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In situ detection of enzymes in yeast
ANALYTICAL
BIOCHEMISTRY
162,384-388
(1987)
In Situ Detection
of Enzymes in Yeast
JOSEPH FARGNOLI AND WILLIAM Waksman Institute of Microbiology, Rutgers-The
SOFER
State University of New Jersey, Piscataway, New Jersey 08854
ReceivedAugust21, 1986 A very simple method that allows for the rapid in situ assay of enzyme activity in yeast is described. Single colonies are collected on sticks (or glass micropipets), or multiple colonies are collected on sandpaper, and crushed onto nitrocellulose filters. The tilters in turn are stained for theenzymeof interestusinga histochemical assay.The method is quantitative and was found to IIIC. work well for four enzymes in yeast. 0 1987Ademic FWSS, KEY WORDS: enzyme assays; gene expression; enzymes; proteins; nitrocellulose; dehydrogenases.
Methods for the in situ detection of enzyme activity in colonies of microorganisms have proved quite useful. In genetic expcriments especially, it is often crucial to be able to detect the presence of a rare colony lacking (or carrying) a particular enzyme activity. For an ongoing project in our laboratory, we needed an in situ assay that could be used on yeast colonies that was applicable to a wide variety of enzymes and that was rapid, sensitive, and quantitative. In the past, such in situ assays have generally made use of histochemical reagents and permeabilized yeast cells. Cells were treated with a variety of physical and chemical agents that allowed entrance of the reagents necessary for appropriate color development. Among the methods used to increase permeability were drying (I), treatment with organic solvents (2), treament with cell wall digesting enzymes (3), and freeze-thawing (4). (See Felix (5) for a review of the methods investigators have used to permeabilize cells.) These methods all worked to varying degrees, but all have significant disadvantages for the purposes outlined above. For example, unless special care is taken to wash the cells after permeabilization, the presence of
0003-2697187
Copyri%t8
$3.00
1987 ty Academic F-ES,Inc. AU rights of reproduction in any form reserved.
384
endogenous substrates may confound the assay. This is true for dehydrogenase assays, for example, where the presence of substrates that can be utilized by a variety of dehydrogenases may allow release of NADH+ (which will yield a positive signal if a tetrazoliumbased assay is being used) regardless of the presence or absence of the enzyme being assayed in the cell. Another potential problem is that some of the methods used for permeabilization may denature the enzyme of interest. A third disadvantage is that all methods that depend on visualizing enzyme activity in whole yeast cells require replication of the original plate because, in general, the permeabilization kills the cells. In this report, we describe an extremely simple method for in situ enzyme detection. We transfer cells to a nitrocellulose filter either individually with an applicator stick or, in the case of multiple colonies, with sandpaper and, by applying a moderate amount of pressure, break the cells, thereby releasing the internal enzymes. Because proteins bind firmly to nitrocellulose filters, the lilters may easily be washed thoroughly to remove lowmolecular-weight substrates, and after washing, activity may be. detected by simple histochemical staining. In addition, the sandpaper
ENZYME
DETECTION
acts like a replica plate, picking up only a fraction of the cells on a plate and leaving the remaining colonies in place for subsequent manipulation. We have tested this method with four yeast enzymes and found that it works well in all cases. MATERIALS
AND METHODS
Yeast strains and growth conditions. Yeast strains 500-l 1 (a ural trpl leu2 adel-11 ADRS-F adrl-1 adm) and Sl50-2b (a leu2-3 leu2-112 trpl-289 ura3-52 his3Al) were gifts of V. MacKay (Zymogen Corp., Seattle, WA). Strain B6748 (CYCl:lacZ CYC7: CYMZ+ ura3-52 his3Al leu2-3 leu2-112 trpl-289 canl-100 cyh2R) was obtained from R. Moerschell (University of Rochester). Strains YAT-44 (a arg6 pho3 pho80) and YAT-42 (a arg6 pho3 pho4) were obtained from R. Kramer (Hoffman-LaRoche, Nutley, NJ). Unless otherwise indicated, all strains were maintained and grown at 30°C on standard YPAD’ (1% yeast extract, 2% bactopeptone, 0.003% adenine sulfate, 2% dextrose) medium on 2% agar plates or in liquid medium. Preparation of staining medium. All enzyme activities were detected using histochemical staining solutions dissolved in agarose. Antidiuretic hormone (ADH) activity. A solution containing 5 ml of nitro blue tetrazolium (50 mg/ml), 1 ml of ethanol, 1 ml of NAD+ (50 mg/ml), and 1 ml of 1 M TrisHCl, pH 8.0, was heated to 50°C and mixed with a 1% agarose solution in TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA) at the same temperature. This solution was poured into two 82-mm plastic petri dishes and allowed to solidify. The plates ’ Abbreviations
used: YPAD, 1% yeast extract, 2%
bactopeptone, 0.003% adenine sulfate, 2% dextrose; TBE, 0.089 M Trig 0.089 M boric acid, 0.002 M EDTA; X-Gal, S-bromo4chloro-3-indolyl-&mactopyranoside; X-P, 5-bromo4chloro-3-indolyl phosphate; ADH, antidiuretic hormone.
IN YEAST
385
may be stored for up to three months in the cold before use. @-Galactosidase activity. One percent agarose was dissolved in 50 IIIM sodium phosphate (pH 7.0), the solution was cooled to 5O”C, and sufficient 5-bromo-4-chloro-3indolyl-@+galactopyranoside (X-Gal) in dimethylformamide was added to make a 50 mg/ml solution. Acid phosphatase activity. One percent agarose was dissolved in 0.09 M sodium citrate, pH 4.8, the solution was cooled to about 5O”C, and 5-bromo-4-chloro-3-indolyl phosphate (X-P) was added to a final concentration of 1 mg/ml. Alkaline phosphatase activity. The same staining solution was used for alkaline phosphatase as for acid phosphatase except that 0.5 M Tris-HCl, pH 9.0, 25 mM MgS04 was substituted for citrate buffer. Stainingprocedures. The main elements of the procedure are shown in Fig. 1. Individual colonies or liquid cultures. Flatheaded wooden applicator sticks (or glass micropipets) were either dipped into liquid cultures or touched to the surface of yeast colonies and firmly pressed onto a strip of nitrocellulose until an indentation on the membrane was visible. Washing was accomplished by vigorously shaking the membrane in three changes of buffer of the same composition as in the staining plate (but without histochemical reagents) after which it was blotted dry between several layers of paper towels. The membrane was then placed on an agarose staining plate and incubated at room temperature (for ADH and acid/alkaline phosphatase) or at 37°C (for p-galactosidase) until a color developed (generally from 5 to 10 min). Whole plates. Sandpaper (3M-L-240; wet or dry) was cut into circles of the same diameter as a petri plate and carefully pressed onto the surface of a plate containing yeast colonies that had grown overnight on YPAD medium. Impressions of the colonies were clearly visible on the surface of the sandpaper
386
FARGNOLI
AND SOFER
Sandpapel
Yeast colonies
a] Transfer and Crush
1
Nitrocellulose
Filter
b] Wash filter, place on substrate
c] Incubate +
cgxl + Enzyme
Stain
FIG. 1. Blotting protocol. Left side-procedure for blotting individual yeast colonies or cells in liquid culture. Right side-procedure for blotting and screening colonies growing on solid media. See Materials and Methods for details.
when it was lifted away. After the sandpaper was placed on a nitrocellulose membrane, the cells were broken and the enzyme was released by applying pressure with a small wooden roller (2-3 times) over the sandpaper/nitrocellulose sandwich. RESULTS
Figure 2 shows the results of crushing various yeast cells on nitrocelhtlose filters and staining for enzyme activity. In all these experiments, colonies were picked from solid media with a glass micropipet and crushed on nitrocellulose as described above. Stain-
ing for ADH, acid phosphatase, and /3-galactosidase is shown. In the first two cases, strains lacking enzyme activity were compared with those with wild-type levels of enzyme. In the case of Bgalactosidase (linked to a CYCl controlling region), a fully derepressed strain was compared to partially derepressed, fully repressed, and @-galactosidase-negative strains. In all cases, the amount of stain that was deposited on the filter seemed to reflect the level of activity of the enzyme in the strain from which it came. Since our goal was to be able to assay many colonies on a single plate all at once, we investigated the possibility of crushing
ENZYME Acid Phosphatase
ADH
+
DETECTION
-
387
IN YEAST
of ADH ranging from 0.25 X lo4 to about 2.0 X lo4 enzyme units. DISCUSSION
+
FIG. 2. Enzyme stain from blotted cells. Colonies grown on solid media were transferred to nitrocellulose using a 0.2-ml glass pipet and were. crushed open as described under Materials and Methods. Strain S 150-2b containing ADH activity (+) and strain 500-l 1 lacking ADH activity (-) were used to stain for ADH. Strains YAT-44 (+) and YAT-42 (-) were used to stain for acid phosphatasc activity. Strain B6748 containing a CYCIZucZ fusion gene was tested for &galactosidase activity under different growth conditions. One set of cells was grown on medium where sucrose was substituted for glucose, which should result in derepression of the CYCl-fucZ (+). A second set of cells was grown on glucose, which partially represses the expression of the CYCI-1acZ gene (+/-). A third set of cells was grown under anaerobic conditions, which leads to almost complete repression of the fusion gene (-). As a further control, strain 500- 11, which completely lacks &galactosidase activity, was also blotted (-).
We were surprised to find that simply applying pressure to yeast cells results in the release of their macromolecular contents. Simply placing the colonies on the nitrocellulose does not seem effective in this regard. In fact, the amount of enzyme released (as measured by the amount of staining) seemed strongly dependent on the amount of pressure applied. Other methods that we could have used in an effort to release enzyme, including drying, freezing, and enzyme treatment, were not as convenient or as simple to use. While the assay worked well for all the enzymes that were tested, it is possible that some enzymes may be denatured on the nitrocellulose and not show activity. In that case, nylon membranes may offer an alternative. Other than enzyme assays, there are other potential applications for this procedure.
many colonies simultaneously on the nitrocellulose. For the experiment shown in Fig. 3, alternating rows of ADH-positive and -negative colonies were placed on a plate and allowed to grow overnight on YPAD medium. The colonies were transfered to nitrocellulose with sandpaper, and the filter was stained for ADH activity. As is evident from the figure, only the ADH-containing colonies left enzyme activity on the filter. To estimate the sensitivity and linearity of the assay, we crushed varying numbers of cells on the nitrocellulose. As can be seen in Fig. 4, the method yields a linear deposition of stain when the initial cell concentration ranged from 0.5 to 4 X lo* cells/ml. This level of staining, determined by scanning similarly sized spots containing known amounts of enzyme, corresponded to a level
FIG. 3. Screening whole plates. Alternating rows of strain S 150-2b (-I-) and 500- 1 1 (-) were grown overnight on standard YPAD medium. These colonies were subsequently transferred and crushed onto a nitrocellulose filter using sandpaper and were stained for ADH activity.
B-Galactosidase
+
+I-
-
388
FARGNOLI
AND SOFER
40,
30..
ADH activity
0.4
0.8
1.2
1.6
Number of cells (x 16’ ) /ml FIG. 4. Blotting sensitivity. A late-log-phase liquid culture of S150-2b cells containing 1.5 X IO-* cells/ml was serially diluted in liquid medium. Cells from each dilution were transferred, crushed, and assayed for ADH activity (inset). The blot was then scanned using a densitometer. Enzyme activity was determined by comparing these values with values obtained from a densitometer scan of known amounts of yeast ADH.
Crushing may provide an alternative way of liberating proteins from the cells so that they may be identified using antibodies (6). In addition, it is possiblethat crushing may release sufficient DNA (or RNA) from colonies so that colony hybridizations (7) might be conveniently carried out. ACKNOWLEDGMENTS The research reported here was supported by NIH Grants GM28791 and ES02920 to W.S.
REFERENCES I. Moshowitz, D. B. (1976) Anal. B&hem. 70,94-99. 2. DeSmet, M. J., Kingma, J., and Witholt, B. (I 978) B&him. Biophys. Acta 506,64-80. 3. Yee, W. S., Decker, R. W., and Brunk, C. F. (1976) Biochim. Biophys. Acta 447,385-390. 4. Souzu, H. (1980) Biochim. Biophys. Acta 603, 13-26. 5. Felix, H. (1982) Anal. B&hem. 120,2 I l-234. 6. Nathon, N., and Lyons, S. (1984) Proc. Natl. Acad. Sci. USA g&7426-7430. 7. Grunstein, M., and Hogness, D. (1975) Proc. Natl. Acad. Sci. USA 72,3961-3965.
BIOCHEMISTRY
162,384-388
(1987)
In Situ Detection
of Enzymes in Yeast
JOSEPH FARGNOLI AND WILLIAM Waksman Institute of Microbiology, Rutgers-The
SOFER
State University of New Jersey, Piscataway, New Jersey 08854
ReceivedAugust21, 1986 A very simple method that allows for the rapid in situ assay of enzyme activity in yeast is described. Single colonies are collected on sticks (or glass micropipets), or multiple colonies are collected on sandpaper, and crushed onto nitrocellulose filters. The tilters in turn are stained for theenzymeof interestusinga histochemical assay.The method is quantitative and was found to IIIC. work well for four enzymes in yeast. 0 1987Ademic FWSS, KEY WORDS: enzyme assays; gene expression; enzymes; proteins; nitrocellulose; dehydrogenases.
Methods for the in situ detection of enzyme activity in colonies of microorganisms have proved quite useful. In genetic expcriments especially, it is often crucial to be able to detect the presence of a rare colony lacking (or carrying) a particular enzyme activity. For an ongoing project in our laboratory, we needed an in situ assay that could be used on yeast colonies that was applicable to a wide variety of enzymes and that was rapid, sensitive, and quantitative. In the past, such in situ assays have generally made use of histochemical reagents and permeabilized yeast cells. Cells were treated with a variety of physical and chemical agents that allowed entrance of the reagents necessary for appropriate color development. Among the methods used to increase permeability were drying (I), treatment with organic solvents (2), treament with cell wall digesting enzymes (3), and freeze-thawing (4). (See Felix (5) for a review of the methods investigators have used to permeabilize cells.) These methods all worked to varying degrees, but all have significant disadvantages for the purposes outlined above. For example, unless special care is taken to wash the cells after permeabilization, the presence of
0003-2697187
Copyri%t8
$3.00
1987 ty Academic F-ES,Inc. AU rights of reproduction in any form reserved.
384
endogenous substrates may confound the assay. This is true for dehydrogenase assays, for example, where the presence of substrates that can be utilized by a variety of dehydrogenases may allow release of NADH+ (which will yield a positive signal if a tetrazoliumbased assay is being used) regardless of the presence or absence of the enzyme being assayed in the cell. Another potential problem is that some of the methods used for permeabilization may denature the enzyme of interest. A third disadvantage is that all methods that depend on visualizing enzyme activity in whole yeast cells require replication of the original plate because, in general, the permeabilization kills the cells. In this report, we describe an extremely simple method for in situ enzyme detection. We transfer cells to a nitrocellulose filter either individually with an applicator stick or, in the case of multiple colonies, with sandpaper and, by applying a moderate amount of pressure, break the cells, thereby releasing the internal enzymes. Because proteins bind firmly to nitrocellulose filters, the lilters may easily be washed thoroughly to remove lowmolecular-weight substrates, and after washing, activity may be. detected by simple histochemical staining. In addition, the sandpaper
ENZYME
DETECTION
acts like a replica plate, picking up only a fraction of the cells on a plate and leaving the remaining colonies in place for subsequent manipulation. We have tested this method with four yeast enzymes and found that it works well in all cases. MATERIALS
AND METHODS
Yeast strains and growth conditions. Yeast strains 500-l 1 (a ural trpl leu2 adel-11 ADRS-F adrl-1 adm) and Sl50-2b (a leu2-3 leu2-112 trpl-289 ura3-52 his3Al) were gifts of V. MacKay (Zymogen Corp., Seattle, WA). Strain B6748 (CYCl:lacZ CYC7: CYMZ+ ura3-52 his3Al leu2-3 leu2-112 trpl-289 canl-100 cyh2R) was obtained from R. Moerschell (University of Rochester). Strains YAT-44 (a arg6 pho3 pho80) and YAT-42 (a arg6 pho3 pho4) were obtained from R. Kramer (Hoffman-LaRoche, Nutley, NJ). Unless otherwise indicated, all strains were maintained and grown at 30°C on standard YPAD’ (1% yeast extract, 2% bactopeptone, 0.003% adenine sulfate, 2% dextrose) medium on 2% agar plates or in liquid medium. Preparation of staining medium. All enzyme activities were detected using histochemical staining solutions dissolved in agarose. Antidiuretic hormone (ADH) activity. A solution containing 5 ml of nitro blue tetrazolium (50 mg/ml), 1 ml of ethanol, 1 ml of NAD+ (50 mg/ml), and 1 ml of 1 M TrisHCl, pH 8.0, was heated to 50°C and mixed with a 1% agarose solution in TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA) at the same temperature. This solution was poured into two 82-mm plastic petri dishes and allowed to solidify. The plates ’ Abbreviations
used: YPAD, 1% yeast extract, 2%
bactopeptone, 0.003% adenine sulfate, 2% dextrose; TBE, 0.089 M Trig 0.089 M boric acid, 0.002 M EDTA; X-Gal, S-bromo4chloro-3-indolyl-&mactopyranoside; X-P, 5-bromo4chloro-3-indolyl phosphate; ADH, antidiuretic hormone.
IN YEAST
385
may be stored for up to three months in the cold before use. @-Galactosidase activity. One percent agarose was dissolved in 50 IIIM sodium phosphate (pH 7.0), the solution was cooled to 5O”C, and sufficient 5-bromo-4-chloro-3indolyl-@+galactopyranoside (X-Gal) in dimethylformamide was added to make a 50 mg/ml solution. Acid phosphatase activity. One percent agarose was dissolved in 0.09 M sodium citrate, pH 4.8, the solution was cooled to about 5O”C, and 5-bromo-4-chloro-3-indolyl phosphate (X-P) was added to a final concentration of 1 mg/ml. Alkaline phosphatase activity. The same staining solution was used for alkaline phosphatase as for acid phosphatase except that 0.5 M Tris-HCl, pH 9.0, 25 mM MgS04 was substituted for citrate buffer. Stainingprocedures. The main elements of the procedure are shown in Fig. 1. Individual colonies or liquid cultures. Flatheaded wooden applicator sticks (or glass micropipets) were either dipped into liquid cultures or touched to the surface of yeast colonies and firmly pressed onto a strip of nitrocellulose until an indentation on the membrane was visible. Washing was accomplished by vigorously shaking the membrane in three changes of buffer of the same composition as in the staining plate (but without histochemical reagents) after which it was blotted dry between several layers of paper towels. The membrane was then placed on an agarose staining plate and incubated at room temperature (for ADH and acid/alkaline phosphatase) or at 37°C (for p-galactosidase) until a color developed (generally from 5 to 10 min). Whole plates. Sandpaper (3M-L-240; wet or dry) was cut into circles of the same diameter as a petri plate and carefully pressed onto the surface of a plate containing yeast colonies that had grown overnight on YPAD medium. Impressions of the colonies were clearly visible on the surface of the sandpaper
386
FARGNOLI
AND SOFER
Sandpapel
Yeast colonies
a] Transfer and Crush
1
Nitrocellulose
Filter
b] Wash filter, place on substrate
c] Incubate +
cgxl + Enzyme
Stain
FIG. 1. Blotting protocol. Left side-procedure for blotting individual yeast colonies or cells in liquid culture. Right side-procedure for blotting and screening colonies growing on solid media. See Materials and Methods for details.
when it was lifted away. After the sandpaper was placed on a nitrocellulose membrane, the cells were broken and the enzyme was released by applying pressure with a small wooden roller (2-3 times) over the sandpaper/nitrocellulose sandwich. RESULTS
Figure 2 shows the results of crushing various yeast cells on nitrocelhtlose filters and staining for enzyme activity. In all these experiments, colonies were picked from solid media with a glass micropipet and crushed on nitrocellulose as described above. Stain-
ing for ADH, acid phosphatase, and /3-galactosidase is shown. In the first two cases, strains lacking enzyme activity were compared with those with wild-type levels of enzyme. In the case of Bgalactosidase (linked to a CYCl controlling region), a fully derepressed strain was compared to partially derepressed, fully repressed, and @-galactosidase-negative strains. In all cases, the amount of stain that was deposited on the filter seemed to reflect the level of activity of the enzyme in the strain from which it came. Since our goal was to be able to assay many colonies on a single plate all at once, we investigated the possibility of crushing
ENZYME Acid Phosphatase
ADH
+
DETECTION
-
387
IN YEAST
of ADH ranging from 0.25 X lo4 to about 2.0 X lo4 enzyme units. DISCUSSION
+
FIG. 2. Enzyme stain from blotted cells. Colonies grown on solid media were transferred to nitrocellulose using a 0.2-ml glass pipet and were. crushed open as described under Materials and Methods. Strain S 150-2b containing ADH activity (+) and strain 500-l 1 lacking ADH activity (-) were used to stain for ADH. Strains YAT-44 (+) and YAT-42 (-) were used to stain for acid phosphatasc activity. Strain B6748 containing a CYCIZucZ fusion gene was tested for &galactosidase activity under different growth conditions. One set of cells was grown on medium where sucrose was substituted for glucose, which should result in derepression of the CYCl-fucZ (+). A second set of cells was grown on glucose, which partially represses the expression of the CYCI-1acZ gene (+/-). A third set of cells was grown under anaerobic conditions, which leads to almost complete repression of the fusion gene (-). As a further control, strain 500- 11, which completely lacks &galactosidase activity, was also blotted (-).
We were surprised to find that simply applying pressure to yeast cells results in the release of their macromolecular contents. Simply placing the colonies on the nitrocellulose does not seem effective in this regard. In fact, the amount of enzyme released (as measured by the amount of staining) seemed strongly dependent on the amount of pressure applied. Other methods that we could have used in an effort to release enzyme, including drying, freezing, and enzyme treatment, were not as convenient or as simple to use. While the assay worked well for all the enzymes that were tested, it is possible that some enzymes may be denatured on the nitrocellulose and not show activity. In that case, nylon membranes may offer an alternative. Other than enzyme assays, there are other potential applications for this procedure.
many colonies simultaneously on the nitrocellulose. For the experiment shown in Fig. 3, alternating rows of ADH-positive and -negative colonies were placed on a plate and allowed to grow overnight on YPAD medium. The colonies were transfered to nitrocellulose with sandpaper, and the filter was stained for ADH activity. As is evident from the figure, only the ADH-containing colonies left enzyme activity on the filter. To estimate the sensitivity and linearity of the assay, we crushed varying numbers of cells on the nitrocellulose. As can be seen in Fig. 4, the method yields a linear deposition of stain when the initial cell concentration ranged from 0.5 to 4 X lo* cells/ml. This level of staining, determined by scanning similarly sized spots containing known amounts of enzyme, corresponded to a level
FIG. 3. Screening whole plates. Alternating rows of strain S 150-2b (-I-) and 500- 1 1 (-) were grown overnight on standard YPAD medium. These colonies were subsequently transferred and crushed onto a nitrocellulose filter using sandpaper and were stained for ADH activity.
B-Galactosidase
+
+I-
-
388
FARGNOLI
AND SOFER
40,
30..
ADH activity
0.4
0.8
1.2
1.6
Number of cells (x 16’ ) /ml FIG. 4. Blotting sensitivity. A late-log-phase liquid culture of S150-2b cells containing 1.5 X IO-* cells/ml was serially diluted in liquid medium. Cells from each dilution were transferred, crushed, and assayed for ADH activity (inset). The blot was then scanned using a densitometer. Enzyme activity was determined by comparing these values with values obtained from a densitometer scan of known amounts of yeast ADH.
Crushing may provide an alternative way of liberating proteins from the cells so that they may be identified using antibodies (6). In addition, it is possiblethat crushing may release sufficient DNA (or RNA) from colonies so that colony hybridizations (7) might be conveniently carried out. ACKNOWLEDGMENTS The research reported here was supported by NIH Grants GM28791 and ES02920 to W.S.
REFERENCES I. Moshowitz, D. B. (1976) Anal. B&hem. 70,94-99. 2. DeSmet, M. J., Kingma, J., and Witholt, B. (I 978) B&him. Biophys. Acta 506,64-80. 3. Yee, W. S., Decker, R. W., and Brunk, C. F. (1976) Biochim. Biophys. Acta 447,385-390. 4. Souzu, H. (1980) Biochim. Biophys. Acta 603, 13-26. 5. Felix, H. (1982) Anal. B&hem. 120,2 I l-234. 6. Nathon, N., and Lyons, S. (1984) Proc. Natl. Acad. Sci. USA g&7426-7430. 7. Grunstein, M., and Hogness, D. (1975) Proc. Natl. Acad. Sci. USA 72,3961-3965.