Effect of irradiation and mutagenic chemicals on the generation of ADH2- and ADH4-constitutive mutants in yeast: the inducibility of Ty transposition by UV and ethyl methanesulfonate

Mutation Research, 229 (1990) 69-77

69

Elsevier MUT 04841

Effect of irradiation and mutagenic chemicals on the generation of ADH2- and ADH4-constitutive mutants in yeast: the inducibility of Ty transposition by UV and ethyl methanesulfonate Cornelia Morawetz and Ulrich Hagen Gesellschaftfftr Strahlen- und Umweltforschung, lnstitut fftr Strahlenbiologie, D-8042 Neuherberg (F.R.G.)

(Received27 September1988) (Revision received7 August 1989) (Accepted 17 October 1989)

Keywords: Ty transposition; Fermentation-defectiveyeast strain

Summary A strain defective in fermentation due to a deletion in the A D H 1 gene was used to generate revertants which are constitutive mutants of the genes A D H 2 and A D H 4 . By analyzing the DNA of the mutants we determined the frequency of Ty insertions into the promoter region of these genes. We found an increase in transposition after UV irradiation and treatment with ethyl methanesulfonate (EMS). Chemical inhibition of DNA synthesis and translation decreased the induced mutant yield and the transposition frequency, whereas inhibition of transcription had no effect. Differences in transposition frequencies between different strains and between the 2 loci lead to the conclusion that not only the transposable element itself but also the insertion sites determine the frequency of Ty transposition to a given locus.

Transposable elements are widespread throughout the living organisms from bacteria or unicellular eukaryotes to plants and animals. They are capable of transposition to new sites in the genome and can promote chromosomal aberrations (for a review see Sankaranarayanan, 1986). The haploid genome of the yeast Saccharomyces cerevisiae harbors up to 35 copies of the retrotransposon Ty. Although the number of these elements seems to be stable in the cell, they can integrate at new places in the genome at a frequency of 10-7-10 -8 per cell (Cameron et al., 1979). Stress factors like adaptive changes, tem-

Correspondence: Dr. C. Morawetz, Gesellschaft fiir Strahlenund Umweltforschung, Institut ftir Strahlenbiologie, D-8042 Neuherberg (F.R.G.).

perature and genotoxic agents seem to enhance the transposition of Ty elements (Adams and Oeller, 1986; Paquin and WiUiamson, 1986; Morawetz, 1987). Ty elements are hot spots for genomic changes such as deletions, inversions and cross-over between non-homologous chromosomes (Roeder and Fink, 1980; Breilmann et al., 1985; Rothstein et al., 1987; Picologlou et al., 1988). Several genes have been identified which are inducible by mutagenic stress. Some examples are genes controlling D N A repair (RAD), DNA polymerase I and D N A ligase. The D D R genes are transcribed at higher levels after exposure of yeast cells to various mutagenic agents (Maga et al., 1986; McClanahan and McEntee, 1984, 1986). The transcription of Ty elements is enhanced up to 20-fold by mutagenic agents (McClanahan and McEntee, 1984; Rolfe, 1985). Boeke et al.

0027-5107/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

70

(1985) have shown that at least 1 mechanism of Ty amplification is the reverse transcription of Ty mRNA (Fulton et al., 1985; Garfinkel et al., 1985). Overexpression of a Ty element mediated by the GAL promoter in the presence of galactose leads to enhanced transposition not only of the GAL-regulated Ty element but also of other Ty elements in the genome (Boeke et al., 1986, 1988). Different genetic systems have been described to quantify Ty transposition events. The system used in the experiments described in this paper was the Ty-mediated overexpression of 2 isoenzymes of alcohol dehydrogenases of yeast. A strain with a defective adhl gene, which is unable to ferment (Ciriacy, 1975), can grow anaerobically on glucose medium after mutations in other genes. These mutants are resistant to antimycin A (Ciriacy, 1976; Paquin and Williamson, 1984, 1986). So far 2 genes have been isolated which cause the described phenotype under the influence of the Ty promoter and enhancer. The ADH2 gene of the ghiconeogenic pathway of yeast is normally repressed in the presence of fermentable carbon sources. It was shown to be transcribed after transposition of a Ty element into its promoter region (Ciriacy and Williamson, 1981). The other gene, called ADH4, was discovered by Paquin and Williamson (1986) by virtue of its Ty-mediated expression. The wild-type allele is a gene which is silent or expressed at a very low level. According to its primary structure and substrate specificity, this gene is not closely related to the group of the other 3 alcohol dehydrogenases of yeast, but rather to the alcohol dehydrogenase gene of the prokaryotic Zymomonas rnobilis. The mutant yield of antimycin A-resistant clones in an appropriate tester strain can be increased by -/-irradiation, UV-irradiation and ethyl methanesulfonate (EMS) treatment (Morawetz, 1987). The frequency of Ty transposition can be increased by -/-irradiation (Morawetz, 1987). In the tested gene locus the relative increase in transposition events was higher than the increase in total mutant frequency. Therefore the inducibility of Ty transposition by other mutagenic agents was tested. Ty insertions at the ADH2 or ADH4 locus can be detected and distinguished from other muta-

tional events by an EcoRI restriction fragment length polymorphism (RFLP) of the DNA of antimycin A-resistant mutant clones in a tester strain with the relevant genotype a d h l - ADH2 adh3 adh4-silent. It has been shown too that after y-irradiation the mutant yield for antimycin A resistance can be increased if the cells are incubated in rich medium prior to plating on selective medium (Morawetz, 1987). We wanted to know whether Ty transposition is affected. Inhibitors of protein, DNA or RNA metabolism could influence transposition events during this incubation period. Cycloheximide, hydroxyurea (HU), actinomycin D and phenanthroline were chosen as inhibitors. The influence of metabolic inhibitors during the treatment with EMS was studied as well. Material and methods

Strains Table 1 shows the strains that were used; in every case the adhl mutation is a deletion spanning - 1 4 0 0 to +40 of the ADH1 structural gene (Willamson et al., 1983). In these strains the ADH4 gene occurs in 2 alleles. These can be distinguished by the EcoRI restriction pattern either giving 1 fragment of 8 kb (ADH4-1) or giving 2 fragments of 5.2 kb and 2.3 kb (ADH4-2). Media and growth conditions Standard yeast YPD medium was made of 1% yeast extract, 2% bacto peptone (Difco), and 2% glucose. For the mutant-selective medium, antimycin A (Serva) was added to a final concentration of 5 ppm. Plates were solidified with 1% agar (Difco). Cells were grown at 28 ° C.

TABLE 1 RELEVANT GENOTYPES OF THE STRAINS USED 46-10 MC31 36-201

adhl A D H 2 A D H 3 A D H 4 - 1 adhl A D H 2 adh3 A D H 4 - 2 adhl adh2 adh3 A D H 4 - 2 ade2 ura3

Mat a Mat a Mat a a

Kindly provided by W. Vogel, GSF-Strahlenbiologie, Neuherberg (F.R.G.).

71 Mutagenesis Cells were grown overnight and harvested at 1-2 x 10 7 cells/ml. After appropriate dilutions, cells were plated on non-selective medium (YPD) or selective antimycin A-containing medium and irradiated directly on the plates. For UV exposure a transilluminator was used (dose rate: 8.5 J / m 2. s). For y-irradiation a 6°Co y source (Canada Ltd) was used at a dose rate of 58 G y / m i n . Chemical treatment with EMS was carried out in phosphate buffer at room temperature at the indicated concentrations for 2 h before plating. For the determination of the survival rates plates were incubated for 4 days, while antimycin A-containing plates were incubated for 7 days at 28°C. To compare the effects of the different agents, equitoxicity doses were chosen. These doses were determined in previous experiments (Morawetz, 1987) to give equal results for survival and the yield of antimycin A-resistant mutants. For each dose 3 non-selective and 6 selective plates were scored. The experiments were repeated at least 3 times. To quantify the transposition frequency from each agent and dose 24 mutants were analyzed for changes in the EcoRI restriction pattern at the ADH2 and A D H 4 loci as described previously (Morawetz, 1987). Mathematical calculations The calculation of mutant yield and mutant frequency has been described by Haynes and Eckardt (1979). For comparison of the frequencies of Ty transposition in the various strains, we define the term 'Transposition number' (T) as T = Nt(x) Nd(x)

Nm(x) Nm(0)

where Nt(x) is the number of mutants at a given dose x with a Ty insertion at the locus analyzed, Nd(x) is the number of antimycin A-resistant mutants screened at this dose (usually 24), Nm(x) is the mutant frequency (number of antimycin A-resistant mutants per survivor) at dose x, Nm(0) is the spontaneous mutant frequency. The values of Nm(x) and Nm(0) were calculated from at least 3 different experiments.

DNA analysis DNA preparation and hybridization have been described previously (Morawetz, 1987). The probe containing the A D H 4 locus was from plasmid pYADH4 (kindly provided by V. Williamson, ARCO Plant Cell Inst., Dublin, CA, U.S.A.). Results Induction and analysis of antimycin A-resistant mutants Overnight-grown cells were harvested at 1-3 x 10 7 cells/ml and treated with UV, y-rays or EMS and then plated on the respective media. Survival and mutant rates were determined as described in Material and methods. Mutants arose at a frequency of 10-100 per 10 v cells. All mutants tested were dominant for antimycin A resistance in a cross with an a d h l - A D H 2 strain. Fig. 1 shows the EcoRI RFLPs obtained after irradiation with either T-rays (a) or UV (b). The mutants were derived from 3 experiments after y-irradiation and 4 experiments with UV-irradiation. It can be seen that the mutants obtained after y-irradiation have more variations in the restriction pattern whereas the mutants obtained after UV-irradiation show a more uniform restriction pattern. Strain 36-201 has a defective A D H 2 structural gene. In this strain only insertions at the A D H 4 locus were expected under the conditions used. Indeed, no Ty insertions were found at the A D H 2 locus (data not shown). To compare the insertion frequencies at the A D H 2 and A D H 4 loci, strains 46-10 and MC31 were used. During the crosses performed to originate the tester strains 1 segregant was tested that gave only a single Ty insertion (at the A D H 4 locus) among 150 antimycin A-resistant mutants. From the results of the autoradiographs and the frequency of antimycin A-resistant mutants the transposition number was calculated for the various treatments (Fig. 2). It can be seen that Ty insertions at the A D H 4 locus are induced by all agents tested. Transposition to the A D H 2 locus, however, is always very rare in these experiments. Therefore the detection of an insertion at this locus would require the analysis of more mutants. Treatments with EMS and T-irradiation show a dose-dependent relationship for the transposition

72 23~

]

the inhibitors were (1) hydroxyurea 760 /~g/ml; (2) cycloheximide 10 # g / m l ; (3) actinomycin D 80 # g / m l ; a fourth sample was taken as a control. To ensure rapid penetration of the agents, especially actinomycin D, into the yeast cells they were incubated for 1 h in the presence of 1% brij 58. This chemical permeabilizes the cell wall (Kuo and Campbell, 1982). Under the conditions used brij 58 had no effect on plating efficiency (data not shown). Strain MC31 was used in these experiments. Cells were irradiated with 100 Gy and resus-

25

Fig. 1. Hybridization of mutants from strain 36-201. EcoRI RFLP obtained with ADH4 probe of antimycin A-resistant mutants from strain 36-201. Mutant lanes with an insertion are indicated by an arrow. (a) Mutants obtained after y-irradiation. (b) Mutants obtained after irradiation with UV. For experimental details see Material and methods.

1"

15

frequency. UV-irradiation, however, shows a decrease in transposition at higher doses. The fraction of Ty-mediated mutants is increased after a dose of 5 J, whereas with 10 J no Ty insertions were detected among 54 mutants.

Effect of metabolic inhibitors on the generation of antimycin A-resistant mutants after y-irradiation In previous experiments it had been found that the yield of antimycin A-resistant mutants can be further increased by incubating y-irradiated cells under growth conditions before plating (Morawetz, 1987). To analyze the specific processes responsible for this effect we used different agents to inhibit biosynthetic pathways. Hydroxyurea was used as an inhibitor of long-patch DNA synthesis. Cycloheximide in spite of its side effects is often used to inhibit protein synthesis. Actinomycin D is known to block transcription. Concentrations of

ill 0

0,5

1

%EMS

2

5

10

30

120 240

UV

Fig. 2. Transposition numbers obtained from strain 46-10 after treatment with UV, y-rays or EMS. Vertical shading, the fraction of mutants with an insertion at the A D H 4 locus; diagonal shading, the fraction of mutants having an insertion at the ADH2 locus; no shading, no detectable insertion. Zero shows the results of the spontaneously grown mutants. EMS is given in % (vol/vol), UV is given in J / c m 2 and ~,-ray dose is given in Gy. For experimental details see Material and methods, where the calculation of the transposition number is also explained.

73

1

.ct

200,t P(%)

0

a contr.

2

8h

i

b "10"7

501

J

contr. act.

40

3O

20

~

~HU

,oY/ 1

o

T

2

~

-8h P

Fig. 3. The effect of metabolic inhibitors on cell growth and mutant formation. Plating efficiency (P, a) and mutant yield (b) from strain MC31 after y-irradiation with 100 Gy followed by incubation at 2 8 ° C for various times in the presence of actinomycin D (act.), hydroxyurea (HU) or cycloheximide (cycl.) and in the control without an additional agent. The value obtained at time zero is derived from mutants plated directly after irradiation. It was calculated from the spontaneously grown mutants. For experimental details see Material and methods.

pended in aliquots of 50 ml YPD medium with brij 58 at a density of 7-106 cells/ml. One sample of 10 ml was taken immediately, the cells were collected by centrifugation and plated after appropriate dilutions on selective and non-selective medium. Further samples were taken after 2, 4 and 8 h. During the first 4 h after irradiation we observed hardly any change in the survival of the cells (Fig. 3a). However, after 8 h there was an increase in the survival of the control cells and

those that were treated with actinomycin D. With hydroxyurea the cells did not divide as determined by plating efficiency and by counting in a Neubauer chamber (data not shown). When cycloheximide was added the number of surviving cells decreased to 60%. The yield of antimycin A-resistant mutants is shown in Fig. 3a. In the control, the mutant yield reached a plateau between 4 and 6 h (see Fig. 3b). The actinomycin D-treated cells showed a lag of 2 h but after 8 h their mutant yield corresponded to that of the control cells. The treatments with hydroxyurea and cycloheximide decreased the mutant yield. Cells treated with hydroxyurea showed a small increase in mutant yield after 2 h. Cycloheximide even decreased the mutant yield obtained by direct plating. There was no increase in the mutant yield for 6 h. In some experiments not a single antimycin A-resistant clone was obtained although the cells did survive the treatment as shown in Fig. 3a. The question arose whether the Ty transposition was influenced during the postincubation. Mutants from time zero and those that were incubated for 4 h were used to determine the transposition number (Fig. 4). After 4 h of incubation without an additional agent not only the mutant yield but also the transposition number was increased. Actinomycin D seemed to have no effect on the generation of Ty-mediated mutants in this test whereas hydroxyurea clearly decreased the transposition number. The incubation with cycloheximide also influenced the Ty transposition. In none of the rare mutants obtained could an alteration in the EcoRI restriction pattern at the ADH2 and A D H 4 loci be found.

The influence of metabolic inhibitors on the induction of transposition by E M S A similar experiment was performed with strain 46-10 to study the effect of metabolic inhibitors on the induction of antimycin A-resistant mutants and Ty transposition during treatment with 1% EMS. In these tests phenanthroline (1,10phenanthroline) was used to inhibit transcription. This chemical has been described as a rapid and effective inhibitor of transcription without any effect on the D N A or protein synthesis under the conditions used (Santiago et al., 1986).

74

3-

T

25-

2.

T: F. M~x~ M(o)

20-

1 15"

direct

plating

contr,

act

cycl

HU

Fig. 4. Transposition to the A D H loci in strain MC31 after treatment with metabolic inhibitors. Transposition numbers obtained after y-irradiation with 100 Gy and direct plating or plating after 4 h of incubation with no additional agent (contr.), actinomycin D (act), cycloheximide (cycl) or hydroxyurea (HU). Cells were incubated in YPD at 28°C, washed twice with water and plated as described in Material and methods, where the calculation of the transposition number is also explained. Vertical shading, the fraction of mutants with an insertion at the A D H 4 locus; diagonal shading, the fraction of mutants having an insertion at the A D H 2 locus; no shading, no detectable insertion.

10'

/llJ fill ¢IIA

II/i f//l fill

5¸

¢11t ¢11t ¢11t ¢lll ¢/11 ¢llJ fill

¢///

'/N

The results are shown in Fig. 5. In this experiment also, EMS increased the mutant frequency and transposition number. With hydroxyurea the mutant frequency and the transposition number obtained with EMS are lower than the values obtained without the mutagenic agent. Phenanthroline had no effect on the transposition number at the A D H 4 locus either in the control or in the EMS-treated cells. Locus-specific differences could be seen in the response towards the mutagenizing agents. Again, the frequency of Ty insertions at the ADH2 locus was very low.

Kontr. 0

1%

HU 0

1°1o

Phen. o I%

Fig. 5. The effect of metabolic inhibitors during treatment with EMS on Ty transposition to the A D H 2 or A D H 4 locus. The transposition numbers T were calculated from antimycin A-resistant mutants of strain 46-10 after incubation with no additional agent (Kontr.), with hydroxyurea (HU) or 1,10phenanthroline (Phen.) in the presence (1%) or absence (0) of EMS. Cells were incubated for 2 h at 28°C, washed twice, plated and scored for mutants as described in Material and methods, where the calculation of the transposition number is also explained. Vertical shading, the fraction of mutants with an insertion at the A D H 4 locus; diagonal shading, the fraction of mutants having an insertion at the A D H 2 locus; no shading no detectable insertion.

75 Discussion

Induction of transposition Ty transcription is influenced by the growth medium. It depends on carbon source, mating type, growth rate and stress (Taguchi et al., 1984; McClanahan and McEntee, 1984; Errede et al., 1985; Roeder et al., 1985; Rolfe, 1985). The correlation between Ty transcription and transposition has been shown by Boeke and coworkers (1985). Therefore we studied the influence of mutagenic agents on the inducibility of Ty transposition. In the experiments described in this paper the Ty transposition frequencies have been determined by changes in the EcoRI RFLP at 2 genetic loci, ADH2 and ADH4. All Ty elements found had 1 or 2 EcoRI sites, none of the insertions analyzed had a BamHI recognition site. This leads to the conclusion that the insertions all belong to the Ty elements of class 1. One of our tester strains had only 1 Ty transposition among 150 resistant mutants. This could be due to a mutation in one of the S P T genes which are necessary for the Ty life cycle (for an overview see Winston et al., 1987). In all other strains used in our experiments 20-50% of the antimycin A-resistant mutants had insertions at the ADH2 or A D H 4 locus. The frequencies and the type of RFLP obtained varied in the experiments. In strain 36-201 greater variations in the RFLP were obtained using y-irradiation whereas UV irradiation resulted in a more uniform restriction pattern. As the mutants were obtained from separate selections we think that y-irradiation and UV-irradiation act differently on the induction of Ty transposition. We explain this result by variations in the promoter region of the Ty elements. This is also confirmed by the fact that the 3 agents gave very different numbers of Ty insertions. The mutant yield for antimycin A resistance increases with the dose. The Ty insertions, however, show this dose dependence only after treatment with EMS and y-irradiation, whereas higher doses of UV show a decrease in Ty transposition. Perhaps UV-irradiation induces other mutations stronger than the Ty transposition or Ty transposition is inhibited by higher doses of UV-irradiation. The different behavior of identical Ty elements under different conditions

has also been described by Picologlou and coworkers (1988). Effects of inhibitors on T-irradiated cells In our experiments an increase in mutant yield as well as in transposition number was found when the yeast cells were incubated under growth conditions between 6°Co y-irradiation and plating on selective medium. Without a metabolic inhibitor the number of mutants increased until 4 h after mutagenization. After this time only a very small increase in mutant yield was detected. This could be explained by the cell division which starts again. Garfinkel and coworkers (1985) showed that the overexpression of a single Ty element promotes transposition of other chromosomal Ty elements in trans. The transposition was increased by transcription and translation of the induced Ty element. Unexpectedly, in our experiments the inhibition of transcription had no effect on mutant yield and transposition number. We do not know whether the intracellular level of Ty mRNA was sufficient to promote mutants or the mutant formation was independent of the level of Ty mRNA. Hydroxyurea as well as cycloheximide had only very little effect on survival, but inhibited Ty integration and other mutations leading to antimycin A resistance. Hydroxyurea prevented the formation of the induced mutants. This agent inhibits the initiation of long-patch DNA synthesis by blocking the enzyme phosphoinosyltransferase (Slater, 1973). Therefore we think that long-patch D N A synthesis was indeed necessary for the formation of the mutants during the postincubation period. The small increase in mutant yield in the first 2 h may be explained by the fact that the initiated DNA replication in S-phase cells was completed to produce the mutations leading to antimycin A resistance. Cycloheximide was the agent that had the strongest effect in this experiment: after incubation with this agent the mutant yield was even below the spontaneous level. However, cycloheximide is reported to interfere with carbon catabolite repression (Entian, 1975). Therefore it is possible that the action of cycloheximide in our test system was an indirect rather than a direct effect. Our test system required a switch from

76 aerobic to a n a e r o b i c growth which could have been an i m p o r t a n t step in the o u t g r o w t h of the mutants. If this was blocked it could also explain the cell killing which occurs between the i r r a d i a tion and the 2 h of p o s t i n c u b a t i o n when the cells of the other tests started to recover. Locus-specific transposition O n e i m p o r t a n t finding in our e x p e r i m e n t s was the locus-specific difference in t r a n s p o s i t i o n frequency. T r a n s p o s i t i o n to the A D H 4 locus was up to 50% of the m u t a n t s analyzed. A t the A D H 2 locus the s p o n t a n e o u s t r a n s p o s i t i o n n u m b e r was very low, in some experiments no insertion was detected. W a r m i n g t o n a n d coworkers (1986) described a hot spot for Ty insertion at the r i g h t / l e f t a r m of c h r o m o s o m e III. It seems that certain regions on the D N A are m o r e susceptible to the ' l a n d i n g ' of a Ty element than others. This leads to the conclusion that T y t r a n s p o s i t i o n d e p e n d s not only on the events at the d o n o r element itself but also on the D N A at the insertional site. F o r most genes the transposition of Ty elements is r e p o r t e d to be m o r e frequent to the p r o m o t e r region. F r o m our experiments and the results p u b l i s h e d b y other groups we have different explanations. (1) Ty insertions are d e p e n d e n t on the specific D N A sequence of a given locus because the integrase can only recognize a n d / o r cut these sequences. (2) Ty insertions are more frequent at a given D N A region because certain proteins are b o u n d or absent at the integration site. F o r e u k a r y o t i c genes it is k n o w n that i n d u c t i o n of t r a n s c r i p t i o n alters the n u c l e o s o m e phasing of the p r o m o t e r region (Lohr, 1983). In contrast to the silent A D H 4 gene the A D H 2 gene is t r a n s c r i b e d at a low level even u n d e r repressed conditions. Bacterial transposons c a n n o t be c o m p a r e d in all cases to the eukaryotic Ty elements, b u t it is r e m a r k a b l e that for Salmonella typhimurium a t r a n s c r i p t i o n a l occlusion for t r a n s p o s o n insertions was f o u n d (Casadeus a n d Roth, 1989). (3) In our test system only those Ty insertions were detected that increase the f o r m a t i o n of the A D H enzyme to the level p r o v i d i n g a n t i m y c i n A resistance. Vogel (1989) found d u r i n g the analysis of a n t i m y c i n A-resistant m u t a n t s that Ty integra-

tion at the A D H 2 gene was restricted to the region between the u p s t r e a m activating sequence ( U A S 1 ) a n d the T A T A box. A t the A D H 4 locus the insertion sites were d i s t r i b u t e d m o r e r a n d o m l y (Vogel, 1989; o u r own u n p u b l i s h e d results). T h e r e f o r e we think that the selected p h e n o t y p e in our test system requires Ty insertions at defined regions at least at the A D H 2 locus. W e are currently sequencing the exact insertion sites of Ty integration sites to get m o r e i n f o r m a t i o n on the influence of integration site a n d gene expression.

Acknowledgements W e t h a n k M. Ciriacy, V.M. W i l l i a m s o n and W. Vogel for p r o v i d i n g yeast strains and plasmids. W e also t h a n k C. Schippel, K. Peters and C. Fischer for their assistance, C. W i n k l e r for writing the c o m p u t e r p r o g r a m for the m a t h e m a t i c a l calculations, as well as W. Vogel a n d F. E c k a r d t for m a n y fruitful discussions a n d their critical reading of the manuscript. This work was d o n e d u r i n g a p o s t d o c t o r a l fellowship of C.M. a n d was s u p p o r t e d by the E u r o p e a n C o m m u n i t y (BI-0085-D(B)) a n d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( H a 100/3-1).

References Adams, J., and P.W. Oeller (1986) Structure of evolving populations of Saccharomyces cerevisiae: adaptive changes are frequently associated with sequence alterations involving mobile elements belonging to the Ty family, Proc. Natl. Acad. Sci. (U.S.A.), 83, 7124-7127. Birnboim, H.K., and J. Doly (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res., 7, 1513 1518. Boeke, J.D., D.J. Garfinkel, C.A. Styles and G.R. Fink (1985) Ty elements transpose through an RNA intermediate, Cell, 40, 491-500. Boeke, J.D., C.A. Styles and G.R. Fink (1986) Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements, Mol. Cell. Biol., 6, 3575 3581. Boeke, J.D., D. Eichinger, D. Castrillon and G.R. Fink (1988) The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Tyl, Mol. Cell. Biol., 8, 1432-1442. Breilmann, D., J. Gafner and M. Ciriacy (1985) Gene conversion and reciprocal exchange in a Ty mediated translocation in yeast, Curr. Genet., 9, 553-560. Cameron, JR., E.Y. Loh and R.W. Davis (1979) Evidence for transposition of dispersed repetitive DNA families in yeast, Cell, 16, 739-751.

77 Casadeus, J., and J.R. Roth (1989) Transcriptional occlusion of transposon targets, Mol. Gen. Genet., 216, 204-209. Ciriacy, M. (1975) Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. I. Isolation and analysis of adhmutants, Mutation Res., 29, 315-320. Ciriacy, M. (1976) Cis-dominant regulatory mutations affecting the formation of glucose-repressible alcohol dehydrogenase (ADHII) in Saccharomyces cerevisiae, Mol. Gen. Genet., 145, 327-333. Ciriacy, M., and V.M. Williamson (1981) Analysis of mutations affecting Ty-mediated gene expression, Mol. Gen. Genet., 182, 159-163. Errede, B., M. Company, J.D. Ferchak, C.D. Hutchinson and W.S. Yarnell (1985) Activation regions in a yeast transposon have homology to mating type control sequences and to mammalian enhancers, Proc. Natl. Acad. Sci. (U.S.A.), 82, 5423-5427. Fulton, A.M., J. Mellor, M.J. Dobson, J. Chester, J.R. Warmington, K.J. Indege, S.G. Oliver, P. dela Paz, W. Wilson, A.J. Kingsman and S.M. Kingsman (1985) Variants within the yeast Ty sequence family encode a class of structurally conserved proteins, Nucleic Acids Res., 13, 4097. Garfinkel, D.J., J.D. Boeke and G.R. Fink (1985) Ty element transposition: reverse transcriptase and virus-like particles, Cell, 42, 507-517. Haynes, R.H., and F. Eckardt (1979) Analysis of dose response patterns in mutation research, Can. J. Genet. Cytol., 21, 277-302. Kuo, C., and J. Campbell (1982) Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system, Proc. Natl. Acad. Sci. (U.S.A.), 79, 4243-4247. Lohr, D. (1983) The chromatin structure of an actively expressed, single copy yeast gene, Nucleic Acids Res., 11, 6755-6773. Maga, J.A., T.A. McClanahan and K. McEntee (1986) Transcriptional regulation of DNA damage responsive (DDR) genes in different tad mutants strains of Saccharomyces cerevisiae, Mol. Gen. Genet., 205, 276-284. McClanahan, T., and K. McEntee (1984) Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage, Mol. Cell. Biol., 4, 2356-2363. McClanahan, T., and K. McEntee (1986) DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae, Mol. Cell. Biol., 6, 90-96. Morawetz, C. (1987) Effect of irradiation and mutagenic chemicals on the generation of ADH2-constitutive mutants in yeast, Mutation Res., 177, 53-60. Paquin, C.E., and V.M. Williamson (1984) Temperature effects on the rate of Ty transposition, Nature (London), 301, 53-55. Paquin, C.E., and V.M. Williamson (1986) Ty insertions at two loci account for most of the spontaneous antimycin A resistance mutations during growth at 15°C of Saccharomyces cerevisiae strains lacking ADH1, Mol. Cell. Biol., 6, 70-79.

Picologlou, S., M.E. Dicig, P. Kovarik and S.W. Liebman (1988) The same configuration of Ty elements promotes different types and frequencies of rearrangements in different yeast strains, Mol. Gen. Genet., 211,272-281. Rigby, P.W.J., M. Dieckmann, C. Rhodes and P. Berg (1977) Labelling deoxynucleic acids to high specific activity in vitro by nick translation, J. Mol. Biol., 113, 237-241. Roeder, G.S., and Fink, G.R. (1980) DNA rearrangements associated with a transposable element in yeast, Cell, 21, 239-249. Roeder, G.S., A.B. Rose and R.E. Pearlman (1985) Transposable element sequences involved in the enhancement of yeast gene expression, Proc. Natl. Acad. Sci. (U.S.A.), 82, 5428-5432. Rolfe, M. (1985) UV-inducible transcripts in Saccharomyces cerevisiae, Curr. Genet., 9, 533-538. Rothstein, R., C. Helms and N. Rosenberg (1987) Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae, Mol. Cell. Biol., 7, 1198-1207. Sankaranarayanan, K. (1986) Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation, genetic risk evaluation in man, Mutation Res., 160, 73-86. Santiago, T.C., l.J. Purvis, A,J.E. Bettany and A.J.P. Brown (1986) The relationship between mRNA stability and length in Saccharomyces cerevisiae, Nucleic Acids Res., 15, 8347-8352. Taguchi, A.K.W., M. Ciriacy and E.T. Young (1984) Carbon source dependence of transposable element-associated gene activation in Saccharomyces cerevisiae, Mol. Cell. Biol., 4, 61-68. Vogel, W. (1989) Entwicklung eines Systems zur lsolierung von Ty-lntegrationen mit dem ADH2- und ADH4-Gen auf Plasmiden in Saccharomyces cerevisiae, Charakterisierung der Integrationen, Thesis, Munich. Warmington, J.R., R. Anwar, C.S. Newlon, R.B. Waring, R.W. Davies, K.J. lnge and S.G. Oliver (1986) A 'hot-spot' for Ty transposition on the left arm of yeast chromosome I11, Nucleic Acids Res., 14, 3475-3485. Williamson, V.M., and C.E. Paquin (1987) Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zyrnornonas mobilis, Mol. Gen. Genet., 209, 374-381. Williamson, V.M., E.T. Young and M. Ciriacy (1981) Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase 1I, Cell, 23, 605-614. Williamson, V.M., D. Beier and E.T. Young (1983) Use of transformation and meiotic gene conversion to generate a yeast strain containing a deletion in the alcohol dehydrogenase I gene, in: P.F. Lurquin and A. Kleinhofs (Eds.), Genetic Engineering in Eukaryotes, Plenum, New York, pp. 21-32. Winston, F., C. DoUard, E.A. Malone, J. Clare, J.G. Kapakos, P. Farabaugh and P.L. Minehart (1987) Three genes are required for trans-activation of Ty-transcription in yeast, Genetics, 115, 649-656.