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Protocol 4.2: Steps of a Genomic Southern Blot
PLANT GENOMIC SOUTHERN BLOTTING
tions of the rDNA clone that represent 102, 103, and 104 copies. Include a molecular weight standard, such as k HindIII-cut DNA. If a
nonradioactive, biotin-labeling detection system will be used, it is useful to use biotin-labeled k HindIII-cut DNA so that the molecular weight
marker can be detected when the hybridized probe is detected.
5. Run the gel; stain and photograph the gel. Prepare a Southern blot of
the gel. Probe the gel with labeled SSRUBISCO or rDNA clones. The
protocols for these procedures are described in Chapter 3.
6. Determine the size of the fragments that hybridize to the probes. Compare the intensity of the hybridization signal of the DNA sample lanes
with the reconstruction lanes to estimate the copy number of the fragments. What differences are observed between the SSRUBISCO and
the rDNA probes? What are the differences between the times needed
to detect a hybridization signal for the two different probes? Examine
the published restriction endonuclease maps for each probe to predict
the sizes of fragments observed. Predict the hybridization results for
tandem duplications of the rDNA probe. How might the protocol be
modified to increase the signal strength for the probe that shows the
less intense signal?
Dellaporta, S. L., Wood, J., and Hicks, J. B. (1983). A plant DNA minipreparation: version
II. Plant Mol. Biol. Rep. 1, 19-21.
Russell, P. J. (1992). "Genetics," 3 ed. Harper Collins, New York.
Ribosomal DNA (rDNA)
Agarwal, M. L., Aldrich, J., Agarwal, A., and Cullis, C. A. (1992). The flax ribosomal RNAencoding genes are arranged in tandem at a single locus interspersed by 'non-rDNA'
sequences. Gene 120, 151-156.
Goldsbrough, P. B., and Cullis, C. A. (1981). Characterisation of the genes for ribosomal RNA
in flax. Nucleic Acids Res. 9, 1301-1309. [Note: This is the source of the rDNA probe.]
Grierson, D. (1982). RNA processing. In "Nucleic Acids and Proteins in Plants II Structure,
Biochemistry and Physiology of Nucleic Acids" (Pathier and Boulter, eds.).
Grierson, D., and Covey, S. N. (1984). "Plant Molecular Biology." Blackie, Glasglow.
Gutell, R. R. (1993). Collection of small subunit (16S-and 16S-like) ribosomal RNA structures.
Nucleic Acids Res. 21, 3051-3054.
Hillis, D. M., and Dixon, M. T. (1991). Ribosomal DNA:Molecular evolution and phylogenetic
inference. Q. Rev. Biol. 66, 411-453.
Ingle, J., and Sinclair, J. (1972). Ribosomal RNA genes and plant development. Nature
(London) 235, 30-32.
Neefs, J.-M., Van de Peer, Y., De Rijk, P., Chapelle, S., and De Wachter, R. (1993). Compilation
of small ribosomal subunit RNA structures. Nucleic Acids Res. 21, 3025-3049.
Olsen, G. J., Overbeek, R., Larsen, N., Marsh, T. L., McCaughey, M. J., Maciukenas, M. A.,
Kuan, W.-M., Macke, T. J., Xing, Y., and Woese, C. R. (1991). The ribosomal database
project. Nucleic Acids Res. 20(Suppl) 2199-2200.
Rogers, S. O., and Bendich, A. J. (1987). Ribosomal RNA genes in plants: Variability in copy
number and in the intergenic spacer. Plant Mol. Biol. 9, 509-520.
Sollner-Webb, B., and Mougey, E. B. (1991). News from the nucleolus: rRNA gene expression.
Trends Biochem. Sci. 16, 58-62.
Steele, S. N., and Ingle, J. (1973). The genes for cytoplasmic ribosomal ribonucleic acid in
higher plants. Plant Phys. 51, 677-684.
RUBISCO (Ribulose Bisphosphate Carboxylase/Oxygenase)
Berry-Lowe, S. L., McKnight, T. D., Shah, D. M., and Meagher, R. B. (1982). The nucleotide
sequence, expression, and evolution of one member of a multigene family encoding
the small subunit of ribulose-l,5-bisphosphate carboxylase in soybean. I. Mol. Appl.
Genet. 1, 483-498.
Cashmore, A. R. (1979). Reiteration frequency of the gene coding for the small subunit of
ribulose-l,5-bisphosphate carboxylase. Cell 17, 383-388.
Kuhlemeier, C., Green, P. J., and Chua, N.-H. (1987). Regulation of gene expression in higher
plants. Annu. Rev. Plant Phys. 38, 221-257.
Mazur, B. J., and Chui, C.-F. (1985). Sequence of genomic DNA for small subunit of RUBISCO
from tobacco. Nucleic Acids Res. 13, 2373-2386. [Note: This is the source of the SS
Moses, P. B., and Chua, N. H. (1988). Light switches for plant genes. Sci. Am. 258, 88-93.
Zhu, G., and Jensen, R. G. (1991). Fallover of ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Plant Physiol. 97, 1354-1358.
RNA PURIFICATION A N D N O R T H E R N
B LOT A N A L Y S I S
RNA Introduction: Overview of Experiment
The isolation and characterization of messenger RNA are important
parts of the study of gene expression of an organism (Farrell, 1993). In
this experiment, RNA is isolated from plants. The majority of RNA species
isolated are ribosomal RNAs and tRNAs (Ausubel et al., 1989). Poly(A) §
RNA can also be isolated. The RNA isolated is separated on the basis of
size using a denaturing formaldehyde agarose gel (Lehrach et al., 1977;
McMaster and Carmichael, 1977). The RNAs are then transferred from the
gel to a membrane~producing a Northern blot (Alwine et al., 1977, 1979;
Thomas, 1980). The Northern blot is then hybridized with a cloned probe
for a gene to determine the steady state levels of mRNA present in the
cell that are complementary to the cloned probe.
The basic protocols needed are presented in this chapter. The student
is to design the exact conditions for examining gene expression. For example, the student might use the cloned probe for the small subunit of ribulose
bisphosphate carboxylase/oxygenase (SSRUBISCO) (see Chapter 4) to look
for expression of SSRUBISCO in seedlings grown in the light and in the
The isolation of RNA presents a challenge because RNases are ubiquitous. Endogenous RNases are quickly inactivated by the phenol/SDS extraction step of the RNA isolation procedure. Glassware and solutions are
treated with agents such as diethyl pyrocarbonate to inactivate RNases.
The yield of RNA can vary depending on the source. Pea seedlings
generally give a high yield, as much as 7 mg of total RNA from 15 g of
PROTOCOL 5.1: RNA EXTRACTION FROM PLANT LEAVES
plant material. Arabidopsis plants may yield as little as 3 mg of total RNA
per 15 g of plant tissue (Ausubel et al., 1989).
RNA Extraction from Plant Leaves
TES: 50 mM Tris; 20 mM EDTA; 50 mM NaC1; pH 8.0
TE: 50 mM Tris; 20 mM EDTA; pH 8.0
2 M LiC1
5 M LiC1
The pheonol has been equilibrated with 3% NaC1 and neutralized
with Tris, and 0.1% 8ohydroxyquinoline has been added. The hydroxyquinoline is an antioxidant that reduces the formation of oxidation products
of phenol such as quinones. The phenol is brought to a neutral pH because
acidic phenol has a tendency to trap single-stranded nucleic acid at the
interface between the phenol and the aqueous phases. Alternatively, if a
molecular biology grade phenol is used, the hydroxyquinoline may be
9 3 M NaOAc (sodium acetate)
9 20% Sarkosyl
RNases are ubiquitous. Great care should be taken to destroy RNases
that might be present on glassware or in solutions. RNases are also present
on the hands. Gloves should be worn during RNA handling steps.
1. Bake all glassware needed, except centrifuge tubes, overnight at 250~
to destroy RNases.
2. Soak glass and plastic centrifuge tubes and centrifuge bottles in sterile
distilled water with 1% (v/v) diethyl pyrocarbonate (DEPC) for 10 to
15 min. Pour off the DEPC. Autoclave the tubes and bottles.
CAUTION: When handling diethyl pyrocarbonate, wear safety goggles,
gloves, and a laboratory coat. Work in a fume hood.
3. Add DEPC to a final concentration of 0.1% (0.1 ml DEPC/IO0 ml solution) to all solutions, including H20. Shake the solutions. Let the solutions with the DEPC stand for 30 min. Autoclave the solutions.
4. Wear latex gloves at all times during the procedures to avoid contamina-