarly mutations in the CRE and CCR4 genes affected ADH2 when its upstream activation sequences were displaced or deleted (Tables

2 and 5). These results imply that CREI, CRE2 and CCR4 affect transcription independent of the up- stream regulatory sequences. T h e above data are also consistent with previous genetic data which suggested that the CRE and CCR4 genes acted independently of ADRl (DENIS 1984) which binds to upstream activa- tion sequences (EISEN et al. 1988). However, it should be noted that upstream sequences both for SUC2

(NEIGEBORN, CELENZA and CARLSON 1987) and ADH2 (Table 5 ) may influence the activity of the CRE and

CCR4 genes in their function. Whether glucose

repression of ADH2 is mediated by CCR4 in addition to ADRl remains unclear. I t is likely, however, that the CRE and CCR4 genes play a very general role in transcription and are not involved specifically in car- bon catabolite repression.

Additional support for the general rule of the CRE and CCR4 genes in transcription derives from the identification of S P T l l and -12 as histone genes

(CLARK-ADAMS et al. 1988) and SPT15 as the “TATA”-binding protein (HAHN et al. 1989; EISEN- M A N N , DOLLARD and WINSTON 1989). These results imply that the class of SPT genes and CCR4 play integral roles in nucleosome structure and the tran- scriptional state. T h e function of the CCR4 and CREl proteins should become clearer from their molecular and biochemical characterization.

One additional interesting feature of CCR4 is that different alleles varied in their abilities to suppress crel-induced ADH2 and his4-912delta expression and to elicit a temperature sensitive defect on non-fermen- tative carbon sources. The observed phenotypes could result from altered protein contacts made by the mu- tated CCR4 proteins or from different structures in- herent at the various promoters (NORRIS, DUNN and

OSLEY 1988). T h e fact that some ccr4 alleles display an effect on his4-912delta but not on nonfermentative growth while others display a reversed phenotype suggest that the differences lie more with CCR4 and

its protein contacts than with differences at the partic- ular promoter.

Other differences in the way the cre alleles affect his4-912delta and ADH2 are also evident. cre muta- tions cause increased ADH2 expression but do not affect wild-type HIS4 expression (FASSLER and WIN- STON 1988; WINSTON et al. 1984). Also, our results indicated that while increased CRE2 dosage could

allow his4-912delta expression (CLARK-ADAMS and

WINSTON 1987) it did not affect ADH2. Finally, in- creased histone gene dosage allows his4-912delta expression (CLARK-ADAMS et al. 1988) but we have observed no effect of increased histone gene dosage on ADH2 expression under glucose growth conditions (data not shown). These results and those concerning the different ccr4 alleles suggest that other factors in addition to the CRE genes influence CRE and CCR4 function.

We would like to thank F. WINSTON for generously providing strains and plasmids relating to SPT genes and his4-922delta expres- sion. We would also like to thank V. WILLIAMSON for plasmids 943

and 943.2 and T. YOUNG for the plasmid containing the ADHI disruption. We appreciate R. VALLARI’S comments on the manu- script and E. LEVIN and C. WEISBART’S secretarial skills. This research was supported by National Science Foundation grants

PCM-831627 1 and DCB-8616045 and HATCH project 291. This research was also supported by Biomedical Research Support Grant 2S07 RR07108-14. Scientific contribution 1583 of the New Hamp- shire Agriculture Experiment Station.

LITERATURE CITED

BEIER, D. R . , A. SLEDZIEWSKI and E. T. YOUNG, 1985 Deletion analysis identifies a region, upstream of the ADH2 gene of

Saccharomyces cereuisiae, which is required for ADRI-mediated derepression. Mol. Cell. Biol. 5: 1743-1749.

BEIER, D. B., and E. T . YOUNG, 1982 Characterization of a regulatory region upstream of the ADR2 locus of S. cereuisiae.

Nature 3 0 0 724-728.

CHERRY, J. R., T . R. JOHNSON, C. DOLLARD, J. R. SHUSTER and C.

L. DENIS, 1989 Cyclic AMP-dependent protein kinase phos- phorylates and inactivates the yeast transcriptional activator ADR1. Cell 5 6 409-419.

CIRIACY, M . , 1975 Genetics of alcohol dehydrogenase in Saccha- romyces cerevisiae. 11. T w o loci controlling synthesis of the glucose-repressible ADH 11. Mol. Gen. Genet. 138: 157-164.

CIRIACY, M. 1979. Isolation and characterization of further cis- and trans-acting regulatory elements involved in the synthesis of glucose-repressible alcohol dehydrogenase (ADH 11) in Saccha- romyces cereuisiae. Mol. Gen. Genet. 176 427-43 1 .

CIRIACY, M., and V. M. WILLIAMSON, 1981 Analysis of mutations affecting Ty-mediated gene expression in Saccharomyces cerevis-

iae. Mol. Gen. Genet. 182: 159-163.

CLARK-ADAMS, C. D., and F. WINSTON, 1987 T h e SPT6 gene is essential for growth and is required for delta-mediated tran- scription in Saccharomyces cereuisiae. Mol. Cell. Biol. 7: 679- 686.

CLARK-ADAMS, C. D., D. NORRIS, M . A. OSLEY, J. S. FASSLER and

F. WINSTON, 1588 Changes in histone gene dosage after transcription in yeast. Genes Dev. 2: 150-159.

DENIS, C. L., 1984 Identification of new genes involved in the regulation of yeast alcohol dehydrogenase 11. Genetics 108:

(9)

ccr4 and @-Mediated Expression 29 1

DENIS, C. L., 1987 T h e effects of ADRZ and CCRZ gene dosage on the regulation of the glucose-repressible alcohol dehydro- genase from Saccharomyces cereuisiae. Mol. Gen. Genet. 208:

101-106.

DENIS, C. L., M. CIRIACY and E. T. YOUNG, 1981 A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydro- genase from Saccharomyces cereuisiae. J. Mol. Biol. 148: 355- 368.

DENIS, C. I.., J. FERCUSON and E. T. YOUNG, 1983 mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cereuiszae decrease upon growth on a nonfermentable carbon source. J. Biol. Chem. 258: 1 165-1 17 1.

DENIS, C. I.., and C. GALLO, 1986 Constitutive RNA synthesis for the yeast activator ADRZ and identification of the ADRZ-5' mutation: implications in posttranslational control of ADRI. Mol. Cell. Biol. 6: 4026-4030.

DENIS, C. L., and E. T . YOUNG, 1983 Isolation and characteriza- tion of the positive regulatory gene ADRZ from Saccharomyces cerevisiae. Mol. Cell. Biol. 3: 360-370.

EISEN, A., W. E. TAYLOR, H. BLUMBERG and E. T. YOUNG, 1988 The yeast regulatory protein ADRl binds in a zinc- dependent manner to the upstream activating sequence of ADH2. Mol. Cell. Biol. 8: 4552-4556.

EISENMANN, D. M., C. DOLLARD and F. WINSTON, 1989 SpT15, the gene encoding the yeast T A T A binding factor TFIID, is required for normal transcription initiation in vivo. Cell 58:

1183-1 191.

FASSLER, J. S . , and F. WINSTON, 1988 Isolation and analysis of a novel class of suppressors of T y insertion mutations in Saccha- romyces cereuisiae. Genetics 118: 203-21 2.

HAHN, S., S. BURATOWSKI, P. A. SHARP and L. GUARENTE,

1989 Isolation of the gene encoding the yeast T A T A binding protein TFIID: a gene identical to the SPT15 suppressor of T y element insertions. Cell 58: 1173-1 181.

Iro, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168.

NEIGERORN, L., J. L. CELENZA and M. CARLSON, 1987 SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae. Mol. Cell. Biol.

7: 672-678.

NEIGEBORN, L., K. RUBIN and M. CARLSON, 1984 Suppressors of

snf2 mutations restore invertase derepression and cause tem- perature-sensitive lethality in yeast. Genetics 112: 741-753.

NORRIS, D., B. DUNN and M. A. OSLEY, 1988 The effect of histone gene deletion on chromatin structure in Saccharomyces cereuis- iae. Science 242: 759-76 1.

SHUSTER, J., J. Y U , D. COX, R. V. L. CHAN, M. SMITH and E.

YOUNG, 1986 ADRZ-mediated regulation of ADH2 requires an inverted repeat sequence. Mol. Cell. Biol. 6: 1894-1902. WILLIAMSON,

v.

M., E. T . YOUNG and M. CIRIACY,

1981 Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase 11. Cell 23: 605-

614.

WILLIAMSON, V. M . , D. COX, E. T. YOUNG, D. W . RUSSELL, and M. SMITH, 1983 Characterization of transposable element-asso- ciated mutations that alter yeast alcohol dehydrogenase I1 expression. Mol. Cell. Biol. 3: 20-3 1.

WINSTON, F . , D. T . CHALEFF, B. VALENT and G. R. FINK, 1984 Mutations affecting Ty-mediated expression of the

Figure

TABLE 1 A
TABLE 2
FIGURE - . W H 2  delta refers pair nun~brrs. Kcstriction  sites fi.;~r~trrs ~.-Deletions;n~d insertions i n  the ;\Dff2promoter
TABLE 3
+2

References

Related documents

Prevalence of depression and its associated factors in patients with type 2 diabetes: A cross-sectional study in Dhaka, Bangladesh.. Definition and diagnosis of diabetes mellitus

Crossreactive antibodies against the proline-rich region of the PspA could explain the high levels of recognition of the PspC Figure 6 Schematic model of the proposed

MDA ⳱ mass drug administration; LF ⳱ lymphatic filariasis; NTD ⳱ neglected tropical diseases; IEC/COMBI ⳱ information education communication/communication for behavioral impact;

III The flow chart contains key words, condition branch names, appropriate. boxes and connecting lines, and also line nlli~bers from the assembly

belangstelling voor een appartement en de belangstelling voor een tussenwoning is erg laag. De mensen in leeftijdsklasse 40 tot 50 jaar zijn nogal verdeeld in de mening ten opzichte

Antara perkara yang disentuh di dalam bab ini adalah maklum balas dari edaran soal selidik dan latar belakang responden, analisis tahap kemajuan dan keberkesanan pelaksanaan

trauma-sensitive strategies including staff self-care, intentional relationship building among school educators and students and/or families, tiered intervention curricula,

If you currently have NetMeeting version 3.0 on your PC you must verify the following then you can skip this section. Bring up NetMeeting as shown below and click on the Tools tab.