[ W E V I E W S

S e x in plants, as in animals, is not a simple process of sperm meets egg. To achieve fertilization, the male gametophyte (pollen grain) must interact with, and grow a pollen tube through, the female pistil in order to deliver sperm. In angiosperms, double fertilization adds additional complexity: two mitotically derived (and hence genetically identical) sperm nuclei sep- arately fertilize the egg and the central cell, resulting in the formation of the zygote and the endosperm (a nutritive tissue for the developing embryo), respectively. Plants exhibit a plethora of genetically characterized hybridization barriers, both within and between species, that can arrest the process at numerous points before and after fertilization.

Plant reproduction research has already been the subject of entire books 1,2, as well as many recent reviews~. This article summarizes the latest results in the area, and points out how the molecular tools we are accumulating can be used to address some un- answered questions.

Male gametogenes i s Figure 1 highlights some of the processes that occur

during microsporogenesis. Different flowering plant species exhibit variations of this generalized scheme. Male gametogenesis starts when a diploid sporophytic cell (the primary sporogenous cell) divides to give rise to the tapetal initial and the sporogenous initial. The sporogenous cells undergo meiosis, giving rise to a tetrad of haploid cells. The individual cells of the tetrad develop into microspores and undergo an asymmetric mitotic division, resulting in a pollen grain containing two cells: a large vegetative cell, and a small generative cell with a condensed nucleus and a reduced amount of cytoplasm. There is some evidence 1,7 that the asym- metry of the first mitotic division is critical for continu- ation of the gametophytic pathway of development, because a variety of treatments (such as cold or col- chicine) that cause a symmetrical division can redirect the microspore towards a sporophytic pathway, eventu- ally resulting in the formation of haploid plantlets. In some plant families, the two-celled pollen grain is released from the anther, and a further mitotic division of the generative cell occurs during pollen tube growth through the pistil, giving rise to two sperm nuclei. In other plant families, the second mitotic division occurs before pollen is released from the plant.

Other anther cell types, in addition to the develop- ing pollen, are essential in microsporogenesis and pollen release. The tapetum is a transient tissue orig- inally derived from the same division that gave rise to the primary sporogenous cell. This tissue lines the locule containing the developing microspores, and is thought to provide nutritive and structural components to the developing pollen grain. For example, the tap, etum secretes callase 2, required for release of micro- spores from the tetrad. The tapetum degenerates dur- ing the later stages of pollen grain development, and some of the tapetal cellular 'debris' is deposited on the surface of the pollen grain. When the pollen has matured, the cell layers of the anther wall undergo structural and degradative changes that facilitate release of the pollen grains from the locule.

Molecular analysis of male gamet0genesis in plants SHIm, HA MCCORMICK

Reproduction in plants rivals the complexity of the process in animals. Recent~, several genes that are expressed at specific stages during male gametogenesis have been cloned These anther-specific genes are providing tools with which to study the cis- and trans- acting factors that regulate gene expression during pollen formatto~ Seque~e analysis of the coding regions of some of these abundant~ expressed anther genes is providing unexpected insights into the cell-cell interactions occurring during gamete formation and fertiUzatio~

There are genetic and molecular data supporting a role for both sporophytic and gametophytic gene expression in the regulation of microsporogenesis. Numerous male-sterile mutations disrupt the process of microsporogenesis 2. The recessive nature of most male-sterile mutations argues that the expression of these genes occurs in diploid cells, and not in the hap- loid developing pollen grain, and several male-sterile mutants show altered tapetal metabolism 2. On the other hand, analysis of chromosome deficiency stocks of maize shows that haploid gene expression is critical for successful completion of microsporogenesis 8. In hypoploid strains of maize that are deficient for a given chromosome arm, two of the microspores of each tetrad are arrested, and the stage of arrest differs depending on which chromosome arm is deleted.

The pollen cell wall is composed of two layers. The protein components of the inner layer (intine) are largely thought to be derived from gametophytic gene expression l, while the protein components of the outer layer (exine) are thought to be produced by the sporophytic tapetal layer. The species-specific sculp- turing patterns of the exine wall are also determined by the sporophyte 1.

Pollen-pistil interactions After pollen is released from the anther, the pollen

grain attaches to the stigmatic surface of the pistil, hydrates and extrudes a tube (an extension of the intine cell wall) that grows toward the ovule through the extracellular matrix of the stylar tissue. Currently, the most popular area of research related to pollen- pistil interactions is self-incompatibility; in contrast, relatively little is known about the molecular cell biology of pollen-pistil interactions during compatible matings. Self-incompatibility is a genetically controlled means of preventing inbreeding, and is determined by one genetic locus with multiple alleles. The incompati- bility phenotype of pollen and pistil must be different for a successful cross to occur. In the gametophytic system of self-incompatibility, the phenotype of the pollen is determined by the genotype of the pollen, while in the sporophytic system, the phenotype of the pollen is determined by the genotype of its diploid parent plant (reviewed in Refs 3, 5).

T1G SEPTEMBER 1991 VOL. 7 NO. 9

,©1~)1 Elsevier Science Publishers Lid (UK) 01(',8 9479/91/$O2 0~ m

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Generalized schematic of male gametogenesis and pollen germination. (A) The meiotic products (microspores) develop into pollen grains. The tapetum deposits components onto the pollen wall. Pollen grains land on the stigmatic surface and germinate in order to deliver sperm to the ovules. (B) Cross-section of anther. (C) Detail showing relationship of anther wall, tapetum and locule.

Pistil-specific S-glycoproteins, which are thought to be involved in self-incompatibility, have been purified and the corresponding genes cloned. The gametophytic and sporophytic S-glycoproteins show no amino acid sequence similarity. Because of the genetics of self- incompatibility, it was speculated that the male com- ponent shares coding region similarity with the pistil S-glycoprotein sequences. Unfortunately, the predicted anther component of either type of self-incompatibility has thus far proved elusive; however, expression in transgenic plants of reporter genes driven by S-glyco- protein promoters suggests that the predicted expression in the tapetum (for sporophytic self-incom- patibility) or in pollen (for gametophytic self- incompatibility) can be obtained 9.

Anther-specific genes Several groups have isolated cDNAs of RNAs that

are preferentially expressed in the anther during gam- etogenesis. The tissue specificity and developmental ex- pression of these genes has been analysed by RNA blot analysis, in situ localization, and by use of chimeric gene constructs in transgenic plants, Sequence similarity searches, immunolocalization and antisense approaches are being used in attempts to gain insights into the possible roles these gene products play during anther development. Table 1 summarizes the current status of analysis of a subset of anther-specific genes, selected to represent a range of species, patterns of cell type expression and the availability of promoter analyses.

Transcriptional regulation The transcriptional regulation conferred by promoter

regions of several anther-specific clones has been analysed by sequence comparisons and by functional analysis in transgenic plants13,16,18,2128 and/or with transient assays via microprojectile bombardment of pollen 13. Except for the two Brassica Bp4 genes 21, whose promoters are very similar, there are at best only limited regions of sequence similarity among pro- rooters that direct similar patterns of gene expression, even within the same species of plant, and among promoters of genes with similar coding regions. For example, there are no obvious sequence similarities between the promoter regions of the tobacco TA29 and TA36 genes ~6, which are both expressed in the tapetum. The coding regions of the LAT56 and LAT59 genes of tomato show 54% amino acid identity ]4, but their promoters share only a small (9 out of 10 bp) region of sequence identity 13. The coding regions of the LAT52 tomato gene 12 and the Zm13 corn gene 2° show 32% amino acid identity, whereas their promoter regions12:9 share only limited regions of similarity.

Detailed functional analyses of several anther pro- moters have been performed13:6:8,2L Briefly, the anther promoters studied thus far are composed of relatively short sequences that are sufficient for proper tissue expression (Table 1) and upstream regions important for higher levels of activity. To date, func- tional analyses have not uncovered any cis-acting regions that suppress expression of anther-specific

TIG SEPTEMBEI~ 1991 VOL. 7 NO. 9

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] R E V I E W S

genes in other tissues. Additional deletion analyses will be required to define precisely the c~-acting sequences necessary for anther specificity, and may reveal com- mon regulatory elements within the minimal promoters.

Now that the promoter analyses have provided reasonably well-defined control regions, the search can begin for trans-acting regulatory factors that inter- act with these promoters. Some of the upstream se- quence elements in these anther-expressed promoters 13 resemble elements found in other plant genes that are expressed in non-anther tissues, and can activate a heterologous core promoter13,16. It is likely that, as in other tissues 29, a combination of general and tissue- specific factors and c/s-acting elements regulate anther gene expression.

Trans-acting factors that interact with anther-specific genes are apparently not restricted to particular species, in that tapetum or pollen promoters isolated from one plant species show the same specificity in transgenic plants of different plant families17,18, 28. One surprising and unexplained exception is the pattem of gene expression in tobacco that is directed by the sporophytic self-incompatibility locus promoter from Brassica 9. The reporter gene is expressed in pollen, rather than in the tapetum as might be expected.

Applications for anther promoters Tissue-specific promoters are useful tools for bio-

technological applications, and for addressing more basic questions of gene regulation. Two groups have elegantly illustrated the uses that can be made of flower- specific promoters. A tapetum-specific promoter (TA29) from tobacco, when fused to either of two RNase genes 17, or to the diphtheria toxin gene 16, could selectively ablate the tapetum, resulting in the produc- tion of male-sterile plants. Such cytotoxic gene con- structs, coupled with a method to r e s t o r e fertility 17, will facilitate the production of hybrid seed in a variety of plants. Expression of the diphtheria toxin gene behind the Brassica S-glycoprotein gene promoter in transgenic tobacco 9 resulted in ablation of both pollen and pistil cells. These results supported the proposal that gene regulation in the two self-incompatibility sys- tems might share common features.

The results of these gene fusion experiments also suggest that cell interactions may play a rote in normal flower development. For example, even though toxin expression driven by the S-glycoprotein promoter was restricted to the transmitting tissue of the pistil, there were phenotypic effects on the other tissues of the pistil9. This may indicate that normal development of

TIG SEPTEMBER 1991 VOt. 7 NO. 9 Iml

~EVIEWS

.I~,T59 M G G P K I K Y S F L F L C I T F A T I I P S L M A H I G R Y D E V W R R R A E E A K E Y A R N I Y E P H P N V T T L A F N N Q K L LAT56 ........................................................................................................ M E Y S Y K I V[~]F I

9612 ............................................................................................ MS T LFF TF S LLLL~A~P LLV I S S I

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FIG[] Amino acid sequences derived from genes encoding ragweed pollen allergens (Ambal.1, AmbaL2 and AmbaI.3), two tomato pollen proteins (IAT59 and IAT56) and one tomato pistil protein (9612). Amino acid identities shared between either LAT56 and/or LAT59 and any one of the ragweed allergen sequences are boxed. The boxed amino acids in the pistil protein, 9612, represent shared identity with any one of the pollen sequences.

the pistil requires cooperation between cell types in that organ. The TA29 promoter-based cytotoxic gene constructs affected only the tapetum; anther wall development appeared normal. This is in contrast to the phenotype of many male-sterile mutants 2, whose anthers are smaller and somewhat shrivelled relative to wild type. Reduced expression of several anther- expressed mRNAs 26,30 (relative to wild-type anthers) was observed on thin sections or northern blots pre- pared from male-sterile anthers, also suggesting a cooperation between cell types in the anther.

Gene transcription and translation in gametogenesis Mature pollen grains are thought to contain a store

of presynthesized mRNAs that will be translated for processes required during and after pollen grain ger- minationl, 6. The first detectable expression of several pollen-expressed genes (e.g. Zm13, IAT52, IAT59) correlates with the first microspore mitosis 18,2s. It is generally thought that transcription after this mitotic division is restricted to the vegetative nucleus 1,6. It is still unknown whether the asymmetric division some- how activates gene expression exclusively in one of the two nuclei resulting from this division. No exper- iments have so far addressed the possibility of trans- lational control of pollen gene expression, although there are certainly analogies for such controls in ani- mal sperm 31 and oocytes, which accumulate stored message for later use. Lastly, it is unexplained why a heat shock response cannot be detected in mature or

germinating pollen grains 32, even though developing pollen is transcriptionally active.

Functions of anther-specific proteins Numerous estimates of gene expression ],4,6 suggest

that many genes are likely to be expressed only in anthers. Such genes might include anther-specific ver- sions of structural proteins or of metabolic pathway enzymes. For example, there are anther- and/or pollen-specific isoforms of 0t- and []-tubulins33. Pollen mother cells of lily specifically express a histone-like protein34 that is similar in amino acid sequence to the histone-like proteins expressed in mammalian testis. Anther-specific genes might also encode proteins that are involved in biosynthetic pathways unique to microsporogenesis. Genes encoding enzymes involved in biosynthesis of sporopollenins - substances only found deposited on the pollen wall - may be ex- amples of the second class 1.

Database searches for sequence similarities with the predicted proteins encoded by anther-specific cDNAs have yielded, variously, nothing significant, the pre- dictable, or the unexpected (Table 1). One unexpected sequence similarity was found for the tomato LAT56 and IAT59 gene products. These proteins show striking sequence similarities to a limited region of the pectate lyases from the bacterial plant pathogen Erwinia 11,]4. This initial finding seemed plausible, because pollen tubes probably require pectin-degrading enzymes dur- ing pollen tube growth and wall synthesis 1. We also

"FIG SEPTEMBEI~ 1991 VOL. 7 NO. 9

[ ] ~ E V I E W S

noticed that an amino-terminal region of the proteins showed sequence similarity to a tryptic peptide from a pollen allergen of Japanese cedar TM, and used this find- ing to support our contention that the LAT56 and LAT59 proteins were likely to be a component of the pollen wall, as are many allergens.

Recently, more extensive sequence similarities (Fig. 2) to the LAT56 and LAT59 protein sequences came to light: they are strikingly similar to the major ragweed pollen allergen (AmbaD 23. Interestingly, these pollen se- quences also bear a striking similarity to the predicted amino acid sequence encoded by 9612, a tomato cDNA of a gene that is predominantly expressed in pistils 27. The fact that plants as diverse as the angiosperms tom- ato and ragweed (dicots) and corn 6 (monocot), as well as Japanese cedar (a gymnosperm), have conserved an open reading frame and its pollen-specific expression suggests a highly conserved role for these proteins. The pattern of regions of high amino acid similarity, inter- spersed with regions of variability, is reminiscent of the patterns seen among pistil-expressed S-glycoproteins from different S-alleles3,5. For S-glycoproteins it has been suggested that the variable regions determine allele specificity and that the conserved regions are important for the structure or function of the proteins. Perhaps the gene products of the pollen-expressed members in Fig. 2 somehow interact with the pistil- expressed member(s?) during compatible pollinations. The extracellular location of the allergens (in the pollen wall) 23 and the predicted location for the 9612 protein 27 (extracellular matrix of the transmitting tissue of the style) is compatible with this hypothesis.

It is also plausible that the LAT56, LAT59, allergen and 9612 proteins merely show sequence similarity because they are pectin-degrading enzymes, and that the conserved regions are important for enzyme struc- ture. It is not yet known whether the corresponding regions from the pectate lyases of Erwinia are import- ant for substrate binding or enzyme activity. We have as yet no evidence that the plant proteins are functional pectate lyases.

It should be informative to determine the se- quences and patterns of expression of analogs of this ragweed allergen group from additional plant species. It may also be possible similarly to analyse other potentially interacting components of pollen and pistil, using other nonrelated allergen sequences24, 25 as start- ing points for gene isolation.

Database searches have also suggested roles in pollen-pistil interactions for other pollen allergens and pistil proteins. A major stylar glycoprotein 35 shows sequence similarity to a [3-glucanase, one of a group of pathogen response (PR) genes. In addition, a major allergen of birch tree pollen 36 shows sequence simi- larity to the product of a PR gene from pea. Since the pistil protein represents about 10% of the stylar extra- cellular matrix proteins, it was suggested that it may play a role in pollen tube guidance35.

It seems reasonable, but is by no means proven, that pollen allergens are important for reasons other than causing allergic reactions in mammals. Bio- chemical characterizations will allow testing of the hypothesis that the proteins interact as components of pollen-pistil recognition.

Conclusions and prospects Mthough only a few anther-specific genes have

been analysed, significant progress has been made in determining the c/s-regulatory regions important for pollen gene expression. Gene probes specific to cer- tain stages of microsporogenesis or cell types are underrepresented; additional probes will be needed in order to attain a more complete understanding of gene regulation during pollen development. Cell separation techniques were helpful in s.tudying gene expression at specific developmental stages of mammalian sper- matogenesis; methods for separating precise develop- mental stages of microspores37 should help add to our repertoire of genes. We still have no clue as to whether there is sperm-specific gene expression near the time of fertilization3S, or if there are female- expressed genes that correlate with the receptivity of the egg. There is no molecular information addressing the question of directed fertilization 38 of the sperm nuclei. Nonetheless, we have interesting leads about proteins that may be important during pollen develop- ment, and can anticipate that further work in the pollen-pistil interaction arena will yield complexities as yet unimagined.

Acknowledgements I thank Keith Hamby, Paul Herzmark, Guy Vancanneyt

and Judy Yamaguchi for helpful comments on the manu- script, Guy Vancanneyt for Figures, and Ron Wells for the Table. Work in my laboratory is supported by USDA CRIS no. 5335-22230-002-00D and by the NSF Center for Plant Developmental Biology, University of California, Berkeley DIR 8719933.

References 1 Giles, K.L. and Prakash, J., eds (1987) Pollen: Cytology

and Development (Int, Rev. Cytol. Vol. 107), Academic Press

2 Kaul, M.L.H. (1988) Male Sterility in HigherPlants, Springer-Verlag

3 Dickinson, H.G. (1990) BioEssays 12, 155-161 4 Drews, G.N. and Goldberg, R.B. (1989) Trends Genet. 5,

256-261 5 Hating, V. et al, (1990) Science 250, 937-941 6 Mascarenhas, J.P. (1990) Annu. Rev. Plant Physiol. Plant

Mol. Biol. 41,317-338 7 Zaki, M.A.M and Dickinson, H.G. Sex. Plant Reprod. (in

press) 8 Kindiger, B., Beckett, J.B and Coe, E.H. Genome(in press) 9 Thorsness, M.K., Kandasamy, M.K., Nasrallah, M.E. and

Nasrallah, J.B. (1991) Dev. Biol. 143, 173-184 10 Ohkawa, J., Okada, N., Shinmyo, A. and Takano, M.

(1989) Proc. Natl Acad. Sci. USA 86, 1239-1243 11 McCormick, S., Twell, D., Vancanneyt, G. and

Yamaguchi, J. in Molecular Biology of Plant Development (Proc. SEB Symposium 45) (Jenkins, G.I. and Schuch, W., eds), Company of Biologists (in press)

12 Twell, D., Wing, R., Yamaguchi, J. and McCormick, S. (1989) Mol. Gen. Genet. 217, 240-245

13 Twell, D. et al. (1991) Genes Dev. 5, 496-507 14 Wing, R.A. etal. (1989) PlantMol. Biol. 14, 17-28 15 Smith, A.G., Gasser, C.S., Budelier, K.A. and Fraley, R.T.

(1990) Mol. Gen. Genet. 222, 9-16 16 Koltunow, A.M. etal. (1990) Plant Cell 2, 1201-1224 17 Mariani, C. etal. (1990)Nature 347, 737-741 18 Guerrero, F.D. et al. (1991) 31101. Gen. Genet. 224,

161-168

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g,~}2

[]~EVIEWS 19 Hamilton, D.A., Bashe, D.M., Stinson, J.R. and

Mascarenhas, J.P. (1989) Sex Plant Reprod. 2, 208-212 20 Hanson, D.D. et al. (1990) Plant Cell 1, 173-179 21 Albani, D. etal. (1990) PlantMol. Biol. 15, 605-622 22 Brown, S.M. and Crouch, M.L. (1990) Plant Cell 2,

263--274 23 Rafner, T. et al. (1991) J. Biol. Chem. 266, 1229-1236 24 Silvanovich, A. et al. (1991)J. Biol. Chem. 266, 1204-1210 25 Singh, M.B. et al. (.1991) Proc. Natl Acad. Sci. USA 88,

1384-1388 26 Domon, C. etal. (1990) PlantMol. BioL 15, 643-646 27 Budelier, K.A., Smith, A.G. and Gasser, C.S. (1990) Mol.

Gen. Genet. 224, 183-192 28 Twell, D., Yamaguchi, J. and McCormick, S. (1990)

Development 109, 705-713 29 Schindler, U. and Cashmore, A.R. (1990) ~ B O J . 9,

3415-3427 30 Ursin, V.M., Yamaguchi, J. and McCormick, S. (1989)

Plant Cell 1,727-736

31 Erickson, R.P. (1990) Trends Genet. 6, 264-269 32 Dupuis, I. and Dumas, C. (1990) Plant Physiol. 94,

665-470 33 Ludwig, S.R., Oppenheimer, D.G., Silflow, C.D.

and Snustad, D.P. (1988) PlantMol. Biol. 10, 311-321

34 Sasaki, Y., Yasuda, H., Ohba, Y. and Harada, H. (1990) Plant Physiol. 94, 1467-1471

35 Ori, N. et al. (1990) EMBOJ. 9, 3429-3436 36 Breiteneder, H. et al. (1989) EMBOJ. 8, 1935-1938 37 Bedinger, P.A. and Edgerton, M.D. (1990) Plant Physiol.

92, 474-479 38 Russell, S.D., Cresti, M. and Dumas, C. (1990) Physiol.

Plant. 80, 669-676

s, MCCORMICK ns IN TH~ USDA/ARS-UC-BESKELEY PLANT GENE EXPRESSION CENTEg 800 BUCHANAN ST, A~a4N~, CA 94710, USJL

~ g ] O O K [ ~ E V I E W S

Between Nucleus and Cytoplasm

by Paul S. >,gutter, Chapman & Hall, 1990. £10.95 (vii + 148 pages) ISBN 0 412 32190 4

The investigation of the transport of molecules across the nuclear envelope is an important area of cell biology because of the relevance of both import and export to other processes, such as the regulation of gene expression. It is also one of the more challeng- ing because of the complexity of the major identified channel in the transport process, the nuclear pore. However, compared with the study of mitochondrial protein import, for example, there is a relative lack of detail and hard fact. Despite this, a large number of review articles and mini-reviews have appeared over the past few years, making this one of the most reviewed fields in cell biology. To produce a textbook on nucteocytoplasmic transport at this time is therefore a challenge, but also a good opportunity to discuss, in depth, topics that the reviews have avoided or treated lightly.

Between Nucleus a n d Cytoplasm is directed at senior undergraduate

and postgraduate biologists, and attempts to integrate the various studies of protein import into and RNA export from the nucleus and structural analyses of the nuclear substructure and nuclear envelope. It deals with methods and tech- niques, the cellular structures involved in nucleocytoplasmic transport, protein and RNA trans- port and finishes with a chapter on the importance of nucleocytoplas- mic transport in hormonal regu- lation of gene expression, carcino- genesis, development and ageing.

In the study of protein import into the nucleus, two broad classes of in vitro system have been de- vised. One uses purified nuclei or nuclear envelope vesicles in a de- fined buffer system. The other, more biochemically complex, utilizes amphibian egg extracts or per- meabilized whole cells. Although specific and efficient transport has been reported in both, the amphib- ian egg extract appears to hold the greatest promise for a biochemical characterization of the transport pro- cess. The extracts are made from Xenopus eggs arrested in the second meiotic metaphase. Extracts made from oocytes arrested at the first meiotic prophase are totally in- capable of supporting protein trans- port. Unfortunately, Agutter repeat- edly confuses the oocyte with the egg, describing experiments from the literature as having used oocyte extracts when he means egg

TIG SEPTEMBER "1991 VOL. 7 NO. 9

extracts. The seminal papers de- scribing the ability of egg extracts to carry out these processes are either miscited or not cited at all, while a number of very important papers that were published later are somewhat overenthusiastically credited with the major contribution. Although this is not as serious as errors of fact, it is nonetheless irritating to those in the field and misleading to those new to it.

The idea that macromolecular transport across the nuclear en- velope is mediated by filamentous structures is an old but attractive one for which supporting evidence is accumulating. However, exper- imental confirmation of such a transport mechanism and the direct identification of any component is currently lacking. Agutter describes a monoclonal antibody that will inhibit the export of poly(A) + RNA from nuclear envelope vesicles and concludes that the antigen is an integral part of the RNA export machinery in which ribonucleopro- tein particles are exported from the nucleus on filamentous structures. However, there is a clear contradic- tion between the biochemical behaviour of the antibody and the subcellular localization of the anti- gen. This discrepancy is glossed over and unpublished data from the author's own laboratory are cited as providing the definitive test of the hypothesis. This specific example illustrates an unfortunate