34. Plasmid DNA sequences hybridize with total DNA isolated from rice root tissues

Plant Molecular Biology research Group MOLBAS, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

During the study of the structure of a single copy gene (gene 9) which is root specifically expressed, we encountered the phenomenon that the organization of its locus is different between green tissues and in vitro grown tissues (cell suspensions). To check whether the organization of the gene 9 locus differed also in the root tissues of rice, total DNA isolated from roots and green tissues was compared by Southern analysis. With a cDNA probe, including vector plasmid sequences, additional hybridizing bands in root DNA were found. We show here that these additional bands were due to hybridization of plasmid sequences to root DNA.

Total DNA was isolated from root and green tissue from the rice varieties Indica IR36 and Japonica T309 according to Hensgens and Van Os-Ruygrok (1989). Tissues were harvested from 10 day old seedlings. Seedlings were grown under non-sterile conditions or were grown from sterilized seeds under aseptic conditions. DNAs were digested with EcoRV and Hind III under standard conditions, electrophorized on a 0.8% agarose gel (1 X TBE). DNA fragments were transferred to Genescreen Plus filters by capillary blotting and hybridized with P³²-labelled probes. Probes were prepared by nicktranslation (Sambrook et al. 1989).

Using a cDNA 9 probe cloned in PUN121 more hybridizing bands were found in DNA isolated from roots (lanes W) than in DNA from green tissue (lanes G). This was observed for both T309 and IR36. Three extra bands of approxiatemately 5,000 basepairs are visible in the EcoRV and Hind III digests of IR36 root DNA. The intensity of the additional bands is similar to those derived from the single copy gene 9. Also for Japonica T309 additional bands were found in the hybridization with root DNA (compare lanes G with W) although they are weaker and have a higher molecular weight than the additional bands observed in IR36 root DNA.

Same digests were hybridized with labelled vector plasmid DNA alone (pannel PUN121 in Fig. 1). Using this probe no hybridization was observed with the DNA isolated from the green tissue (G lanes). All previously observed extra bands hybridized with the plasmid probe.

DNA isolated from seedlings which were aseptically grown from sterilized seeds gave identical results (not shown). The observed additional bands in DNA isolated from roots are therefore not due to bacterial contamination. Also Blue- script vectors and PBR322 hybridized with root DNA although the signals found with PBR322 are much weaker (not shown).

We can only speculate about what causes the hybridization of plasmid se- quences with root DNA. Bacterial contamination was ruled out and no hybridi- zation was found with leaf DNA. Therefore, root DNA differs from DNA isolated from the rest of the rice plant. The hybridizing sequences might have a different and more homogenous degree of methylation in root tissue than in the rest of the plant. Alternatively the hybridizing sequences might be present in an amplified form in roots only (eg. mitochondrial DNA) and are therefore only detected in root DNA.

We did not determine which sequence of the various plasmids was responsible for hybridization. Since hybridizations were performed under standard conditions the observed phenomenon emphasizes that care should be taken in screening of potential rice transformants by Southern or dot blot analysis with probes

Fig.1 2.5 ug total DNA isolated from leaves (G) and root (W) from Oryza sativa Indica IR36 and from Japonica T309 was digested with HindIII (H) and EcoRV (EV). Digests were separated on 0.8% agarose (TBE) gel and transferred to GeneScreenPlus according to Southern (1975). After UV crosslinking, the filter was hybridized with a rice cDNA9 probe specific for root tissues and with the vector alone (PUN121). Hybridization was performed as described by Memelink et al. 1987. M\1\ and M\2\ are molecular weight marker DNAs.

containing plasmid sequences. Especially when DNA isolated from root tissue or from tissue prone to root formation is being analysed.

Hensgens, L. A. M. and P. E. Van Os-Ruygrok, 1989. Isolation of RNA and DNA from different rice tissues. RGN 6: 163-168.

Memelink, J., J.H.C. Hoge, and R.A. Schilperoort, 1987. Cytokinin stress changes the developmental regulation of several defence-related genes in tobacco. EMBO Journal 6: 3579-3583.

Nilsson, B., M. Uhlen, S. Josephson, S. Gatenbeck, and L. Philipson, 1983. An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nuc. Acid Res. 11: 8019-8030.

Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989. Molecular Cloning. A laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press. Southern, E., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.