5S-RNA genes of barley are located on the second chromosome

A. Kolchinsky, V. Kanazin, C. Kole*, and E. Ananiev
Laboratory of Plant Molecular Genetics
N.I. Vavilov Institute of General Genetics of
the USSR Academy of Sciences
Gubkin str., 3
Moscow B-333, USSR
*On leave from Irissa University of Agriculture and Technology, Bhubaneswar-751003, India.


The barley-wheat chromosome addition lines (AD-lines) were obtained by Islam et al. (1981). Each of them contains one barley chromosome. However, we know of only a few cases when they were used for the localization of barley genes (see, for example, Bohlmann et al., 1988; Belostotsky et al., 1989). This fact can be explained by the difficulties in the growing and cytological control of AD-lines and some technical problems. However, molecular markers are now widely used in genome studies, and AD-lines are proving to be extremely useful for their mapping.

Here we report the mapping of barley genes coding for ribosomal 5S- RNA using AD-lines. These lines were kindly sent to us by Dr. Islam. They were grown, with constant monitoring of karyotype and reasonable fertility, at relatively high temperature and moderate photoperiod. DNA was isolated from 7-8 days old shoots using 50 mM Tris-Oh, 0.2 M EDTA, pH 9.0, 0.5% SDS, 200 microg/ml protease at 55 °C and further purified using routine protocol. Barley genes coding for ribosomal 5S-RNA were cloned earlier in our lab (Khvyrleva et al., 1988). DNA was digested with BamHI or MvaI according to manufacturer's instructions ("Ferment", Vilnius), purified, concentrated, and applied on non-denaturing 5% polyacrylamide gel (Tris-borate buffer). After electrophoresis DNA was transferred on the membrane "Hybond" (Amersham) using the electro-blotting device Bio-Rad. DN was cross-linked to the membrane by UV-treatment and hybridized with P32 -labelled probe. In order to prepare probes, cloned fragments were cut out from the vector, isolated by electrophoresis, and 50-100 ng were labelled using the random primer procedure (Feinberg and Vogelstein, 1983).

The result of this experiment is shown on Fig. 1. We used here the restriction enzyme MvaI which cuts the 5S RNA genes of barley and most of 5S genes of wheat once per repeat and is not sensitive to methylation. DNA from different barley cultivars gives a known pattern (Khvyrleva et al., 1988); two major repeats, 301 and 450 bp long, are seen in the lower part of the lanes. Their relative content in studied cultivars varies significantly. Upper bands correspond to the repeats which deviate from canonical sequence, and the shortest of them is 900 bp long.

The wheat cultivar 'Chinese Spring' is a parental strain for AD-lines. It gives two sets of bands corresponding to unit repeats grouping around 410 and 500 bp (Gerlach and Dyer, 1980). This fine heterogeneity of wheat 5S repeat could not be shown earlier by agarose electrophoresis.

Fig. 1 shows clearly that only the AD-2 line contains the bands 301 and 900 bp corresponding to barley 5S genes. The 450 bp band of the barley moved too close to wheat bands and is not seen at this resolution.

To check the identity of our AD-lines, the radioactive probe was washed away from the filter and it was reprobed with a P32 labeled fragment of the repeat coding for the major ribosomal RNAs cloned earlier in our lab (Fig. 2) (Khvyrleva et al., 1987). Though it shows some cross-hybridization with wheat DNA, there are barley-specific bands seen only in the AD-lines 6 and 7. This experiment agrees well with the known localization of barley ribosomal genes on the chromosomes 6 and 7 (Appels et al., 1980) and with the known fact of different organization of ribosomal clusters in two nucleolar organizers (Appels and Moran, 1984).

Another way to show the presence of barley chromosomes in the AD- lines is to hybridize their DNA with a barley-specific repeat kindly donated to us by Dr. Metzlaff (Junghans and Metzlaff, 1988). The result of this experiment is shown on Fig. 3. It proves the conservation of barley chromosome in our stocks of AD-lines.

We conclude that the cluster of 5S genes is located on chromosome 2 of barley and that 5S genes are absent from chromosomes 1, 3, 4, 6, and 7. No statement can be made now concerning chromosome 5, because the corresponding AD-line was not produced.

Fig. 1. DNA digests Betzes barley (B), Chinese Spring wheat (W), and Barley-wheat additions cut with restriction enzyme MvaI, which cuts the 5S RNA genes of barley into 301, 450 and 900 bp segments and probed within radioactive 5S RNA of barley used.

Fig. 2. DNA digests of Betzes barley (B), Chinese Spring wheat (W) and barley-wheat addition lines cut with MvaI and probed with a repeat segment of the major barley ribosomal RNAs.

Fig. 3. DNA digests of Betzes barley (B), Chinese Spring wheat (W), and barley-wheat addition lines cut with MvaI and probed with a barley- specific repeat.

References:

Appels, R., W.L. Gerlach, E.S. Dennis, H. Swift, and W.J. Peacock. 1980. Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNA in cereals. Chromosoma 78:293-311.

Appels, R., and L.B. Moran. 1984. Molecular analysis of alien chromatin introduced into wheat. In Gene Manipulation in Plant Improvement. 16th Stadler Genet. Symp. New-York, London.

Belostotsky, D.A., N.V. Milshina, and E.V. Ananiev. 1989. Character- ization of relic DNA from barley genome. Theor. Appl. Genet. (submitted).

Bohlmann, H., S. Clausen, S. Behnke, C. Giese, U. Reimann-Philipp, G. Schrader, V. Barkholt, and K. Apel. 1988. Leaf-specific thionins of barley - a novel class of cell-wall proteins. EMBO J. 7:1559-1565.

Feinberg, A.P., and B. Vogelstein. 1983. A technique of radiolabelling DNA fragments to high specific activity. Anal. Biochem. 132:6-13.

Gerlach, V.L., and T.A. Dyer. 1980. Sequence organization of the repeat units in the nucleus of wheat which contain 5S rRNA genes. Nucl. Acids Res. 8:4851-4865.

Islam, A.K.M.R., K. Shepherd, and D.H.B. Sparrow. 1981. Isolation and characterization of euplasmic wheat-barley chromosome addition lines. Heredity 46:161-174.

Junghans, H., and M. Metzlaff. 1988. Genome specific highly repeated sequences of Hordeum vulgare. Cloning, sequencing and squash dot test. Theor. Appl. Genet. 76:728-736.

Khvyrleva, C.D., A.E. Chernyshov, S.S. Bochkanov, E. Yu. Yakovleva, and E.V. Ananiev. 1987. Intervariety polymorphism and organization of 18S-26S rRNA genes in barley. Genetika (USSR) 23:717-724.

Khvyrleva, C.D., A.K. Gazumyan, and E.V. Ananiev. 1988. Organization and the primary nucleotide sequence of the 5S rRNA genes of barley. Genetika (USSR) 25:1830-1840.


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