JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 472-481 Copyright © 1977 American Society for Microbiology
Vol. 129, No. 1 Printed in U.S.A.
Deoxyribonucleic Acid Sequence Organization of a Yeast Plasmid DENNIS M. LIVINGSTON* AND HANNAH L. KLEIN Department of Genetics, University of Washington, Seattle, Washington 98195
Received for publication 23 June 1976
Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-,um DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-,um DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed. DNA preparation. Cells were converted to spheroplasts with glusulase (Endo Laboratories) according to the method of Peterson et al. (20) with minor modifications (19). After glusulase digestion the spheroplasts were centrifuged at 3,000 x g for 5 min. The pellet was resuspended in sorbitol-citrate-ethylenediaminetetraacetic acid (EDTA) buffer (19) at a final concentration of 2 x 1010 spheroplasts/ml. Two-micrometer DNA was prepared by a modification of the procedure of Hirt (14). The solution of spheroplasts was added dropwise to an equal volume of 50 mM Tris-hydrochloride, 10 mM EDTA, 2% wt/ vol sodium dodecyl sulfate, pH 8.0, in a centrifuge tube (15 by 105 mm). The tube was then capped with parafilm and slowly inverted 10 times. Next, 5 M NaCl was added to a final concentration of 1 M and again mixed by inverting the tube 10 times. This solution was permitted to stand on ice for 16 h before being subjected to centrifugation at 27,000 x g for 1 h. A 5.0-ml portion of the supernatant solution was combined with 6.0 g of CsCl (Harshaw), 1.0 ml of 10 mg per ml ethidium bromide solution (Sigma), and suffilcient buffer (10 mM Tris-hydrochloride, 1 mM EDTA, pH 7.5) to bring the total weight of the solution to 13.60 g. The mixture was then subjected to centrifugation at 33,000 rpm for 60 h in a Spinco 40 rotor. Fractions were collected and analyzed for DNA content (10). The fractions containing 2-j,m DNA were pooled, and ethidium bromide was removed by three successive extractions with equal amounts of isopropanol:water (5:1). The fractions MATERIALS AND METHODS were dialyzed against 10 mM Tris-hydrochloride, 1 Cell growth. Saccharomyces cerevisiae A364A was mM EDTA, pH 7.5, and stored at 0°C. Enzyme digestions. The restriction endonuclease grown at 30°C in synthetic medium containing per liter: 6.7 g of yeast nitrogen base without amino Pst was purified from Providencia stuartii 164 by the acids (Difco), 20 g of glucose, 40 mg each of histidine. procedure of Smith and Davies (unpublished obsertyrosine, and lysine, 20 mg of adenine, 10 mg of vations). The restriction nucleases EcoRI and HincII uracil, and 1 mCi of [6-3H]uracil (New England Nu- were purchased from New England Bio Labs. Reaction mixtures contained 100 mM Tris-hydroclear). Cells in late log phase were collected by chloride (pH 7.4), 5 mM MgCl2, 50 mM NaCl, 10 mM centrifugation. 472
The yeast Saccharomyces cerevisiae is a simple eukaryote with a genome of seventeen linkage groups and a haploid deoxyribonucleic acid (DNA) content of 9 x 109 to 10 x 109 daltons (P. Whitney, personal communication). In addition to nuclear and mitochondrial DNA, yeast cells also contain a circular 2-,um plasmid DNA (25) called 2-/Am DNA. Measurement of the contour lengths of circular 2-/um DNA and linear fragments of nuclear DNA in a preparation of total yeast DNA by electron microscopy indicates that there are approximately 100 copies of 2-/im DNA per cell (4). The 2-,um DNA kinetic complexity of 6,000 base pairs is in agreement with the analytical complexity of molecular weight 4.0 x 106 (18). This indicates that there is no significant intermolecular sequence heterogeneity and little intramolecular repetitive DNA. Two-micrometer DNA is not found in isolated nuclei (4) or in mitochondria (3). Recently, Griffiths et al. (8) and also Guerineau et al. (9) have isolated mutations conferring resistance to venturicidin and triethyl tin which segregate as if on a cytoplasmic element. Because these mutations exhibit no genetic linkage to any known mitochondrial markers, the possibility exists that these resistance genes may be located on 2,um DNA.
YEAST PLASMID DNA
VOL. 129, 1977
/8-mercaptoethanol, 100 /ig of gelatin per ml, 10 jig of ribonuclease per ml (bovine pancreas, five times crystallized, Sigma), and between 1.0 and 2.0 ,ug of DNA in a total volume of 100 ,ul. An appropriate amount of restriction nucleases was added to complete digestions in 2 h at 37°C. Agarose gel electrophoresis. A vertical slab gel electrophoresis apparatus with gel dimensions of 4.5 by 6 by 0.25 inches and sample wells measuring 0.2 inch in width was employed. Gels were run at a potential of 2 V/cm according to the method of Greene et al. (7). Gel concentrations were either 0.7 or 1.6% agarose. For size determinations, markers of EcoRI-digested phage X and phage P22 DNA were used (13). Electron microscopy. Foldback and heteroduplex molecules were prepared according to the method of Davis et al. (6) as modified by Cech and Hearst (2). DNA samples in 50 mM sodium phosphate buffer (pH 6.9) were denatured by incubation in 0.11 M NaOH for 5 min at 37°C followed by an additional 5 min at room temperature. The solution was then adjusted to pH 8.5 and made 100 mM Tris, 10 mM EDTA, and 0.18 ionic strength (IL) with a TrisEDTA-NaCl solution. The renaturation temperature was made equivalent to 550C by the addition of an equal volume of formamide (recrystallized from Matheson, Coleman & Bell, 99% pure). Renaturation was carried out to Cot 5 x 10-4 to form intramolecular hybrids and to Cot 5 x 10-2 to form heteroduplexes. After renaturation, the hyperphase was diluted in 50% formamide-50 mM Tris-hydrochloride (pH 8.5), and cytochrome c (horse heart type VI, Sigma) was added to 80 ,g/ml. The hyperphase was spread onto a hypophase of 20% formamide-5 mM Tris-hydrochloride (pH 8.5). Grids were stained with uranyl acetate and then rotary shadowed with Pt-Pd (80:20). A thin carbon film was evaporated onto the grids after metal shadowing to improve the film stability prior to examination in a Philips 300 electron microscope. Electron micrographs were taken at a magnification of 20,000 x. Contour lengths were measured with a Graf/Pen model GP-3 digitizer connected to a Hewlett-Packard calculator. The measured lengths were calibrated with OX 174 singlestrand circles of a molecular length of 5.25 kilobases (kb) and T7 linear duplexes of a molecular length of 37.9-kb pairs.
RESULTS Two-micrometer DNA contains an inverted repeat. A nontandom inverted repetition has been identified by electron microscopic examination of denatured and briefly reannealed 2,um DNA. Any 2-,um duplex circle containing a single-strand interruption (open circle) can be denatured to produce a linear and a circular single-strand molecule. Upon renaturation to Cot 5 x 10-4, only intramolecular reannealing occurs as diagrammed in Fig. 1. The closed single-strand circle renatures to form a dumbbell-shaped structure having two single-strand loops connected by a duplex segment. Examples of these structures are shown in Fig. 2a-c. The
473
denaturation
/\
reassociation
I FIG. 1.Frf
c
a Irf
b b
b
b
c'
c
c
e
d
d
el
FIG. 1. Formation offoldback molecules. A circular molecule with an inverted repeat contains four domains: two inverted repeated sequences (jagged lines b-c) and two different interstitial segments, A (segment a-f) and B (segment d-e). Circular molecules with single-strand interruptions denature to form a single-strand linear plus a single-strand circular molecule. Upon renaturation of the inverted repeat-foldback molecules form with either one or two single-strand loops. In this figure the placement of the single-strand interruption has been made arbitrarily between markers d and e.
single-strand linear molecule sometimes renatures to give a duplex segment with a singlestrand loop at one end and two single-strand tails at the other end, similar to those pictured in Fig. 2d-f. Since the open circles arose by random single-strand interruptions of superhelical molecules, it was not possible to use this collection of open circles to determine the location of the inverted repeat with respect to a unique site.
474
LIVINGSTON AND KLEIN
J. BACTERIOL.
*.4 ;
*,,~,
v, ,
FIG. 2. Electron micrographs offoldback molecules. Panels (a) through (c) are dumbbell-shaped molecules formed by renaturing single-strand circles released upon denaturation of2-,um DNA containing single-strand interruptions. Panels (d) through (fp are foldback products ofPst-cleaved 2-,un molecules. Denaturation and renaturation were carried out as described under Materials and Methods. The bar in section (d) represents 1 kb.
To determine the location of the inverted repeat with respect to a reference point, a collection of nonpermuted linear molecules was produced by digesting 2-,Im DNA with the restriction endonuclease Pst. Figure 4b shows that this enzyme makes one double-strand break in 2-,um DNA. An electron microscopic analysis of Pst-cleaved molecules which had been denatured and then reannealed to permit only intramolecular renaturation revealed that all the molecules (>95%) form the structures shown in Fig. 2d-f. If the inverted repeat occupies a unique location, then the loop, stem, and tail segments of the molecules pictured in Fig. 2d-f should have unique contour lengths. Figure 3a shows the distribution of loop lengths. The average loop length of 116 molecules is 1.89 ± 0.13 kb. The duplex stem-length distribution is
shown in Fig. 3b. The mean stem length is 0.64 ± 0.07 kb. There is greater variation in the single-strand tail lengths, possibly because of exonuclease contamination. Therefore, we have analyzed the tail lengths of those molecules that are at least 85% of the 2-,um molecule duplex length. Tail-length distributions are shown in Fig. 3c. The tail lengths are 1.30 + 0.18 and 0.80 ± 0.18 kb. The sum of the tail lengths (2.10 ± 0.18 kb) is in good agreement with the length of the larger loop (2.06 ± 0.17) determined by measuring 50 dumbbell-shaped molecules (Fig. 2a-c), and we therefore assume that the subset (43/116) of foldback molecules used to measure the tail lengths represents the entire population. These results show that 2,um DNA molecules contain an inverted repeat, and that the inverted repeated sequences are
YEAST PLASMID DNA
VOL. 129, 1977
n) 4)
m
-V
z
Length (kb)
FIG. 3. Observed length distributions of segments of the Pst-cleaved foldback molecules. Panel (a) shows the distribution of single-strand loop lengths. The mean size is 1.89 + 0.13 kb with a sample size of 116. Panel (b) shows the distribution of duplex stem lengths. The mean size is 0.64 + 0.07 kb with a sample size of 119. Panel (c) shows the distribution of single-strand tail sizes. Only foldback molecules with a contour length of at least 85% of the monomer duplex length are included. The mean size of the short tail ( ) is 0.80 + 0.18 kb. The mean size of the long tail (- - -) is 1.30 + 0.18 kb. The sample size is 43.
located at specific sites on the 2-,um DNA molecule. The molecule can therefore be considered to consist of four domains, the two repeated sequences and two different interstitial segments as diagrammed in Fig. 1. Two forms of 2-,um DNA. Figure 4c shows that the cleavage of 2-,tm DNA by the restriction enzyme EcoRI results in four fragments. The sum of the molecular weight of these fragments is 8.0 x 106, twice the molecular weight of 2-,um DNA. Figure 4d shows that HincII produces a similar pattern with four bands of different sizes. The cleavage of 2-,um DNA into four fragments that total twice the molecular weight of a single molecule by both of these endonucleases suggests that two forms of 2-,um DNA exist, each having the same molecular weight and each having two EcoRI and two HinclI sites. A plausible explanation of the two forms of 2,um DNA is that both contain the same se-
475
quence information, but that the arrangement of this information differs in the two forms. Sharp et al. (23), Kleckner et al. (16), Sheldrick and Berthelot (24), and Hsu and Davidson (15) have noted that a reciprocal recombination event between inverted repeat sequences reorients one interstitial sequence with respect to the other. When this event occurs in a 2-,um DNA molecule, the result, shown in Fig. 5, is a 1800 reorientation of one of the interstitial sequences. The four EcoRI fragments can be explained by postulating that in each 2-,um DNA molecule the restriction endonuclease produces two breaks, one in each of the two interstitial sequences. The largest and smallest of the four fragments derive from one form of 2-,um DNA, whereas the two intermediate-size fragments are the restriction products of the second (inverted) form of 2-,um DNA. The two EcoRI sites cannot lie within only one of the two interstitial sequences or within the inverted repeated sequences. In either of these cases only two, instead of four, restriction fragments would be found after EcoRI digestion. Indeed, electron microscopic analysis of denatured and briefly renatured EcoRI fragments does not show any foldback molecules. This demonstrates that each EcoRI fragment contains only one unit of the repeated sequences and that the two EcoRI sites must be in different interstitial segments. The same model will explain the HinclI data. Heteroduplex analysis. That the two interstitial segments exist in two different orientations as postulated in Fig. 5 has been shown by an examination of heteroduplex molecules. This has been achieved by denaturation and renaturation of Pst-cleaved 2-,4m DNA to a 0ot allowing intermolecular reassociation to occur. The expected structures are diagrammed in Fig. 6. A type I heteroduplex has a singlestrand bubble, the length of which should equal the loop length of the Pst-cleaved foldback molecules. Type II heteroduplexes should have single-strand tails at each end which are the same as the single-strand tails of Pst-cleaved foldback molecules. Figure 7 shows two electron micrographs of typical type I heteroduplexes. Twenty-four type I heteroduplexes have been photographed and measured. The strands of the single-strand bubble are 1.56 + 0.34 kb. This is in agreement with the 1.89-kb loop length value of the Pst-cleaved foldback molecules (Fig. 3). Although some type II heteroduplexes have been observed, these have not been analyzed because of variability in the number and length of single-strand tails. However, the identification of the two types of heteroduplexes shows that part of the 2-,nm genome is
476
J. BACTERIOL.
LIVINGSTON AND KLEIN
a b
c d
f
e
q
h
j
k
60
40
60
4.48 3.73
373
3.73
4.19
373
3.29
329 3.0306 .6 3358 306 335 38
2.0
2.40
2.11
2 12
188
1.88
1.21
1 21
2 11
30
S
1.96 1.73
1.0
_
_ 0 .8 1.21 °
-.5
C
1_21
~~~~~~~~~~~~~~~~~~~~
'C
0.6 _
Vol. 129, No. 1 Printed in U.S.A.
Deoxyribonucleic Acid Sequence Organization of a Yeast Plasmid DENNIS M. LIVINGSTON* AND HANNAH L. KLEIN Department of Genetics, University of Washington, Seattle, Washington 98195
Received for publication 23 June 1976
Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-,um DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-,um DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed. DNA preparation. Cells were converted to spheroplasts with glusulase (Endo Laboratories) according to the method of Peterson et al. (20) with minor modifications (19). After glusulase digestion the spheroplasts were centrifuged at 3,000 x g for 5 min. The pellet was resuspended in sorbitol-citrate-ethylenediaminetetraacetic acid (EDTA) buffer (19) at a final concentration of 2 x 1010 spheroplasts/ml. Two-micrometer DNA was prepared by a modification of the procedure of Hirt (14). The solution of spheroplasts was added dropwise to an equal volume of 50 mM Tris-hydrochloride, 10 mM EDTA, 2% wt/ vol sodium dodecyl sulfate, pH 8.0, in a centrifuge tube (15 by 105 mm). The tube was then capped with parafilm and slowly inverted 10 times. Next, 5 M NaCl was added to a final concentration of 1 M and again mixed by inverting the tube 10 times. This solution was permitted to stand on ice for 16 h before being subjected to centrifugation at 27,000 x g for 1 h. A 5.0-ml portion of the supernatant solution was combined with 6.0 g of CsCl (Harshaw), 1.0 ml of 10 mg per ml ethidium bromide solution (Sigma), and suffilcient buffer (10 mM Tris-hydrochloride, 1 mM EDTA, pH 7.5) to bring the total weight of the solution to 13.60 g. The mixture was then subjected to centrifugation at 33,000 rpm for 60 h in a Spinco 40 rotor. Fractions were collected and analyzed for DNA content (10). The fractions containing 2-j,m DNA were pooled, and ethidium bromide was removed by three successive extractions with equal amounts of isopropanol:water (5:1). The fractions MATERIALS AND METHODS were dialyzed against 10 mM Tris-hydrochloride, 1 Cell growth. Saccharomyces cerevisiae A364A was mM EDTA, pH 7.5, and stored at 0°C. Enzyme digestions. The restriction endonuclease grown at 30°C in synthetic medium containing per liter: 6.7 g of yeast nitrogen base without amino Pst was purified from Providencia stuartii 164 by the acids (Difco), 20 g of glucose, 40 mg each of histidine. procedure of Smith and Davies (unpublished obsertyrosine, and lysine, 20 mg of adenine, 10 mg of vations). The restriction nucleases EcoRI and HincII uracil, and 1 mCi of [6-3H]uracil (New England Nu- were purchased from New England Bio Labs. Reaction mixtures contained 100 mM Tris-hydroclear). Cells in late log phase were collected by chloride (pH 7.4), 5 mM MgCl2, 50 mM NaCl, 10 mM centrifugation. 472
The yeast Saccharomyces cerevisiae is a simple eukaryote with a genome of seventeen linkage groups and a haploid deoxyribonucleic acid (DNA) content of 9 x 109 to 10 x 109 daltons (P. Whitney, personal communication). In addition to nuclear and mitochondrial DNA, yeast cells also contain a circular 2-,um plasmid DNA (25) called 2-/Am DNA. Measurement of the contour lengths of circular 2-/um DNA and linear fragments of nuclear DNA in a preparation of total yeast DNA by electron microscopy indicates that there are approximately 100 copies of 2-/im DNA per cell (4). The 2-,um DNA kinetic complexity of 6,000 base pairs is in agreement with the analytical complexity of molecular weight 4.0 x 106 (18). This indicates that there is no significant intermolecular sequence heterogeneity and little intramolecular repetitive DNA. Two-micrometer DNA is not found in isolated nuclei (4) or in mitochondria (3). Recently, Griffiths et al. (8) and also Guerineau et al. (9) have isolated mutations conferring resistance to venturicidin and triethyl tin which segregate as if on a cytoplasmic element. Because these mutations exhibit no genetic linkage to any known mitochondrial markers, the possibility exists that these resistance genes may be located on 2,um DNA.
YEAST PLASMID DNA
VOL. 129, 1977
/8-mercaptoethanol, 100 /ig of gelatin per ml, 10 jig of ribonuclease per ml (bovine pancreas, five times crystallized, Sigma), and between 1.0 and 2.0 ,ug of DNA in a total volume of 100 ,ul. An appropriate amount of restriction nucleases was added to complete digestions in 2 h at 37°C. Agarose gel electrophoresis. A vertical slab gel electrophoresis apparatus with gel dimensions of 4.5 by 6 by 0.25 inches and sample wells measuring 0.2 inch in width was employed. Gels were run at a potential of 2 V/cm according to the method of Greene et al. (7). Gel concentrations were either 0.7 or 1.6% agarose. For size determinations, markers of EcoRI-digested phage X and phage P22 DNA were used (13). Electron microscopy. Foldback and heteroduplex molecules were prepared according to the method of Davis et al. (6) as modified by Cech and Hearst (2). DNA samples in 50 mM sodium phosphate buffer (pH 6.9) were denatured by incubation in 0.11 M NaOH for 5 min at 37°C followed by an additional 5 min at room temperature. The solution was then adjusted to pH 8.5 and made 100 mM Tris, 10 mM EDTA, and 0.18 ionic strength (IL) with a TrisEDTA-NaCl solution. The renaturation temperature was made equivalent to 550C by the addition of an equal volume of formamide (recrystallized from Matheson, Coleman & Bell, 99% pure). Renaturation was carried out to Cot 5 x 10-4 to form intramolecular hybrids and to Cot 5 x 10-2 to form heteroduplexes. After renaturation, the hyperphase was diluted in 50% formamide-50 mM Tris-hydrochloride (pH 8.5), and cytochrome c (horse heart type VI, Sigma) was added to 80 ,g/ml. The hyperphase was spread onto a hypophase of 20% formamide-5 mM Tris-hydrochloride (pH 8.5). Grids were stained with uranyl acetate and then rotary shadowed with Pt-Pd (80:20). A thin carbon film was evaporated onto the grids after metal shadowing to improve the film stability prior to examination in a Philips 300 electron microscope. Electron micrographs were taken at a magnification of 20,000 x. Contour lengths were measured with a Graf/Pen model GP-3 digitizer connected to a Hewlett-Packard calculator. The measured lengths were calibrated with OX 174 singlestrand circles of a molecular length of 5.25 kilobases (kb) and T7 linear duplexes of a molecular length of 37.9-kb pairs.
RESULTS Two-micrometer DNA contains an inverted repeat. A nontandom inverted repetition has been identified by electron microscopic examination of denatured and briefly reannealed 2,um DNA. Any 2-,um duplex circle containing a single-strand interruption (open circle) can be denatured to produce a linear and a circular single-strand molecule. Upon renaturation to Cot 5 x 10-4, only intramolecular reannealing occurs as diagrammed in Fig. 1. The closed single-strand circle renatures to form a dumbbell-shaped structure having two single-strand loops connected by a duplex segment. Examples of these structures are shown in Fig. 2a-c. The
473
denaturation
/\
reassociation
I FIG. 1.Frf
c
a Irf
b b
b
b
c'
c
c
e
d
d
el
FIG. 1. Formation offoldback molecules. A circular molecule with an inverted repeat contains four domains: two inverted repeated sequences (jagged lines b-c) and two different interstitial segments, A (segment a-f) and B (segment d-e). Circular molecules with single-strand interruptions denature to form a single-strand linear plus a single-strand circular molecule. Upon renaturation of the inverted repeat-foldback molecules form with either one or two single-strand loops. In this figure the placement of the single-strand interruption has been made arbitrarily between markers d and e.
single-strand linear molecule sometimes renatures to give a duplex segment with a singlestrand loop at one end and two single-strand tails at the other end, similar to those pictured in Fig. 2d-f. Since the open circles arose by random single-strand interruptions of superhelical molecules, it was not possible to use this collection of open circles to determine the location of the inverted repeat with respect to a unique site.
474
LIVINGSTON AND KLEIN
J. BACTERIOL.
*.4 ;
*,,~,
v, ,
FIG. 2. Electron micrographs offoldback molecules. Panels (a) through (c) are dumbbell-shaped molecules formed by renaturing single-strand circles released upon denaturation of2-,um DNA containing single-strand interruptions. Panels (d) through (fp are foldback products ofPst-cleaved 2-,un molecules. Denaturation and renaturation were carried out as described under Materials and Methods. The bar in section (d) represents 1 kb.
To determine the location of the inverted repeat with respect to a reference point, a collection of nonpermuted linear molecules was produced by digesting 2-,Im DNA with the restriction endonuclease Pst. Figure 4b shows that this enzyme makes one double-strand break in 2-,um DNA. An electron microscopic analysis of Pst-cleaved molecules which had been denatured and then reannealed to permit only intramolecular renaturation revealed that all the molecules (>95%) form the structures shown in Fig. 2d-f. If the inverted repeat occupies a unique location, then the loop, stem, and tail segments of the molecules pictured in Fig. 2d-f should have unique contour lengths. Figure 3a shows the distribution of loop lengths. The average loop length of 116 molecules is 1.89 ± 0.13 kb. The duplex stem-length distribution is
shown in Fig. 3b. The mean stem length is 0.64 ± 0.07 kb. There is greater variation in the single-strand tail lengths, possibly because of exonuclease contamination. Therefore, we have analyzed the tail lengths of those molecules that are at least 85% of the 2-,um molecule duplex length. Tail-length distributions are shown in Fig. 3c. The tail lengths are 1.30 + 0.18 and 0.80 ± 0.18 kb. The sum of the tail lengths (2.10 ± 0.18 kb) is in good agreement with the length of the larger loop (2.06 ± 0.17) determined by measuring 50 dumbbell-shaped molecules (Fig. 2a-c), and we therefore assume that the subset (43/116) of foldback molecules used to measure the tail lengths represents the entire population. These results show that 2,um DNA molecules contain an inverted repeat, and that the inverted repeated sequences are
YEAST PLASMID DNA
VOL. 129, 1977
n) 4)
m
-V
z
Length (kb)
FIG. 3. Observed length distributions of segments of the Pst-cleaved foldback molecules. Panel (a) shows the distribution of single-strand loop lengths. The mean size is 1.89 + 0.13 kb with a sample size of 116. Panel (b) shows the distribution of duplex stem lengths. The mean size is 0.64 + 0.07 kb with a sample size of 119. Panel (c) shows the distribution of single-strand tail sizes. Only foldback molecules with a contour length of at least 85% of the monomer duplex length are included. The mean size of the short tail ( ) is 0.80 + 0.18 kb. The mean size of the long tail (- - -) is 1.30 + 0.18 kb. The sample size is 43.
located at specific sites on the 2-,um DNA molecule. The molecule can therefore be considered to consist of four domains, the two repeated sequences and two different interstitial segments as diagrammed in Fig. 1. Two forms of 2-,um DNA. Figure 4c shows that the cleavage of 2-,tm DNA by the restriction enzyme EcoRI results in four fragments. The sum of the molecular weight of these fragments is 8.0 x 106, twice the molecular weight of 2-,um DNA. Figure 4d shows that HincII produces a similar pattern with four bands of different sizes. The cleavage of 2-,um DNA into four fragments that total twice the molecular weight of a single molecule by both of these endonucleases suggests that two forms of 2-,um DNA exist, each having the same molecular weight and each having two EcoRI and two HinclI sites. A plausible explanation of the two forms of 2,um DNA is that both contain the same se-
475
quence information, but that the arrangement of this information differs in the two forms. Sharp et al. (23), Kleckner et al. (16), Sheldrick and Berthelot (24), and Hsu and Davidson (15) have noted that a reciprocal recombination event between inverted repeat sequences reorients one interstitial sequence with respect to the other. When this event occurs in a 2-,um DNA molecule, the result, shown in Fig. 5, is a 1800 reorientation of one of the interstitial sequences. The four EcoRI fragments can be explained by postulating that in each 2-,um DNA molecule the restriction endonuclease produces two breaks, one in each of the two interstitial sequences. The largest and smallest of the four fragments derive from one form of 2-,um DNA, whereas the two intermediate-size fragments are the restriction products of the second (inverted) form of 2-,um DNA. The two EcoRI sites cannot lie within only one of the two interstitial sequences or within the inverted repeated sequences. In either of these cases only two, instead of four, restriction fragments would be found after EcoRI digestion. Indeed, electron microscopic analysis of denatured and briefly renatured EcoRI fragments does not show any foldback molecules. This demonstrates that each EcoRI fragment contains only one unit of the repeated sequences and that the two EcoRI sites must be in different interstitial segments. The same model will explain the HinclI data. Heteroduplex analysis. That the two interstitial segments exist in two different orientations as postulated in Fig. 5 has been shown by an examination of heteroduplex molecules. This has been achieved by denaturation and renaturation of Pst-cleaved 2-,4m DNA to a 0ot allowing intermolecular reassociation to occur. The expected structures are diagrammed in Fig. 6. A type I heteroduplex has a singlestrand bubble, the length of which should equal the loop length of the Pst-cleaved foldback molecules. Type II heteroduplexes should have single-strand tails at each end which are the same as the single-strand tails of Pst-cleaved foldback molecules. Figure 7 shows two electron micrographs of typical type I heteroduplexes. Twenty-four type I heteroduplexes have been photographed and measured. The strands of the single-strand bubble are 1.56 + 0.34 kb. This is in agreement with the 1.89-kb loop length value of the Pst-cleaved foldback molecules (Fig. 3). Although some type II heteroduplexes have been observed, these have not been analyzed because of variability in the number and length of single-strand tails. However, the identification of the two types of heteroduplexes shows that part of the 2-,nm genome is
476
J. BACTERIOL.
LIVINGSTON AND KLEIN
a b
c d
f
e
q
h
j
k
60
40
60
4.48 3.73
373
3.73
4.19
373
3.29
329 3.0306 .6 3358 306 335 38
2.0
2.40
2.11
2 12
188
1.88
1.21
1 21
2 11
30
S
1.96 1.73
1.0
_
_ 0 .8 1.21 °
-.5
C
1_21
~~~~~~~~~~~~~~~~~~~~
'C
0.6 _
