Somatic genetic analysis of the expression of cell surface molecules

T I G - - J a n u a r y 1988, Vol. 4, no. 1

review Somatic genetic analysis of " ' / the expression of cell surface molecules

There is considerable evidence that cell surface molecules that are differentially expressed on particular subpopulations of cells mediate the interaction of the cell with its environment. Some of these cell surface molecules are receptors for growth factors 1, while others deterRobert Hyman mine the adhesion or homing of cells to target organsz's or function as Many mutations that affect the biosynthesis and expression of cell surface molecules specialized effector molecules arepotentially lethal in vivo. Somatic cellgeneticsprovides a means of isolating novel (for example, the T-cell receptor mutants and studying their effects. This approach has been applied to study the genes complex) 4. The isolation and analysis important in mediating the cell surface expression of the routine Thy-1 glycoprotein, a of specific mutants provides a way to moleculepresent on subsets of cells within the hemat@oietic system. Nine classes of probe the function of cell membrane mutants with no known in vivo counterpart have been identified and studied. molecules and the mechanisms responsible for controlling the 30% carbohydrate by weight), of unknown function, expression of these molecules on the cell surface. coded for by a structural gene on chromosome 9. Two alternative antigenic forms of Thy-1 (Thy-l.1 and ThyUsing somatic cell mutants to analyse the 1.2) are determined by an arginine/giutamine interchange expression of cell surface molecules Although numerous polymorphisms have been at position 89 (Ref. 15). The sequence of the mouse Thy-1 gene predicts a detected, mutations occurring in vivo that result in the loss or abnormal expression of particular cell surface protein of 143 amino acids that has a hydrophobic molecules are rare a's'6. The analysis of somatic cell transmembrane-like region at its carboxy terminus16. mutants, coupled with the use of molecular techniques to The mature protein, however, lacks this hydrophobic introduce specific gene constructions into appropriate region and is anchored to the membrane by a glycophosmutant cell fines, provides a complementary approach to phoh'pid structure attached to the carboxy-terminal cysstudies on mutants occurring in vivo. Cultured cells may teine at position 112 (Ref. 17). This giycophospholipid be permissive for mutations that would be lethal in the structure appears similar to the phosphatidylinositolintact animal. Also, celi fines with one copy of a mutant containing glycophospholipids that function as structural gene that cannot be expressed andno copies of membrane-anchoring components for a number of the wild-type gene provide a permissive host into which eukaryotic cell surface molecules, in p~rticular alkaline normal or mutant gene constructions can be introduced phosphatase, acetylcholinesterase and the variant surface giycoproteins of trypanosomes TM. and their expression analysed7-9. The Thyol glycoprotein provides a favorable sy,,cem Selection for or against expression of a specific cell for somatic genetic analysis. Many Thy-1 ÷ T-cell lymmembrane molecule in cultured cells often results in the isolation of mutations in genes other than the structural phomas have been established in culture. Selection for gene encoding the molecule selected against. Analysis of rare Thy-1- somatic cell mutants can be carried out by such mutants provides information on genes that may treating cultured cells with anti-Thy-1 antibody and potentially control the cell surface expression of the complement and growing out the surviving cells1°. Since molecule of interest. Somatic cell mutants may define two antigenic forms of Thy-1 have been identified, genes acting at post-translational steps that are critical independent mutants can be grouped into complemenmfor the cell surface expression of particular moleculesI°. tion classes by somatic cell fusion analysis. Similarly, the Mutants of this type con be used to analyse biosynthetic dominance of each mutation can be studied by fusion of pathways involved in post-translational modifications of mutant with wild-type cells. Nine classes of Thy-1the molecule in question and to identify potential control mutants have been characterized (Table 1). mechanisms that might be @fflcult to analyse by other means. Also, somatic cell mutants may define genes that Mutations that affect biosynthesis of the act to regulate the expression of specific cell surface glycophospholipid membrane anchor Six mutant classes show a recessive behavior in molecules at the level of RNA transcription or processing11'Iz. These latter mutants may provide evi- hybrids to wild-type cell lines and define genes that act at dence for sequences that act in c/s to regulate the points in the post-translational processing of the Thy-1 expression of structural genes, or they may define genes polypeptide required for expression of the glycoprotein that act in tram to regulate the lineage-specific expres- on the cell surface. The Class E mutant is the best sion of genes coding for particular cell surface molecules. understood mutant of this type. Early studies showed that the defect defined by the Class E mutant affected the biosynthesis of high mannose oligosaccharides. The Thy- 1 glycoprotein and Thy- 1 m u t a n t s Somatic cell mutants of murine T-cell lymphomas with defect was pleiotropic in that all cell surface glycoproteins defects in the expression of the Thy-1 glycoprotein had mutant high mannose oligosaccharides; however, illustrate this experimental approach. In the mouse, only certain cell surface molecules, in particular Thy-1 Thy-1 is a major cell surface glycoprotein of thymocytes and Ly 6, were not expressed on the cell surface of the and T lymphocytes, is present in small amounts on mutant cell linex°. Further studies showed that the Class E mutant defect hemaatop4oietic progenitors, and is absent on mature B affected the biosynthesis of the preformed lipidcells • . The murine Thy-1 glycoprotein is a small molecule (112 amino acids), heavily glycosylated (about oligosaccharide donor that is added as a unit to the ~)1988, ~ P u b l i c a ~ m , Cambfid~ 0 1 6 8 - ~ 0 0

TIG--January 1988, Vol. 4, no. 1

iews Table 1. Properties of Thy-1 - mutant classes

Class Na

Dominance Molecular basis of defect in hybrids to wild typeb AffectS glycophospholipid R synthesis or attachment, Cterminal peptide cleavage

Ref.

19

A

11

B

1

R

Affects glycophospholipid synthesis or attachment

20

C

1

R

Affects glycophospholipid synthesis or attachment, Cterminal peptide cleavage

20

D E

3 Ic

CD R

Thy-1 structural gene

9

Block in synthesis of dolicholp.mannose; affects lipidoligosaccharide assembly and glycophospholipid synthesis or attachment and C-terminal peptide cleavage

19, 21 22

F

1

R

Affects glycophospholipid synthesis or attachment, Cterminal peptide cleavage

20

G

1

D

Transcription or processing of Thy-1 mRNA

11

H

1

R

Affects glycophospholipid synthesis or attachment, Cterminal peptide cleavage

20

Id

1

ND

Methylation of sites in Thy-1 structural gene

12

aN, number of independent mutants isolated. "R, mutant phenotype recessive; CD, mutant phenotype co-dominant; D, mutant phenotype dominant; ND, not determined. CMultiple isolations from one cell line. . dComplementation analysis not performeo. Classification inferred from the phenotype of the mutant.

nascent polypeptide chain as the first step in the biosynthesis of N-linked oligosaccharides (Fig. 1). The Class E mutant is unable to synthesize dolichol-P-mannose from GDP-mannose and dolichol-P (Ref. 21). Dolichol-Pmannose is the donor of the last four mannose residues present in the lipid--oligosacchafide moiety. Consequently a lipid-oligosaccharide unit containing five, rather than the normal nine, mannose residues is synthesized by the Class E mutant and is added to protein. This truncated oligosaccharide intermediate can be further processed to yield normal complex N-linked oligosaccharides, but the high mannose N-linked oligosaccharide chains are mutant1°. Since the defect in the Class E mutant affected all glycoproteins containing N-linked high mannose oligosaccharides, the question remained why only certain glycoproteins were not expressed on the surface of the Class E mutant. There was evidence from studies of the envelope glycoprotein of vesicular stomatitis virus grown in wildtype and Class E mutant cells that folding of nascent proteins did not occur normally in the mutant cells23. Most of the Class E mutant Thy-1 glycoprotein appeared to be retained within the mutant cell, where it was degraded19'24, and one possibility for the failure of Thy-1 to be expressed on the cell surface was that Thy-1 was representative of a class of m~lecule in which proper glycosylation was necessary for the molecule to attain a stable conformation in which it could be transported to the cell surface rather than being degraded.

The demonstration that Thy-1 and other molecules sensitive to the defect defined by the Class E mutant cell are anchored to the membrane through a glycophospholipid has led to a re-examination of this point. Recent studies 19'2°'22 have shown that all of the mutants that define genes that act post-translationally (Classes A, B, C, E, F and H) have defects that involve assembly or attachment of the glycophospholipid. Phosphatidylinositol-specific phospholipase C cleaves the diacyiglycerolglycosylphosphoinositol linkage of the glycophospholipid structure anchoring the Thy-1 polypeptide to the cell membrane is. After enzymatic cleavage, the bulk of the Thy-1 glycoprotein from wild-type cells partitions into the aqueous phase .of a Triton X-114 solution, whereas before cleavage the bulk of the wild-type Thy-1 glycoprotein partitions into the detergent phase. The Thy-1 molecules of Class A, C, E, F and H mutants, however, are detergent soluble both before and after treatment with phospholipase C (Refs 19, 20 and 22). This point will be discussed further below. Furthermore, the Thy-1 glycoproteins of several of these mutant classes do not label with [3H]-palmitic acid, in contrast to the Thy-1 glycoproteins from the corresponding wild-type cell lines2°'22. These two observations indicate either that an abnormal glycophospholipid structure must be added to the Thy-1 glycoprotein of the mutant cell lines or that the glycophospholipid is not added in the mutants but the mutant Thy-1 glycoprotein is detergent soluble due to some further defect. The evidence supports the latter view and indicates that the mutant cell lines contain at least some of the Cterminal portion of the polypeptide that is normally cleaved during glycophospholipid addition. As discussed above, although the Thy-1 glycoprotein of Class A, C, E and H mutants does not contain a glycophospholipid structure sensitive to phospholipase C, the bulk of the mutant Thy-1 glycoprotein is detergent soluble19'2°. Furthermore the Thy-1 glycoprotein of the mutant but not of the corresponding wild-type cell lines can be metabolically labelled by tryptophan, which is found only at position 124 in the putative hydrophobic transmembrane-like region that is normally cleaved from the molecule at the time of the addition of the glycophospholipid. This observation implies that at least a portion of this C-terminal region of the Thy-1 polypeptide is not cleaved in the mutant cell lines. If so, then there must be some linkage between addition of the glycophospholipid and cleavage of the C-terminal 31 amino acids. It is possible that a single enzyme catalyses both peptide cleavage and glycophospholipid addition. However, there is evidence that the two events can be dissociated under some circumstances, since the Class B mutant synthesizes large amounts of a water-soluble Thy-1 molecule in which cleavage of the C-terminal portion of the polypeptide appears to have occurred but addition of the glycophospholipid structure appears blocked2°. Since the primary defect in the Class E mutant is the inability to synthesize dolichol-P-mannose, there is the strong implication that dolichol-P-mannose functions directly or indirectly as a mannosyl donor during the biosynthesis of the glycophospholipid structure, which is known to contain mannose 1~.1s. The latter structure, like the lipid-oligosaccharide, is probably synthesized as a unit and added to the nascent polypeptide within 1-2 min of translation ls,2s. While the primary basis of mutant classes A, B, C, F and H is less well defined, these

review

TIG--January 1988, Vol. 4, no. 1 mutant classes must also identify other enzymes that function in the biosynthesis or addition of the glycophospholipid membrane anchor structure. The techniques of molecular biology make the isolation of the relevant structural genes defined by these complementation groups potentially feasible.

DOLICHOL-P

~

UDP-GLcNAc

DOL-P-P-GLCNAc UDP-GLCNAc~ OOL-F-P.-(GLCNAC)2 GDP-MAN -- t DOL-P-P-(GLCNAC)2- MAN

Mutations that affect Thy- 1 gene transcription or RNA processing The cell-type specific expression of I /MAN Thy-1 within the hematopoietic system DOL-P- P- (GLCNAC)2- MAN~ implies that the Thy-1 structural gene is GDP- MAN | MAN-MAN-MAN + //2DOL-P-MAN reguLated, presumably at the level of DOL-P / MaNThy-1 gene transcription. Three mutant OOL-P-P-(GLCNAC)2- MAN~ classes provide information relevant to MAN- MAN- MAN these putative transcriptional control mechanisms. Mutants of classes D and I MAN/MAN_MAN define regions of the Thy-1 structural gene important for gene transcription. OOL-P-P- (GLCNAC)2-MAN/" ~'MAN-MAN The only Class D mutant that has been characterized at a molecular level9 has a MAN- MAN-MAN-GLU-GLU.-GLU major structural rearrangement of one copy of the Thy-1 gene: deletion of the /MAN entire 5' end of the gene including the two alternatively used promoters. The DOL-P-P- (GLCNAC)2-MAN,~MAN_MAN_MAN.GLU_GLU_GLU other copy of the Thy-1 gene has been deleted in this mutant. This Class D Fig. 1. The biosynthesis of the lipid-linked oligosaccharide in wild-type and mutant is useful as a permissive Thy-1- Class E mutant cells. The Class E mutant is unable to synthesize dolichoI-Phost into which various normal and mannose and consequently addition of the sixth mannose residue (outlined) mutant Thy-1 gene constructions can be and subsequent mannose residues does not occur. introduced. It has been shown that an 8.2 kb EcoRI fragment including the Thy-1 structural gene is expressed at wild-type levels in to all other mutant classes examined, the Thy-1- mutant this mutant7, indicating that this region contains all phenotype is dominant when Class G mutant cells are sequences necessary for expression, although not neces- hybridized to wild-type cell lines. Thy-1 cell surface sarily for tissue-specific regulation, of the Thy-1 struc- expression and Thy-1 mRNA synthesis are very low in most hybrid cells, although a small, variable proportion of tural gene. The Class I mutant is of considerable potential hybrid cells do express detectable Thy-I on their surinterest. This mutant does not accumulate detectable face. Revertants that express wild-type levels of Thy-1 Thy-1 mRNA, as determined by northern blotting analy- glycoprotein derived from both parental cell lines can be sis; however, revertants that express Thy-1 mRNA can isolated from these Thy-1- hybrids by fluorescencebe isolated at high frequency after treatment with 5- activated cell sorting. Thy-1 + revertants have also been deoxyazacytidine1~. Analysis of this mutant and its rever- isolated from the mutant cell line itself and a second rant indicates that extinction of Thy-1 mRNA expression generation mutant has been derived from the revertant is correlated with methylation at sites at the 5' end of the which has properties identical to those of the first Thy-1 gene and with the loss of a DNase I hypersensitive generation mutant. The Class G mutant, therefore, site located at the border of exon la. The properties of defines a gene that acts in trans to regulate expression of the Class I mutant support the hypothesis that hypo- the Thy-1 structural gene. Whether the gene defined by the Class G mutant acts methylation of regions at the 5' end of the Thy-1 gene is required for gene expression and that changes in methyl- at the level of transcription or RNA processing has not ation can regulate the expression of the gene lz'~s. Since been definitely determined, but it has been shown that hypomethylation is observed in Thy-1- tissues in trans- the mutant does not accumulate significant quantities of genic animalsze, however, the state of methylation of the Thy-1 nuclear RNA (R. Hyman and K. Cunningham, Thy-1 gene cannot, alone, account for its tissue-specific unpublished). Also, the action of the gene appears to be expression. It would be of some interest to determine quite specific to Thy-1 since the expression of a number whether the expression of other cell surface molecules is of other cell surface markers is identical in mutant and altered in the Class I mutant because dexamethasone- revertant cell lines. No differences between the mutant resistant variant cell lines whose basis may also revolve and revertant cell lines in the structure of the Thy-1 gene changes in methylation have been reported to have lower or in the immediately surrounding region have been (but nonzero) levels of several surface molecules, includ- detected by Southern blotting; and, in contrast to the Class I mutant, a high frequency of Thy-1 + revertants has ing Thy-1 (Ref. 27). The Class G mutant11 has a unique phenotype. This not been observed after treatment of the Class G mutant mutant does not accumulate detectable Thy-1 mRNA as with 5-azacytidine. At this point the properties of the Class G mutant are determined by northern blotting analysis, but irl contrast

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r iews

TIG--January 1988, Vol. 4, no. 1

Gallatin, M. et al. (1986) Cell 44, 673-680 Kishimoto, T. et al. (1987)Cell 50, 193--202 Allison, J. and Lanier, L. (1987) Annu. Rev. Immunol. 5, 503-540 Jones, P., Murphy, D. and McDevitt, H. (1981)Immunogeuctics 12, 321-337 6 Davis, G. et al. (1986) Cell 45, 15--24 7 Evans, G. et al. (1984)Proc. Nati Acad. Sci. USA 81, 5532-5536 8 Gunter, K. et aL (1987)Nature 326, 505-507 9 Evans, G., Hyman, R. and Lewis, K. (1987)Immunogeuctic$ 25, Perspective 28-34 The analysis of the Thy-1- somatic cell mutants 10 Hyman, R. (1985)Biochem. ]. 225, 27--40 described above illustrates the usefulness of this experi- 11 Hyman, R. and Cunningham, K. (1986) Immunogenetics 23, 312mental paradigm in studying the control of the expression 12 321 Sheller, M. and Gunter, K. (1987)]. ImmunoL 138, 3503--3512 of cell surface molecules. Clearly somatic cell genetic 13 Basch, R. and Berman, J. (1982) Eur. J. Immunol. 12, 359-364 methods permit the isolation and study of many mutant 14 Muller-Sieburg, C., Whiflock, C. and Weissman, I. (1986) Cell 44, 653-662 genes that would be impossible to obtain and analyse if one were restricted to mutant screening in animal 15 Williams, A. and Gagnon, J. (1982) Science 216, 696-703 16 Seki, T. et al. (1985)Science 227, 649--651 populations. 17 Tse, A., Barclay, A., Watts, A. and Williams, A. (1985) Science The ultimate objective is to understand what controls 230, 1003-1008 the expression of a particular gene in a particular cell 18 Cross, G. (1987)Cell 48, 179-181 population. For Thy-1 glycoprotein, the analysis of 19 Conzelmann, A., Spiazzi, A., Hyman, R. and Bron, C. (1986) 5, 3291-3296 mutants obtained by somatic cell genetics provides 20 EMBOJ. Conzelmarm, A., Spiazzi, A., Bron, C. and Hyman, R. Mol. Cell. information complementary to studies using Biol. (in press) transfection7,s and transgenic animals ~8'29. Each 21 Chapman, A., Fujimoto, K. and Kornfeld, S. (1980)J. Biol. Chem. 255, 4441-4446 approach provides a somewhat different perspective on the problem. Each has so far provided mainly descriptive 22 Fatemi, S. and Tartakoff, A. (1986) Cell 46, 653-657 Gibson, R., Kornfeld, S. and Schlesinger, S. (1981)J. Biol. Chem. information. To progress past this point, it is likely that 23 256, 456-462 there must be a merging of these three ap~proaches with 24 Trowbridge, I., Hyman, R. and Mazanskas, C. (1978) Cell 14, 2132 those of cell and developmental biology~u to develop a means whereby the factors that govern a specific choice 25 Conzelmann, A., Spiazzi, A. and Bron, C. (1987)Biocbem. J. 246, can be studied in a biologically relevant, but experimen- 26 605--610 Kolsto, A-B. et al. (1986)Nucleic Adds Res. 14, 9667-9678 tally approachable, system. 27 MacLeod, C., Hays, E., Hyman, R. and Bourgeois, S. (1984) CaucerRes. 44, 1784--1790 Acknowledgements 28 Kollias, G. etal. (1987) Proc. NatlAcad. Sd. USA 84, 1492-1496 I thank Don Anson, Jayne Lesley and lan Trowbridge 29 Gordon, J. et al. (1987) Cell 50, 445-452 for comments on the manuscript. My work was sup- 30 Adkins, B. et al. (1987)Annu. Rev. Immunol. 5, 325--365

compatible with molecular models encompassing both positive and negative control. Since the mutant is defined by lack of antigen expression, approaches to identify and clone the gene defined by the Class G mutant present a number of uncertainties, although several approaches are being investigated.

2 3 4 5

ported by National Cancer Institute Grant CA-13287. References 1 Greaves, M., ed. (1984)Monoclonal Antibodies to Receptors: Probes for Receptor Structure and Function, Chapman & Hall

R. Hyman is at the Department of Cancer Biolosy, The Salk Institute, Box 85800, San Diego, CA 92138, USA.

Protein-DNA interactions in genetic recombination

~

The association of DNA molecules leading to new linkage relationships between genes or parts of genes is one of the fundamental processes of biology. Genetic exchanges can be of two types: (1) general genetic reStephen C. West combination between homologous chromosomes, or (2) recombination The formation of DNA-protein complexes that are capable of interaction with other that does not require extensive DNA molecules is necessary for efficient genetic recombination. How do such homology, e.g. site-specific recomcomplexes form, and how are homologous DNA sequences brought into alignmenP bination or end-to-end ligation Physical and biochemical studies of recombination enzymes from bacteria indicate events. In this review, the focus will that these proteins provide the structural framework within which the genetic be directed towards the enzymes of exchanges occur. general genetic recombination. Genetic experiments first provided evidence that exchanges occurred by the formation In Escherichia coli, a number of mutants have been of heteroduplex DNA in which one strand was provided isolated that have reduced recombination frequencies. by each parent. This exchange of strands leads to the Cells with mutations in the recA gene are completely covalent connection of the two parental DNA duplexes by defective in homologous recombination. Others, such as means of a crossover or Holliday structure 1 (Fig. 1). The those with mutations in recB, recC, recF, rec], recN, crossover may move along the DNA to generate recO, recQ and ruv have defects that vary according to the long regions of heteroduplex. Where exchanges occur type of genetic cross. between mutant sites, the base mismatches that arise in The product of the recA gene is RecA protein, the the heteroduplex region may be recognized and acted enzyme that may be regarded as the 'recombinase' since upon by repair enzymes that correct mispairing and result it catalyses homologous pairing and strand exchanges in gene conversion. resulting in the formation of heteroduplex DNA. The (~)1988,ELsevierPublications,Cambridge 0168- 9525/88/$02.00