Analysis of the junction between ribosomal RNA genes and single-copy chromosomal sequences in the yeast Saccharomyces cerevisiae

Cell, Vol. 26. 355-364,

February

1962,

Copyright

0 1962

by MIT

Analysis of the Junction between Ribosomal RNA Genes and Single-Copy Chromosomal Sequences in the Yeast Saccharomyces cerevisiae Timothy J. Zamb* and Thomas Department of Microbiology University of Chicago 920 East 58th Street Chicago, Illinois 60637

D. P&es

Summary The yeast Saccharomyces cerevisiae has a single tandem array of 100 ribosomal RNA (rRNA) genes. We have cloned and characterized a junction between the centromere-distal end of this array and the adjacent single-copy chromosomal sequences. We have shown that the junction occurs within the nontranscribed region of the repeat. By mapping the junction, we have found that the 35s rRNA precursor is transcribed toward the centromere while the 5s rRNA is transcribed away from the centromere. We have also shown that different yeast strains can have different single-copy sequences at the junction. Introduction All eucaryotes that have been examined contain repeated genes. Often these repeated genes are arranged in tandem arrays flanked by other types of DNA sequences. Although there is considerable information concerning the structure of repeated genes, the junction between a tandem array of transcribed genes and single-copy DNA sequences has not been previously analyzed. In the experiments described below, we examined the junction between the ribosomal RNA genes and the single-copy sequences of the yeast chromosomal DNA. The yeast Saccharomyces cerevisiae has about 100 ribosomal RNA genes per haploid genome (Schweizer et al., 1969). Each gene contains 9 kb of DNA and encodes four species of rRNA; the 25S, 18S, 5.8s and 5s rRNAs (Bell et al., 1977; Philippsen et al., 1978). The 25S, 18s and 5.8s species are transcribed as a single 35s precursor that is later processed (Udem and Warner, 1972). The 5s rRNA is transcribed separately (Figure 1) and in the opposite direction from the 35s precursor (Maxam et al., 1977; Valenzuela et al., 1977; Kramer et al., 1978). Most yeast strains contain rRNA genes that have seven Eco RI sites per repeat (Bell et al., 1977; Cramer et al., 1977; Nath and Bollon et al., 1977; Petes et al., 1978a; Philippsen et al., 1978). The seven Eco RI fragments derived from the rDNA are designated A through G, A being the largest and G the smallest. The arrangement of these fragments within the repeat and the position of the transcripts relative to these fragments is shown in Figure 1 (based l Present address: Molecular Genetics, Minnetonka. Minnesota 55343.

Inc.. 10320

Bren Road East,

on data of Bell et al., 1977; Philippsen et al., 1978, and others). The type of rRNA gene shown in Figure 1 has been called form I rDNA (Petes et al., 1978a). In other studies, a haploid yeast strain with a different type of rDNA (form II) was observed (Petes et al., 1978a). The size and restriction endonuclease map of the form II gene is very similar to that of form I, one difference being a deletion of the Eco RI site between Eco RI fragments B and E. Thus, in form II rRNA genes, there are only six Eco RI fragments, X’ (consisting of B and E sequences), A, C, D, F and G. The form l-form II heterogeneity has been used in several genetic studies of the rDNA. In one study, a haploid strain containing form I rDNA was crossed to a haploid containing form II (Petes and Botstein, 1977). When the resulting diploid was sporulated, in most of the tetrads, two spores contained form I rDNA and two spores contained form II. Thus, even though there are about 100 rRNA genes, they generally show the segregation pattern expected for single-copy genes. This experiment shows that most of the rRNA genes are located on a single chromosome; a more detailed mapping study showed these genes were located on chromosome XII (Petes, 1979a). The 2:2 segregation of form I and form II also shows that meiotic recombination between nonsister arrays of rRNA genes occurs infrequently (Petes and Botstein, 19771, although later experiments showed that sisterstrand recombination between rRNA genes occurs frequently (Pete& 1980; Szostak and Wu, 1980). It is not known whether the rRNA genes of yeast are arranged in a single tandem array. Studies of rDNA in which density gradients (Cramer et al., 19721, restriction enzymes (Cramer et al., 1977; Petes et al., 1978a) and electron microscopy of R-looped rDNA (Kaback and Davidson, 1980) are used have indicated extensive clustering (a minimum of 20 rRNA genes in tandem). The genetic demonstration that the rRNA genes segregate as a single unit (Petes and Botstein, 1977; Pete& 1979a, 1979b) suggests that all chromosomal rRNA repeats are within a single cluster. When the rRNA genes were mapped to chromosome XII (Petes, 1979a, 1979b), their relative position was found to be centromere-asp5-ga12-rRNA gene cluster-ura4. Since the rRNA gene cluster is flanked on two sides by single-copy genes, this tandem array must have at least two junctions with non-rDNA sequences. One approach to the analysis of these junctions is to isolate recombinant plasmids that contain restriction fragments derived from the rDNA repeat and restriction fragments of unique mobility. In a previous analysis of a collection of 75 recombinant plasmids that hybridized to yeast rRNA (Petes et al., 1978a), one plasmid (pY1 rB10) was found that contained restriction fragments of the sizes expected for the rDNA repeat as well as a fragment of unique mobility. This plasmid had Eco RI fragments the same size as rDNA fragments A, E and F, as well as a fourth

Cell 356

El

B I

Figure

fS/ I I

1. Restriction

C

lD( I I

and Transcription

A

lFlEl II I

B * I 5s

lG I

Map of the Form I rRNA Gene

The map is based on the data of Sell et al. 1977; Philippsen et al. 1978; and others. The letters A-G represent the seven fragments obtained by treatment of rDNA with Eco RI. The positions of the Eco RI restriction site are indicated on the upper horizontal line by short vertical lines. As indicated by the large arrow, the 3% rRNA precursor is transcribed from left to right. The thin portions of the arrow represent transcribed spacer. The 5s rRNA is transcribed in the opposite direction from the other rRNA species.

Eco RI fragment (X”) approximately 500 bp in length. For this report, we renamed the X” fragment Jl to indicate a potential junction fragment. In a rescreening of the same recombinant plasmid collection by S. Fuhrmann, a recombinant plasmid (pY1 rG11) was found that had Eco RI restriction fragments of the sizes C and G, as well as a fragment of 2650 bp, which we call J2. (In the first screen of the recombinant plasmids [Petes et al., 1978a], pY1 rG11 was misclassified as having B, C and G.) The fragments Jl and J2 could represent junctions between the rRNA genes and other chromosomal sequences, heterogeneity of restriction sites within the rDNA or an artifact of the cloning procedure. We show that neither Jl nor J2 is the result of cloning artifacts. The J2 fragment represents the centromere-distal junction of the rRNA genes with single-copy chromosomal sequences. The detailed physical and genetic characterization of Jl and J2 are described below. Results Physical Characterization of Putative Junction Fragments Jl and 52 The Eco RI fragments Ji and J2 were classified as putative junction fragments because they were present in recombinant plasmids (Jl in plasmid pY1 rB10, and J2 in plasmid pY1 rG11) that hybridized to yeast rRNA but had electrophoretic mobilities that were different from those Eco RI fragments characteristic of the rDNA (Petes et al., 1978a). As a first step in their characterization, we made restriction maps of the plasmids containing these fragments (Figure 2). A comparison with the restriction map of the rDNA Eco RI fragment B is also shown in this figure. ,To ensure that Jl and J2 were not artifacts of the cloning procedure, we isolated each fragment and, in separate experiments, used them as hybridization probes in a Southern analysis (Southern, 1975) of an Eco RI digest of genomic DNA. The collection of recombinant plasmids from which pY1 rBl0 and pY1 rG11 were derived was constructed with DNA isolated from a dlploid strain +D4 (Petes et al., 1978a). The diploid +D4 was constructed by mating

a form I rDNA haploid (A364a) to a form II rDNA haploid (2262). In our analysis of Eco RI-treated genomic DNA, we examined hybridization to both A364a and 2262. As shown in Figure 3A, when the Jl fragment is used as a hybridization probe, several positions of hybridization are found in A364a and 2262. In A364a, the form I strain, there is strong hybridization at the position of the Eco RI B rDNA fragment and weak hybridization at the expected position for Jl. When Jl is hybridized to DNA isolated from 2262, the form II strain, strong hybridization is seen at the position of X’ and weak hybridization at the position of Jl . These results indicate that the Jl fragment has homology to Eco RI rDNA fragments B and X’ and that Jl is present in both A364a and 2262. In some experiments in which 2262 is hybridized with Jl , a weak band is observed at the position of Eco RI fragment B. This weak hybridization to B in 2262 is observed because all strains that have form II rRNA genes also have a small fraction (about 5%) of form I genes (our unpublished data; J. H. Cramer, personal communication). The weakness of hybridization of Jl relative to the B and X’ fragments presumably reflects the high copy number of B and X’. To confirm that the faint band of hybridization at the position of Jl corresponded to the Jl fragment of the plasmid, we also performed Hind Ill-Eco RI double digests of the DNA isolated from A364a and 2262. The position of hybridization in the double-digested DNA shifted by the expected amount (data not shown). We conclude that Jl is not an artifact of the cloning procedure. Similar experiments were also performed with J2. When the J2 fragment was labeled and hybridized to Eco RI-treated DNA of A364a and 2262, intense hybridization was observed at the position of the B fragment in A364a and the X’ fragment of 2262 (Figure 38). This result shows that J2 contains B-like sequences; since X’ contains both B and E sequences (Petes et al., 1978a), the hybridization of J2 to X’ is expected. In the A364a DNA, a band of hybridization is present at the expected position of J2; this band is missing in DNA from 2262. We also performed two other types of experiments to determine whether J2 sequences were present in 2262. First, we repeated the Southern analysis with J2 as a probe, using DNA from A364a and 2262 that had been treated with both Eco RI and Hpa I. Since Hpa I cleaves both B and X’ but does not cleave J2 (Figure 2) the distance between the expected position of hybridization of J2 and the intense hybridization to B and X’ increases. In these double digests, we observed the J2 band in A364a. In the 2262 strain, no band was observed at the position of J2. The second type of experiment to detect J2 sequence in 2262 involved a subfragment of J2 that hybridizes exclusively to single-copy sequences. To determine which restriction sites to use to prepare such subfragments, we used the detailed restriction

Ribosomal 357

DNA Junctions

A

in Yeast

F

pYlrGll

E

B

l

G

j --

v

bjk

c

81 All

Ikb

Figure 2. Restriction Maps Compared to the Restriction

of the Plasmids pY1 rB10 and pY1 rG11 Map of the Form I rRNA Gene

In this diagram, the middle horizontal line represents the restriction map of a portion of the rRNA gene: Eco RI fragment D is not shown. The thickest portion of the horizontal lines represents DNA shown to hybridize with rDNA. The horizontal lines of intermediate thickness in Jl and J2 represent regions that are not yet classified as rDNA or non-rDNA. The thinnest horizontal lines represent single-copy nonrDNA sequences (as determined by Southern hybridization experiments). Complete restriction maps of the inserts in pYlrBl0 and pY1 rG11 were made only with Eco RI. For other enzymes, only the Jl and J2 restriction fragments were examined. The enzymes Pvu I (6). Hind Ill (1). Hpa I(J). Sma I (A) and Pst I (i) were used with both Jl and J2: with Pvu II (8) and Hae Ill CO), only the sites within the Blike portion of J2 were examined. The restriction sites mapped in B have been described previously (Petes et al., 1981). We determined the location of the 5s rRNA within the J2 fragment by a Southern analysis, using 5s rRNA provided by M. McMahon.

maps of Jl and J2 shown in Figure 2. Several points concerning these maps are worth mentioning. First, both Jl and J2 contain restriction sites in common with Eco RI fragment B. Second, Jl contains a different portion of B than J2. (We found in Southern blotting experiments that Jl and J2 do not crosshybridize [data not shown].) Third, the sum of the B sequences from Jl and those from J2 may be nearly equal to an intact B. We estimate that between 63% and 83% of B is present in Jl and J2 (Figure 2). Fourth, the J2 fragment includes the sequences complementary to 5s rRNA as determined by Southern blots of the pY1 rG11 plasmid with 32P-labeled 5s rRNA as a hybridization probe (data not shown). Fifth, the position at which rDNA adjoins non-rDNA in the J2 fragment (and probably also in the Jl fragment) is within the nontranscribed region of the gene. In the J2 fragment, there is a Pvu I site that is not present in Eco RI B (Figure 2). If this site is located in single-copy sequences, then the 400 bp Pvu I-Eco RI subfragment of J2 should hybridize exclusively to nonrDNA sequences. When we hybridized this fragment to an Eco RI digest of A364a DNA, only one strong band of hybridization at the position of J2 was observed (Figure 3~). The J2 fragment therefore is clearly a junction between rDNA and non-rDNA. Since the unique portion of J2 hybridizes to a single band in restriction digests with Eco RI, Pst I, Bgl I, Pvu I, Hpa I, Bgl II, Sma I and Sal I, the non-rDNA component of J2, is probably single-copy DNA. The faint band observed in the A364a genome that hybridizes at the

position of Eco RI C is the result of an intentional contamination of the J2 probe with a small amount of labeled Eco RI C DNA, which gave us an internal standard to compare the intensity of hybridization of J2 in A364a and 2262. When the Pvu I-Eco RI subfragment of J2 was hybridized to the form II rDNA strain 2262, no hybridization was observed (Figure 3C). The chromosomal DNA sequences adjacent to form II rDNA therefore must be different from those adjacent to form I. To confirm this result, we examined the meiotic segregation of the J2 fragment in a diploid strain (TP20) that was heterozygous for the form l-form II heterogeneity. When this strain was sporulated, most of the tetrads segregated two spores with form I rDNA to two spores with form II rDNA (Petes, 1979b3). For four such tetrads, we isolated DNA from spore cultures and hybridized the samples to the Pvu I-Eco RI fragment of J2. As expected, all form I spores hybridized to the probe and all form II spores failed to hybridize (Figure 4). In addition to confirming the conclusion that some form II strains lack the J2 sequence, these results show that J2 is genetically linked to form I rDNA and is therefore on chromosome XII. The probability that the observed segregation pattern of J2 and form I rDNA reflects random segregation rather than linkage is (3’ or 0.0008. We have been unable to prepare a subfragment of Jl that hybridizes exclusively to non-rDNA sequences. The Jl fragment is small and has no sites different from Eco RI fragment B other than the one Eco RI site (Figure 2). We cannot, therefore, exclude the possibility that this fragment represents a rearrangement internal to Eco RI fragment B found in some repeats but not others. Three arguments suggest Jl is more likely to be a junction fragment than an internal rearrangement of B. First, the intensity of hybridization of Jl in a Southern blot is approximately that expected for single-copy sequences. Second, we have separated rDNA from non-rDNA sequences of A364a in cesium chloride density gradients, using the procedure described previously (Petes et al., 1978a). The Jl fragment hybridizes to both rDNA and nonrDNA sequences (data not shown), which is the expected behavior for a junction fragment. Third, the Jl fragment has the correct transcriptional polarity to represent the opposite end of the rRNA gene tandem array from J2 (Figure 5). The arrangement of restriction fragments in pY1 rG11 is C-G-J2 (Figure 2). Since the 35s rRNA transcript initiates in the B fragment and continues in the direction G-C-D-A-F-E (Figure 11, the 35s rRNA is being transcribed away from J2 junction. The arrangement of restriction fragments in pY1 rB10 is A-F-E-J1 Transcription of the 35s rRNA, therefore, should be toward the Jl junction. In summary, we have demonstrated that J2 is a junction of rDNA with single-copy chromosomal se-

Cell 356

Figure 3. Hybridization Fragments to Genomic

of DNA

rDNA

Junction

(A) We used 20 X 10’ cpm f3’P) of purified Jl fragment to probe Eco RI digests of either A364a DNA (lane 1) or 2262 DNA (lane 2). After a one-day fld) exposure, a single band was observed at the position of Eco RI fragment B in A364a (2.22 kb) and at the position of Eco RI fragment X’ in 2262 (2.76 kb). Afler 14 days of exposure, an additional band at the position of Jl (500 bp) was found in both A364a and 2262. (6) We used 15 x 10’ cpm f3*P) of purified J2 fragment to probe Eco RI digests of DNA isolated from either A364a (lane 1) or 2262 (lane 2). In the A364a lane, two bands were detected. one at the expected position of J2 (2.65 kb). and one at the expected position of Eco RI fragment B. In the 2262 lane, only a single band was observed (at the position expected for Eco RI fragment X’). (C) A Pvu I-Eco RI subfragment of J2 was isolated and labeled with 32P. A mixture of 10 x 10’ cpm of this fragment and 10’ cpm of Eco RI rDNA fragment C was used to probe Eco RI digests of A364a Although DNA from both strains hybridized to Eco RI C, only A364a DNA hybridized to the J2 subfragment.

quences. The structure of the recombinant indicates that the major 3% rRNA species scribed away from this junction.

plasmid is tran-

Mapping of the 52 Junction The analysis described above showed that J2 represents one end of the rRNA gene cluster but did not show whether J2 was at the centromere-proximal or centromere-distal end. In principle, one could map the position of J2 relative to the centromere by using a diploid strain that is heterozygous for J2 (a form Iform II heterozygote), heterozygous for an insertion of a selectable gene within the rDNA (Szostak and Wu, 1979; Pete% 1980) and heterozygous for the centromere-linked chromosome XII marker ga12 (Petes, 197Qb), and then determining relative map positions by a three-point cross analysis. In general, in a cross involving three heterozygous markers (A, B and C), it is easiest to establish the order when the distance between A and B is similar to the distance between B and C. The difficulty in analyzing the position of J2 by a three-point cross is that the ga/2 locus is loosely linked to the rRNA gene cluster (Pete% 197Sb) and, since there is little nonsister meiotic recombination within the rDNA (Petes and Botstein, 1977), J2 would be expected to be tightly linked to an insertion within the rDNA. An unambiguous map order, therefore, would be extremely difficult to determine. To circumvent these problems, we used a two-step procedure. First, we examined genetic markers previously localized on chromosome XII to find one that showed tighter genetic linkage to the rDNA than did ga12. Second, we isolated recombinant DNA molecules that contained both DNA sequences overlapping with J2 and single-copy sequences that were further

(lane 1) and 2262

(lane 2) DNA.

from the rDNA than J2. By methods that will be described in detail below, we used these cloned sequences to construct yeast strains with a selectable marker near but not at J2. Using these strains, we then carried out three-point crosses to establish that J2 represents the centromere-distal junction of the rDNA. In our search for a genetic marker that was tightly linked to the rDNA, we used yeast strains that contained the selectable gene LEUP integrated in the rDNA. Such strains can be constructed by transforming a yeast strain that is mutant at the leu2 locus (on chromosome Ill) with a recombinant plasmid that has the wild-type LEU2 gene as well as yeast rDNA sequences (Szostak and Wu, 1979; Pete% 1980). Since recombinant plasmids integrate into the genome by sequence homology (Hinnen et al., 1978), most Leu+ transformants (leucine prototrophs) contain the LEU2+ gene integrated into the rDNA. In describing transformants that are mutant at the normal leu2 locus but contain a LEU2+ gene within the rDNA, we will use the notation leu2 rDNA::LEU2+. Using strains containing LEU2+ insertions in the rDNA, we looked for linkage between the rDNA and two genetic loci (car2 and pep3) that had been previously mapped to chromosome XII. The car2 mutation, previously mapped to chromosome XII by F. Hilger and R. Mortimer (personal communication), showed no meiotic linkage to the rDNA. When the diploid strain 2303 (genotype described in Experimental Procedures section) that was heterozygous for the LElJ2 insertion in the rDNA and heterozygous for the car2 mutation was sporulated, 11 of the asci were parental ditype (PD), 11 were nonparental ditype (NPD) and 38 were tetratype (T). Since meiotic linkage is indicated by a statis-

Ribosomal 359

DNA Junctions

in Yeast

(

Figure

35s

5. Proposed

5,5

Structure

-+

of the rRNA

35s

Gene Tandem

5s

Array

Capital letters are Eco RI fragments of form I rDNA. The directions of transcription of the 35s rRNA precursor and the 5s rRNA gene are indicated by arrows.

Figure 4. Hybridization of TP20

of Unique

DNA from J2 to Meiotic

Segregants

Eco RI-Hind Ill double digests of total nuclear DNA from the four spores of the TP20-45 tetrad (Table 1) were probed with the 0.42 kb Pvu I subfragment of J2. We added 15 x 10’ cpm ?P) of the J2Pvu I probe, plus 0.15 X 1O’cpm c3*P) of the Eco RI rDNA A fragment as a size standard. The 2.04 kb band is the large Hind Ill subfragment of 6 or X’ (Figure 2). The 1.64 kb band is the large Hind Ill subfragment of the Eco RI rDNA C fragment. The B and C subfragments hybridize because the J2 Pvu I probe is contaminated with small amounts of C sequences and the B portion of J2 (from pY1 rG11). Spores in lanes a and b are form II rDNA; spores in lanes c and d are form I rDNA.

tically significant excess of PD tetrads over NPD tetrads (Mortimer and Hawthorne, 19691, it is clear that car2 is not closely linked to the rDNA. The results obtained with the pep3 mutation were more useful. This mutation had been previously mapped onto chromosome XII by E. Jones (personal communication). To examine the linkage between pep3 and other chromosomal loci, we mated the haploid strain TPl 01-3a (leu2 [form I rDNA: :LEUP+] PfP3’ ga12) with the strain PEPld (leu2 pep3 GALP+). The resulting diploid (2301) was sporulated, and tetrads were analyzed by standard techniques (Mortimer and Hawthorne, 1969). The data from this analysis are shown in Table 1. It is evident that pep3

is closely linked (11 centifvlorgans) to the rRNA gene cluster. The map order of the genes that is required to make the map distances additive is centromere-ga/2pep3-rDNA. As the next step in the mapping of J2, we constructed yeast strains that had a selectable marker further from the rRNA gene cluster than J2. The first step in this construction was to isolate recombinant DNA molecules containing sequences that overlapped J2. Using the single-copy Pvu I-Eco RI fragment of J2 as a hybridization probe, we analyzed a collection of recombinant bacteriophages given to us by J. Woolford. This collection was constructed by limited Eco RI digestion of DNA isolated from the yeast strain A364a (J. Woolford and M. Rosbash, personal communication). The yeast DNA was inserted into the X bacteriophage vector Charon 4A (Blattner et al., 1977). We purified recombinant phages that crosshybridized with J2 (Benton and Davis, 1977) and analyzed the recombinant DNA by treatment with Eco RI. One of these phages (XZl , Figure 6) contained the J2 fragment and three other Eco RI fragments, K2 (1 .O kb), L2 (1.6 kb) and M2 (7.5 kb). The M2 fragment was purified, labeled with 32P and used in a second series of plaque hybridization experiments. A recombinant phage AZ2 (Figure 6) detected in this experiment contained M2 and a large Eco RI fragment N2 (11.3 kb). In a Southern analysis of Eco RI-treated A364a DNA, each of the fragments (K2 through N2) hybridizes to a single band of the appropriate molecular weight. None shows significant homology to the rDNA or other repeated sequences. Since the total length of these overlapping fragments is about 22 kb, this result suggests that J2 represents the true end of the array rather than a small insertion of single-copy DNA in the middle of the rRNA gene cluster. Stable yeast transformants are often the result of a homologous recombination event between the plasmid and the chromosome (Hinnen et al., 1978). In order to insert a selectable genetic marker near (but not at) J2, we constructed a recombinant plasmid (pZ1) that had the M2 fragment inserted into pBR322 at the Eco RI site. The plasmid pZ1 also had a Hind Ill fragment containing the wild-type URA3’ gene (Bach et al., 1979) inserted in the Hind Ill site of pBR322. This plasmid was transformed into a diploid strain (SSUlO) that was homozygous for mutations at the leu2 and ura3 loci, and was heterozygous for the form l-form II heterogeneity. Transformants that were Ura+

Cell 360

Table 1, Mapping of the Gene Order Gene Cluster in the Diploid 2301

Genetic

Interval

ga12-pep3

PD

NPD

Length of Interval kMY

T

71

9

192

45

216

1

53

11

ga/P-[rDNA::LEU2+]

61

17

194

54

was calculated

D

pYlrGll

pep3-[rDNA::LEU2+]

a The genetic distance Perkins (1949).

J2 GC

of ga12, pep3 and the rRNA

according

to the equation

M2

Our findings concerning the sequences at the junction between the rRNA genes and single-copy chromosomal DNA have several interesting implications relating to the evolution and function of repeated genes.

750 M2

N2

of

were isolated, and one of these transformants, SSUl O(pZ1 I#1 , was sporulated. In 18 of 18 tetrads analyzed, we observed 2:2 segregation for the Ura+ phenotype. For four of these tetrads, we isolated rDNA and analyzed the Eco RI restriction pattern. In all four tetrads, the Ura+ spores had form I rDNA and the uraspores had form II. This is the result expected if pZ1 integrated by homology near the junction of the form I rDNA cluster of SSUl O(pZ1 I#1 . The expected distance between J2 and the URA3’ insertion is approximately 10.5 kb, the sum of Eco RI fragments K2, L2 and M2 (Figure 6). The orientation of this URA3’ insertion relative to other markers on chromosome XII was then determined. A spore derived from SSUlO(pZ1 I#1 , SSUl O(pZ1 )#l -16d (/eu2 ura3 [M2: :pZl -URA3+] PEP3+ form I rDNA) was crossed to the strain Z30145b (ura3 leu2 [form I rDNA::LEUP+] pep3). The diploid strain (2302) was therefore heterozygous for the pep3 mutation, heterozygous for an insertion within the rDNA and heterozygous for an insertion of URA3’ in the single-copy sequences near J2. The data for the tetrad analysis of this strain are shown in Table 2. These data clearly demonstrate that the order of the genetic markers is centromere+ep3rDNA-URA3+ insertion. Since the URA3’ insertion is near the J2 junction, J2 must represent the centromere-distal junction. These data, taken together with those summarized in Figure 5, show that the 35s rRNA must be transcribed toward the centromere of chromosome XII, and the 5s rRNA must be transcribed away from the centromere. The complete map of markers on chromosome XII, as determined in these studies, is given in Figure 7. The mapping of the Jl fragment must be regarded as preliminary since we have not excluded the possibility that Jl is a rearrangement internal to the rRNA gene cluster. Discussion

~2 K2

J2 ah6

mioS

2.63

XZI

II 25

Figure 6. Recombinant jacent to J2

750

DNA Molecules

Containing

Sequences

Ad-

Thick horizontal lines represent ribosomal DNA; thin horizontal lines, unique single-copy DNA: intermediate horizontal lines, nonspecified DNA. Vertical lines show Eco RI cleavage sites: hZ1 was isolated by hybridization to the 400 bp unique DNA Pvu I-Eco RI subfragment of J2; X22 was isolated by hybridization to the M2 Eco RI fragment of Ml. Numbers below the lines are sizes of Eco RI fragments measured in kilobases.

Structure and Evolution of the Junction Both the centromere-distal junction J2 and the putative centromere-proximal junction Jl involve a fusion within the nontranscribed region of the ribosomal DNA. Both these junctional rRNA repeats are likely to be functional. The fraction of the rRNA repeat that is not transcribed can be calculated since the initiation and termination sites for the 35s rRNA precursor have been mapped. The initiation site is about 50 bp from the Eco RI site separating Eco RI fragments B and G (Bayev et al., 1980). The termination site is about 90 bp from the Eco RI site that separates the B and E fragments (Veldman et al., 1980). From these data and estimates of the length of rRNA gene (Philippsen et al., 1978), one can calculate that the nontranscribed region of rRNA repeat is about 25% of the total length. Assuming no selection against the location of the breakpoints, we calculate that the probability that two random junctions will be in this region is 0.06. Another interesting feature of these junctions is that the sum of the B-like sequences in Jl and J2 may nearly equal an intact B (Figure 2). A number of lower eucaryotes, s&h as Paramecium (Findley and Gall, 1978), have circular extrachromosomal rDNA molecules. One simple explanation of our observations is that the current Saccharomyces cerevisiae strains evolved from an organism that had extrachromosomal circular rDNA. In this organism, a single circular rDNA molecule recombined with sequences on chromosome XII. For this gene to be functional, the recombination event would have to occur within the nontranscribed region. Subsequently, a tandem array of rRNA genes could be generated by integration of other circular DNA molecules into the chromosomal copy. Such integration events would not change the original junctions. Although this hypothesis satisfactorily explains a number of our observations, it does not explain why the joints in J2 and Jl do not abut exactly. One possibility is that mutational

Ribosomal 361

DNA Junctions

in Yeast

Table 2. Genetic Mapping of pep3, an Insertion of LEUZ in the rDNA [rDNA::LEUP] and an Insertion of URA3’ in the Single-Copy Chromosomal Sequences Distal to J2 [M2::URA3’]

t

3lcM

PD

NPD

T

Length of Interval mw

pepS-(rDNA::LEU2+]

111

1

26

12

[rDNA::LEUP+]-[M2::Uf?A3’]

126

1

11

6

98

2

35

17

Genetic

Interval

pep3-[M2::URA3+]

18cM

asp 5 32cM gal2

+ K2

L2

M2

32

K2

L2

M2

!s@ I

45cM

/-/’

LEU2+ 11 CM

changes at the junction during scured the original breakpoints.

6CM

evolution

/ / /

M2

CONA --T&F--’

? t

a This analysis was carried out in the diploid 2302. ‘The genetic distance was calculated according to the equation of Perkins (1949). The only diploid chromosome composition consistent with these recombination intervals is: 12

mak 5

have

ob-

Single-Copy Sequences at the Junction The most surprising result concerning the single-copy sequences at the J2 junction was that different yeast strains can have totally different sequences at this position. This difference appears to correlate with whether a strain has form I or form II rDNA. Of three independent form I strains, all had J2 sequences (T. Zamb, unpublished data); the only well characterized form II strain (2262) lacked these sequences. This result has two possible interpretations; either the single-copy sequences adjacent to form I rDNA were deleted in the form II strains, or the form II strains have a substitution of novel single-copy sequences at the junction that are not present in form I strains. We have preliminary evidence, based on a Southern blot analysis, of the existence of an Eco RI fragment (2.7 kb in size) that hybridizes to Eco RI B and is allelic to J2. We have not been able to clone this junction. We also have preliminary evidence that 2262 does not hybridize to Eco RI fragment K2 but does hybridize to M2 and N2. We are attempting to determine whether L2 hybridizes to the 2.7 kb putative junction fragment. Hybridization between these fragments would indicate that 2262 has a simple deletion relative to A364a.. Whether 2262 has a deletion or substitution at the J2 end of the array, the observation that form I and form II tandem arrays have different centromere-distal junction sequences suggests either that these sequences are unimportant in controlling the expression of the cluster or that quite different sequences can play the same role. It was unexpected that both form I and form II strains would have Jl One of the Eco RI sites that defines the Ji fragment is the Eco RI site that separates the

I IcM 3cMl

pep/Y’

\

Figure

7. Genetic

\

\

\\

\

\\

\

\

\

\

\\

Map of the Yeast Chromosome

XII

The solid line represents map distances determined from tetrad analyses. The dashed line indicates linkage demonstrated by mitotic linkage analysis. The order of the markers in parentheses has not yet been determined. The circle in the diagram represents the chromosome XII centrometre. The map distances tin centiklorgans) for the intervals mak 7 2-centromere. centromere-asp5 and asp&gal2 were obtained from Mortimer and Schild (1980). The genetic length of the rDNA tandem array was also determined previously (Petes, 1979b). Other intervals were measured in this study.

Band E sequences (Figure 2). Since this site is absent from form II rDNA, it is surprising that 2262 has a cross-hybridizing sequence of the same size. One obvious possibility is that one of the small number of form I rRNA genes present in 2262 is at the Jl junction.

Cell 362

Although junctions between a repeated eucaryotic gene and single-copy chromosomal DNA have not been characterized previously, a junction between a satellite DNA of Drosophila and the repeated copia gene has been analyzed (Carlson and Brutlag, 1978). This junction may have resulted from the transposable copia element inserting into the tandemly repeated satellite sequence. The satellite sequences adjacent to the copia element showed considerable divergence from the consensus satellite sequences. If sequence homogeneity among repeated genes is conserved by repeated cycles of unequal recombination (Smith, 1973) then sequence divergence at the junction is expected (discussed by Brutlag, 1980). The restriction pattern of the rRNA genes adjacent to Jl and J2 (Figure 2) is identical to that of the consensus rRNA repeat. Two possible explanations of this result exist. One explanation is that for these yeast genes mechanisms other than unequal recombination may be responsible for maintaining sequence homogeneity. One such mechanism that has been demonstrated experimentally is intrachromosomal gene conversion, the nonreciprocal transfer of information from one repeat to another (Klein and Petes, 1981; Jackson and Fink, 1981; S. C. Falco and D. Botstein, personal communication). An alternative explanation is that the sequences are kept identical by selection. We have been able to find only two junction-containing plasmids of the rRNA genes within a large collection of recombinant plasmids. We have estimated that there is an 85% chance that a single-copy gene will be present in this collection (Petes et al., 1978b). In Southern blot analysis, using hybridization probes from all portions of the repeat, we have not found any additional putative junction fragments. This result suggests that there is only a single tandem array of rRNA genes. In our strains, we have also been unable to detect orphons, single copies of the repeat separate from the tandem array. In one yeast strain, orphon sequences hybridizing to a cloned rDNA from Xenopus laevis have been reported (Childs et al., 1981). The lack of these sequences in our strain may reflect a strain difference, a difference in the hybridization conditions or a difference in the hybridization probes. We believe that the most likely interpretation of the available data is that all chromosomal rRNA genes are located in a single tandem array and transcribed in a single direction (35s rRNA transcribed toward the centromere). In some strains, a small fraction (fewer than five copies per cell) of the rDNA may be extrachromosomal (Szostak and Wu, 1979). Meiotic Recombination between Nonsister Tandem Arrays In previous experiments, meiotic recombination between nonsister tandem arrays was assayed in a diploid that was heterozygous for the form l-form II heterogeneity (Petes and Botstein, 1977; Petes, 1979b). Recombination was detected by finding

spores that had a mixture of form I and form II rDNA. We found that approximately 5% of the tetrads had recombined between nonsister tandem arrays (Petes, 1979b). The expected frequency of recombination based on the number of recombination events per genome and the fraction of the genome that is rDNA (Petes and Botstein, 1977) was 1 OO-fold higher. We could not rule out in these experiments the possibility that meiotic recombination was suppressed because of the form l-form II heterogeneity. This possibility, however, is excluded by the data of Table 2. The recombination distance between the URA3+ insertion on the centromere-distal side of the array and the pep3 mutation on the centromere-proximal side of the array is 17 centiMorgans. A distance of 17 centiMorgans indicates that 34% of the tetrads have had a recombination event in this interval. Since the expected frequency of recombination in the rDNA is five events per cell (Petes and Botstein, 1977) meiotic recombination is suppressed by at least a factor of 15. The recombination distance of 17 centiMorgans is a maximum estimate of the amount of recombination in the rDNA, since exchange can also occur in the single-copy sequences between pep3 and the URA3+ insert. Since the diploid analyzed in this cross was homozygous for form I rDNA, the observed recombination suppression is not a function of the form l-form II heterogeneity. Experimental

Procedures

Yeast Strains The genotypes of A364a and 2262 have been described previously (Petes and Sotstein. 1977). The diploid strain +D4 (provided by L. Hartwell) was constructed by mating A364a with 2262. The genotype of the diploid TP20 has also been reported previously (Pete% 1979b). The car2 genetic marker was provided by F. Hilger in the strain Be266 (a car2 ga12 ura4). In the mapping of the car2 mutation, we used two strains, CAR1 la (a leu2 car2 ma4 gal2 form I rDNA) and TPl 014b (a /eu2 his4 tfpl ga12 [form I rDNA: :LEU2 ‘I). The notation [form I rDNA::LEU:!‘] indicates that the wild-type LEU2 gene was inserted into the rDNA by transformation of a leu2- strain with a recombinant plasmid containing the LEU2+ gene (Pete& 1960). The diploid 2303 is the result of a cross between TPl 01-4b and CAR1 1 a. We obtained the genetic marker pep3 (from E. Jones) in the strain MET1 25A-273 (a frp I gal2 pep3- 72). We mapped the pep3 gene by using the haploid strains PEPld (U ade2 his4 lys2 ufa3 leu2 pep3 form I rDNA) and TPl Ol-3a (a his4 trpl leu2 ga/2 [form I rDNA:: LEU2+]). The diploid 2301 was obtained by mating PEPld with TPI Ol-3a. To insert a selectable gene WRA3’) near the J2 junction, we transformed the diploid strain SSUlO (constructed by S. SmolikUtlaut) with the recombinant plasmid pZ1. The genotype of SSUlO is: (I HIS5 HIS4 LYSl 1 leu2 ura3 ASP5 gal2 form I rDNA ------a his5 his4 lysl 1 leu2 ura3 asp5 gal2 form II rDNA The URA + transformed diploid was named SSU 1 OpZl #l For mapping the position of the pZ1 insertion, we used two haploid strains, SSUlOpZl#l-16d (a his4 leu2 ura3 asp5 ga12 form I rDNA [M2:: pZ1 -URA’]) and Z301-45b (a ade2 his4 leu2 ura3 pep3 [form I rDNA: :LEUP’D. The diploid 2302 was constructed by mating these two haploid strains. Media Most media used in these experiments were standard (Sherman et al., 1972). The only nonstandard medium used was M-ornithine. The

Ribosomal 363

DNA Junctions

in Yeast

recipe for this medium (F. Hilger. personal communication) is: yeast nitrogen base (Difco) without amino acids and without ammonium sulfate (1 g). dextrose (12 g), ornithine (0.6 g), agar (12 g) and 600 ml water. Since ornithine is not heat-stable, it was added after the medium was sterilized.

marked “advertisement” in accordance solely to indicate this fact. Received

August

3. 1981;

revised

with 18 U.S.C.

November

Section

1734

2. 1981

References Genetic Analysis Tetrad analysis was performed by standard techniques (Mortimer and Hawthorne, 1969). The pep3 gene was scored on YPG plates at 37% (E. Jones, personal communication), and car2 was scored on M-ornithine plates (F. Hilger, personal communication). Yeast transformation was performed according to the method of Hinnen et al. (1978). Recombinant Plasmid and Recombinant Bacteriophage Collections The recombinant plasmids used in these studies have been described previously (Petes et al., 1978a, 1978b). The collection was made by inserting randomly sheared fragments of DNA (isolated from the yeast strain +D4) into the Eco RI site of the plasmid pMB9. The recombinant bacteriophage collection was provided by J. Woolford and M. Rosbash. The collection was constructed by inserting partial Eco RI DNA fragments from the yeast strain A364a into the A vector Charon 4A (Blattner et al., 1977). Construction of Recombinant Plasmid pZ1 The plasmid pZ1 was constructed by inserting the Eco RI fragment M2 from the recombinant bacteriophage AZ2 into the Eco RI site of the recombinant plasmid MB1 068. The plasmid MB1 068 (provided by S. C. Falco) contains the wild-type URA3 gene of yeast inserted as a Hind Ill fragment (Bach et al., 1979) in the Hind Ill site of pBR322. For the construction of pZ1, the MB1 068 DNA was treated sequentially with Eco RI (Miles Laboratory) and calf intestine alkaline phosphatase (Davis et al., 1980). The enzyme was removed by phenol and ether extraction. The treated MB1068 DNA was mixed with Eco RI-treated pZ1 DNA, and T4 DNA ligase was added (Davis et al., 1980). The ligation mixture was then used to transform the ampicillinsensitive and tetracycline-sensitive E. coli strain C600 to ampicillin resistance (Mandel and Higa, 1970). DNA Isolation Yeast DNA was isolated by use of cesium chloride gradients that contained the fluorescent dye Hoechst 33258 (Petes et al., 1977; D. H. Williamson, personal communication). DNA from recombinant plasmids and recombinant bacteriophages were isolated by standard procedures (Davis et al., 1980). In those experiments in which a DNA restriction fragment was purified, the procedure of Given and Kieff (1978) was used. Southern Analysis and Plaque Hybrfdizatlon For restriction analysis of DNA, the buffers and other conditions recommended by the supplier were used. Following agarose gel electrophoresis. fragments were transferred to nitrocellulose (Southern, 1975). These filters were hybridized to 32P-labeled DNA probes that had been prepared by nick translation (Schachat and Hogness, 1973). The conditions were similar to those used by Botchan et al. (1976) except that dextran sulfate was included in some reactions (Wahl et al., 1979). Hybridization was detected with Kodak XR-5 film and Du Pont Cronex intensifying screens. Plaque hybridization was performed by the procedure of Benton and Davis (1977). Acknowledgments We thank D. Stamenkovich and Drs. Jones and Hilger studies. We also thank Drs. and Smolik-Utlaut for their The work was supported Health, the National Cancer Winchell Cancer Fund. The costs of publication payment of page charges.

and C. Desich for technical assistance for providing yeast strains used in these Farber, Klapholtz. Klein, Giroux, Wagstaff comments on the manuscript. by grants from the National Institutes of Institute and the Damon Runyon-Walter of this article were defrayed in part by the This article must therefore be hereby

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