Plant Molecular Biology 35: 313–321, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Analysis of multiple copies of geminiviral DNA in the genome of four closely
related Nicotiana species suggest a unique integration event
Mark K. Ashby
, Andrew Warry
, Eduardo R. Bejarano
, Alaa Khashoggi
and Conrad P. Lichtenstein
School of Biological Sciences, Queen Mary and Westﬁeld College, University of London, London E1 4NS, UK
author for correspondence);
Department of Biochemistry, Imperial College of Science, Technology & Medicine,
London SW7 2AZ, UK;
Advanced Technologies (Cambridge) Ltd., Cambridge Science Park, Cambridge CB4
4WA, UK; present addresses:
Department of Biology, University College London, London WC1E 6BT, UK;
Entomology and Nematology Department, IACR-Rothamsted, Harpenden, Herts , AL5 2JQ, UK;
etica, Departmento de Biolog
ia Celular y Gen
etica, Campus Universitario de Teatinos 29017, M
Received 23 December 1996; accepted in revised form 16 May 1997
Key words: integration, geminivirus, plant genomes, evolution
Previously, we discovered multiple direct repeats of geminivirus-related DNA (GRD) sequences clustered at a
single chromosomal position in Nicotiana tabacum (tobacco). Here we show that, in addition to tobacco, multiple
copies of these elements occur in the genomes of three related Nicotiana species, all in the section Tomentosae:
N. tomentosiformis, N. tomentosa and N. kawakamii, but not in 9 other more distantly related Nicotiana species,
nor in various other solanaceous and non-solanaecous plants. DNA sequence analysis of 18 GRD copies reveal
4 distinct, but highly related, sub-families: GRD5, GRD3 and GRD53 in tobacco; GRD5 in N. tomentosiformis
and N. kawakamii; and GRD2 in N. tomentosa. In addition to novel sequences, all elements share signiﬁcant but
varying lengths of DNA sequence similarity with the geminiviral replication origin plus the adjacent rep gene.
There is extended sequence similarity to REP protein at the deduced amino acid sequence level, including motifs
associated with other rolling circle replication proteins. Our data suggest that all GRD elements descend from a
unique geminiviral integration event, most likely in a common ancestor of these Tomentosae species.
Integration of viral DNA into the host nuclear gen-
ome is well known in bacterial and animal systems.
In plants, most viruses have RNA genomes and can
acquire host sequences by RNA recombination with
host transcripts , but integration of DNA viruses
into the plant nuclear genome was unknown until we
discovered, in the course of antisense experiments
, geminivirus-related DNA (GRD) sequences in
the Nicotiana tabacum (tobacco) nuclear genome .
Geminiviruses are insect-transmitted single-stranded
(ss) DNA viruses which infect a wide range of mono-
Thenucleotidesequencedatareportedwillappear in theEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers U81299–U81311.
cotyledonous and dicotyledonous plants . They
have mono- or bi-partite genomic components and,
during infection, the viral DNA is transported to the
nucleus where replication takes place via a double-
stranded (ds) DNA intermediate.
GRD (1) is found as three related repeat classes,
GRD5, GRD53 and GRD3, clustered asmultiple direct
repeats at a single locus on the T4 homologues, which
are a pair of small sub-metacentric tobacco chromo-
somes; (2) most closely resembles the family of New
World bipartite geminiviruses; (3) comprises a part of
the geminiviral rep (formerly called al1) gene, encod-
ing an essential replication protein, plus the adjacent
REP recognition site, the origin of initiation of viral
rolling circle replication [1, 10, 16].
Gr.: 201001999 , PIPS Nr. 141263 BIO2KAP
plan3791.tex; 22/08/1997; 8:15; v.7; p.1