TIC, - - November 1986

belicase is a key regulated protein in normal cell cyde control The large T antigens of all the papovavinmes are almost certainly helicases but it will also be important to look for helicases among the products of other DNA viruses. If vitally encoded helicases are a common feature of eulmryote DNA viruses they coMd represent an ~portant new target for antiviral drugs.

Sadly, the discovery of this new activity ofT, wbile shedding new light on its role in viral replication, probably does little toinform us about its action as an oncogene. Mutants of T antigen that lack ATPase ~ and origin binding activit~ can still transform cells with apparently

the same efficiency as the wild- type vires and so far no biochemical activity of T antigen has been linked directly to its transforming action.

References 1 Ri~, P. w, J. ~md Line, D. P.

(1983) in Ad~st~ /~ Viral O~ob~ (VoL 3) (Klein, G., ed.), pp. 31-57, P, ave~ Press

2 T~an, R. (1978)CeUI& 165-179 3 Shortle, D. R., Margols]u~,

R.F. and Nathans, D. (1979) Proc. Nag Aem£ SoL USA 76, 6128-6131

4 Te~meyer, P. (1972) ]. l//roL 10. 591-598

5 Stahl, H., Droge, P., Zentgraf,

FL and Knippers, R. (1985) ]. Viral. 54, 473-482

6Stal~ IL, Droge, P. and Ks~oem, R. (1986) EMBO]. 5, 1939-1944

7 Giacherio, D. and Hager, L. p. (1979)]. Biol. C/m~. 254, 8113- 8116

S Clar~ IL, Lane, D. P. andT'~m, R. (1981) J. Biol. Chem. 256, 11854-11858

9 Mapm, M. M. and Gluzman, Y. (1984) MoL Call BIOL 4,1125- 1133

10 Matson, S. W. (1986) ]. Biol. Chem. 261, 10169-10175

11 LeBowitz, J. and McMacke~ R. (1986)]. BioL Chem. 261, 4738- 4748

/~ Baker~ T. ~, Se~n~ K., Funne~ B. E. and Komber~ A. (1986) Cell 45, 53-64

13 ~ K (1982)DNA

monitor ROI~IIO~ (supplement), W. H. Freennan

14 van der Ende, A., Baker, T. A., O~lwa, T. and Komberg, A. (1985)Pmc. Na~Acad. SoL USA 82, 395~-,,~

15 Matson, S. W., Tabor, S. an~ Richardson, C.C. (1983) ]. Biol. Chem.. 258, 14017- 14024

1.6 Vm'~tc-s~,, bL, Silver, L. and NossM, N.G. (1982).I. Biol. C/~s. 257, 12426-12434

17 Murakm~ Y., Wobbe, C. R., W~sbach, L., Dean, F. B. and Hurwitz, J. (1986) P~. Na~ Amd. Sc/. USA 83, 2869-2873

18 Hubscber, U. and Stalder, IL (1985) Nudd¢ ,4c/~ Res. 13, 5471-5483 Pr[ves, C., Covey, L., Schel]er, A. and Gluzman, Y. (1983) MoL CelL BioL 3, 1958-1966

/9

Insecticide resistance is a most important problem in both agri- celture and medicine. However, until now very little information has been available on the mech- anisms of resistance at the genetic and molecular levels t. Recently, an association be- tween resistance to temephos Jan organopbesphate (OP) insec- ticide], quantitative increase in the activity of a detoM]~ estemse and specific DNA se- quence amplification has been descrbed in several field strahm (and laboratory strains denved from the OP-resistant field col- lection) of mosquitoes from the C ~ ~ complex and in the housefly Musca ~ ~ .

The OP-resistant strains Cu- ~ S54 from the south of

Fmnce:-:-s and Cu/~ q ~ e - )'mc/ah~ TEM-R, Wdlow and Coachella of California 4, contain elevated doses of either estemse A or esterase B which are coded by two di~erer~ Ioc~ (Est-3 and Est.2, respectively). These est- erase actiw'lies are barely or not detectable in the corresponding suscepffole strains. Similarly, a resistant strain of Musca domes- t~a produces much more glum- thione-S-transfemse, and pos- s~ly an estemse, than the susceptible strain ~.

In the TEM-R swain, it has been observed that esterase activity roughly doubles from one homozygous resistant strain to another, and that high levels of resist~mce are not maintained in the absence of selection pres- sure s, suggesfingunstable ampli- fication of the esterase B gene as the mechanism of resistance. These features are very similar to those previously described in

Gene amplification in pesti c ide- res i sta n t

insects O. Hyrien and G. Buttin

U ~ & G b ~ I K S o ~ lmgtat P asJmv, ~ me& Dr R ~z, 757"~ Pafis, CeAex IS, France.

insecticide-resistant strains of the aph~ M y , ~ ~ , where to~ carboxytesterase activity was found to increase by a factor of two between each of seven resistant varmnts ?.

U'sb~ the tedmique of Ren- mson s, Mond~s et ~. detected amplilied DNA h-agmunts in the l~.nome of resistant mosquito and housefly strains ~. This tech- nique allows detection of re- peated DNA fralpnents with no need for any specific probe. DNA is digested with a resection endoundease and then radio- labelled and electrophoresed in agm~se. Two cydes of demtum~on/remtura~ov/fn ~h~ digestion with S1 nudease are performed, p r e s ~ only the repetitive f~agments that can effidently rearmS; these are detected as discrete hands by automdiography.

Comparison of amplified DNA patterns between susceptible and resistant strains of mos- quitoes and houseflies revealed the following. (1) The patterns of major repeated sequences of several ssmp~le mosquito swains are the same, be they C. ~ m or C. v~e/mciatm. (2) Resistant strains do possess

amplified fragments. (3) These additional trends are specific for the overproduced enzyu~ In the C. ~ - fmdatm strains Tem-R, W'dlow and Coachelb, which overpro- duce estemse B, the snne sb: addilional EcoRI fragments are found to be specifically an~olified, although to different degrees. The degree of ampli~catian correlates with the deip'ee of estemse overproduction. In C.

$54, which overproduces esterase A, two dilferem bands are detected. These exl)eri- ments demonstrate that ;pe~c gene amplification is associated with the resistant phenoqq~e but do not prove that the anq2Med DNA codes for the detoxifying enzyme.

This has now been achieved in the case of the TEM-R resistant strata 9. Screening of a TEM-R cI)NA expression library with an antiserum raised against est- erase BI yielded a cDNA insert able to select by hybridization a mRNA that codes for eaterase BL Esterase B! is 500 times more abundant in OP-resistant TEM-R than in susceptible S- LAB mosquitoes t°. Hybridiza- tion of this cDNA to Southern

~1986. Elsevier Sc~ Pul2ishersB.V., Amsterdam 0168-~.200

blots and slot blots of TEM-R and S-LAB DNAs demonstrated a 250-fold amplification of a genomic 2.1 kb EcoRl fragment in TEM-R DNA. This is the ssme size as one of the sixEcoRl fragments detected by the re- natmation technique in agarose gels z. The identity of both fragments was confirmed by hybridization with the cDNA for esterase B19. 'Ibis dearly showed that the amplified DNA detected by in gel renaturation indeed contained the esterase B1 gene.

It follows that the amplified DNA detected in the other resistant strains of mosquitoes and houseflies probably contains the gene for the detoxifying enzyme. Thus, gene amplifica- tion may be a widespread medmsm in the acquisition of pesticide resistance.

Interestingly, Shah et ¢d. ~ have recently reported a 20-fold amplMcalion of the 5-enol- pymvylshikimate 3-phosphate (EPSP) synthase in a Petunia ~ cell line resistant to the herbicide glyphosate. They fur- ther demonstrated that resistant trmmgenic plants could be oh- tamed by reg~emtion from calli tmnsfonned with a chimeric, overproducing EPSP synthase gene. These reports should lead to a more effective mamgement ~,f insecticide and herbicide use, and suggest the possib'dity of engineering pesticide resistance in ecologically useful animals.

So far, gene amplification has only been well documented as a medmnism of drug resistance in cultured cells, where it is widely investigated, and in human tu- rnouts, where it accounts for

275

monitor either methotrexate or multi- drug resistance ~z. Only in one case has it been shown to occur in a normal tissue d a whole animal: mice treated with cad- midm at concentrations live to tent imes higher than that required to induce mmdm~ transcription of the MT-I gene showed a two- to threefold increase in MT-1 gene concen- tration specific to liver nuclear DNA ]3.

Pestidde-resistant insects now offer new convenient models for the study of gene amplification in reproducing animals evolving in field conditions and to solve questions that could not be easily answered using cultured cells, for instance, transmission and modi- fication of an amplified array of genes at meiosis.

Formation of multigene fami- lies can be accounted for by gene

amplification followed by evo- lution of the newly created gene duster. But bow they are established as permanent sU'uc- tures conferring a selective ad- vantage is unknown. Thu~'; it has been proposed" that the multigene family of the whiter flounder's antifreeze protein was acquired by gene amplification as a response to the relatively rapid ocean cooling at the onset of the Cenozoic ice age. Gene ampli- fication in field populations of mosquitoes facing a brutal pesti- cide selection represents an attractive experimental system to approach such questions.

References I Brattsten, L. B., Holyoke,

C.W., Jr, Leeper, J.R. and. Raffa, K. F. (1986)Science 231,

1255-1260 2 Mouch~s, C., Foumier, D.,

Raymond, M., Magnin, M., BergS, J-B., Pasteur, N. and Georghiou, G.P. (1985)C.R. AcccL Sd. Pans Set. I l l 301, 695-700

3 Pasteur, N., Iseki, A. and Georghiou, G.P. (1981)B/o- chem. Goner 19, 909-919

4 Georghiou, G. P., Pasteur, N. and Hawley, M.K. (1980) ]. Econ. £ntomol. 73, 301--305

5 Clark, g. G., Shanman, N. A., Dauterman, W. C. and Hayaolm, T. (1984) Pes~. B ~ / m . Physiol. 22, 51-59

6 Pasteur, N., Georghinu, G. P. and lseki, A. (1984) GUt S~I. £vol. 16, 271-284

? Devonshire, A. L and Sawicld, R. M. (1979) Nature 280, 140- 141

8 Roninson, I. B. (1983) NJz/dc AddsRes. 11, 5413-5431

9 Mouch~s, C., Pasteur, N.,

TIC, - - November 1986

BergS, J-B., Hyden, O., Raymond, M., Robert de Saint Vincent, B., de Silvestri, M. and Georghiou, G. P. (1986)Sc/e~ 233, 778-780

lOMouch~s, C., Magnin, M., Berg6, J-B., de Siivestri, M., Beyssat' V., Pasteur, N. and Georgldou, G.P. Prec. Nati Accd. Sd. USA On press)

I1 Shah, D. M., Horsch, IL B., Kiee, H.J., Kishore, G.M., Winter, J.A., Turner, N.E., Hironaka, C.M., Sanders, P.R., Gasser, C. S., Aykent, S., Siegel, N. R., Rogers, S. G. and Fraley, R. T. (1986) Sdence 233, 478-481

12 Stark, G. R. (1986)CamccvSuw. 5,1-23

13 Koropa~ck, J., Wmni~ R., Wiese, E., Heschl, M., Gedamu, L. and Duerksen, J. (1985) N~¢/¢/c Adds Res. 13, 5423- 5439

14 Scott, G. K., Hew, C. L and Davies, P. L. (1985) Proc. Natl Ac.ad. St'/. USA 82, 2613-2617

In multicelluiar organisms, com- munication between non-adja- cent cells is frequently mediated bypeptide hormones. Hormones inform the target cells about conditions in the environment, and induce the target cell to respond appropriately. Peptide hormones also play a role in intercellular communication in some unicellular eukaryotes. The mating factors produced by the two sexes, or mating types, of the yeast Sacc~romyces cevn~s~ are among the best studied examples. In this case, the hormones inform cells of one mating type of the presence of cells of the opposite mating type within the vicinity, and induce processes in the target cell that are required for mating. The mating factors of this yeast are known as a-factor, a 13-amino acid hormone made by cells of the a mating type, and a.factor, an ll.amino acid hormone made by cells of the a mating type. Each mating factor acts to arrest cells of the opposite mating type at the poi~t in the cell cycle known as start. Start is also the point at which cells arrest their growth and division when nu- trient limited. Although a-factor and o~-factor have no sequence similarity, recent results from a number of laboratories demon- strate dose parallels between the response of a cells to a- factor and the response of a cells to a-factor. This article high- lights these results and de- scribes evidence indicating that, despite the absolute specificity

Regulation of-cell division by peptide

hormones of Saccharomyces

cerevisiae Jasper Rine

D@artmntofBioch~nis~, UnivmityofCalifomi¢ Be~dey, CA94720, USA.

of the action d each factor on cells of the opposite mating type, both factors trigger a common internal signal that mediates growth arrest. For a more thor- ough discussion of the many interesting aspects of matmg- factor biology, see Ref. 1.

Our present understanding of the role of poptide hormones in the mating of S. cevevisiae is rooted in the genetic analysis of mutations that block mating. Genes defined by these mu- tations are referred to as STE genes, indicating that the mu- tants are sterile. Most STE mutations block the mating of both a cells and c~ cells. How- ever, a few ste mutations specific- ally block the mating of a cells while others specifically block the mating of a cells. Since the two ma~g factors are very different chemically, one might expect the

mating.type-specific ste mum. tions to be dominated by genes dedicated to the synthesis of, and response to, one or the other mating factor. By similar argu. ments, some of the non-specific ste mutations may define genes that act in steps common to the mating.factor response in cells of either mating type. This broad generalization is supported by several lines of evidence. For example, ST£2 is an a.spedfic gene that, by both genetic and biochemical criteria, encodes the a.factor receptor ~. S'unllady, STE3 is an a-spedfic gene that appears to encode the a-factor receptoP ,9. Although the deduced amino acid sequences of the STE2 and STE3 proteins are not homologous, secondary structure predictions suggest that both proteins have very similar stmc- tures, with seven potential trans.

membrane domains and a hydro- phih'c carboxyl terminus Cs. The other mating-type-specific ST£ genes identified to date are all involved in the synthesis or processing of the corresponding mating factor.

Studies of the expression of ST£2 and ST£3 indicate that these genes are regulated in several different ways. Expres- sion of the genes is regulated by the products of the mating-type locus. STE2 mRNA is present only in a cells and ST£3 mRNA is present only in a cells. Surpdsingiy, the level of ST£3 mRNA is induced by a-factor and the level of ST£2 mRNA is induced by a-factox ~'s. Thus, both hormones appear to induce the synthesis of higher levels of their own receptor. In the case of STE3, it has been shown that induction of additional ST£3 mRNA by a-factor is a primary response since induction occurs in the absence of protein syn- thesis and with rapid kinetics. The reasons for induction of additional receptor by mating factors is unclear. Perhaps the strength of the signal that can be generated by the basal level of receptors is insuffident to elidt the entire spectnun of mating- factor-induced events, and ad- ditional receptors may be re- quired. A two-phase response to mating factors may allow the morphological changes de- scribed below to occur at low factor concentrations, yet ensure that a cell does not become arrested before there is a

2 7 6 ~) 1906, Elsevim" Sdence Publishers B.V., Amsterdam 0168- 9525/{Fo~.200