Autonomously Replicating Macronuclear DNA Pieces Are the Physical Basis of Genetic Coassortment Groups in Tetrahymena thermophila

Corresponding author: Eileen P. Hamilton, Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106., ehamilto{at}lifesci.lscf.ucsb.edu (E-mail)

Communicating editor: S. L. ALLEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces. These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus. In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other. This phenomenon is called phenotypic assortment. We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece. The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups. Thus, coassortment allows the mapping of the somatic genome by purely genetic means. The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.

HERITABLY reconfigured somatic genomes are rare but have wide phylogenetic distribution among eukaryotes. Such rearrangements include chromosome fragmentation (breakage of a chromosome into multiple pieces), chromosome elimination (loss of entire germline chromosomes), and chromatin diminution (loss of DNA through internal deletion; TOBLER 1986 ). Somatic chromosome fragmentation in the parasitic nematode Ascaris (MULLER et al. 1996 ), discovered by Boveri in the last century, provided graphic evidence for Weissmann's germ-line theory. Fragmentation of germline-derived chromosomes and chromatin diminution occur in every ciliated protozoan so far investigated (PRESCOTT 1994 ).

Ciliated protozoa are unicellular eukaryotes that maintain two related and differentiated genomes within a common cytoplasm: the micronucleus (MIC) and the macronucleus (MAC). The micronucleus is diploid, lacks gene expression, and is the germline of the cell. The macronucleus contains the somatic genome, which is fragmented, polyploid, and actively expressed. Site-specific chromosome fragmentation and amplification occur during conjugation, when a mitotic sister of the micronucleus differentiates into a new macronucleus (COYNE et al. 1996 ). In the ciliate Tetrahymena thermophila, the MAC consists of 200–300 identifiable autonomously replicating pieces (ARPs, also known as MAC chromosomes; ALTSCHULER and YAO 1985 ; CONOVER and BRUNK 1986 ), derived from the five pairs of germline-derived chromosomes. With the exception of the 9000-copy rDNA macronuclear piece, the ploidy of the bulk MAC DNA is estimated at an average of 45 copies of each ARP per G1 MAC. This estimate is based on the kinetics of phenotypic assortment (ALLEN and NANNEY 1958 ; ORIAS and FLACKS 1975 ; DOERDER et al. 1992 and see below) and is supported by measurements of amount and kinetic complexity of MIC and MAC DNA (WOODARD et al. 1972 ; YAO and GOROVSKY 1974 ).

Attempts to show any cellular apparatus capable of ensuring the regular distribution of daughter ARP copies, e.g., kinetochores, have failed (DAVIDSON and LAFOUNTAIN 1975 ). Moreover, early genetic work in Tetrahymena showed that when cells with a MAC heterozygous at a given locus undergo vegetative multiplication, subclones that irreversibly express the phenotype associated with homozygotes for either of the alternative alleles arise. This phenomenon, called phenotypic assortment, is well explained by a model of random distribution of allele copies (ALLEN and NANNEY 1958 ; ORIAS and FLACKS 1975 ). In principle, two extreme models can be envisioned to explain the random distribution of allele copies: (1) the random distribution of ARP copies themselves or (2) the regular (mitotic-like) distribution of ARP copies, accompanied by extensive MAC recombination, which randomizes the distribution of allele copies.

We have recently identified an extensive collection of DNA polymorphisms between inbred strains B and C3 of T. thermophila using the randomly amplified polymorphic DNA (RAPD) method (WILLIAMS et al. 1990 ). The mapping of these RAPDs and the identification of meiotic linkage groups in the MIC (ORIAS 1997 ) allowed us to study the assortment behavior of many loci in the MAC of double heterozygotes. This led to the discovery of pairs of loci, closely linked in the micronucleus, that show a highly correlated pattern of macronuclear assortment. In other words, assortants with parental allele combinations at both loci are much more frequent than those of recombinant type (LONGCOR et al. 1996 ). This phenomenon was called coassortment, and a set of loci that coassort with one another in all combinations defines a coassortment group. We next sought to determine the physical basis for a coassortment group. Labeled DNA probes from two loci (1PM8 and 1KF2) in the PM8 coassortment group were shown to hybridize to MAC ARPs of identical size in inbred strain B cells. With both probes, a strong hybridization signal was observed at 1.2 Mb and a fainter signal at 1.0 Mb. In contrast, DNA probe from a neighboring locus (1KN3), which assorted independently of the other two, hybridized to an ARP with a different size (1.0 Mb) and restriction pattern. These observations led us to the hypothesis that MAC ARPs are the physical basis of coassortment groups.

We have more recently discovered a size polymorphism between inbred strains B and C3 in the PM8 ARP, reported in this article, and we (WICKERT et al. 2000 ) have identified two more loci, 1EO3R and 1JO9, that coassort with 1PM8 and 1KF2 (Fig 1). These advances have allowed us to investigate the relationship between the assortment of the ARP size polymorphism and the assortment of the loci believed to lie on it. In this article, we provide experimental evidence for the random distribution of ARP copies during vegetative multiplication and decisive genetic evidence that the 1.2 Mb ARP is the physical correlate of the PM8 coassortment group. Our results strongly support the hypothesis that MAC ARPs are the physical basis of coassortment groups in Tetrahymena. These findings provide a firmer foundation for mapping the fragmented somatic genome of Tetrahymena by purely genetic means. The coassortment map of the left arm of chromosome 1 (WICKERT et al. 2000 ) strongly supports the idea that coassortment groups indeed provide a fundamental genetic view of the Tetrahymena MAC genome.

View larger version (35K): In this window In a new window Download PPT slide   Figure 1. The PM8 coassortment group. Data for this figure are taken from WICKERT et al. 2000 . (A) Segment of the map of MIC chromosome 1L containing the four loci that comprise the PM8 coassortment group. (B) Data demonstrating coassortment of the four loci with one another. Names of terminal assortant strains are written vertically; the SB prefix has been omitted. 1 and 0 represent presence and absence of B-derived polymorphic band for 1PM8, 1KF2, and 1JO9 and absence or presence of the C3-derived band for 1EO3R, respectively. Data for terminal assortants of recombinant type are shaded. LOD values against independent assortment range from 4.4 (1PM8 vs. 1JO9 or 1EO3R) to 14.5 (1J09 vs. 1EO3R).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, culture, and crosses:
Routine methods used in our lab for Tetrahymena storage, culture, and genetic crosses have been described (ORIAS and BRUNS 1975 ; HAMILTON and ORIAS 1999 ; ORIAS et al. 1999 ). Tetrahymena strains used in crosses first reported in this article are shown in Table 1. All the DNA polymorphisms used here are RAPDs (WILLIAMS et al. 1990 ) between inbred strains B and C3 (BRICKNER et al. 1996 ). Genetic methods for working with RAPD polymorphisms in Tetrahymena have been described (LYNCH et al. 1995 ; BRICKNER et al. 1996 ; LONGCOR et al. 1996 ). Tetrahymena RAPD loci have a leading "1" to indicate their identification in this laboratory. The names of RAPDs for which the polymorphic band is templated by C3 DNA end in R, while absence of a final R indicates one for which the polymorphic band is templated by B DNA (ALLEN et al. 1998 ). ARPs and coassortment groups (PM8 in this work) are named after one RAPD in the group, omitting its leading 1.

Two panels of B/C3 terminal assortants were used. One panel (cell lines SB1805–1840) consists of 36 terminal assortants described in LONGCOR et al. 1996 , passaged further so as to reach at least 500 fissions. The other panel (cell lines SB4210–4221) consists of 12 additional assortants obtained by crossing strains SB1969 and SB3546 (see Table 1) and passaging progeny lines for at least 350 fissions. Given the age of these lines, there is at least 95% probability that assortment will be complete at any given locus (DOERDER et al. 1975 ). "Young" progeny are mixtures of progeny derived from more than 1000 conjugating pairs and maintained in mass culture for roughly 20 fissions after conjugation. Young B homozygotes were obtained by crossing strains SB1969 and SB210. Strains SB3539 and SB3546 were crossed to obtain the young C3 homozygotes, and SB1969 and SB3546 were crossed to make the young B/C3 heterozygotes.

RAPD polymerase chain reactions:
The conditions used for RAPD PCR were as in WILLIAMS et al. 1990 . Reactions (25 µl) were set up in siliconized, 600-µl-capacity microfuge tubes with standard wall thickness. The primers were 10-mers available from Operon Technologies, Inc. Reaction mixtures for RAPD PCR amplification were as described in LYNCH et al. 1995 , except that the reactions for 1PM8 contained 5 mM (instead of 2.5 mM) MgCl₂ to maximize contrast in the RAPD polymorphic band. Primers and polymorphic band sizes (kb) for the RAPDs used in this work are: 1PM8, B17 and B20, 0.5; 1KF2, A2 and C6, 0.6; 1EO3R, A19 and B4, 0.4; 1JO9, A11 and D11, 0.2.

Coassortment tests:
These tests were done with the two panels of B/C3 terminal assortants described above. DNA from each member was subjected to RAPD PCR amplification to identify the RAPD allele fixed in each terminal assortant. Two loci are said to coassort when the alleles from a single parent are found together in statistical excess over the recombinant types among the terminal assortants. LOD > 3 (i.e., log of the odds against independent assortment >3, or odds >1000:1) was used as the threshold of statistical significance. For more detailed descriptions, see LONGCOR et al. 1996 and WICKERT et al. 2000 .

Probing pulsed-field gel blots with cloned RAPD probes:
Polymorphic RAPD bands were cloned as described in LONGCOR et al. 1996 . The authenticity of each insert was confirmed by hybridization to a Southern blot of RAPD products from a panel of Tetrahymena strains in which the RAPD locus was meiotically segregating (see LYNCH et al. 1995 ). To separate ARPs, living cells were embedded in agarose plugs and lysed as in CHAU and ORIAS 1996 . The DNA plugs were inserted in the wells of a 1% agarose gel in 1x TAFE buffer (10 mM Tris-acetate and 0.5 mM EDTA, free acid) and were subjected to electrophoresis using a Beckman (Fullerton, CA) Geneline transverse alternating field electrophoresis (TAFE) apparatus. The pulse program was as follows: Stage 1—30 min, 170 mA, 4 sec in each field direction; stage 2—22 hr, 175 mA, 50 sec in each field direction. Micronuclear DNA stays in the wells under these conditions. The pulsed-field gels were blotted to nylon membranes (Nytran-Plus, Schleicher & Schuell, Keene, NH) by standard Southern transfer procedures. After UV crosslinking, the membrane was probed with cloned RAPD DNA. ³²P-labeled probes were made using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis), purified with a "push column" (Stratagene, San Diego), and quantitated with a scintillation counter. Membranes were prehybridized for 2 hr at 45° in 6x SSC, 5x Denhardt's, 20 mM Tris pH 8.0, 0.125% SDS, 2 mM EDTA, and 27 mg/ml salmon sperm DNA. Boiled probe (1 x 10⁶ cpm/ml) was added and allowed to hybridize overnight at 65°. Membranes were given two 15-min washes in 3x SSC, 0.2% SDS and one 30-min wash in 0.1% SSC, 0.2% SDS, all at 55°, and were then autoradiographed using X-OMAT AR X-ray film (Kodak, Rochester, NY).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

An ARP size polymorphism between inbred strains B and C3 undergoes phenotypic assortment:
Four loci (1JO9, 1EO3R, 1KF2, and 1PM8) that map to MIC chromosome 1L coassort with one another in the MAC and define the PM8 coassortment group (LONGCOR et al. 1996 ; WICKERT et al. 2000 ). The relevant segment of MIC chromosome 1L and the data that establish the MAC coassortment of these four loci are shown in Fig 1. We now report the discovery of an ARP size polymorphism. It was detected after probing a pulsed-field blot containing DNA from young homozygotes of inbred strains B and C3, using labeled DNA from the 1PM8 locus. An autoradiogram is shown in Fig 2A. The probe hybridized to a 1.2-Mb band in the B DNA lane and a 0.9-Mb band in the C3 DNA lane. The B vs. C3 difference defines an ARP size polymorphism between these two inbred strains. Young B/C3 heterozygotes showed a mixture of bands of both sizes, whether tested in mixed population (Fig 2A) or tested as single-cell lines subcloned by 20 fissions of age (not shown).

View larger version (48K): In this window In a new window Download PPT slide   Figure 2. The four loci in coassortment group PM8 are carried in the same MAC ARP. All parts represent autoradiograms of Southern blots of pulsed-field gels of intact whole cell DNAs. (A) A PM8 ARP size polymorphism. DNAs are from C3 homozygotes, B homozygotes, and B/C3 heterozygotes, probed with labeled cloned 1PM8 genomic DNA. All DNAs were prepared from mass cultures less than 20 fissions old (see MATERIALS AND METHODS). (B–E) Cloned DNAs from the four loci of the 1PM8 coassortment group hybridize to ARP homologues of identical size. Lanes 1–6: Blotted DNA from terminal assortant cell lines SB4212–SB4217, respectively. Each cell line was initiated with a different B/C3 heterozygote and subcloned after asexual propagation for roughly 350 fissions. The blot was successively stripped and probed with labeled cloned DNA of the four RAPDs indicated.

If the size polymorphs are derived from homologous MIC genetic segments in inbred strains B and C3 and if their copies are randomly distributed during MAC division, they should assort from one another in the course of asexual cell multiplication. To test this prediction, the DNA of nearly 50 terminal assortant lines of B/C3 heterozygotes (see MATERIALS AND METHODS) was subjected to pulsed-field electrophoresis and Southern blots were probed with cloned 1PM8 DNA. Results are illustrated in Fig 2B. We can summarize the results as follows (Fig 3):

Half of the assortants (23) showed exclusively the 0.9-Mb band characteristic of inbred strain C3. The other half (24) showed one or more bands characteristic of inbred strain B: (a) the 1.2-Mb band previously described (LONGCOR et al. 1996 and Fig 2A of this article); (b) the 1.0-Mb band previously observed in inbred strain B lines maintained as stock cultures in our laboratory (LONGCOR et al. 1996 ); and (c) a novel 1.4-Mb band, seen in one heterozygous cell line (1807 in Fig 3), likely also to be of B origin (see below and DISCUSSION section).

View larger version (35K): In this window In a new window Download PPT slide   Figure 3. Assortment patterns of loci in the PM8 coassortment group and of sizes of PM8 ARP polymorphs in panels of B/C3 heterozygotes. Names of terminal assortant strains are written vertically; the SB prefix has been omitted. Key to RAPD symbols: For 1PM8, 1KF2, and 1JO9, (B) B-allele-determined band present, (C) B-allele-determined band absent; for 1EO3R, (B) C3-allele-determined band absent, (C) C3-allele-determined band present. Key to ARP size symbols: Presence of ARP of corresponding size is shown with a B or a C, depending on the known or suspected B or C3 origin (see DISCUSSION). No ARP size data are available for SB1819. Recomb., simplest events generating terminal assortants of apparent recombinant type. (D) Deletion of B alleles (see DISCUSSION section); (X) normal crossing over; (C) gene conversion. LOD against independent assortment of PM8 ARP homologues (assuming that 1.0 and 1.4 bands are B derived) for the 1JO9/1EO3R RAPD alleles is 12.1.

Since the C3-derived polymorph (0.9 Mb) completely assorted from the group of putatively B-derived polymorphs (1.2, 1.0, and 1.4 Mb), we conclude that the ARP size polymorphism observed here between inbred strains B and C3 is genetically determined.

Identical hybridization patterns were observed when the blot shown in Fig 2B was successively stripped and reprobed with labeled 1KF2, 1EO3R, or 1JO9 DNA (Fig 2, C–E). The identity and specificity of the hybridization pattern, involving three different band sizes in different assortants, is compelling physical evidence that all four RAPD loci are carried on the same MAC ARP.

The PM8 ARP size polymorphism genetically coassorts with the RAPDs of the PM8 coassortment group:
If the ARP is the physical basis of the coassortment group, the assortment pattern of the ARP size polymorphic forms should be correlated with that of the B and C3 alleles of all RAPDs carried on the ARP. The results are summarized in Fig 3 and examples are shown in Fig 4. Among the 24 terminal assortants showing ARP bands putatively derived from inbred strain B, all carried the B allele at the 1JO9 and 1EO3R loci. Conversely, all but one of 23 assortants having the 0.9 Mb ARP form carried the C3 allele at the 1JO9 and 1EO3R loci. (The sole exception, line SB1818, carried the B allele at both loci, presumably as a result of macronuclear crossing over between B and C3 homologous copies of the PM8 ARP.) Thus alleles at these two loci strongly coassort with the B- and C3-derived polymorphs of the PM8 ARP (LOD against independent assortment = 12.1), supporting strongly as well the putative B origin of the 1.0- and 1.4-Mb polymorphs. Highly statistically significant coassortment is also observed with the other two RAPDs in the PM8 coassortment group: 1KF2 (LOD = 5.6) and 1PM8 (LOD = 4.8). The unambiguous coassortment of the ARP polymorphs with the alleles of the four loci provides conclusive molecular genetic evidence that the PM8 ARP is the physical basis of the PM8 coassortment group. Furthermore, the parallel assortment behavior of the ARP size polymorphs and alleles at genetic loci (whose random distribution in Tetrahymena has been well characterized experimentally) provides strong experimental evidence that the ARP copies themselves are randomly distributed at MAC division.

View larger version (61K): In this window In a new window Download PPT slide   Figure 4. Coassortment of PM8 ARP size polymorphs with the RAPDs of the 1PM8 coassortment group. (A) Autoradiogram of a Southern blot of a pulsed-field gel of intact whole-cell DNA from stock cultures of inbred strain B and C3 (controls) and from terminal assortants SB1810–17, probed with labeled cloned 1PM8 genomic DNA. (B) Ethidium bromide-stained agarose gels of PCR products obtained from the same strains using primers specific for each of the RAPDs (polymorphic bands indicated by arrows). Symbols below each RAPD gel: B and C represent B and C3 alleles; represents the putative deletion of the B allele, rather than its replacement by the C3 allele (see text). Assortants showing faint bands at the location of the 1EO3R and 1JO9 polymorphic bands were scored as band negative. The faint band is attributed to PCR amplification from the band-positive allele maintained by mitotic division in the MIC DNA, which constitutes a few percent of the whole-cell-DNA preparation.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Two rigorous lines of evidence lead us to conclude that the PM8 ARP is the physical basis of the PM8 coassortment group, as hypothesized by LONGCOR et al. 1996 :

1. Four distinct RAPD loci that coassort with one another were found to reside on macronuclear ARPs of identical size. Moreover, this correspondence was observed among terminal assortants showing bands of four sizes due to the existence and assortment of heritably distinct ARP size polymorphs.

2. ARP size polymorphs coassort with the B and C3 alleles of each RAPD whose DNA hybridizes to the ARP. Thus we conclude that the coassortment of two loci reflects their MAC synteny.

This relationship between coassortment groups and ARPs appears to be a general feature of MAC genetics: we have made analogous physical observations with two other groups of coassorting loci (E. P. HAMILTON, L. WONG and E. ORIAS, unpublished observations; S. L. ALLEN, L. WONG, E. ORIAS and E. P. HAMILTON, unpublished observations). The converse proposition, i.e., that loci in the same ARP should coassort, need not always be true: in theory, MAC recombination (DEAK and DOERDER 1998 ) or deletion (of the type observed in the PM8 ARP; see below and WICKERT et al. 2000 ) could occasionally have local intensity high enough to break up coassortment groups. Regardless, we conclude that coassortment provides a general genetic strategy for positively identifying loci that are incorporated into the same MAC DNA piece during MAC differentiation and thus for mapping the MAC by purely genetic means.

The appearance of multiple forms of the inbred strain B ARP homologue was unexpected. Two lines of evidence strongly suggest the B origin of the 1.0- and 1.4-Mb bands observed in our 500-fission-old heterozygotes: (1) We have observed the 1.0 Mb band in old clones of inbred strain B (LONGCOR et al. 1996 ) and (2) the 1.0- and 1.4-Mb bands correlate perfectly with the presence of B alleles at the two loci presumed (from the MIC map, Fig 1) to lie in the same half of the PM8 ARP. To explain these and other unpublished observations we propose the following simple model for their appearance. The 1.0-Mb band arises as a consequence of a post-MAC-differentiation deletion of a roughly 200-kb tandem repeat in the B-specified 1.2-Mb ARP. Loss of the repeat is generally accompanied by the loss of the polymorphic copies of both the 1PM8 and 1KF2 RAPD loci. The 1.0- and 1.4-Mb polymorphs could both result from unequal crossing over between two misaligned copies of the 1.2-Mb B ARP homologue. The 1.0-Mb polymorph could equally well (and perhaps more likely) arise from an intramolecular crossover that excises one copy of the tandem repeat. The preceding model accounts for the following observations reported here:

• The late generation of both 1.0- and 1.4-Mb bands, neither of which are detectable in young B homozygotes and young B/C3 heterozygotes.

• The assortment of the 1.0-Mb band from the 1.2-Mb band, as expected from genetically distinct polymorphs.

• The incomplete assortment of the 1.0- or 1.4-Mb bands from the 1.2-Mb band in four heterozygous lines (Fig 3) is consistent with their late (post-MAC-differentiation) generation. This observation stands in contrast with the complete assortment of B vs. C3 polymorphs, which are generated during MAC differentiation, observed in all the heterozygotes examined.

• The absence of growth selection against assortants pure for the 1.0-Mb polymorph that might have been expected from the loss of 200 kb of DNA.

Additional work will be needed for a detailed understanding of these deletion events, but the phenomenon has already been useful in providing more discriminating evidence for the correspondence between the genetic coassortment group and a physical ARP.

The overall colinearity of the MIC and MAC maps suggests an uncomplicated model of ARP formation, i.e., exclusively by fragmentation (WICKERT et al. 2000 ). This colinearity, taken together with the ability to identify an ARP restriction pattern (LONGCOR et al. 1996 ), suggests an approach to making a physical map of the genome: identifying, for every ARP, which ARPs and in which orientation are derived from the adjacent segments in the MIC. Furthermore, the correspondence between coassortment groups and ARPs described here provides an important way in which to relate the physical map to the genetic map. Efforts to systematically clone at least one RAPD of every coassortment group, to identify the size and restriction pattern of the MAC chromosome piece that corresponds to the coassortment group, and to sequence the RAPD clone are now underway. This information will ultimately be useful in relating the genetic map, the physical map, and the genome sequence.

Mapping a fragmented somatic genome by purely genetic means is so far possible only in Tetrahymena. A maximum-likelihood map detailing the correspondence between the map location of loci on the left arm of MIC chromosome 1L and the coassortment groups formed by these loci in the MAC is shown in WICKERT et al. 2000 . Analogous coassortment mapping of the other chromosomes is underway. The phenomenon of coassortment generates tools useful for molecular genetics. For example, the ability to genetically map a mutant locus to a coassortment group tagged with a DNA polymorphism and to relate the group to a physical ARP confers the potential to narrow down the location of the locus to an identifiable DNA segment that comprises, on the average, roughly 0.3% of the genome. This should become useful for cloning mutant genes of interest, and more so in the future, as more coassortment groups become identified, more Tetrahymena ARPs become tagged with a DNA polymorphism, and a more complete genome physical map and sequence become available.

	FOOTNOTES

¹ Present address: Operon Technologies, Inc., 1000 Atlantic Ave., Suite 108, Alameda, CA 94501.
² Present address: AMGEN Inc., 1 Amgen Center Dr., Thousand Oaks, CA 91320-1718.
³ Present address: Protein Pathways Inc., 1145 Gayley Ave., Suite 304, Los Angeles, CA 90024.
⁴ Present address: Biology Department, Loyola Marymount University, Los Angeles, CA 90045.

	ACKNOWLEDGMENTS

We thank Louise Clarke, Ruth Finkelstein, Tim Lynch, John Merriam, Daniel E. Morse, and Joel Rothman, at UCSB, for thoughtful comments on an earlier draft of the manuscript. This work was supported by a grant (RR09231) from the National Institutes of Health National Research Resource Center to E.O. and Loyola Marymount University sabbatical support to V.M.

Manuscript received February 8, 2000; Accepted for publication March 31, 2000.

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