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Protocol 4.2: Steps of a Genomic Southern Blot

Protocol 4.2: Steps of a Genomic Southern Blot

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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?



References

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.

Suggested Reading



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.



REFERENCES



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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

RUBISCO probe.]

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

dark.

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

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PROTOCOL 5.1: RNA EXTRACTION FROM PLANT LEAVES



203



plant material. Arabidopsis plants may yield as little as 3 mg of total RNA

per 15 g of plant tissue (Ausubel et al., 1989).



PROTOCOL 5.1:

RNA Extraction from Plant Leaves

Solutions

9

9

9

9

9



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

1:1 phenol:chloroform



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

omitted.

9 3 M NaOAc (sodium acetate)

9 20% Sarkosyl



Preparation

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-



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