Coassortment of genetic loci during macronuclear division in Tetrahymena thermophila

Europ . J. Protisto!' 32, Supp!. l: 85-89 (1996) October 31, 1996

European Journal of

PROTISTOLOGY

Coassortment of Genetic Loci During Macronuclear Division in Tetrahymena thermophila Megan A. Longcor, Steven A. Wickert, Miu-Fun Chau, and Eduardo Orias Department of Molecular, Cellular and Developmental Biology University of California at Santa Barbara, Santa Barbara, CA 93106 USA

SUMMARY Our aim was to detect coassortment, a macronuclear form of genetic linkage, in Tetrahymena thermophila. A set of 36 multiplied heterozygous caryonides, derived from a cross between inbred strains Band C3, were allowed to multiply vegetatively for over 500 fissions to ensure the near completion of phenotypic assortment. Each assortant was classified as having the B- or C3- derived allele at the mating type and cycloheximide resistance loci and at polymorphic DNA (RAPD) loci. A pair of loci was considered to coassort if the set of assortants showed a statistically significant excess of parental over recombinant allele combinations. Two coassortment groups were identified. Two loci in one of these groups, lKF2 and lPM8, were shown to be on the same macronuclear autonomously replicating piece (ARP), i. e., syntenic, by hybridization. lKN3, separated from lPM8 by 8.2 cM in the micronucleus, failed to coassort with lKF2 or lPM8 and was shown to be on a different ARP. Our results are consistent with the hypothesis that macronuclear ARPs are the molecular basis of coassortment groups. The low frequency of somatic macro nuclear recomb inants observed in these coassortment groups may allow the efficient and comprehensive mapping of loci in the Tetrahymena genome to macronuclear ARPs by purely genetic means .

Introduction As is typical of ciliates, Tetrahymena thermophila cells maintain a germinal micronucleus (MIC) and a somatic macronucleus (MAC), a unique phenomenon known as nuclear dimorphism. The polyploid MAC is differentiated from a mitotic sister of the diploid MIC after genetic material is exchanged between two cells temporarily united in conjugation [reviewed in 5 and 17]. The differentiated MAC contains about 300 different chromosome species, terminated by telomeric sequences, that were generated by the site-specific fragmentation and rearrangement of the five chromosomes in the MIC [22,23]. These autonomously-replicating DNA pieces (ARPs) are amplified to make the MAC approximately 45-ploid. The MAC divides "amitotically": there are no kinetochores [8] or other detectable mechanisms for regular segregation of chro© 1996 by Gustav Fischer Verlag

mosomes. During cell division, allele copies at a locus are distributed at random to the two daughter MACs [20]. The resulting genetic drift in allelic ratios after successive fissions causes the heterozygous MAC to eventually become homogeneous for one of the two alternative alleles at a given locus. This general phenomenon of the Tetrahymena MAC, now known as phenotypic assortment [reviewed in 9 and 16], was discovered and analyzed by Allen and Nanney [1] and Schensted [25] in the context of loci mating type differentiation. Coassortment of during MAC division has not been previously reported [5], presumably because of the paucity of mapped genetic markers. The meiotic mapping of many DNA polymorphisms [4, 13] obtained by the Random Amplified Polymorphic DNA (RAPD) method [21] provided favorable conditions for a new search for coassortment and its molecular basis. We report here the discovery 0932-4739-96-0032-0085$3 .50-0

86 . M. A. Longcor, 5. A. Wickert, M.-F. Chau, and E. Orias of two MAC coassortment groups derived from the right and left arms of M IC chromosome 1 and 2, respectively. One coassortment group was shown by hy brid izatio n to correspond to a pair of relate d MAC ARPs. The frequency of MAC recombination within each of t he tw o coassortment groups is very low.

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M aterial an d M ethods Routine methods used in our lab for Tetrahymena storage, culture and genetics have been described [11, 18, 19]. A panel of terminal assortants was isolated as follows. F1 s heterozygo us for RAPD markers were constructed by crossing strains CU369 (inbred strain B derivative carry ing a mutation at the ChxA locus, which confers resistance to cycloheximide [ 26]) and C3-3585 (wild type inbred strain C3), as described in Bleyman et al. [3]. From the above cross, 400 pairs were isolated. Four subclones were initiated from each cycloheximide-resistant pair culture (synclone), transferred by replication for 12 transfers and tested for mating type. Two subclones, expressing mating types I and IV (or I and VII) were kept for each of 18 synclones. (Mating type I is diagnostic for the mat-3 allele present in strain C3, while mating types IV and VII are diagnostic for the mat -2 allele of strain B [15]. These lines were named 5B1805-5B1840 and were frozen under liquid nitrogen . These cultures were thawed out and trans ferred by replication in 96-well plates for a total of 74 additional transfers (roughly an additional 500 fissions). A single cell was then isolated from each of these lines to generate a panel of 36 "terminal assortants." More than 99% of these cultures are expected to be pure for either allele at any given locus [10]. We tested these lines for allele purity using IBR2, a codominant RAPD polymorphism on MIC chromosome 5 (primer : Cl Z, 1.0 Kb band, mapped by Brandie Rocci in our lab); no mixed clones were detected (data not shown) . Tests for MAC coassortment were done as follows. The terminally assorted allele at each locus (B or C3) was first determined for each of the 36 assortants described above . RAPD methods, PCR reagents and loci used here have been previously described [13]. All primers used were from standard Operon Technologies Inc. RAPD primer kits. For the mat locus in the MAC, we used the mating types I and IVI VII as diagnostic of mating type determination by C3 and B alleles, respectively. To test for coassortment, the frequency of recombinant types within the panel of assortants was determ ined for every pairwise combination of loci. On ly statistically significant coassortm ents (LOD score > 3) were accepted. The MAPMAKER program 112] was used as an aid in searching for coassortment. Cloning and verifying RAPD polymorphic DNA was done as described in Lynch et al. [13], except that lKF2 DNA was cloned in the pZEROTM-l vector (Invitrogen Corp .) after treatment with Klenow fragment (Promega). Methods related to pulsed field gel electrophoresis were as described in Chau and Orias [7]. Hybridization methods and conditions were as described in Lynch et al. [13]. DNA embedded in agarose was digested with restriction enzymes as follows. Plugs stored in 0.5 M EDTA were cut to well size, soakad twice in excess TE (10 mM Tris, 1 mM EDTA, pH 7.6 ) for 30 min at room temperature and incubated in the appropriate restriction enzyme buffer for 30 min at 4 °C. The restriction buffer was replaced, 20-50 units of restriction enzyme were added, the plugs were incubated at least overnight at

Fig. 1. Segments of the curren t maximum likelihood maps of the loci investigated here. 2L RAPD loci are described in Lynch et al. [13]. lR loci (except for IMLJ) are described in Brickner et al. [4] and Allen et al. [2]. 1MLJ (primers: A2, A6, 0.4 Kb band) was identified and mapped by M. L. and E. O. in our lab. The scale relating centiMorgans (cM) to Kilobase pairs (Kb) is based on a preliminary estimate for the mat neighbor hood in chromosome 2L [13].

appropriate temperatures and the reaction was termina ted by soaking the plugs in TE for 1 hat 4 °C.

Res ults Closely linked loci in the ma t linkage group in MIC chromosome 2L (Fig. 1) were first chosen to look for MAC coassort ment. T he presence or absence of a band, corresponding to either the B or C3 allele, was scored for all RAPD loci in eac h assortant. A typical assortment test is illustrated in Fig. 2 . All the results are summarized in Fig . 3. Fig.3a shows that R APD s lKF2 and IPM8 coassort in the MAC. T hey show

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Fig. 2. Phenotypic assortmen t of two RAPDs, lKF2 on 2L (0.6 Kb) and IML4R on 4L (0.9 Kb). Primers: A2 and C6. Template DNA: Lanes Band C3, parental strai n DNAs; other lanes, members of the terminal assorta nt panel. B/C3 heterozygotes tested soon after conjugation show the band for each RAPD (not shown) . The two loci assort independently; assortants 5B1805, 08, 11 and 14 are recombinants. A very weak 1KF2 band, variably observed in some assortants, is interpreted to be templated by the B-derived copy in the MIC.

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Fig. 3. MAC assortment pattern of RAPD loci on MIC chromosomes 1Rand 21. Genotypes at each RAPD locus for 36 assortants (SB1805-SB1840) are listed horizontally in groups of four. Key to genotypes. 1 and 0: B- and C3-derived allele, respectively; blank space: undetermined. Dot: allele identity to the RAPD listed above it; used only among members of the same coassortment group. Only one assortant shows a recombination event within either coassortment group (SB1826, cag-l ML3 group) . Strictly speaking, our results test assortment not for the mat locus but for the MAC mating type determination (mtd [24]) locus. It is likely (but not proven) that mtd is the differentiated MAC form of the MIC mat locus.

an identical pattern of allele assortment, i. e., all assortants are of parental type. These results define cagIPM8 (lPM8 MAC coassortment group) (Coassortment is the MAC analog of MIC meiotic linkage; to avoid confusion, we suggest that when used without qualification, the term linkage should refer exclusively to MIC meiotic linkage). lKN3 and mat do not belong to this coassortment group, as neither coassorted significantly with lKF2 or 1 PM8 (Fig.3a). RAPD uic» maps between lKF2 and IPM8 in 2L , yet it assorted the B allele predominantly (Fig. 3a). This is explained by the finding that lEal is in MIC-specific DNA, i. e., DNA internally deleted during MAC differentiation, based on the hybridization of cloned lEal probe to Southern blots of purified MIC and MAC DNA (data not shown) and confirmed by RAPD mapping with whole cell DNA from nullisomic strains [Merriam and Orias, unpublished observations] . Thus, the lEal dominant band is being phys ically ternplated by the relatively small amount of MIC DNA present in the whole-cell DNA preparations.

Loci on the 1PM8 coassortment group are located on the same MA CARP We next tested the hypothesis that loci that coassort are on the same MAC ARP. Fig.4a shows T. thermophila whole cell DNA digested to completion with various rare cutting restriction enzymes and fractionated on a TAFE (pulsed field) agarose gel. Figs. 4b-d show a Southern blot of the gel in Fig. 4a probed separately with 32p labelled lKN3, IPM8, and lKF2 cloned DNA. The results show that IPM8 and lKF2 are on the same MAC ARPs, i. e., they are syntenic, since

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Fig. 4. Evidence that lKF2 and IPM8 are located on the same MAC ARP. Lane Y: Yeast (Saccharomyces Corevisiae) chromosomes. Left scale: sizes of selected yeast chromosomes in Megabase pairs (Mb). Lanes 1 and 2: untreated DNA. Right scale: estimated size of the sources of radioactive signal in the auto radiographs. Lanes 3-5: DNA digested with NotI, SmaI and SadI, respectively. (a) Relevant segment of a photograph of an ethidium bromide stained TAFE gel of whole cell DNA of strain SBI969 (derived from inbred strain B). Pulsed field conditions: Stage 1: A=B=4s, 170 rnA, 30 min; stage 2: A=B =70s, 150 rnA, 24 h (where A and B are the transverse alternating pulses). (b-d) Southern blot of gel in (a), separately hybridized with cloned PCR band from lKF2, IPM8 and lKN3 , respectively. The blot was stripped and checked for loss of signal between successive hybridizations. The hybridization signals in panels band c are superimposable with one another in the original autoradiographs. The 1KF2 and IPM8 probes do not cross-hybri dize with one another (data not shown). The smearing at the bottom of the gel (the tip is seen at the bottom of the auto radiograph) is probably due to some partial degradation .

both probes give identical hybridization patterns. Both probes label two ARPs of different size (approximately 1.2 and 1.0 Mb) with roughly the same relative difference in intensity. Comparison of Figs. 4c and 4d shows that 1PM8 and lKN3, the closest markers flanking the mat locus, are on separate MAC ARPs: the 1 PM8 ARP was not cut by SmaI or SadI in the same way as the lKN3 ARP. ThereMIC eM

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Fig. 5. Model relating MAC genetic coassortment to MAC ARPs and to the genetic map of MIC chromosome 21. Solid thick line: segment of the mat linkage group in the MIC (see Fig. 1). The white rectangle (of arbitrary length) represents DNA, including the lEOl RAPD, deleted from MAC DNA during MAC differentiation. Shaded thick lines: MAC ARPs carrying the MIC RAPD loci shown . Two parallel thick lines (heavy and light shading) represent the two ARPs that hybridize with lKF2 and IPM8 DNA. They are arbitrarily shown as if derived from the same MIC segment . Dotted horizontal lines: unknown ARP boundaries. Dotted vertical lines: putative correspondence between MIC and MAC location of loci. Distances between MIC loci have been arbitrarily decreased by 15% in the MAC ARPs to reflect the loss of MIC DNA during MAC differentiation. The mat ARP (question mark) has not yet been molecularly identified.

88 . M. A. Longcor, S. A. Wickert, M.-F. Chau, and E. Orias

fore, at least one chromosome breakage site [23] must occur in the MIC somewhere between these two loci. The same conclusion applies to 1KN3 and 1KF2.

MAC coassortment over a 30cM segment of MfC chromosome 1R We have also identified five loci in MIC chromosome 1R that coassort with one another: 1KN2, 1ML3, 1J014, 1JB22 and 1JB8 (Fig.3b). These five loci define the 1ML3 coassortment group (cag-1ML3). This group does not include ChxA and 1J015 (Fig. 1 and 3b). Additional work is required to determine whether the MIC segment carrying 1JP12a and 1J018 becomes part of cag-1ML3. These markers assort to the B-derived band phenotype exclusively (Fig.3b) and could be physically absent from the MAC, i. e., be MIC-limited, like 1EO1 in 2L. As in cag-1PM8, the frequency of coassortment in cag-1 ML3 is very high: only one out of the 36 terminal assortants tested (5B1826) shows a recombination event in the entire group of five loci (coassortment frequencies = 97 -100%; Fig. 3b). The cag-1ML3 extends over a DNA segment corresponding to more than 30 meiotic cM (perhaps 600 Kb [13] and Fig. 1) in the MIC.

Discussion The availability of genetically characterized and mapped RAPD DNA polymorphisms in T. thermophifa allowed us to provide here the first complete demonstration that allelic DNA loss is the molecular basis for phenotypic assortment at a genetic locus. It also enabled us to detect coassortment in the MAC and to delineate coassortment groups. Furthermore, we have related a MAC coassortment group to a physical linkage on a MAC ARP: DNA probes from coassorting loci 1PM8 and 1KF2 both hybridize to MAC ARPs of identical size and response to three restriction enzymes used (Fig.4a). In contrast, the probe from 1KN3, a locus which failed to coassort with 1KF2 and 1PM8, hybridizes with a MAC ARP of different size and restriction pattern. All these findings are consistent with the idea that MAC ARPs are the molecular basis of genetic coassortment groups in Tetrahymena. The model in Fig. 5 illustrates this correspondence. Interestingly, the 1PM8 and 1KF2 cloned DNA probes each hybridized to two MAC ARPs, one at 1.2 Mb and the other at 1.0 Mb. Possible explanations for this observation are alternative deletion [22] or alternative fragmentation of MIC 2L DNA [see ref. 6 for an example in Paramecium]; both types of events would have occurred during MAC differentiation. Another possibility is the presence of a duplicated DNA segment in the MIC. A possible inequality of copy number of these two ARPs is intriguing because of the questions it raises regarding ARP copy number control and gene balance.

Recombinants were obtained in the two MAC coas sortment groups with low frequency. Only a single recombinant was observed among 36 segregants over a combined segment representing almost 40 cM of MIC DNA, which may represent about 800 Kb [13]. This suggests that, for these coassortment groups, the frequency of somatic recombination in the MAC (on a per copy per fission basis) may be at least 2 orders of magnitude lower than in MIC meiosis. We do not know the basis for this difference. If this low frequency of MAC recombination is typical, then previous failures to observe MAC coassortment in Tetrahymena [5] must be attributed to having tested loci on different MAC ARPs. The frequency of somatic recombinants described here does not appear to be strikingly different from that in the rDNA ARP [27] after correcting for appropriate differences (copy number, distance and replication advantage). The low frequency of MAC recombination should be very useful for making a map of MAC ARPs in the Tetrahymena genome by the simple genetic assignment of loci to coassortment groups. Furthermore, finding coassortment of a mutant gene with a RAPD already placed on an ARP of known size could facilitate cloning of the gene by complementation. Fewer Tetrahymena transformants would need to be screened if a minilibrary made from a limited region of a pulse field gel is used instead of a whole genome library. For a phenotypically identified locus that fails to coassort with any mapped RAPD, the MAC ARP size can be determined by finding a coassorting RAPD by "bulk segregant analysis" [13, 14], i. e., by screening pooled DNA from several phenotypic assortants expressing the alternative alleles of the locus in question [with new RAPD primer combinations].

Acknowledgements We dedicate this article to Professor Koichi Hiwatashi - a distinguished ciliate biologist and mentor, a warm and generous human being and a dear friend. We thank Judy Orias and Mary Baum for excellent technical help; Xueyu Shen and Dr. Eileen Hamilton for helpful advice; Dr. Virginia Merriam for sharing unpublished observations; and Drs. Sally L. Allen, Lea K. Bleyman and Virginia Merriam for valuable comments on the manuscript. This research was partially supported by a UCSB Howard Hughes Medical Institute Undergraduate Research Fellowship to M. L. and by grants from NIH (RR 09231) and American Cancer Society (MV552) to E. O.

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15 Nanney D. L., Caughey P. A., and Tefankjian A. (1955): The genetic control of mating type potentialities in Tetrahymena pyriformis. Genetics, 40, 668-680. 16 Nanney D. L. and Preparata R. M. (1979): Genetic evidence concerning the structure of the Tetrahymena thermophila macronucleus. J. Protozool., 26, 2 -9. 17 Orias E. (1986): Ciliate conjugation. In: Gall j. G. (ed.): Molecular biology of the ciliated Protozoa, pp. 45 - 84. Academic Press, New York. 18 Orias E. and Baum M. P. (1984): Mating type differentiation in Tetrahymena thermophila: strong influence of delayed refeeding of conjugating pairs. Devel. Genet., 4, 145-158. 19 Orias E. and Bruns P. j. (1975): Induction and isolation of mutants in Tetrahymena . In: Prescott D. M. (ed.): Methods in cell biology, vol. 13, 247- 282. Academic Press, New York. 20 Orias E. and Flacks M. (1975): Macronuclear genetics of Tetrahymena. I. Random distribution of macronuclear gene copies. Genetics, 79, 187-206. 21 Williams j. G. K., Kubelik A. R., Livak K. j ., Rafalski j. A., and Tingey S. V. (1990) : DNA polymorphisms amplified by arbitrary primers are useful genetic markers. Nucl. Acids Res., 18, 6531-6535. 22 Yao M.-C. (1989): Site-specific chromosome breakage and DNA deletion in ciliates. In: Berg D. E. and Howe M. M. (eds): Mobile DNA, pp. 715 -734. American Society for Microbiologists, Washington DC. 23 Yao M.-C., Yao C. H., and Monks B. (1990): The controlling sequence for site-specific chromosome breakage in Tetrahymena. Cell, 63, 763-772. 24 Orias E. (1981): Probable somatic DNA rearrangements in mating type determination in Tetrahymena tbermophila: a review and a model. Devel. Genet., 2, 185 - 202. 25 Schensted I. V. (1958): Appendix: Model of subnuclear segregation in the macronucleus of ciliates. Amer. Naturalist, 92, 161-170. 26 Bruns P. j. and Cassidy-Hanley D. (1993): Tetrahymena thermophila. In: O'Brien S. j. (ed.): Genetic maps: locus maps of complex genomes, 6th ed., pp. 2175 -2179. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. 27 Lovlie A., Haller B. L., and Orias E. (1988): Molecular evidence for somatic recombination in the ribosomal DNA of Tetrahymena thermophila. Proc. Natl. Acad. Sci. U. S. A., 85, 5156-5160.

Key words: DNA amplification - DNA rearrangement - Genetic map - Nuclear differentiation - Chromosome fragmentation Eduardo Orias, Department of Molecular, Cellular and Developmental Biolog University of California at Santa Barbara, Santa Barbara, CA 93106, USA