Inverted repeats in chloroplast DNA from the genus Zea

Chloroplast DNAs (ctDNAs) of corn and among teosintes differ by restriction endonuclease fragment pattern analyses (D. H. Timothy, C. S. Levings III, D. R. Pring, M. F. Conde, and J. L. Kermicle, Proc. Nat. Acad. Sci. USA 76:4220-4224, 1979). Additionally, the patterns of the differences formed a hierarchical structure of taxonomic validity when compared to the conventional biosystematic classifications of the taxa. Those groupings were a reflection of the differences in the number and position of hexanucleotide sequences in the ctDNA molecules of each taxa. Electron microscopy of ctDNA from corn, lettuce, and spinach has shown that each contains a large sequence repeated one time in reverse polarity, and that the structure of the inverted sequence is highly conserved in the plants (R. Kolodner and K. K. Tewari, Proc. Nat. Acad. Sci. USA 76:41-45, 1979). In that study, the inverted segment was approximately 16% of the native length of the three ctDNAs. The repeated sequence in corn is about 22,500 base pairs long (R. Kolodner and K. K. Tewari, Biochim. Biophys. Acta 402:372-390, 1975). The structure of the corn ctDNA (J. R. Bedbrook, R. Kolodner and L. Bogorad, Cell 11:739-750, 1977) is such that one copy of the two sets of chloroplast specific rRNA genes (J. R. Thomas and K. K. Tewari, Proc. Nat. Acad. Sci. USA 71:3147-3151, 1974) is found in each of the inverted sequences of the ctDNA. This preliminary report deals with additional characterization of ctDNA in the genus Zea.

Preparations of corn and teosinte ctDNAs were denatured, neutralized, allowed to self-renature, and examined by electron microscopy. Molecular conformation was that of a small single-stranded DNA loop, separated from a large single-stranded loop by a large duplex region (Figure 1). The lengths of these portions of the partially duplexed ctDNA molecules are summarized in Table 1. Included in these data are measurements of single-stranded loops and duplex segments resulting from broken molecules as illustrated in Fig. 1B and of either single-stranded loop with the duplex segment. Only the measurements from complete and unbroken loops and duplex segments are reported.

Figure 1.

Table 1. Length measurements on self-renatured ctDNA molecules in Zea.
Taxa Small loop, f X units* Double segment, f X units Large loop, f X units Duplex segment in % of total molecule length
Corn 2.32 ± 0.04 3.90 ± 0.05 15.24 ± 0.32 15.4
  (7) (10) (6)  
Central Plateau 2.35 ± 0.07 4.10 ± 0.06 14.75 ± 0.74 16.2
  (19) (15) (2)  
Huehuetenango 2.23 ± 0.04 3.93 ± 0.07 14.03 ± 0.20 16.3
  (9) (9) (3)  
Guatemala 2.35 ± 0.07 4.05 ± 0.05 15.51 ± 0.31 15.5
  (12) (12) (7)  
Perennial (4N) 2.26 ± 0.14 4.08 ± 0.07 15.55 ± 0.60 15.7
  (10) (10) (6)  

*Lengths are reported as a ratio in f X units, using the double-stranded or single-stranded forms of the bacteriophage f X 174 as an internal standard against the respective double- or single-stranded portions of the ctDNA molecules. Numbers of molecule portions measured are enclosed in ( ).

Although previous work using restriction endonucleases has distinguished evolutionary differences among ctDNAs of teosinte and corn, the number of molecules reported here with self-renaturation are insufficient to make those distinctions. Additionally, the specificity and resolution of the two techniques is somewhat different. However, it is now apparent that both corn and teosinte ctDNA genomes contain a sequence repeated once in reverse polarity, that each sequence is approximately 16% of the native length of the ctDNA, and that the sequence is highly conserved. Although we have no evidence that the inverted ctDNA sequence in teosinte codes for the chloroplast rRNA genes, as in corn, the similarity in the conformation of the corn and teosinte ctDNAs allows the speculation that the teosinte inverted repeat has a similar function to that of corn.

D. H. Timothy, W. W. L. Hu, and C. S. Levings, III

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