Hybridization studies of Barley Stripe Mosaic Virus cDNA clones to virus induced maize mutants

Some multicomponent RNA virus strains of Barley Stripe Mosaic Virus (BSMV) or Wheat Streak Mosaic Virus (WSMV) are known to act as mutagenic agents in corn and induce mutations unspecifically (G. F. Sprague et al., Science 141:1052, 1963). The majority of these mutations are stable, although an unstable mutation has been identified recently (P. Friedemann and P. A. Peterson, Mol. Gen. Genet. 187:19, 1982).

Whether this mutagenic effect is correlated with the integration of viral sequences into the maize genome was tested by Southern type hybridization experiments. Therefore, cDNA clones from the BSMV four component strain Argentina mild (kindly provided by M. Brakke, Lincoln) were constructed and used to probe virus induced mutant maize lines. The two mutant lines tested were originally isolated as aberrant ratio lines 874-30 and 74, 287-4 by G. F. Sprague (G. F. Sprague and H. H. McKinney, Genetics 54:1287, 1967) and further characterized by M. K. Brakke, R. G. Samson, and W. A. Compton (Genetics 99:481, 1981) and P. Friedemann and P. A. Peterson (Mol. Gen. Genet. 187:19, 1982).

The cDNA clones homologous to BSMV RNA sequences were constructed by standard procedures (U. Wienand et al., Nucl. Acid Res. 6:2707, 1979) and characterized by hybrid released-translation and Northern hybridization. Two non cross-hybridizing cDNA clones homologous to BSMV RNA component I (720 and 520 base pairs in length) were identified, as well as clones homologous to RNA component II (1150 base pairs) and RNA component III (480 base pairs). No clone homologous to RNA component IV could be identified.

In a Southern experiment these clones were used to probe maize wild type DNA (a color converted W22 line) and the two mutant DNAs described above. The EcoR1 digested DNAs were separated on 0.8% agarose gels, transferred to nitrocellulose and probed with each of the four nick translated cDNA clones. Although hybridization conditions were used to detect single gene sequences, no hybridization could be seen.

Since the cDNA clones represented only part of the viral genome (the size of the RNA component varies between 2200 and 2800 nucleotides), total four-component BSMV RNA was also used as a radioactive probe for hybridization. Three bands could be seen in all the maize lines tested after hybridization of the 32P labeled RNA to the EcoR1 digested DNA. Two of these could be competed with rRNA from maize endosperm. Using total barley RNA as competitor in the hybridization experiment, no bands could be detected any longer. Thus the hybridization signals seemed to be due to contamination of plant RNA in the virus preparation.

From both DNA and RNA hybridization experiments we conclude that there are no viral sequences present in the mutant maize stocks tested.

Udo Wienand, Peter Peterson and Heinz Saedler

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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