~]~I~VIEWS Cell 50, 779-788 23 Wasserman, S.A., Dungan, J.M. and Cozzarelli, N.R. (1985) Science 229, 171-174 24 Stark, W.M., Sherratt, DO. and Boocock, M.R. (1989) Cell

58, 779-790 25 Sanderson, M.R. et al. (1990) Cell63, 1323-1329 2 6 Pollock, T.J. and Nash, H.A. (1983) J. Mol. BioL 170, 1-18 2 7 Stark, W.M., Grindley, N.D.F., Hatfull, G.F. and Boocock, M.R. (1991) EMBOJ. 10, 3541-3548 28 Kanaar, R. et al. (1990) Cell 62, 353-366 29 Cowart, M., Benkovic, S.J. and Nash, H.A. (1991)J. Mol. Biol. 220, 621--629

Hypotrichs contain both a germ-line nucleus (micronucleus) and a somatic nucleus (macronucleus) (Fig. 1). Following cell mating a copy of the micronucleus gives rise to a new macronucleus, and the old macronucleus is destroyed. The micronuclear genome has evolved remarkable features in its DNA organization that are altered during the development of a micronucleus into a macronucleus. For example, all genes in the micronuclear genome are interrupted by 'nonfunctional' sequences that are spliced out of the DNA to make functional macronuclear genes during development1, 2. Functional segments within some micronuclear genes are scrambled, and these segments are spliced into a different order during development to yield functional macronuclear gene#, 4. Mso, massive elimination of micronuclear DNA sequences yields a macronucleus with a much lower sequence complexity 5. Finally, during processing every macronuclear gene comes to reside in a physically separate, small DNA molecule. Each different DNA molecule is amplified to about 1000 copies in the DNA-rich macronucleus. The multiple events in the reconstruction of micronuclear genes into macronuclear genes after cell mating in the hypotrichs are the subject of this review. Space limitations preclude inclusion of related DNA processing in Tetrahymena. The reader is referred to excellent papers by Yao and colleagues6, 7.

The micronucleus and its DNA The DNA in micronuclear chromosomes has the very high molecular weight (more than 1000 kbp) typical of eukaryotic chromosomes. Genes occur in groups along the DNA molecule8, 9, with short sequences between genes within a group, and much longer repetitive and unique sequences between groups of genes. The genes in micronuclear DNA are silent in vegetatively growing cells; for example, no RNA synthesis is detectable by autoradiography. The micronucleus undergoes meiosis when cells mate. An outline of the nuclear events of mating is shown in Fig. 2. Cells in a mated pair exchange haploid micronuclei. An exchanged micronucleus fuses with a stationary micronucleus to make a new diploid micronucleus in each cell. The cells separate, and the new micronucleus divides by mitosis without cell division. One daughter micronucleus remains as the germ-line nucleus, and the other develops into a new

30 Nash, H.A. and Pollock, T.J. (1983) J. Mol. Biol. 170, 19-38 31 Duckett, D.R. et al. (1988) Cell 55, 79-89 32 Dr6ge, P., Hatfull, G.F., Grindley, N.D.F. and Cozzarelli, N.R. (1990) Proc. Natl Acad. Sci. USA 87, 5336-5340 33 Chen, J-W., Lee, J. and Jayaram, M. (1992) Cell 69, 647-658 34 Kim, S.H., Moitoso de Vargas, L., Nunes-Diiby, S.E. and Landy, A. (1990) Cell 63, 773-781 W.M. STARK, M.R. BOOCOCK AND D.J. SHERRAIT ARE IN THE DEPARTMENTOF GENETIC~ UNIVERSITYOF GLASGOW, CHURCH STREET,, GLASGOW, UK GI I 5JS.

The unusual organization and processing of gen0mic DNA in hyp0trich0us ciliates DAVID M. PRESCO'IT Hypotrichs are a large group of ciliate species that cut, splice, reorder and eliminate DNA sequences to an extraordinary extent during their sexual life cycle. Such DNA processing occurs when a ciliate converts a copy of its germ-line nucleus into a somatic nucleus after cell mating. somatic macronucleus over several days. While the new macronucleus is forming, the old macronuclei are degraded, along with leftover diploid and haploid micronuclei. Little is known about this precisely selective destruction of the superfluous nuclei.

The macronucleus and its DNA The organization of DNA is dramatically changed during development of a micronucleus into a macronucleus. In the macronucleus all the DNA occurs as short molecules ranging in size from a few hundred base pairs to about 15 000 bp with an average size of around 2200 bp (Ref. 10), varying slightly from one hypotrich species to another. Each DNA molecule contains a single transcription unit, or gene (Fig. 3). The coding region of the gene is preceded by a leader region typically of about 50 to a few hundred base pairs and is followed by a few hundred base pairs of trailer sequence. The leader and trailer contain sequences that function in the initiation and termination of transcription, but little is known about promoter function or regulation of transcription of any hypotrich gene. The ends of every gene-sized molecule are capped with an identical telomeric sequence made of repeats of 5'dC4da43' (Ref. 11). The complementary chain, composed of 3'dG4T45', extends to form a 3' single-stranded overhang of 16 bases in Oxytricha species. The telomeric sequences are important for replicating the ends of the gene-sized molecules la.

TIO DECEMBER1992 VOL.8 NO. 12 ©1992 Elsevier Science Publishers Ltd (UK)

~-'~EVIEWS Making macronuclear DNA from micronuclear DNA

iiii~i{iiiiiiiiiiiiliiiii! ~iii~i!~i!!!iil~i~ii!~;~ii~ii~

FIGH An Oxytricha stained by the Feulgen technique to show the two compact, transcriptionally inactive micronuclei and the two DNA-rich, transcriptionally active macronuclei. Bar represents 20 ~m.

Mi

Internal eliminated sequences that interrupt micronuclear genes

°

Ma (a)

(d)

Immediately after cell mating, macronuclear development begins with multiple rounds of replication of chromosomes in one micronucleus to form polytene chromosomes 13. Gene segments in the polytene chromosomes are cut and spliced, the chromosomes are then destroyed by transection through all the inter-bands, genes are excised from the chromosomes, and superfluous DNA sequences are selectively destroyed by an unknown mechanism. Telomeric sequences are added to the excised genes TM, and the genes are amplified many fold by several cycles of DNA replication to yield a mature macronucleus. Vegetative cell reproduction then resumes. The gene-sized molecules are created by excision of genes from the micronuclear chromosomes. In the process, all repetitious sequences and all nongenic unique sequences, accounting for about 95% of the germ-line DNA complexity in Oxytricha, are eliminated 5. Thus, the gene-sized macronuclear DNA molecules represent only about 5% of the germ-line DNA sequences. These sequences encode all the nuclear RNA needed for vegetative growth. The macronucleus contains roughly 24 000 different molecules, each with an average of about 1000 copies, although some, such as the molecules encoding rRNA, are differentially amplified further to about 100 000 copies 2. Altogether, a macronucleus in O. nova contains about 2.5 x 107 gene-sized molecules.

(c)

(b)

(e)

(f)

FIG[] Simplified diagram of nuclear events during mating in Oxytricba. The two micronuclei (Mi) are equivalent, and the two macronuclei (Ma) are equivalent. Only one micronucleus is shown undergoing meiosis. Open small circles: diploid micronuclei. Black or hatched small circles: haploid micronuclei. Part-black, part-hatched circles: new diploid micronuclei, or developing macronucleus. (a) A vegetative cell. (b) Mating cells, with meiotic products of one micronucleus in each cell. (c) A new diploid micronucleus in a cell that has mated. (d) Mitosis of the new micronucleus and degradation of all other nuclei. (e) Development of 6ne of the new micronuclei into a macronucleus. (f) Division of the micronucleus and the newly developed macronucleus to produce a mature, vegetative cell. DNA replication bands are shown in the macronuclei. Reproduced, with permission, from Ref. 26.

Since m a c r o n u d e a r genes are derived from micronuclear genes during development, macronuclear genes should have counterparts that are matched exactly to the base pair in micronuclear DNA. Thus, the 746 bp sequence of the macronuclear C2 gene of 0. nova occurs perfectly in a cloned micronuclear copy 15. However, in addition to the perfect correspondence, the micronuclear copy of the C2 gene contains an additional 130 bp distributed in three blocks within the gene (Fig. 4). A block of 49 bp interrupts the coding region (open reading frame, ORF), a second block of 49 bp of different sequence interrupts the gene b e y o n d the 3' end of the ORF within the transcribed part of the trailer, and a third block of 32 bp of different sequence interrupts the nontranscribed trailer of the C2 gene. During g e n o m e processing in development these three sequence blocks - called internal eliminated sequences, or IESs - are excised to make a functional macronuclear gene. They separate four segments of the C2 gene that are retained and spliced to make the macronuclear gene copy; these retained segments are designated as macronuclear destined sequences, or MDSs. All the 20 genes characterized so far in Oxytricha nova and Euplotes crassus contain IESs, usually several to many. Given their frequency per unit length of DNA, the micronuclear g e n o m e must contain over 50 000 IESs 16, all of which are removed and destroyed during macronuclear development. IESs are single-copy AT-rich sequences, ranging in size from 14 to 548 bp. They seem to be more or less randomly scattered in genes, with no apparent pattern in relation to each other or to the MDSs that they separate.

TIG DECEMBER1992 VOL.8 NO. 12

i4C

I-flWIF.WS LEADER

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11

FIGE Generalized diagram of macronudear DNA molecules of Oxytricha. Each molecule has a single transcription unit (gene coding region) flanked by a leader, a trailer, and telomeric inverted repeats. The origin of IESs, why they arose or how they inserted into micronuclear genes during evolution are not known. Since they occur in very distantly related hypotrichs (Euplotes and Oxytricha), they presumably represent an evolutionarily old phenomenon. It is possible that lESs are dynamic elements that undergo insertion into and deletion from germ-line genes continuously, even in contemporary hypotrichs.

Removal of lESs from genes

TEC1 elements are excised as circles, apparently by a mechanism similar to that for IES removal. One copy of the 5'TAY repeat is retained in an adjacent MDS, and the excised TEC1 element contains two copies of the 5'TAY repeat separated by 10 bp, suggesting that excision is by a staggered cut, fill-in of the singlestranded tails, and ligation into a circle. Oxytricha fallax contains about 1900 copies of a transposonlike element about 4 kbp long called TBE1 (ReL 21). The TBEls are precisely excised and eliminated during macronuclear development 22. Oxytricha fallax does not mate successfully in laboratory cultures, and therefore the timing of TBE1 removal and destruction is not known. In summary, during evolution the micronuclear genome has been subjected to extensive intrusion by a variety of lESs, transposonlike elements and repetitive sequences with no known functions, all of which are precisely and completely cleared from the DNA to make the highly compact genome of the macronucleus.

Because many of the lESs identified so far interrupt amino acid coding regions of genes, exact excision to the base pair is essential. All IESs have a 2-19 bp direct repeat at their ends that is presumed to participate in the precise guidance of cutting and splicing 1. Excision of an IES leaves one copy of the direct repeat in the macronuclear DNA molecule. Since for most lESs the retained copy of the direct repeat is part of a gene coding sequence, the repeats were presumably present before insertion of the lESs in evolution. The second copy of the direct repeat might have been creScrambling of MDSs in micronuclear genes ated by a duplication event during insertion, or, less likely, it might have been part of the IES before its Gene expression in the micronucleus of hypotrichs arrival at the insertion site. In E. crassus the repeats are has been disabled by the insertion of lESs into most, if 2-4 bp and always contain the dimer 5'TAY, and any not all, genes. For some micronuclear genes the disadditional bases in the direct repeat are variable from ability in function has been made even more severe by one IES to another 1. In O. nova the 2-19 bp direct additional structural disruptions in which MDSs are in repeats lack the 5'TAY consensus and show no the wrong order. The full sequences of micronuclear and macronuclear copies of seven genes in O. nova discernible sequence pattern. have been compared so far. In five of these, the MDSs IESs, at least the longer ones, are removed as are present in the same order in both copies, but are circular molecules during the polytene chromosome stage 17. The excised circle also contains two copies of simply separated by lESs in the micronuclear copy. In the direct repeat, separated by 10 bp derived from the the genes encoding actin I and the 0t telomere-binding adjacent MDSs. This strongly implies that IES removal is by staggered cuts in which C2 micronuclear DNA the single-stranded overhangs are subsequently filled in copy of the direct excised IES and one duplication of 10 MDSsl, 17.

to create the third repeat (two in the in the gene) and the bp from adjacent

MDSs 1

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Transposonlike elements in micronuclear DNA and their elimination In addition to the short lESs, micronuclear DNA of E. crassus contains two related families of transposonlike elements called TEC1 (Refs 18, 1 9 ) a n d TEC2 (Ref. 20), each about 5.3 kbp long and present in about 30000 copies 1. These are excised and destroyed early in the polytene chromosome stage at least 10 h before excision of the IESs.

C2 macronuclear gene 1 b,'S

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FIG~I~ Diagram of the C2 gene in the micronucleus and in the macronucleus of O. nova. The gene is made up of four MDSs separated by three lESs, TIC DECEMBER1992 rot. 8 NO. 12 t41

~EVIEWS

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Diagram of the actin I gene in the micronucleus of O. nova with a proposed recombination model for unscrambling of the MDSs and removal of the lESs during macronu~lear development. (a) The scrambled micronuclear gene. (b) Folding of the micronuclear gene to align the eight pairs of repeat sequences at MDS-IES junctions. (c) Recombination between the members of each repeat, resulting in splicing of the nine MDSs, positioning of telomeric addition sites (TASs) to the termini of the actin I DNA molecule, displacement of lESs 5, 6, 7 and 8 to flanking positions and excision of IESs 1, 2, 3 and 4. (d) Excision of the spliced gene from its chromosome and addition of telomeres. The excised molecule has been drawn in the reverse polarity to conform to conventional orientation. TIG DECEMBER1992 VOL. 8 NO. 12

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[~EVII~WS protein (cxTBP), not only are MDSs separated by IESs but the order of MDSs in the micronucleus is scrambled. The two detectable copies of the actin I gene in the micronucleus of O. nova each consist of eight lESs (ranging in length from 14 bp to 110 bp) that separate nine MDSs in a scrambled linear order 23 (Fig. 5a). The two copies differ in about 3% of nucleotides but encode identical polypeptides; they may be allelic. In the macronucleus the lESs have been removed from the actin I gene, and the nine MDSs have been spliced in numerical order (Fig. 5d), which produces a molecule capable of encoding the actin I protein. In addition MDS 2, which encodes amino acids, must be inverted when it joins MDSs 1 and 3 to produce a correct ORF. Only the two largest MDSs (3 and 4) are in the correct positions relative to one another, with an IES with a direct repeat of AATC at the two MDS-IES junctions. Reordering of all other MDSs is guided by repeat sequences of 9-13 bp at MDS-IES junctions (Fig. 6). For example, in the scrambled copy of the gene, MDS 5 is interposed between MDSs 6 and 7 (Fig. 5a). The sequence at the left end of MDS 5 is matched in a direct orientation by the same sequence at the right end of MDS 4; the sequence at the right end of MDS 5 is matched in direct orientation by the same sequence at the left end of MDS 6 (Fig. 6). If the members in each pair of direct repeats were aligned with one another and recombination occurred, MDSs 4, 5 and 6 would be joined in the correct order, with accompanying elimination of IESs and one copy of the repeat in each pair of repeats. The remaining MDSs would be correctly spliced in the same way except for MDS 2. Each end of MDS 2 has a sequence that is matched in inverted orientation with sequences at the right end of MDS 1 and the left end of MDS 3. Inversion of MDS 2 converts the inverted repeats into direct repeats (Fig. 6), which, when followed by alignment and recombination, would splice MDS 2 into its correct position and orientation between MDSs 1 and 3. Mignment of all eight pairs of repeat sequences at MDS--IES junctions requires that the segment of micronuclear DNA containing the actin I gene be folded into the complicated configuration shown in Fig. 5b, if all the MDSs are to be unscrambled simultaneously. Alternatively, MDSs might be unscrambled individually in a temporal progression. In any case, recombination as shown in Fig. 5 would unscramble the gene, excising IESs 1, 2, 3 and 4 and displacing IESs 5, 6, 7 and 8 to flanking positions at the ends of the reordered gene (Fig. 5c). Excision of the actin I gene is assumed to be a separate, subsequent event that takes place after unscrambling (Fig. 5d). The only detectable copies (at least four) of the c~TBP gene in the micronucleus of O. nova consist of 14 MDSs in a scrambled order separated by 13 IESs (Fig. 7a) 4. Pairs of direct repeats ranging from 3 bp to 19 bp occur at all MDS-IES junctions. As in the actin I gene, IESs with shorter repeats (3-5 bp) separate the nonscrambled MDSs, but those involved in unscrambling are longer (6-19 bp). Scrambling has segregated all but two MDSs (13 and 14) into odd and even numbered groups, so that alignment of pairs of direct

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F1Gr~ Pairs of repeat sequences at IES-MDSjunctions that are presumed to guide elimination of IESs and reordering of MDSs in the actin I gene of O. nova during macronuclear development. TAS indicates the 5' and 3' telomere addition sites. Below, MDS 2 is shown in the inverted orientation (macronuclear orientation), with reading of the complementary strand. This inversion creates direct repeats that match with the right end of MDS 1 and the left end of MDS 3. repeats (there are no inverted MDSs and hence no inverted repeats) produces the concentric folded pattern of the micronuclear ~TBP gene shown in Fig. 7b. Recombination would remove IESs 1-11 as a single circular piece of DNA, remove IESs 12 and 13 separately, and splice the 14 MDSs into an czTBP gene that matches precisely the sequence determined for the macronuclear copy (Fig. 7c) 24. An additional step would be required to excise the unscrambled gene from the chromosome, followed by synthesis of telomeric repeats (Fig. 7d). The patterns of scrambling for the actin I gene and the czTBP gene are quite different. The actin I gene has an inverted MDS and the 0tTBP gene is scrambled in a regular distribution of odd and even numbered MDSs, which may be a clue about the origin of scrambling of the czTBP gene.

Summary of DNA processing during development The many changes in DNA that contribute to the development of a macronucleus from a micronucleus are arranged in Fig. 8 according to current temporal estimates. Development begins with multiple rounds of DNA replication, making polytene chromosomes. TEC and TBE elements and IESs are successively removed from polytene chromosomes and destroyed in Euplotes and Oxytricha. Destruction of other repetitious and unique spacer sequences probably occurs as a part of the massive destruction of DNA 25 that follows transection of the polytene chromosomes through all interbands. The major loss of DNA sequence is likely to occur in concert with excision of genes from chromosomes, followed shortly by telomere synthesis on the ends of excised genes. The excised, gene-sized molecules are then amplified many fold (to an average of about 1000 copies) by four to six rapid rounds of replication. Finally, the finished macronucleus divides without cell division (the germ-line micronucleus has divided earlier) so that there are two of each nucleus per Oxytricha (Fig. 1), and the cell resumes vegetative proliferation with a generation time of approximately 8 h.

TIG DECEMBER1992 VOL. 8 NO. 12

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Diagram of the gene encoding aTBP in the micronucleus of O. nova with a proposed recombination model for unscrambling of the MDSs and removal of the lESs during macronuclear development. (a) The scrambled micronuclear gene. (b) Proposed folding of the micronuclear gene as dictated by the pairs of repeat sequences at the MDS-IES junctions. (c) Resolution of the folded molecule as a result of recombination between the two members of each pair of direct repeats at MDS-IES junctions. All the lESs have been excised and the MDSs spliced in an unscrambled order. (d) Excision of the gene from the chromosome and addition of telomeres.

Evolution of gene scrambling What advantage, if any, accrues to a hypotrich by gene scrambling is not known, and what might have driven the evolution of scrambling is obscure. Scrambling may be a ful~ctionally neutral event in relation to the organism's life cycle, but it does d e m a n d the existence of a molect~lar mechanism for precise

unscrambling in the making of a macronucleus. Unscrambling might be achieved by the same mechanism that removes IESs from nonscrambled genes. ] Scrambled MDSs in the actin I and aTBP genes are in every case separated by lESs with pairs of repeats of 6-19 b p at all scrambled MDS-IES junctions. This suggests that lESs and repeats were essential

TIG DECEMBER1992 vot. 8 NO. 12

m

F~EVIEWS participants in scrambling events N e w diploid micronucleus in evolution. IES invasion into a gene may have preceded MDS osis scrambling or IES insertion may have occurred simultaneously with MDS reordering. In either case, creation of pairs of repeats, presumably by duplication of short Permanent segments of the gene, was essenPolytenization of micronucleus chromosomes tial w h e n scrambling occurred in order to enable unscrambling during macronuclear development. Polymerase chain reaction Removal of TEC elements (PCR) analysis of the uncloned micronuclear actin I gene in two Breakup of other species of Oxytricha, O. Removal of chromosomes IESs t r i f a l l a x and O. sp. (?) WR (M. DuBois and D. Prescott, u n p u b FIGffl Massive lished), reveals that the gene is elimination A flow chart of events during of nongenic scrambled in a pattern similar to the four days of macronuclear DNA Excision of that in O. nova. This means that development. The timing of genes from scrambling of the actin I gene chromosomes removal of TBE elements in probably occurred before diverO. fallax is unknown. The Addition of gence of the three species in evoltiming of the major elimination telomeres ution. PCR analysis of the ~TBP of sequence complexity, such as unique sequence spacer DNA, is gene in O. trifallax detects only 4 to 5 rounds of replication of presumed to coincide with the nonscrambled copies of the gene gene-sized molecules massive reduction in total DNA (J. Mitcham and D. Prescott, amount at the time of polytene unpublished), which means that chromosome breakup. Mature macronucleus scrambling of the CzTBP gene in O. nova probably occurred after divergence from the other two 11 Klobutcher, L.A., Swanton, M.T., Donini, P. and Prescott, species. This implies that gene scrambling may be an D.M. (1981) Proc. Natl Acad. Sci. USA 78, 3015-3019 ongoing evolutionary process in Oxytricha. 12 Zahler, A.M. and Prescott, D.M. (1988) Nucleic Acids Res. 16, 6953--6972 Acknowledgements This work is supported by NIGMS grant no. GM19199 13 Ammermann, D. (1965) Arch. Protistenkd 108, 109-152 14 Roth, M. and Prescott, D.M. (1985) Cell41, 411-417 and by grant no. 3184 from The Council For Tobacco 15 Klobutcher, L.A., Jahn, C.L. and Prescott, D.M. (1984) Cell Research (USA) Inc. to D.M. Prescott. I thank Michelle DuBois and Aenoch Lynn for helpful suggestions and prep36, 1045-1055 aration of illustrations. 16 Ribas-Aparicio, R.M. et al. (1987) Genes De>. 1,323-336 17 Tausta, S.L. and Klobutcher, L.A. (1989) Cell 59, 1019-1026 References 18 Baird, S.E., Fino, G.M., Tausta, S.L. and Klobutcher, L.A. 1 Klobutcher, L.A. and Jahn, C.L. (1991) Curr. Opin. Genet. (1989) Mol. Cell. Biol. 9, 3793-3807 De>. 1,397-403 19 Jahn, C.L., Krikau, M.F. and Shyman, S. (1989) Cell 59, 2 Klobutcher, L.A. and Prescott, D.M. (1986) in The 1009-1018 Molecular Biology of Ciliated Protozoa (Gall, J.G., ed.), 20 Jahn, C.L., Nilles, L.A. and Krikau, M.F. (1988) pp. 111-154, Academic Press J. Protozool. 35, 590-601 3 Prescott, D.M. and Greslin, A.F. (1992) De>. Genet. 13, 21 Herrick, G. et al. (1985) Cell 43, 759-768 66-74 22 Hunter, D.J., Williams, K., Cartinhour, S. and Herrick, G. 4 Mitcham, J.L., Lynn, A.J. and Prescott, D.M. (1992) Genes (1989) Genes De>. 3, 2101-2112 Dev. 6, 788-800 23 Greslin, A.F. et al. (1989) Proc. Natl Acad. Sci. USA 86, 5 Lauth, M.R., Spear, B.B., Heumann, J. and Prescott, D.M. 6264-6268 (1976) Cell 7, 67-74 24 Gray, J.T., Celander, D.W., Price, C.M. and Cech, T.R. 6 Yao, M.C. (1989) in MobileDNA (Berg, D.E. and Howe, (1991) Cell 67, 807-814 MM., eds), pp. 715-734, American Society for 25 Ammermann, D., Steinbrfick, G., von Berger, L. and Microbiology Hennig, W. (1974) Chromosoma 45, 401-419 7 Yao, M-C., Yao, C-H. and Monks, B. (1990) Cell 63, 26 Prescott, D.M. (1992) BioEssays 14, 317-324 763-772 8 Boswell, R.E., Jahn, C.L., Greslin, A.F. and Prescott, D.M. (1983) Nucleic Acids Res. 11, 3651-3663 9 Klobutcher, L.A., Vailonis-Walsh, A.M., Cahill, K. and D.M. PRESCOTt iS IN THE DEPARTMENT OF MOLECULAR, Ribas-Aparicio, R.M. (1986) Mol. Cell. Biol. 6, 3606--3613 CELLULAR AND DEVELOPMENTAL BIOLOGY, UNIVERSITY OF 10 Swanton, M.T., Heumann, J.M and Prescott, DM. (1980) COLORADO,BOULOE~ C O 8 0 3 0 9 - 0 3 4 7, USA. Chromosoma 77, 217-227

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TIG DECEMBER1992 VOL. 8 NO. 12

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