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Cucurbit Genetics Cooperative Report 12:84-85 (article 35) 1989

Determination of Molecular Weight of Chloroplast DNA of Cucurbita pepo L. using Different Restriction Enzymes

H.T. Lim and C. Boyer

Department of Horticulture, Pennsylvania State University, University Park, PA 16802

Chloroplasts contain their own complement of DNA as well as protein synthesis apparatus. The chloroplast DNA (CpDNA) exists as covalently closed circular molecular molecules, ranging in size from 120 to 180 kilobase pairs (kbp) in flowering plant species (4). Chloroplasts, however, are not autonomous: the biogenesis of chloroplasts requires the coordinative expression of both specific nuclear genes and chloroplast genes.

In order to understand mechanisms that control the expression of nuclear and chloroplast genes, one prerequisite is the ability of physically purify the chloroplast DNA and to know genetic organization of the chloroplast DNA. The first introductory study for estimating molecular weight among members of Cucurbitaceae was conducted by Juvik and Palmer (3). However, only the ranges and numbers of fragments produced by different restriction endonucleases were reported. In this report, a rapid method of restriction enzyme analysis of the squash cpDNA is described in some detail and the size of C. pepo chloroplast genome is estimated.

Chloroplast Isolation. Squash (Cucurbita pepo L.) chloroplasts were extracted from young leaves according to the protocol of Gounaris et. al (1) with following modifications. The crude extraction of chloroplasts was resuspended in homogenized buffer and collected by centrifugation at 1500 X g for 15 min. instead of using discontinuous sucrose gradient centrifugation to purify chloroplasts (2, 3), a continuous sucrose gradient was used to remove contaminating nuclear DNA. The resuspended pellet was loaded onto a 30 to 60% w/v gradient of sucrose, and spun at 100,000 X g in a SW-27 rotor at 4°C for 1 hr. The chloroplast bands were collected, diluted with an equal volume of TE buffer, and centrifuged at 2,500 X g for 5 min.

Isolation of cpDNA. The chloroplast pellet was resuspended in 5 ml of the homogenization buffer,m to which 1/10 volumes of 1 mg/ml RNAse A and 2 ml of 10% s/v sodium sarkosinate were added. The suspension was incubated at room temperature for 30 min. for chloroplast lysis. The DNA sample was extracted with an equal volume of buffer-saturated phenol, three times with 4 ml of phenol and 2 ml of chloroform, and twice with water-saturated n-butanol. DNA was precipitated at -70°C for 1 hr by adding 1 ml of 7.5 M ammonium acetate and 2.5 volumes of absolute ethanol. The precipitated DNA pellet was washed with 70% ethanol, dried under nitrogen gas, and dissolved in TE buffer and stored at -20°C.

Digestion of cpDNA with restriction endonucleases. The chloroplast DNA were digested with selected restriction endonucleases under the conditions recommended by the suppliers. Restriction fragments of plastid DNA were separated by electrophoresis in 0.5-1.7% agarose gels, depending on the size of fragments.

Table 1. Numbers, sizes (in Kbp) and stoichiometries (brackets) of squash cpDNA restriction fragments generated by different endonuclease restriction enzymes.

 

Restriction enzyme

Fragment nos.

Sal

Pvu II

Bgl I

Sac II

Pst I

1

47.7 (2x)

57.3

47.5

29 (2x)

29.9 (2x)

2

26.3

28.6

35.3

25.4

25.2

3

21.1

19.5

22.8

20

21.0

4

18.8

16.2

21.1

5

13.6

5

2.4 (2x)

14.2

11.1

16.6

11.9

6

--

10.5

7.4

15.3

10.0

7

--

7.9

6.5 (2x)

12.8

8.4

8

--

6.1 (2x)

4.3 (2x)

10.7

6.1

9

--
--
--

5.4

4.3 (2x)

10

--
--
--

1.6

1.4

11

--
--
--
--

0.6

Total

166.4

166.4

166.7

166.2

166.5

The previously reported method for cpDNA isolation is very time-consuming and tedious (2, 3). The proposed method was modified to avoid the pronase treatment and CsCl density centrifugation, which are replaced with phenol and phenol/chloroform treatment (1).

The length of the restriction fragments was easily determined by calibrating the gel. This was done by running Lambda DNA digested with Hind III and Zho I in another slot of the same gel. The molecular weights of fragments larger than 30 kpb were estimated as the sum of subfragments derived from second digestion. For the five enzymes reported, the size of C. pepo DNA is estimated at 166 Kbp (Table 1) and work is in progress to prepare a detailed restriction enzyme map for C. pepo cpDNA.

Literature Cited

  1. Gounaris, I., C.B. Michalowski, J.J. Bohnert and C. A. Price. 1986. Restriction and gene maps of plastid DNA from Capsicum annuum. Current Genet. 12:219-24.
  2. Palmer, J.D. 1988. Isolation and structural analysis of chloroplast DNA. In: Weisbach, A. and Weisbach, H. (ed) Methods for Plant Molecular Biology. Academic Press, pp 105-124.
  3. Juvik J.A. and Palmer, J.D. 1984. Potential of restriction endonuclease analysis of chloroplast DNA for the determination of phytogenic relationships among members of Cucurbitaceae. Cucurbit Genetics Coop. Rpt. 7:66-68.
  4. Palmer, J.D. 1982. Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativus. Nucl. Acids Res. 10:1593-1605.
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