TIG - - ApN11986

Plant geneticists have tradition- ally chosen economically import- ant species such as maize, tomatO, pea and barley as their experimental material. Unfortu- nately, these species are m several respects not ideal for genetic studies: the generation times are long and relatively large amounts of space are required for cultivation. In con- trast, the small crucifer Ara- b/dops/s possesses a number of attributes that make it particularly well-suited as a model species for genetic studies 1. Chief among these are its small size and short generation time. When grown in continuous illumination, plants will produce the first seeds in less than six weeks so the generations can be rapidly -~dvanced. For some purposes such as mutagenized F2 (M2) seed produ~Ydo~ the plants can be grown at a density as high as 10 plunts/cm z so the large nmnber of individuals required for most genetic experiments can be handled relatively easily. When isolating mutations which are expressed at germination or in seedlings, as many as 10000 individuals can be screened in a single 90 mm Petri dish containing defined agar mediun~ Despite its small size, Arabidopsis is a typical dicotyledenous ~.'.ant (?ig. 1) and should serve as a good nmdel species for the study of a wide range of fumlamental problems.

Genetics and mutagenesis Because Arabidopsis is seif-fer~ale and spontaneously

outcrosses at a low frequency (c, 10 -4) it is easy to maintain genetically stable lines. Sexual crosses between lines can be readily accomplished by hand emasculation and pollination. Because the flowers are ~mall, low power (3x) magnifying glasses are useful Approximately 20-30 crosses can be perfon~eO l~r hour and each hand-pollinated silique ~¢oduces an average of 20 seeds.

In situations where a large amount of hli,brid seed is required, male-sterile lines canting the nuclear ms mutation are available as an alternative to hand emasculation. Because a single plant may produce thousands of seeds in less than eight weeks it is very easy to create segregating populations and to amplify mutant seed stocks.

Mutagenesis can be effectively accomplished by soaking seeds in a solution of a chemical mutagen such as ethyl methane sulfonate, or by treating with ionizing radiation before sowing 2. The objective of this treatment is to induce (beterozygous) mutations in the several cells which will ultimately give rise to the reproductive structure of the plant 2. This M1 generation is allowed to self.fertilize and the resulting M2 progeny are screened for mutants. Any heterozygous mutations present in a sector of the M1 plant destined to develop in a flower will be homozygous in 25% of the seeds produced by that flower. Techniques for estimating the mutation frequency per genome have been developed z. How- ever, we have found that for many loci, a mutation resulting in loss of function can be recovered by screening approximately 2000 M2 plants 3-5.

There is, as yet, no evidence for a naturally occurring transposable elenlent inArabidopsis. However, there is

reviews The mutants of Arabidopsis

M. A. Estelle and C. R. Somerville

The small oucifer Arabidopsis tbnllaqa (L.) Hey~. has many advantages as a model system for plant molecular gmelics. A s~stantial base of genetic informalion already exists and a diverse collec#ion of mutants have been isolated and us~ to address a number of questions in plant biochemistry and physiolo~. The molecular analysis of

these mutants s l~ ld be fadlitated by a relatively small and simple genome.

Fig. I. Arabidopsis thali- arm (L.) HSY~. The plant is a rosette with between 2 and 130 leaves. The don- gated shoot may bear be- tween 0 and 45 leaves below the priszipal inflor- escent. The size and aumber of leaves varies depending on race and emvironmental conditions.

Y

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~) 1986, Elsevier Science Publ~ters B.V., Amsterdam 0168 - 9525/86/$0.200 89

reviews a report of a recessive nuclear mutation (ira) which causes variegated leaves e, and of a nuclear mutation (chin) which acts as a cMomplast mutator 7. Because of the potential importance of transposon mutagenesis, mutants of this type may merit further analysis.

Genome organization in Arabidopsis Arabidopsis is a diploid organisw with a haploid

chromosome number of five. A detailed genetic map with approximately 75 loci has been assembled and a complete set of Risomics have been constructed, several of which have been used to map centromeres s. Because the chromosomes are very small, Arabidopsis is a poor organism for cytological studies. In contrast, several features of the genome suggest that Arabidopsis may be very well suited to molecular genetics. A study of DNA reassociation kinetics revealed that theArabidopsis haploid genome comprises only 70000 kb, the smallest plant genome known and only five times the size of the yeast genome 9'm. These studies also showed that only 10-14% of the nuclear genome is composed of repetitive DNA sequences and that dispersed repeat families are rare. This is a very small repetitive DNA fraction compared to other commonly used organisms such as pea, in which approximately 75% of the genome consists of repeated sequences 1°.

Because of the small genome only 16 000 ~ clones with average inserts of 20 kb need to be screened in order to achieve a 99% probability of isolating a specific unique DNA sequence from an Arabidopsis genomic library. Similarly, using a suitable vector with an average insert of 25 kb, only 14 000 transformation events would be sufficient to ensure a 99% probability of generating a transformant carrying a specific DNA sequence. This may eventually permit the cloning of genes by complementing mutant phenotypes. Since there is virtually no dispersed repetitive DNA it should be possible to clone genes by successive isolation of overlapping genomic clones, providing that the gene has been identified by a mutation, and that the mutation has been mapped close to a cloned DNA sequence which can serve as an orientation point. Meyerowitz and coworkers are attempting to create a collection of mapped orientation points by using restriction site fragment length polymorpl~sms to genetically map cloned DNA sequences u. These and related techniques may make it possible to clone any Arabidopsis gene which can be identified by mutations. Thus, the biochemical basis of many otherwise intractable mutations may eventually be determined by working backwards from the gene. It is this possibility, perhaps more than any other, which underscores the unique possibilities for using the molecular genetics studies of Arabidopsis to solve fundamental problems in plant biology.

Tissue culture of Arabidopsis A substantial amount of effort has been invested in

attempting to identify the optimal conditions for growth of Arabidopsis in tissue culture and regeneration of fertile shoots (see Refs 12-16 and references therein). In general, establishing vigorous callus or suspension cultures from most parts of the plant is straightforward. Depending on the age and source of the callus, shoots can generally be obtained on 10-60% of calii which have been in culture less than six months (Ref. 13 and

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references therein), although subculture through a series of media appears to be necessary ~z'ls. Our experience has been that, although regeneration can be obtained by following published procedures is, the frequency of regeneration appears to be very susceptible to uncontrolled variation which renders the process unreliable. We believe that additional research is needed in this area.

There has been a report of regeneration of shoots from protoplasts of Arabidopsis 14 and (abnormal) plants have been obtained following fusion of Arabidopsis and Brassica campestris protoplasts ~e. Thus, although these procedures have not yet been reproduced in another laboratory, it may be possible to implement direct transfonnatiorL Somatic embryogenesis has also been observed in Arabidopsis ~-2 and haploid plants have been regenerated from anther culture 15 suggesting that it may be possible to establish a system for somatic cell genetics in Arabidopsis. Considered as a whole, these isolated reports suggest that, althoughArabidopsis may not be the ideal organism for tissue culture studies, the technical problems in implementing tissue culture techniques are not iasurmountable.

Transformation Arabidopsis is susceptl"ole to infection with Agro-

bacterium tumefadeas 17. Recently, we have used a disarmed Ti-plasmid to introduce the Zea mays transposable element Mul into Arabidopsis (Zhang and Somerville, unpublished) by minor modifications of the leaf disc transformation protocol of Horsch et al. is and also by the callus transformation protocol of Muller et aL lo Both methods give rise to a low frequency of transformants using the NPTH gene of pMON200 (Ref. 18) as the selectable marker and 25 ltg/ml ofG418 as the selective agent. Only 5-7% of the tissue fragments or calli were transformed by this approach, but this is considered adequate for many purposes and can undoubtedly be improved. We have regenerated fertile transformed plants and, although we have not yet determined if Mul is functional in Arabid~psis, it is apparent that transformation is feasible. In our experience, the major technical difficulty has been in obtaining routine regeneration of the transformed tissue.

The mutants of Arabidopsis Mutations at more than 200 loci have been isolated in

Arabidopsis s'2°. Many of these can be obtained from the Arabidopsis collection maintained by Dr A. R. Kranz, Botanisches Institute, J. W. Goethe-Universit~t, D- 6000 Frankfurt. A majority of the 76 mapped mutations s confer readily-apparent phenotypes such as loss of surface wax, altered flower structure, leaf morphology or color, silique morphology, trichome morphology, growth habit or seed color. Very tittle is known about most of these mutations apart from their map position. Hopefully, the development of techniques for isolating genes by complementation and chromosome walking will stimulate new interest in these mutations. At present, the mutations most extensively characterized are those in which the primary biochemical lesion has been demonstrated or inferred. These fall into a number of distinct classes which serve to illustrate the variety of approaches which can be successfully used to isolate mutants in Arabidopsis.

TIG --- April 1986

Mutants isolated by direct selection The small size of Arabid@sis seed and seedlings makes

it possible to screen very large numbers of individuals for resistance to toxic compounds, allowing recovery of mutants altered in diverse aspects of ceUnlar, function.

Key elements of most well-developed genetic systems are genes which confer a selectable phenotype by the presence or absence of normal function. Such genes are essential for DNA-mediated transformation and may also be used in various kinds of promoter fusion experiments. The enzyme alcohol dehydrogenase is an attractive selectable marker because it is possible to select for and against the presence of activity by treating with acetalde'nyde or ailyl alcohol, respectively. This system has been extensively characterized in maize where the induction of Adh gene expression has been shown to occur in response to anaerobiosis. In Arabid~sis, Adh activity increases under anaerobic conditions, ill tissues exposed to the plant growth regulator 2, 4-dichlorophenoxyacetic acid (2, 4-D) and in germinating seeds 21. Mutants lacking a~cohol dehydro- genase activity have been isolated by siwply tr~ting M z seeds with an allyl alcohol solution prior to germination. In addition to being useful in the study of Adh gene expression these mutants might be used as recipients in transformation experiments with a wild-type Adh gene as the selectable marker. The Ara6idopsis Adh gene has been cloned by us'rag the maize Adhl gene as a heterologous probe 2z.

Another class of genes which show promise as selectable markers are those which confer resistance to herbicides. Sulfonylurea herbicides such as chlorosul- furon have been shown to inhibit acetolactate synthase, the first enzyme of the branci~d chain amino acid pathway m. Mutants resistant to chlorosulfuron were isolated in tobacco tissue cultures. Resistance was correlated with an altered sensitivity of the enzyme to the herbicide in vitro ~3. We have recently isolated several mutants of Arabidopsis which are highly resistant to chlorosulfuron by simply placing 300 000 IVI2 seeds or,

Petri plates containing a defined agar medium and herbicide (Hanglm and Somerville, unpublished). As in the tobacco mutants, the acetolactate synthase activity in extracts of the Arabidopsis mutants is resistant to inhibition by chlorosulfuron, suggesting that an altered enzyme is responsible for the resistance of the whole plant. The putative acetolactate synthase (a/s) gene has been cloned from the resistant line using a v~d-type a/s gene generously provided by B. Mazur (Du Pont). The a/s gene from the resistant mutant is now being tested for its ability to transform Arabidopsis and other species to a herbicide-resistant phenotype.

One of the most common selective systems in plants is the isolation of mutants lacking nitrate reductase activity by selection for resistance to chlorate, due to the mutants' inability to reduce chlorate to chlorite, a cell toxin. This selection scheme was first adapted for use with plants using Arabid@sis and a relatively large number of chlorate-resistant mutants are available. Mutations in which chlorate resistance is associated with reduced nitrate reductase activity have been assigned to seven loci u. However, the great majority of chlorate- resistant mutations fall into one complementation group (ckl-1) which is associated with reduced nitrate uptake u. These mutants should prove very useful for the genetic dissection of this complex biochemical

reviews process, and in characterizing the nitrate-induced genes which have recently been cloned by differential hybridization techniques (N. Crawford and lh Da;~, pers. commun.).

The problem of the modes of action of phytohormones has also been approached by isolating selectable mutations. Mutants resistant to the amdn analog, 2,4-D (Refs 25 and Estelle and Somerville, unpublished), abscisic acid ze and ethylene (Estelle arid Bleecker, unpublished), have recently been isolated. Koorneef and coworkers have also isolated a number of mutant lines deficient in gibberellin (GA) biosynthesis 27. Mutants of

deficient in GA biosynthesis have been instru- mental in elucidating the pathway of GA biosynthesis zs. A subset of theArabid~.~ gibberellin mutants, called non- germinating GA-dwarfs, requires exogenous gibbereUin to overcome abs~'sic acid-induced dormancy. By selecting for phenotypic revertants from mutagenized non-germinating GA-dwarf seed, several mutants with reduced levels of abscisic acid were isolated 29. None of the phytohormone mutants have been characterized biochemically. However, it seems likely that at least some of these willbe in loci of relevance to understanding the molecular basis of phytohormone action. We believe that these are the kinds of mutations which may prove particularly useful when we have established a facile system of molecular analysis in Arabidopsis.

The isolation of mutants by direct assay Because a mutation causing a specific loss of function

may be present in as few as 2000 M2 plants, it is practical to consider isolating mutants by simply assaying individual plants for the absence of a specific metabolite or enzyme activity. Plants are well-suited to this approach because a leaf can be removed in order to perform a destructive assay without killing the whole organisn~ For example, mutants of Arabid~sis with altered membrane composition were isolated using gas ~ t o g r a p h y to exan'&-ie the fatty acid composition of a single leaf from each of 2000 M2 plants. Eight mutants with altered membrane composition were recovered and placed in five complementation groups 3. Mutations at three of the loci caused the loss of specific desaturase activities and mutations at the other two loci affected head group composition. One of the mutants was completely missing an unusual fatty acid called A-trans- hexadec~-mic acid which is present in the thylakoid membranes of all plants, suggesting that it might have some specific and important role in photosynthesis. However, because this mutant is functionally indistin- guishable from the wild-type, this role must be a subtle one 3°. Similarly, the other mutants have proved useful in evaluating theories concerning the pathways of lipid biosynthesis and the functional significance of membrane lipid composition in a wide variety of contexts.

The analysis of biochemically defined mutations has also been useful in studies of starch metabolism and function. In addition to its function as a storage molecule, roles for starch have been proposed in the regulation of photosynthesis and dark respiration, stomatal function and response to gravity. In order to investigate these poss~ilities, several mutants of Arabid@sis have been isolated which are unable to synthesize starch 4. These mutants were isolated by removing a leaf from each plant and assaying for starch by simply staining the

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reviews detached leaf with iodine solution, One of the starchless mutants was shown to lack the chloroplast phospho- ghcomutase isoenzyme, the enzyme responsible for the first step in the conversion of glucose 6-phosphate to starch. The mutation has interesting effects on photosynthesis and respiration 4, and has been useful in critically re-evaluating a well-established theory, that the primary gravity-sensing structure is a starch-filled plastid called a statolith. The starchless mutant has a similar gravitropic response to that of wild-type, indicating that starch is not a prerequisite for gravitropism (Caspar, Pickard and Somerville, un- published).

The starchless mutants provide a good example of the many unexploited opportunities to apply simple genetic techniques to problems of longstanding interest in plant biology. It was straightforward to determine the biochemical lesion, the mutants were easy to isolate and can be used for many diverse experimental purposes.

Lethal mutations Many of the functions one might wish to investigate by

mutant analysis are undoubtedly essential for viability. Mutations which affect these functions will be lethal and cannot be recovered in conventional screens. The standard approach to this problem in plant genetics is to score the progeny of heterozygous lines for segregating lethal mutations. The only well-developed example of this approach in Arabidopsis appears to be the isolation of embryo-lethal mutants which arrest at various stages of development 31. In addition to providing useful material for the study of plant embryogenesis, these studies have been informative with respect to the isolation of lethal mutants in general. Meinke and coworkers 3t have found that some of the embryo-lethal mutations affect the growth of the pollen tube, suggesting that many mutations may be lost in the gametophytic generation.

The other, more famih'ar approach to the isolation of mutants affecting indispensable functions is to define permissive and restrictive conditions. This approach has been used in Arabidopsis to isolate mutants with defects in photorespiratory metabolism 5. These mutants are indistinguishable from the wild-type when grown in an atmosphere enriched with relatively high levels of COz but are inviable in air. The basis for the effect is that high levels of CO2 competitively inhibit ribulose bisphosphate oxygenase activity, the first enzymatic activity associated with photorespiratory metabolism. The primary biochemical lesion has been established in approximately 70 mutants affecting seven loci ~z. In addition to simple blocks in the photorespiratory pathway, mutants were found which lack the activity of enzymes involved in nitrogen recycling, and in one instance a mutant deficient in the transport of amino and organic acids into the chloroplast was f,"~d to lack a chloroplast envelope polypeptide 33. The characteriza- tion of this mutant illustrates the point that the small size of the organism need not preclude detailed analysis of subceliular function.

The most frequent class of conditional lethal mutations in microorganisms are auxotrophs. By contrast, apart from mutations at several loci which confer thiamine auxotrophy x, auxotrophs are conspicu- ously absent in Arabid@sis in spite Of the fact that pro2tracted searches for auxotrophs have been carded out. We think it possible that this may simply be due to

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the nutritional autonomy of the developing embryo. Thus, for example, an embryo which is unable to sythesize an amino acid due to a mutation in the relevant biosynthetic pathway may not receive this compound from the maternal tissue and would fail to develop.

We have also explored the possibility of using temperature to define restrictive and permissive conditions and have isolated a number of mutants which grow at 22°C but not at 30°(; (Estelle and Somerville, unpublished). Thus, the approach is feasible and may be particularly useful when coupled with a specific secondary screen which would permit identification of the mutants of interest.

Prospects About ten years ago the merits of Arabidopsis were

convincingly summarized without much apparent effect on the number of ~boratories using the organism I. Since that time plant genetics has been invigorated by the prospect of applying molecular techniques to bott~ basic and applied problems in plant biology. Although it .~,eems both likely and appropriate that organisms of ecouomic importance such as maize and soybeans will continue to attract substantial attention as experimental orgam'sms, we consider it likely that Arabidopsis will become established as the preferred organism for basic studies of molecular genetics. It is to be hoped that the increase in number of laboratories using Arabid@sis will not disrupt the free exchange of information and materials which seems essential to the creation of a community akin to those which developed maize, yeast and Drosophila genetics.

Acknowledgements We thank E. Meyerowitz for communicating

unpublished results and B. Moffatt, G. Haughn, W. Laing and V. Walbot for helpful comments. This work was supported in part by grants from the US Department of Energy (AC02-76ER01338) and the US Department of Agriculture (83-CRCE-l-1290).

References ! Redei, G. P. (1975)Annu. Rev. Genet, 9, 111-127 2 Redei, G. P. (1975) Genetic Manipulations in Higher Plants

(Ledoux, L., ed.), pp. 329-350, Plenum Press 3 Browse, J., McCourt, P. and Somerville, C. R. (1985)Science 227,

763-765 4 Caspar, T., Huber, S. C. and Somerville, C. R. (1985) Plant

Physiol. 79, 11-18 5 Somerville, C. R. and Ogren, W. L. (1979)Nature 280~ 833-836 6 Redei, G. P. (1967)Genetics 56, 431-443 7 Redei, G. P. (1973)Murat. Res. 18, 149-162 8 Koornneef, M., Van Eden, J., Hanhart, C.J., Stam, P.,

Bracksma, F. J. and Feenstra, W. J. (1983)]. Hered. 74, 265-272 9 Leutwiler, L. S., Hough-Evans, B. and Meyerowitz, E. M. (1984)

Mol. Gen. Genet. 194, 15-23 10 Prmtt, R. E. and Meyerowitz, E. M. (1986) ]. Mol. Biol. 187,

169-184 11 Meyerowitz, E. M. and Pruitt, R.E. (1985)Science 229,

1214-1218 12 Huang, B. C. and Yeoman, M. M. (1984) Plant Sd. Lett. 33,

353-363 13 Negrutiu, I. andJacobs, M. (1978)Z Pflr .... qtThysiol. 90, 431-441 14 Xuan, L. T. and Menczel, L. (1980).. i j ,.enphysiol. 96, 77-80 15 Keathley, D. E. and Scholl, R. L. (1963) Z. Pflanzenphysiol. 112,

24?-255 16 Gleba, Y. Y. and Hoffman, F. (1980) Planta 149, 112-117 17 Aerts, M., Jacobs, M., Hernalsteens, J. P., yon Montagu, M. and

Schell, J. (1979) Plant Sci. Lett. 17, 43-50 18 Horsch, R., Fry, S., Hoffmann, N., Eichholtz, D., Rogers, S. and

Fraley, R. (1985)Science 227, 1229-1231

TIG - - Apn'! 1986

19 Muller, A., M:~mm, T. and Lurquin, P. F. (1984) B/ochem. Biophys. Res. Commun. 123, 458-462

20 McKelvie, A. D. (1962)Rad/~ Bot. 1, ?Z$3--PA1 21 Dolfems, IL, Madmix, G. andJacobs, M, (1985)Mot Gen. Genet.

199, 256-264 Chang, C. and Meyerowitz, E. M. Proc. NaiIAcad. S~ USA (in press)

2.3 CImleff, R. S. and Manvais, C. J. (1984) Sc~m~e 224, 1443-1445 24 Bmaksma, F.J. and Feenstra, W. J. (1982)Theor. Appl. Goner. 64,

83-90 25 Mirm, J. L, OMen, G. M., Iverson, T. H. and bhher, E. P.

(1984) Phys~ol. Plant. 69, 516-522 26 Koornneef, M., Reuling, G. and Kmmsen, C. M. (1984) Physiol.

Plat. 61, 377--383 27 Koonmeef, M. and vander Veen, J. H. (1980) Theor. Appl. Gent.

~, 257-263

reviews 28 Spray, C., Phinney, B.O., Gasldn, P., Gilmour, S.J. and

MacM/II~ J. (1984) P/am~ 160, 464-468 29 Koomeef, M., Jorm, M. L., Brinkhorst-van der Swan, D. L. C.

and Karssen. C. M. (1982) Theor. Appl. Geaet. 61, 38,5-393 30 McCourt, P., Browse, J., Watson, J., Amtzem C.J. and

Somerville, C. R. (1985) Plant Physiol. 78, 853-858 31 Meinke, D. W. (1985) Theor..4~!. G~-,'~L 69, ~ 2 32 Somerville, C. R. (1984) Oxford Surveys of Piant Molecular and

Cell B~/o~ (Miflin, B.J., ed.), Vol. 1, pp. 102-133, Oxford University Press

33 Somerville, S. C. and Somerville, C. R. (1985)P/antSci. Lett. 37, 217-220

M. A. Estdle and C. R. Somerville are at the MSU-DOE Pla~ Research Labom!ory, Michigan Stale Universily, East Lansing, MI 48824, USA.

A tun~-@°point in cancer re- search has been the realization that normal cellular genes have the potential to h~duce neephsia. Such genes are now collectively called proto-oncogenes, a term rapidly gaining wide acceptan._~ce among cancer biologists ~,2. On- cogenes were first discovered through the agency of retro- viruses, which by far are the richest source of potent carcino- gens s, and over the last few years it has been firmly estab- lished that the transforming genes of retroviruses have been acquired from the genomes of normal cells 4.s. The search for proto- oncogenes has widened with the very successful approach of using DNA transfection6"7; to date, nearly two dozen proto-oncogenes have been identified and the list is growing a. It seems a miracle that a cell remains normal in the face of such potenEal adversity. Why has the cell in its evolutionary ~sdom not lost such potentially-lethal genes? The answer to these contradic- tions lies in delineath~g the role of proto-oncogenes in normal cellular metabolic processes.

In this review, I describe the salient features of the multifaceted proto-oncogene c-los, wl~ch is expressed durin a g cell growth, cell differentiation and develop- ment s. The viral homologue, v-los, "was identified as the resident transforming gene of FBJ-murine osteo- sarcoma virus which induces bone tumors in mice 14.

Molecular archi tec ture of the l o s gene Elucidation of the complete molecular structure of the

los gene preceded knowledge of its expression in a variety of cell types. This was propitious because it is essential to know its detailed genomic organization in order to grasp the subtle and complex regulation of its expression. To date, two retroviruses containing thefos oncogene have been identified, namely FBJ-murine sarcoma virus (FBJ-MSV) and FBR-MSV (Refs 9 and 10). The complete nucleotide sequences of their provirai DNAs have been deduced u. In addition, the nudeotide sequence of the cellular progenitor of thefos gene from mouse and human cells has also been determined u'lz. Figure I illustrates the organization of viral and cellular fos genes and their deduced products. The major features can be summarized as follows.

FBJ-MSV (1) FBJ-MSV provirai DNA contains 4026 nucleo-

01986. ElsevierScience~B.V.. ~ 0 1 6 8 - ~ . 2 0 0

Proto-oncogene los: a multifaceted gene

Inder M. Verma

Both v-fos and c-fos proteins, despite t ier altered carbozyl terminus, are nuclear and can Iransfonn fibroblasls in vitro. The c-fos gene is highly inducible and is expressed during development, differentiation and growth. Exln'ession of c-fos Irtotein exhiMts complex regulation involving interacgon with 3' sum-ceding sequcr~es of c-fos RNA.

tides, including two long terminal repeats (LTRs~ of 617 nucleotides each, 1639 nucleotides of acquired ~:ellular sequences (v-los), and a portion of the envelope (env) gene. (2) Both the initiation and termination codons of the v-fos protein are within the acquired sequences that encode a protein of 381 amino acids, having a molecular weight of 49601. (3) In cells transformed by FBJ-MSV, a phosphoprotein with an aplurent Mr of 55 000 (p55) on SDS polyacrylamide gel electropltoresis (SDS-PAGE) has been identified as the transforming protein. The discrepancy between the observed size and the size predicted by sequence analysis is probably due to the unusual amino acid composition of thefos protein (10% proline), since the v-los protein expressed in bacteria has a similar mobility on SDS-PAGE 13.

Proto-oncogene fos (1) The sequences in the c-los gene that are

homologous to those in the v-los gene are mterrupted by four regions of non-homology, three of which represent bona fide inUons. (2) The fourth region (104 nudeotides), which is present in both mouse and human c-los genes, represents sequences that have been deleted during the biogenesis of the v-los gene. (3) The c-los protein has 380 amino acids, which is remarkably sindlar to the size of the v-los protein (381 amino acids) (the additional 104 nucleotides in the c-los gene ~ p t s do not increase the predicted size of the c-fos proteins, because of a switch to a different reading frame). (4) In the first 332 amino acids, the v-los and mouse c-los proteins differ at only five residues, while the remaining 48 amino acids of the c-los protein are encoded in a different reading frame from that in the v-fos protein. Thus the v-los and c-los proteins, though largely similar, have different carboxyl termini (Fig. lb). (5) Despite their different carboxyl termini, both the

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