Vol. 134, No. 1
OF BACTERIOLOGY, Apr. 1978, p. 295-305 0021-9193/78/0134-0295$02.00/0 Copyright © 1978 American Society for Microbiology
JOURNAL
Printed in U.S.A.
Characterization of Two Types of Yeast Ribosomal DNA Genes THOMAS D. PETES,t* LYNNA M. HEREFORD,' AND KONSTANTIN G. SKRYABIN2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154'; and Institute of Molecular Biology, U.S.S.R. Academy of Sciences, Moscow 117312, U.S.S.R.2 Received for publication 31 October 1977
The intragenic organization of ribosomal DNA from a diploid strain of Sacchacerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 x 106 daltons), A (1.77 x 106 daltons), B (1.48 x 106 daltons), C (1.22 x 106 daltons), D (0.39 x 106 daltons), E (0.36 x 106 daltons), F (0.22 x 106 daltons), and G (0.17 x 106 daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: romyces
E
B
type I:
F A
...CGBEFADCGBEF...
D
Xi tYPe II:
...
C.CGX'F AD C GX'F. ...
G C
F A D
in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered. In the yeast Saccharomyces cerevisiae, as in other eucaryotes, the genes that code for rRNA species are present in many copies in the genome. There are about 100 to 140 copies of these repeating units in haploid yeast cells (24). Each of these units codes for four rRNA species: the 25S, 18S, 5.8S, and 5S rRNA's (1, 16, 20, 30). The arrangement of the ribosomal genes on the yeast chromosomes has also been studied. They have been shown to be tandemly arranged in clusters of at least 10 to 30 units (4). Evidence has also been presented that many of the ribosomal genes may be on chromosome I (7, 8). In this paper, we report on another method for analyzing the organization and homogeneity of the yeast rRNA genes. This method was to analyze with restriction enzymes a large number of recombinant DNA molecules containing randomly sheared fragments of yeast ribosomal
DNA (rDNA). The results indicated that the yeast strain from which the recombinant DNA molecules were constructed contained two types of rDNA genes in approximately equal amounts. The type I rDNA genes contained seven sites for the site-specific endonuclease EcoRI. The spacing of these sites was found to be identical to that first described by Cramer et al. (5) and recently by others (1; P. Philippsen and R. W. Davis, personal communication). The second form of the rDNA gene (type II) was found to contain only six sites recognized by EcoRI. Restriction maps for both type I and type II rDNA genes are presented below. In addition, we also examined the degree of clustering of the rDNA genes.
MATERLALS AND METHODS Construction of recombinant molecules with insertions of yeast rDNA. The details of the cont Present address: Department of Microbiology, University struction of the recombinant molecules with insertions of yeast DNA are presented elsewhere (T. D. Petes, of Chicago, Chicago, IL 60637. 295
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J. Broach, P. Wensink, L. M. Hereford, G. Fink, and D. Botstein, submitted for publication). Sheared fragments of yeast nuclear DNA from the diploid strain +D4 were inserted into the EcoRI-cleaved bacterial plasmid pMB9 by using deoxyadenylate-deoxythymidylate (dA-dT) homopolymer linkers (13). The pMB9 plasmid contains a single EcoRI restriction site and carries genes coding for tetracycline resistance (R. Rodriguez, R. Bolivar, H. Goodman, H. Boyer, and M. Betlach, Proc. ICN-UCLA Symp., in press). Other details of the structure of the recombinant DNA molecules are given in Results. Isolation of plasmid DNA. Clones were used to inoculate cultures in LB broth (11) containing tetracycline (10 ,ug/ml) to a cell density of 109 cells per ml. Chloramphenicol was then added to a concentration of 0.15 mg/ml. Incubation at 37°C was continued for 24 h. Lysis and cesium chloride-ethidium bromide density centrifugation were done as described by Clewell and Helinski (3). Ethidium bromide was extracted from the plasmid DNA with isoamyl alcohol (25), and the DNA was dialyzed against 0.001 M tris(hydroxymethyl)aminomethane (Tris)-0.001 M
This strain was inoculated into 400 ml of YEPD medium (18) and grown at 30°C to a density of about 5 x 107 cells per ml. Cells were disrupted by agitation with glass beads, using a Vortex mixer (T. D. Petes, L. M. Hereford, and D. Botstein, Cold Spring Harbor Symp. Quant. Biol., in press). The cell lysate was then centrifuged at 15,000 rpm for 30 min (Sorvall SS-34 rotor). Portions of the supernatant (1 to 4 ml) were centrifuged to density equilibrium in a fluorescent dyecesium chloride density gradient. This procedure followed that of Williamson and Fennel (31), except the dye Hoechst 33258 was used (D. H. Williamson, personal communication). The rDNA, the bottom fluorescent band in the gradient, was removed by side puncture. Hybridization of the X' fragment to rDNA. One of the EcoRI restriction fragments of the rDNA gene (the X' fragment) was purified by agarose gel electrophoresis (26) and labeled with [32P]dCTP by "nick translation" (23). rDNA from the +D4 strain was then treated with EcoRI and run on a 1.4% agarose gel (26). The DNA fragments were then transferred to cellulose nitrate filters as described by Southern (28). The 32p_ labeled X' fragment was then hybridized to these filters. The position of hybridization was detected by autoradiography (2), using Kodak XR-5 film and Kodak regular intensifying screens. After the position of hybridization was determined, the labeled X' fragment was removed from the cellulose nitrate strip by incubating the strip in water at 60°C for 10 min. The strip was then dried in a vacuum over at 70°C for 2 h. 32P-labeled DNA (23) from the plasmid pYlA12 (which contained the EcoRI fragments A, C, D, F, G, and X') was then hybridized to the same strip. The positions at which these fragments hybridized were determined by autoradiography (2) and acted as controls to identify the position of hybridization of the X' fragment.
ethylene-diaminetetraacetate (pH 8). Restriction analysis of recombinant molecules. Most of the restriction analysis was performed with the EcoRI enzyme (Miles Research Products). The buffer used in these experiments contained 0.1 M Tris, 0.05 M NaCl, and 0.01 M MgCl2 (pH 7.5). The DNA was digested for 1 h at 37°C. In other experiments, either HindIII (Miles Research Products) or BgIII (provided by Geoff Yarranton, Massachusetts Institute of Technology, and New England Biolabs) was used. For the experiments with HindIII, the reaction mixture contained 6 mM Tris, 6 mM MgC92, and 50 mM NaCl (pH 7.5). The buffer for BgiII (G. Yarranton, personal communication) contained the same components as the HindIlI buffer and, in addition, 6 mM f8-mercaptoethanol. Both HindIII and BglII digestions were performed at 37°C for 1 h. Sequential digestions were performed first with either BglII or HindIII, followed by digestion with EcoRI. After the first reaction, Tris buffer (pH 7.5) was added to a concentration of 100 mM for the EcoRI reaction. The sizes of the restriction fragments were measured on 1.4% agarose gels (26). The gels were calibrated by measuring the mobility of restriction fragments of adenovirus or lambda DNA (G. Weinstock, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1977). Gels were photographed under short-wavelength UV illumination with either Polaroid 55 or 57 film. In one set of experiments, the products of a restriction nuclease digest were run on an agarose gel, eluted, redigested, and rerun on an agarose gel. The elution procedure used in these experiments was that developed by McDonell et al. (14). It was found that, after elution of the fragment, approximately a 50-fold excess of EcoRI had to be used to complete digestion. Isolation of yeast rDNA. The yeast strain used in these experiments was the diploid +D4 (provided by L. H. Hartwell, University of Washington). The genotype of this diploid is: a adel ural gall ade2 tyrl his7 lys2 + + + a adel ural gall + + + + his5 lysl l eu2
RESULTS EcoRI digestion of yeast nuclear DNA. Nuclear DNA was isolated from the yeast strain +D4 and digested with the EcoRI enzyme. The fragments were analyzed on agarose gels. Although most of the fragments produced by such a digest were heterodisperse, several bands were seen (Fig. la). These bands presumably represent sequences that are reiterated in the yeast genome. Yeast rDNA can be separated from the other nuclear DNA by centrifugation in density equilibrium gradients (4, 32). To identify which of the bands in Fig. la were fragments generated within the reiterated ribosomal genes, we isolated yeast rDNA. Eight prominent bands were observed after treatment of rDNA with EcoRI. These eight bands have been labeled X', A, B, C, D, E, F, and G. Only four of these bands (X', A, B, and C) are well visualized in Fig. lb. Bands other than these eight (of higher molecular weight) were also found on the gels; they represent DNA fragments generated by cleaving the
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C,,--
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recombinant clones containing insertions of yeast nuclear DNA have been constructed (T. D. Petes, J. Broach, L. M. Hereford, P. Wensink, G. Fink, and D. Botstein, in preparation). The recombinant plasmids have been constructed by connecting randomly sheared yeast DNA to an EcoRI-cleaved bacterial plasmid, pMB9, using homopolymer connectors of dA's and dT's (13). Two thousand of the 2,500 recombinant clones were examined by hybridization of colonies (9) to purified yeast 32P-labeled 18S and 25S rRNA (Petes et al., submitted for publication). In two separate experiments, approximately 5% of the 2,000 clones hybridized to yeast rRNA. Seventyfive of these clones were examined by restriction
FIG. 1. EcoRI restriction pattern of total yeast nuclear DNA (a) and partially purified yeast rDNA (b) from diploid strain +D4. Total nuclear DNA or partially purified rDNA was isolated by preparative density gradient centrifugation. The isolated DNA was treated with EcoRI and analyzed by agarose gelethidium bromide electrophoresis (26). The gel in (a) was run for a shorter time than that in (b).
analysis. The analysis was done by restriction digest of individual plasmids with EcoRI. Since the single EcoRI restriction site in plasmid pMB9 is removed by insertion of yeast rDNA, all EcoRI sites in the recombinant molecule are within the yeast DNA segment. All recombinant plasmids with two or more EcoRI sites, after treatment with EcoRI, will yield one large DNA fragment (containing all of the pMB9 sequences, the "A's and T's" junction, and some attached yeast DNA) and one or more fragments that contain only yeast DNA. The fragments containing only yeast DNA should have the same electrophoretic mobilities as the EcoRI fragments from yeast chromosomal DNA that has not been cloned. One can ask, therefore, whether the recombinant clones containing yeast rDNA have EcoRI fragments with the same mobilities as the reiterated fragments found in a digest of yeast nuclear DNA. As shown in Fig. 2, EcoRI digests of recombinant plasmids often produced fragments that comigrated with reiterated ge-
2-,m circular yeast plasmid (12; T. D. Petes, unpublished data). The molecular weights of the fragments (X' to G), calculated by calibrating the gels with restriction fragments of lambda DNA and adenovirus DNA, were (x106): X', 1.87; A, 1.77; B, 1.48; C, 1.22; D, 0.39; E, 0.36; F, 0.22; and G, 0.17. The stoichiometric ratio of these fragments, as determined by densitometer tracings of photographic negatives, was approximately 0.5:1:0.5:1:1:0.5:1:1. The explanation for these unequal ratios will be discussed in a later section of this paper. To confirm the assignment of the eight reiterated EcoRI fragments to the rDNA genes and to map the order of fragments within the rDNA genes, we analyzed bacterial plasmids containing insertions of yeast rDNA. Analysis of plasmids containing insertions of yeast rDNA. Twenty-five hundred
nome sequences. A summary of the analysis of the 75 clones is shown in Table 1. All eight of the EcoRI fragments shown in Fig. lb were found in the clones. These fragments, then, must be part of the ribosomal gene, since each of the fragments was observed in five or more independently derived clones shown to hybridize to yeast rRNA. Seventy-two of the 75 clones contained at least one of these fragments. Only one of the recombinant clones (pYlrBlO) contained an EcoRI fragment that had a mobility different from that of X', A, B, C, D, E, F, G, or pMB9 fragment. As will be discussed in a later section, this fragment may represent a junction between yeast rDNA and unique DNA. No clones that contained all eight fragments (X', A, B, C, D, E, F, and G) were observed. Two clones were analyzed (pYlrG4 and pYlrl9) that contained seven fragments with a duplication. For example, pYlrlO had the fragments A, B, C,
F
G
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quences (10 kilobases; Petes et al., in preparation). Mapping the EcoRI fragments of the yeast ribosomal gene. We attempted to order TABLE 1. EcoRI fragments of yeast rDNA observed in recombinant DNA plasmidsa Clone
FIG. 2. EcoRI restriction patterns of yeast rDNA and recombinant plasmids containing insertions of yeast rDNA. (a) Yeast rDNA isolated from +D4. The lines drawn to the left of (a) represent the positions of migration of fragments X, A, B, C, D, E, F, and G (with X' at the top of the gel and the other fragments in alphabetical order). (b to e) Four recombinant plasmids containing insertions of yeast rDNA were isolated, treated with EcoRI, and analyzed on agarose gels. Each of these plasmids contain one large fragment (which has the pMB9 sequences) and several small fragments (which are identical in electrophoretic mobility to the EcoRI fragments of yeast rDNA). (b) pYlrA12 (fragments X, A, C, D, F, and G). (c) pYlrG4 (fragments A, B, C, D, E, F, and G). (d) pYlrI3 (fragments A and D). (e) pYlrH9 (fragments A, E, and F).
D, E, and F and two copies of G. Retransformation showed that all of these fragments were within a single plasmid molecule. Clones containing such duplications were unstable. After about 25 generations of growth, plasmids that had deleted the duplicated segment outgrew the original plasmid. This may explain why the average size of the inserted rDNA in the recombinant molecules (4.9 kilobases) was less than the average size of inserted nonribosomal yeast se-
1. pYlrBl2, pYlrC4, pYlrGlO 2. pYlrE8 3. pYlrB9, pYlrC5, pYlrF5, pYlrF9, pYlrGl, pYlrG3, pYlrJl 4. pYlrB5, pYlrB6, pYlrB7, pYlrC6, pYlrCl2, pYlrD5, pYlrE5, pYlrF3 5. pYlrBll, pYlrIl 6. pYlrI3 7. pYlrE9 8. pYlrF2 9. pYlrD3 pYlrH4, pYlrH3, 10. pYlrG7, pYlrH5 11. pYlrD6, pYlrI6 12. pYlrF7 13. pYlrB8, pYlrC2, pYlrF6, pYlrH9 14. pYlrGll 15. pYlrCll 16. pYlrA3, pYlrA7, pYlrAlO, pYlrEll, pYlrD8, pYlrC3, pYlrEl2, pYlrHIl, pYlrI2, pYlrJ3 17. pYlrIlO 18. pYlrFlO 19. pYlrC9 20. pYlrG6 21. pYlrBlO 22. pYlrGl2 23. pYlrB3, pYlrC7, pYlrG8 24. pYlrG2 25. pYlrBI 26. pYlrA8, pYlrE6, pYlrCl,
EcoRI restriction fragments of yeast rDNA found in the cloned plasmids None C D F
G A, D A, F B, G C, D E, F A, C, D A, D, F A, E, F B, C, G B, E, F C, D, G F, G, X' A, B, E, F A, C, D, F A, D, E, F A, E, F, X" B, C, E, G B, E, F, G C, F, G, X' A, C, D, E, F A, C, D, F, G
pYlrFl2, pYlrH2, pYlrI7, pYlrI8 C, D, F, G, X' A, B, D, E, F, G A, C, D, E, F, G pYlrH10 A, C, D, F, G, X' pYlrAl2, pYlrG5 pYlrFll B, C, D, E, F, G A, B, C, 2xD, E, F, pYlrG4 G 33. pYlrI9 A, B, C, D, E, F, 2xG a Plasmid DNA was isolated from 75 clones that had hybridized to yeast rRNA. These plasmids were treated with EcoRI and analyzed on agarose gels. All plasmids contained at least one large fragment (greater than 3.2 x 106 daltons), which contained the pMB9 DNA sequences. In addition, most other plasmids contained one or more fragments with the same electrophoretic mobility as EcoRI fragments produced by digesting yeast rDNA (Fig. 2). These plasmid fragments, therefore, were labeled with the same designations used for the fragments of the rDNA genes (A, B, C, D, E, F, G, and X'). Only one plasmid (pYlrB10) contained a fragment with a different mobility (labeled X"). Two plasmids (pYlrG4 and pYlrI9) contained the seven fragments A, B, C, D, E, F, and G plus a duplication of one fragment. The few cases in which clones with the same set of restriction fragments are clustered (pYlrB5 to pYlrB7 and pYlrH2 to pYlrH4) are likely to represent cross-contamination of rDNA clones within the microtiter dishes.
27. 28. 29. 30. 31. 32.
pYlrF8 pYlrFl, pYlrH7
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the eight EcoRI fragments of the yeast ribo- with these five fragments. One explanation for somal gene by using the recombinant plasmids. these results is that yeast strain +D4 contains The principle of the mapping technique is to two types of ribosomal genes that have different determine which EcoRI fragments are found restriction maps. One type of gene (type 1) contogether in clones with a small number of EcoRI tains the seven EcoRI fragments: A, B, C, D, E, fragments. This mapping method is illustrated F, and G. The second type (type II) contains the in Table 2. Although only six clones were used six fragments: X', A, C, D, F, and G. The map to construct the map, the structures of all clones given in Table 2, therefore, represents a map of (except those containing X' or X" fragments) the type I ribosomal genes. The above interpretation makes the predicwere consistent with this map. It should be pointed out that the circularity of the restriction tion that fragment X' should be equivalent to map does not necessarily indicate that individual fragment B plus E. We have directly demonrDNA genes are circular; a circular restricton strated this by hybridization of labeled X' to map would also be obtained if the rDNA genes Southern gels (28) containing EcoRI-treated rDNA from strain +D4. Purified X' hybridized were clustered in tandem arrays (4, 29). The clones containing X' did not fit with the to B and E as well as X' (Fig. 3b). The hybridimap shown in Table 2. For example, clone zation data were consistent with other experipYlrllO contained fragments X', F, and G, in- mental observations. Fragment X' was never dicating that X' must be next to fragments F observed in clones that contained B or E. The and/or G within the ribosomal gene. The map molecular weight of the X' fragment was apin Table 2, however, indicates that F is next to proximately equal to the sum of the B and E A and E and that G is next to B and C. In fragments. The stoichiometry of the EcoRI fragaddition, we found that there were no clones ments generated by cleavage of rDNA from +D4 that contained the EcoRI fragments B and E in indicated that X', B, and E were present in the addition to X'. Fragment X' was, however, found cell about half as frequently as A, C, D, F, and in some clones in association with A, C, D, F, G. This suggests that type I and type II riboand G; B and E were also found in association somal genes were present in approximately equal numbers of copies in +D4. Finally, when the TABLE 2. Derivation of the EcoRI restriction map haploid parental strains of the yeast diploid +D4 I rDNA the genesa for type were examined (21; Petes et al., in press), we Fragments prothat one parent contained only the type I found duced after EcoRI Derived map" Clone no. ribosomal genes and one parent carried only the treatmentb type II ribosomal genes. This last result shows GB G, B pYlrF2 that X' was not simply the product of incomplete GB(EF) B, E, F pYlrCll cleavage at the EcoRI site separating B and E. CGBEF B, C, E, G pYlrG12 Since the X' fragment appeared to be an alCGBEFA A, B, E, F pYlrFlO temative form of the B and E fragments, we CGBEFAD A, B, D, E, F, G pYlrFl expected that it would have the same position as B and E in the rDNA gene. Since there were B,C,D,E,F,G pYlrFll C GB E only five clones that contained the X' fragment, An unambiguous map of plasmids containing yeast an unambiguous map could not be derived by rDNA (excluding those with an X' or X" fragment) the technique illustrated in Table 2. As an altercan be constructed from the data given in Table 1. native approach, we mapped the order of the The principle of the mapping procedure is to assume EcoRI fragments in the clone pYlrA12 (which that the EcoRI fragments found together in the recombinant plasmid are adjacent to each other in the contains the fragments X', A, C, D, F, and G) by rDNA gene in vivo and that all rDNA genes have the using the restriction enzymes EcoRI, BglII, and same order of fragments. For example, since plasmid HindIII. Restriction analysis of plasmid pYlrA12. pYlrF2 contains only the B and G fragments, these fragments must be adjacent to each other in the rDNA The number and size of fragments produced by gene. Since pYlrC7 has fragments B, E, F, fragments digesting the plasmid with the enzymes EcoRI, E and F (unordered with respect to each other) must BglII, and HindIII in various combinations are be on the other side of B from the G fragment, and so shown in Table 3. Sample gels of digestion prodforth. It is likely from other data (4, 29) that the yeast ucts of the plasmid are shown in Fig. 4. rDNA genes are tandemly arranged and, therefore, a Digestion of the plasmid with EcoRI yielded circular "map" does not require circular DNA moleseven fragments: the pMB9 portion of the molcules for its origin. b Not including the EcoRI fragment containing ecule and fragments with the mobilities of X', A, C, D, F, and G. To keep the nomenclature conpMB9 sequences. cFragments in parentheses are not ordered with sistent for all digestion fragments, the EcoRI fragments will be designated Rl through R7 in respect to each other. a
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the plasmid with HindIII and EcoRI in the same reaction mixture. Eleven fragments were produced; two fragments (HR10 and HR11) had identical mobilities. The presence of these two fragments was detected by quantitative densitometer tracings of gel photographs. These tracings showed that there was twice the amount of DNA with a molecular weight of 0.13 x 106 than that expected if there was a 1:1 ratio of this fragment to the other fragments in the pYlrA12 plasmid. The mobilities of three EcoRI fragments (Rl, R2, and R4) were changed by digestion with HindIII. Since there are four sites for cleavage of the plasmid with HindIII, one of the EcoRI fragments must contain two HindlIl sites. Seven fragments resulting from the HindIII-EcoRI digest had mobilities different from those of fragments produced by EcoRI alone (HR1, HR3, HR4, HR5, HR6, HR10, and HR11). Fragment HR1 must be a cleavage product of Rl since HR1 is larger than any of the other EcoRI fragments. Since the size difference between HR1 and Rl is about 0.8 x 106 daltons, both HR3 and HR4 are too large to be the second cleavage product of Rl. Fragment HR2 is the same size as fragment R3. Since no combination of HindIII-EcoRI fragments can be found that gives the molecular weights expected if HR2 were a cleavage product of the Rl or R2, HR2 is equivalent to R3. Fragment HR3 must be derived from R2 since HR3 is too large to be derived from R4. Similarly, HR4 must represent a cleavage product of R4. Fragment HR5 is too large to represent a second cleavage product of either R2 or R4 and, therefore, is derived from Rl. For similar reasons, fragment HR6 is the
A
B ~ ~
DE
abb FIG. 3. Hybridization of the X' fragment to rDNA from strain +D4. (a) Hybridization of 32P-labeled pYlrA12 DNA to EcoRI-cut rDNA. Fragments Fand G are not visible because they had run off the gel. (b) Hybridization of 32P-labeled purified X' fragment to EcoRI-cut rDNA.
TABLE 3. Molecular weights of restriction fragments from recombinant plasmid pYlrA12a Enzrme(s) Enzyme(s)
Fragments produced
Size of fragments
(xl0r6 daltons)
EcoRI
R1-R7
HindIII
H1-H4 Bi and B2 HRl-HR1lb
3.9, 1.87, 1.77, 1.22, 0.39, 0.22, 0.17 3.9, 3.25, 1.75, 0.68 7.6, 2.8 3.1, 1.77, 1.47, 1.10, 0.65, 0.46, 0.39, 0.22, 0.17, 0.13,
HindIII and
HB1-HB6
BglII and BglII
3.25, 2.60, 1.50, 1.41, 0.68,
BRl-BR9
BglII HindIII and EcoRI
0.13
order of their size. Rl was the largest fragment and included the pMB9 sequences. The seven EcoRI fragments were ordered by analyzing the clone with other restriction enzymes. Four fragments were produced after digestion with HindIII. Since pMB9 contains a single HindIII site located 310 base pairs from the EcoRI site (15), the remaining three HindIII sites must be within the yeast DNA insertion. The locations of the HindIII sites within the EcoRI fragments were determined by cleaving
0.24
3.9, 1.88, 1.23, 1.11, 0.69, EcoRI 0.40, 0.22, 0.13, 0.04 aPlasmid DNA was isolated and treated with EcoRI, HindIII, or BglII. Double-digestion experiments with all combinations of two restriction enzymes were also done. The molecular weights of the fragments were measured by agaroseethidium bromide gel electrophoresis. bThe presence of two fragments (HR10 and HR11) with the same molecular weight (0.13 x 106) was detected by densitometer tracing of the photographic negative taken of the gel.
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to be HR1, HR5, and either HR10 or HR11. The information obtained from these experiments is summarized in Fig. 5. Given the polarity imposed by the location of the HindIII sites, there are eight possible circular orientations of the three fragments shown in Fig. 5. Six of the orientations can be eliminated from consideration by the data shown in Table 3. For example, the right side of the R4 fragment (as drawn in Fig. 5) cannot be oriented toward the right side of Ri. If these two fragments are adjacent to each other in this orientation, then the distance between HindIII sites is 0.26 x 10' daltons. No such fragment was observed in the HindIII digest. There is also no way to interpose any combination of fragments R3, R5, R6, and R7 between Ri and R4 in this orientation so that the sum of the fragments equals the molecular weight of Hi, H2, or H3. Consequently, this orientation is inconsistent with the data. Only two orientations (Fig. 6) are consistent with the 31 3 .1
.1 0.13
R(2) |
I
I
0.46 R(4)H
FIG. 4. Restriction analysis of the plasmid pYlrA12. Plasmid DNA was isolated, treated with one of the restriction enzymes, and analyzed on agarose gels. (a) EcoRI-treated DNA; (b) HindII- treated DNA. The top band is a doublet which is not resolved unless the gel is run further; (c) BglII-treated DNA.
0.13 FIG. 5. Location of HindIII restriction sites within three EcoRI fragments of plasmid pYlrA12. The short, thick vertical lines represent the ends of the EcoRI fragments. The thin vertical lines indicate the HindIII sites. The zig-zag lines in the Rl fragment show the approximate position of the dA-dT linkers. The numbers given under each fragment are the molecular weights (xlO').
second cleavage product of R2. Since R4 is 1.22 x 10' daltons and HR4 is 1.10 x 106 daltons, the expected size of the second cleavage product of R(2) (2) /^~~~~~
OF BACTERIOLOGY, Apr. 1978, p. 295-305 0021-9193/78/0134-0295$02.00/0 Copyright © 1978 American Society for Microbiology
JOURNAL
Printed in U.S.A.
Characterization of Two Types of Yeast Ribosomal DNA Genes THOMAS D. PETES,t* LYNNA M. HEREFORD,' AND KONSTANTIN G. SKRYABIN2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154'; and Institute of Molecular Biology, U.S.S.R. Academy of Sciences, Moscow 117312, U.S.S.R.2 Received for publication 31 October 1977
The intragenic organization of ribosomal DNA from a diploid strain of Sacchacerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 x 106 daltons), A (1.77 x 106 daltons), B (1.48 x 106 daltons), C (1.22 x 106 daltons), D (0.39 x 106 daltons), E (0.36 x 106 daltons), F (0.22 x 106 daltons), and G (0.17 x 106 daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: romyces
E
B
type I:
F A
...CGBEFADCGBEF...
D
Xi tYPe II:
...
C.CGX'F AD C GX'F. ...
G C
F A D
in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered. In the yeast Saccharomyces cerevisiae, as in other eucaryotes, the genes that code for rRNA species are present in many copies in the genome. There are about 100 to 140 copies of these repeating units in haploid yeast cells (24). Each of these units codes for four rRNA species: the 25S, 18S, 5.8S, and 5S rRNA's (1, 16, 20, 30). The arrangement of the ribosomal genes on the yeast chromosomes has also been studied. They have been shown to be tandemly arranged in clusters of at least 10 to 30 units (4). Evidence has also been presented that many of the ribosomal genes may be on chromosome I (7, 8). In this paper, we report on another method for analyzing the organization and homogeneity of the yeast rRNA genes. This method was to analyze with restriction enzymes a large number of recombinant DNA molecules containing randomly sheared fragments of yeast ribosomal
DNA (rDNA). The results indicated that the yeast strain from which the recombinant DNA molecules were constructed contained two types of rDNA genes in approximately equal amounts. The type I rDNA genes contained seven sites for the site-specific endonuclease EcoRI. The spacing of these sites was found to be identical to that first described by Cramer et al. (5) and recently by others (1; P. Philippsen and R. W. Davis, personal communication). The second form of the rDNA gene (type II) was found to contain only six sites recognized by EcoRI. Restriction maps for both type I and type II rDNA genes are presented below. In addition, we also examined the degree of clustering of the rDNA genes.
MATERLALS AND METHODS Construction of recombinant molecules with insertions of yeast rDNA. The details of the cont Present address: Department of Microbiology, University struction of the recombinant molecules with insertions of yeast DNA are presented elsewhere (T. D. Petes, of Chicago, Chicago, IL 60637. 295
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J. Broach, P. Wensink, L. M. Hereford, G. Fink, and D. Botstein, submitted for publication). Sheared fragments of yeast nuclear DNA from the diploid strain +D4 were inserted into the EcoRI-cleaved bacterial plasmid pMB9 by using deoxyadenylate-deoxythymidylate (dA-dT) homopolymer linkers (13). The pMB9 plasmid contains a single EcoRI restriction site and carries genes coding for tetracycline resistance (R. Rodriguez, R. Bolivar, H. Goodman, H. Boyer, and M. Betlach, Proc. ICN-UCLA Symp., in press). Other details of the structure of the recombinant DNA molecules are given in Results. Isolation of plasmid DNA. Clones were used to inoculate cultures in LB broth (11) containing tetracycline (10 ,ug/ml) to a cell density of 109 cells per ml. Chloramphenicol was then added to a concentration of 0.15 mg/ml. Incubation at 37°C was continued for 24 h. Lysis and cesium chloride-ethidium bromide density centrifugation were done as described by Clewell and Helinski (3). Ethidium bromide was extracted from the plasmid DNA with isoamyl alcohol (25), and the DNA was dialyzed against 0.001 M tris(hydroxymethyl)aminomethane (Tris)-0.001 M
This strain was inoculated into 400 ml of YEPD medium (18) and grown at 30°C to a density of about 5 x 107 cells per ml. Cells were disrupted by agitation with glass beads, using a Vortex mixer (T. D. Petes, L. M. Hereford, and D. Botstein, Cold Spring Harbor Symp. Quant. Biol., in press). The cell lysate was then centrifuged at 15,000 rpm for 30 min (Sorvall SS-34 rotor). Portions of the supernatant (1 to 4 ml) were centrifuged to density equilibrium in a fluorescent dyecesium chloride density gradient. This procedure followed that of Williamson and Fennel (31), except the dye Hoechst 33258 was used (D. H. Williamson, personal communication). The rDNA, the bottom fluorescent band in the gradient, was removed by side puncture. Hybridization of the X' fragment to rDNA. One of the EcoRI restriction fragments of the rDNA gene (the X' fragment) was purified by agarose gel electrophoresis (26) and labeled with [32P]dCTP by "nick translation" (23). rDNA from the +D4 strain was then treated with EcoRI and run on a 1.4% agarose gel (26). The DNA fragments were then transferred to cellulose nitrate filters as described by Southern (28). The 32p_ labeled X' fragment was then hybridized to these filters. The position of hybridization was detected by autoradiography (2), using Kodak XR-5 film and Kodak regular intensifying screens. After the position of hybridization was determined, the labeled X' fragment was removed from the cellulose nitrate strip by incubating the strip in water at 60°C for 10 min. The strip was then dried in a vacuum over at 70°C for 2 h. 32P-labeled DNA (23) from the plasmid pYlA12 (which contained the EcoRI fragments A, C, D, F, G, and X') was then hybridized to the same strip. The positions at which these fragments hybridized were determined by autoradiography (2) and acted as controls to identify the position of hybridization of the X' fragment.
ethylene-diaminetetraacetate (pH 8). Restriction analysis of recombinant molecules. Most of the restriction analysis was performed with the EcoRI enzyme (Miles Research Products). The buffer used in these experiments contained 0.1 M Tris, 0.05 M NaCl, and 0.01 M MgCl2 (pH 7.5). The DNA was digested for 1 h at 37°C. In other experiments, either HindIII (Miles Research Products) or BgIII (provided by Geoff Yarranton, Massachusetts Institute of Technology, and New England Biolabs) was used. For the experiments with HindIII, the reaction mixture contained 6 mM Tris, 6 mM MgC92, and 50 mM NaCl (pH 7.5). The buffer for BgiII (G. Yarranton, personal communication) contained the same components as the HindIlI buffer and, in addition, 6 mM f8-mercaptoethanol. Both HindIII and BglII digestions were performed at 37°C for 1 h. Sequential digestions were performed first with either BglII or HindIII, followed by digestion with EcoRI. After the first reaction, Tris buffer (pH 7.5) was added to a concentration of 100 mM for the EcoRI reaction. The sizes of the restriction fragments were measured on 1.4% agarose gels (26). The gels were calibrated by measuring the mobility of restriction fragments of adenovirus or lambda DNA (G. Weinstock, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1977). Gels were photographed under short-wavelength UV illumination with either Polaroid 55 or 57 film. In one set of experiments, the products of a restriction nuclease digest were run on an agarose gel, eluted, redigested, and rerun on an agarose gel. The elution procedure used in these experiments was that developed by McDonell et al. (14). It was found that, after elution of the fragment, approximately a 50-fold excess of EcoRI had to be used to complete digestion. Isolation of yeast rDNA. The yeast strain used in these experiments was the diploid +D4 (provided by L. H. Hartwell, University of Washington). The genotype of this diploid is: a adel ural gall ade2 tyrl his7 lys2 + + + a adel ural gall + + + + his5 lysl l eu2
RESULTS EcoRI digestion of yeast nuclear DNA. Nuclear DNA was isolated from the yeast strain +D4 and digested with the EcoRI enzyme. The fragments were analyzed on agarose gels. Although most of the fragments produced by such a digest were heterodisperse, several bands were seen (Fig. la). These bands presumably represent sequences that are reiterated in the yeast genome. Yeast rDNA can be separated from the other nuclear DNA by centrifugation in density equilibrium gradients (4, 32). To identify which of the bands in Fig. la were fragments generated within the reiterated ribosomal genes, we isolated yeast rDNA. Eight prominent bands were observed after treatment of rDNA with EcoRI. These eight bands have been labeled X', A, B, C, D, E, F, and G. Only four of these bands (X', A, B, and C) are well visualized in Fig. lb. Bands other than these eight (of higher molecular weight) were also found on the gels; they represent DNA fragments generated by cleaving the
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recombinant clones containing insertions of yeast nuclear DNA have been constructed (T. D. Petes, J. Broach, L. M. Hereford, P. Wensink, G. Fink, and D. Botstein, in preparation). The recombinant plasmids have been constructed by connecting randomly sheared yeast DNA to an EcoRI-cleaved bacterial plasmid, pMB9, using homopolymer connectors of dA's and dT's (13). Two thousand of the 2,500 recombinant clones were examined by hybridization of colonies (9) to purified yeast 32P-labeled 18S and 25S rRNA (Petes et al., submitted for publication). In two separate experiments, approximately 5% of the 2,000 clones hybridized to yeast rRNA. Seventyfive of these clones were examined by restriction
FIG. 1. EcoRI restriction pattern of total yeast nuclear DNA (a) and partially purified yeast rDNA (b) from diploid strain +D4. Total nuclear DNA or partially purified rDNA was isolated by preparative density gradient centrifugation. The isolated DNA was treated with EcoRI and analyzed by agarose gelethidium bromide electrophoresis (26). The gel in (a) was run for a shorter time than that in (b).
analysis. The analysis was done by restriction digest of individual plasmids with EcoRI. Since the single EcoRI restriction site in plasmid pMB9 is removed by insertion of yeast rDNA, all EcoRI sites in the recombinant molecule are within the yeast DNA segment. All recombinant plasmids with two or more EcoRI sites, after treatment with EcoRI, will yield one large DNA fragment (containing all of the pMB9 sequences, the "A's and T's" junction, and some attached yeast DNA) and one or more fragments that contain only yeast DNA. The fragments containing only yeast DNA should have the same electrophoretic mobilities as the EcoRI fragments from yeast chromosomal DNA that has not been cloned. One can ask, therefore, whether the recombinant clones containing yeast rDNA have EcoRI fragments with the same mobilities as the reiterated fragments found in a digest of yeast nuclear DNA. As shown in Fig. 2, EcoRI digests of recombinant plasmids often produced fragments that comigrated with reiterated ge-
2-,m circular yeast plasmid (12; T. D. Petes, unpublished data). The molecular weights of the fragments (X' to G), calculated by calibrating the gels with restriction fragments of lambda DNA and adenovirus DNA, were (x106): X', 1.87; A, 1.77; B, 1.48; C, 1.22; D, 0.39; E, 0.36; F, 0.22; and G, 0.17. The stoichiometric ratio of these fragments, as determined by densitometer tracings of photographic negatives, was approximately 0.5:1:0.5:1:1:0.5:1:1. The explanation for these unequal ratios will be discussed in a later section of this paper. To confirm the assignment of the eight reiterated EcoRI fragments to the rDNA genes and to map the order of fragments within the rDNA genes, we analyzed bacterial plasmids containing insertions of yeast rDNA. Analysis of plasmids containing insertions of yeast rDNA. Twenty-five hundred
nome sequences. A summary of the analysis of the 75 clones is shown in Table 1. All eight of the EcoRI fragments shown in Fig. lb were found in the clones. These fragments, then, must be part of the ribosomal gene, since each of the fragments was observed in five or more independently derived clones shown to hybridize to yeast rRNA. Seventy-two of the 75 clones contained at least one of these fragments. Only one of the recombinant clones (pYlrBlO) contained an EcoRI fragment that had a mobility different from that of X', A, B, C, D, E, F, G, or pMB9 fragment. As will be discussed in a later section, this fragment may represent a junction between yeast rDNA and unique DNA. No clones that contained all eight fragments (X', A, B, C, D, E, F, and G) were observed. Two clones were analyzed (pYlrG4 and pYlrl9) that contained seven fragments with a duplication. For example, pYlrlO had the fragments A, B, C,
F
G
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quences (10 kilobases; Petes et al., in preparation). Mapping the EcoRI fragments of the yeast ribosomal gene. We attempted to order TABLE 1. EcoRI fragments of yeast rDNA observed in recombinant DNA plasmidsa Clone
FIG. 2. EcoRI restriction patterns of yeast rDNA and recombinant plasmids containing insertions of yeast rDNA. (a) Yeast rDNA isolated from +D4. The lines drawn to the left of (a) represent the positions of migration of fragments X, A, B, C, D, E, F, and G (with X' at the top of the gel and the other fragments in alphabetical order). (b to e) Four recombinant plasmids containing insertions of yeast rDNA were isolated, treated with EcoRI, and analyzed on agarose gels. Each of these plasmids contain one large fragment (which has the pMB9 sequences) and several small fragments (which are identical in electrophoretic mobility to the EcoRI fragments of yeast rDNA). (b) pYlrA12 (fragments X, A, C, D, F, and G). (c) pYlrG4 (fragments A, B, C, D, E, F, and G). (d) pYlrI3 (fragments A and D). (e) pYlrH9 (fragments A, E, and F).
D, E, and F and two copies of G. Retransformation showed that all of these fragments were within a single plasmid molecule. Clones containing such duplications were unstable. After about 25 generations of growth, plasmids that had deleted the duplicated segment outgrew the original plasmid. This may explain why the average size of the inserted rDNA in the recombinant molecules (4.9 kilobases) was less than the average size of inserted nonribosomal yeast se-
1. pYlrBl2, pYlrC4, pYlrGlO 2. pYlrE8 3. pYlrB9, pYlrC5, pYlrF5, pYlrF9, pYlrGl, pYlrG3, pYlrJl 4. pYlrB5, pYlrB6, pYlrB7, pYlrC6, pYlrCl2, pYlrD5, pYlrE5, pYlrF3 5. pYlrBll, pYlrIl 6. pYlrI3 7. pYlrE9 8. pYlrF2 9. pYlrD3 pYlrH4, pYlrH3, 10. pYlrG7, pYlrH5 11. pYlrD6, pYlrI6 12. pYlrF7 13. pYlrB8, pYlrC2, pYlrF6, pYlrH9 14. pYlrGll 15. pYlrCll 16. pYlrA3, pYlrA7, pYlrAlO, pYlrEll, pYlrD8, pYlrC3, pYlrEl2, pYlrHIl, pYlrI2, pYlrJ3 17. pYlrIlO 18. pYlrFlO 19. pYlrC9 20. pYlrG6 21. pYlrBlO 22. pYlrGl2 23. pYlrB3, pYlrC7, pYlrG8 24. pYlrG2 25. pYlrBI 26. pYlrA8, pYlrE6, pYlrCl,
EcoRI restriction fragments of yeast rDNA found in the cloned plasmids None C D F
G A, D A, F B, G C, D E, F A, C, D A, D, F A, E, F B, C, G B, E, F C, D, G F, G, X' A, B, E, F A, C, D, F A, D, E, F A, E, F, X" B, C, E, G B, E, F, G C, F, G, X' A, C, D, E, F A, C, D, F, G
pYlrFl2, pYlrH2, pYlrI7, pYlrI8 C, D, F, G, X' A, B, D, E, F, G A, C, D, E, F, G pYlrH10 A, C, D, F, G, X' pYlrAl2, pYlrG5 pYlrFll B, C, D, E, F, G A, B, C, 2xD, E, F, pYlrG4 G 33. pYlrI9 A, B, C, D, E, F, 2xG a Plasmid DNA was isolated from 75 clones that had hybridized to yeast rRNA. These plasmids were treated with EcoRI and analyzed on agarose gels. All plasmids contained at least one large fragment (greater than 3.2 x 106 daltons), which contained the pMB9 DNA sequences. In addition, most other plasmids contained one or more fragments with the same electrophoretic mobility as EcoRI fragments produced by digesting yeast rDNA (Fig. 2). These plasmid fragments, therefore, were labeled with the same designations used for the fragments of the rDNA genes (A, B, C, D, E, F, G, and X'). Only one plasmid (pYlrB10) contained a fragment with a different mobility (labeled X"). Two plasmids (pYlrG4 and pYlrI9) contained the seven fragments A, B, C, D, E, F, and G plus a duplication of one fragment. The few cases in which clones with the same set of restriction fragments are clustered (pYlrB5 to pYlrB7 and pYlrH2 to pYlrH4) are likely to represent cross-contamination of rDNA clones within the microtiter dishes.
27. 28. 29. 30. 31. 32.
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the eight EcoRI fragments of the yeast ribo- with these five fragments. One explanation for somal gene by using the recombinant plasmids. these results is that yeast strain +D4 contains The principle of the mapping technique is to two types of ribosomal genes that have different determine which EcoRI fragments are found restriction maps. One type of gene (type 1) contogether in clones with a small number of EcoRI tains the seven EcoRI fragments: A, B, C, D, E, fragments. This mapping method is illustrated F, and G. The second type (type II) contains the in Table 2. Although only six clones were used six fragments: X', A, C, D, F, and G. The map to construct the map, the structures of all clones given in Table 2, therefore, represents a map of (except those containing X' or X" fragments) the type I ribosomal genes. The above interpretation makes the predicwere consistent with this map. It should be pointed out that the circularity of the restriction tion that fragment X' should be equivalent to map does not necessarily indicate that individual fragment B plus E. We have directly demonrDNA genes are circular; a circular restricton strated this by hybridization of labeled X' to map would also be obtained if the rDNA genes Southern gels (28) containing EcoRI-treated rDNA from strain +D4. Purified X' hybridized were clustered in tandem arrays (4, 29). The clones containing X' did not fit with the to B and E as well as X' (Fig. 3b). The hybridimap shown in Table 2. For example, clone zation data were consistent with other experipYlrllO contained fragments X', F, and G, in- mental observations. Fragment X' was never dicating that X' must be next to fragments F observed in clones that contained B or E. The and/or G within the ribosomal gene. The map molecular weight of the X' fragment was apin Table 2, however, indicates that F is next to proximately equal to the sum of the B and E A and E and that G is next to B and C. In fragments. The stoichiometry of the EcoRI fragaddition, we found that there were no clones ments generated by cleavage of rDNA from +D4 that contained the EcoRI fragments B and E in indicated that X', B, and E were present in the addition to X'. Fragment X' was, however, found cell about half as frequently as A, C, D, F, and in some clones in association with A, C, D, F, G. This suggests that type I and type II riboand G; B and E were also found in association somal genes were present in approximately equal numbers of copies in +D4. Finally, when the TABLE 2. Derivation of the EcoRI restriction map haploid parental strains of the yeast diploid +D4 I rDNA the genesa for type were examined (21; Petes et al., in press), we Fragments prothat one parent contained only the type I found duced after EcoRI Derived map" Clone no. ribosomal genes and one parent carried only the treatmentb type II ribosomal genes. This last result shows GB G, B pYlrF2 that X' was not simply the product of incomplete GB(EF) B, E, F pYlrCll cleavage at the EcoRI site separating B and E. CGBEF B, C, E, G pYlrG12 Since the X' fragment appeared to be an alCGBEFA A, B, E, F pYlrFlO temative form of the B and E fragments, we CGBEFAD A, B, D, E, F, G pYlrFl expected that it would have the same position as B and E in the rDNA gene. Since there were B,C,D,E,F,G pYlrFll C GB E only five clones that contained the X' fragment, An unambiguous map of plasmids containing yeast an unambiguous map could not be derived by rDNA (excluding those with an X' or X" fragment) the technique illustrated in Table 2. As an altercan be constructed from the data given in Table 1. native approach, we mapped the order of the The principle of the mapping procedure is to assume EcoRI fragments in the clone pYlrA12 (which that the EcoRI fragments found together in the recombinant plasmid are adjacent to each other in the contains the fragments X', A, C, D, F, and G) by rDNA gene in vivo and that all rDNA genes have the using the restriction enzymes EcoRI, BglII, and same order of fragments. For example, since plasmid HindIII. Restriction analysis of plasmid pYlrA12. pYlrF2 contains only the B and G fragments, these fragments must be adjacent to each other in the rDNA The number and size of fragments produced by gene. Since pYlrC7 has fragments B, E, F, fragments digesting the plasmid with the enzymes EcoRI, E and F (unordered with respect to each other) must BglII, and HindIII in various combinations are be on the other side of B from the G fragment, and so shown in Table 3. Sample gels of digestion prodforth. It is likely from other data (4, 29) that the yeast ucts of the plasmid are shown in Fig. 4. rDNA genes are tandemly arranged and, therefore, a Digestion of the plasmid with EcoRI yielded circular "map" does not require circular DNA moleseven fragments: the pMB9 portion of the molcules for its origin. b Not including the EcoRI fragment containing ecule and fragments with the mobilities of X', A, C, D, F, and G. To keep the nomenclature conpMB9 sequences. cFragments in parentheses are not ordered with sistent for all digestion fragments, the EcoRI fragments will be designated Rl through R7 in respect to each other. a
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the plasmid with HindIII and EcoRI in the same reaction mixture. Eleven fragments were produced; two fragments (HR10 and HR11) had identical mobilities. The presence of these two fragments was detected by quantitative densitometer tracings of gel photographs. These tracings showed that there was twice the amount of DNA with a molecular weight of 0.13 x 106 than that expected if there was a 1:1 ratio of this fragment to the other fragments in the pYlrA12 plasmid. The mobilities of three EcoRI fragments (Rl, R2, and R4) were changed by digestion with HindIII. Since there are four sites for cleavage of the plasmid with HindIII, one of the EcoRI fragments must contain two HindlIl sites. Seven fragments resulting from the HindIII-EcoRI digest had mobilities different from those of fragments produced by EcoRI alone (HR1, HR3, HR4, HR5, HR6, HR10, and HR11). Fragment HR1 must be a cleavage product of Rl since HR1 is larger than any of the other EcoRI fragments. Since the size difference between HR1 and Rl is about 0.8 x 106 daltons, both HR3 and HR4 are too large to be the second cleavage product of Rl. Fragment HR2 is the same size as fragment R3. Since no combination of HindIII-EcoRI fragments can be found that gives the molecular weights expected if HR2 were a cleavage product of the Rl or R2, HR2 is equivalent to R3. Fragment HR3 must be derived from R2 since HR3 is too large to be derived from R4. Similarly, HR4 must represent a cleavage product of R4. Fragment HR5 is too large to represent a second cleavage product of either R2 or R4 and, therefore, is derived from Rl. For similar reasons, fragment HR6 is the
A
B ~ ~
DE
abb FIG. 3. Hybridization of the X' fragment to rDNA from strain +D4. (a) Hybridization of 32P-labeled pYlrA12 DNA to EcoRI-cut rDNA. Fragments Fand G are not visible because they had run off the gel. (b) Hybridization of 32P-labeled purified X' fragment to EcoRI-cut rDNA.
TABLE 3. Molecular weights of restriction fragments from recombinant plasmid pYlrA12a Enzrme(s) Enzyme(s)
Fragments produced
Size of fragments
(xl0r6 daltons)
EcoRI
R1-R7
HindIII
H1-H4 Bi and B2 HRl-HR1lb
3.9, 1.87, 1.77, 1.22, 0.39, 0.22, 0.17 3.9, 3.25, 1.75, 0.68 7.6, 2.8 3.1, 1.77, 1.47, 1.10, 0.65, 0.46, 0.39, 0.22, 0.17, 0.13,
HindIII and
HB1-HB6
BglII and BglII
3.25, 2.60, 1.50, 1.41, 0.68,
BRl-BR9
BglII HindIII and EcoRI
0.13
order of their size. Rl was the largest fragment and included the pMB9 sequences. The seven EcoRI fragments were ordered by analyzing the clone with other restriction enzymes. Four fragments were produced after digestion with HindIII. Since pMB9 contains a single HindIII site located 310 base pairs from the EcoRI site (15), the remaining three HindIII sites must be within the yeast DNA insertion. The locations of the HindIII sites within the EcoRI fragments were determined by cleaving
0.24
3.9, 1.88, 1.23, 1.11, 0.69, EcoRI 0.40, 0.22, 0.13, 0.04 aPlasmid DNA was isolated and treated with EcoRI, HindIII, or BglII. Double-digestion experiments with all combinations of two restriction enzymes were also done. The molecular weights of the fragments were measured by agaroseethidium bromide gel electrophoresis. bThe presence of two fragments (HR10 and HR11) with the same molecular weight (0.13 x 106) was detected by densitometer tracing of the photographic negative taken of the gel.
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to be HR1, HR5, and either HR10 or HR11. The information obtained from these experiments is summarized in Fig. 5. Given the polarity imposed by the location of the HindIII sites, there are eight possible circular orientations of the three fragments shown in Fig. 5. Six of the orientations can be eliminated from consideration by the data shown in Table 3. For example, the right side of the R4 fragment (as drawn in Fig. 5) cannot be oriented toward the right side of Ri. If these two fragments are adjacent to each other in this orientation, then the distance between HindIII sites is 0.26 x 10' daltons. No such fragment was observed in the HindIII digest. There is also no way to interpose any combination of fragments R3, R5, R6, and R7 between Ri and R4 in this orientation so that the sum of the fragments equals the molecular weight of Hi, H2, or H3. Consequently, this orientation is inconsistent with the data. Only two orientations (Fig. 6) are consistent with the 31 3 .1
.1 0.13
R(2) |
I
I
0.46 R(4)H
FIG. 4. Restriction analysis of the plasmid pYlrA12. Plasmid DNA was isolated, treated with one of the restriction enzymes, and analyzed on agarose gels. (a) EcoRI-treated DNA; (b) HindII- treated DNA. The top band is a doublet which is not resolved unless the gel is run further; (c) BglII-treated DNA.
0.13 FIG. 5. Location of HindIII restriction sites within three EcoRI fragments of plasmid pYlrA12. The short, thick vertical lines represent the ends of the EcoRI fragments. The thin vertical lines indicate the HindIII sites. The zig-zag lines in the Rl fragment show the approximate position of the dA-dT linkers. The numbers given under each fragment are the molecular weights (xlO').
second cleavage product of R2. Since R4 is 1.22 x 10' daltons and HR4 is 1.10 x 106 daltons, the expected size of the second cleavage product of R(2) (2) /^~~~~~
