Intergenic regions of banana bunchy top virus

The invention relates a DNA molecule which is a partial fragment of an intergenic region of a BBTV component or alternatively which DNA molecule is derived from said intergenic region whereby the DNA molecule is capable of promoting, enhancing, regulating, or modifying transcription of a non-BBTV gene.

FIELD OF INVENTION 
This invention relates to DNA sequences of banana bunchy top virus (BBTV) 
and, in particular, to the intergenic regions of components 1 to 6. 
BACKGROUND ART 
Banana bunchy top disease (BBTD) is the most important virus disease of 
bananas (Dale, 1987, Advances in Virus Research 33 301-325), The disease 
is widespread in Asia and the South Pacific and has limited distribution 
in Australia and Africa. It has not been reported from the Americas. The 
disease was originally assumed to be caused by a luteovirus as it was 
persistently aphid transmitted but not mechanically transmitted, induced 
yellows type symptoms and infected plants that had damaged phloem. 
However, recently 18-20 nm isometric virus-like particles (VLPs) have been 
purified from infected plants and have been demonstrated to be associated 
with the disease (Harding et al., 1991, Journal of General Virology 72 
225-230; Thomas & Dietzgen, 1991, Journal of General Virology 72 217-224; 
Wu and Su, 1990, Journal of Phytopathology 128 153-160). Harding et al 
(Harding et al., 1991, Journal of General Virology 72 225-230; Harding et 
al., 1993, Journal of General Virology 74 323-328) have isolated circular, 
single-stranded DNA of about 1 kb from these VLPs and cloned and sequenced 
one ssDNA component. This component, known as BBTV DNA component 1, had 
one large open reading frame (ORF) in the virion sense and encoded a 
putative replicase. This component was transmitted with the disease via 
aphids. 
In Burns et al., 1994, Arch Virol. 137 371-380, they report the cloning and 
sequencing of a second component of BBTV. They found that a 93 nucleotide 
sequence was strongly conserved between the two ssDNA genomic components 
of BBTV. Two outwardly extending degenerate primers were designed from 
this sequence and used in a polymerase chain reaction (PCR) with DNA 
extracted from purified BBTV virions. PCR amplified products consisting of 
at least seven distinct bands all approximately 1 kb and possibly 
representing full-length BBTV dsDNA were resolved. The PCR amplified 
products were cloned and the clones screened by restriction enzyme 
analysis. Four distinct restriction analysis groups were identified. This 
reference concluded that the genome of BBTV contains at least five 
components and that BBTV belongs to a previously undescribed group of 
plant viruses which may also contain subterranean clover stunt virus. 
In Karan et al., 1994, Journal of General Virology 75 3541-3546, mention is 
made of BBTV component 1 from isolates from 10 different countries being 
cloned and sequenced and the sequences were subsequently aligned and 
compared. This analysis indicated two groups: the South Pacific group 
(isolates from Australia, Burundi, Egypt, Fiji India, Tonga and Western 
Samoa) and the Asian group (isolates from the Philippines, Taiwan and 
Vietnam). The mean sequence difference within each group was 1.9 to 3.0% 
and between isolates from the two groups were approximately 10%, but some 
parts of the sequences differed more than others. However, the protein 
encoded by the major open reading frame differed by approximately 5%. The 
region from the beginning of the stem-loop sequence to the potential TATA 
box was identical in all isolates except for a two nucleotide change in 
the Western Samoan isolate and a single change in that of the NSW isolate. 
These results, together with other evidence, suggest that BBTV has spread 
to bananas after the initial movement of bananas from the Asian Pacific 
regions to Africa and the Americas. 
In Xie et al., 1995, Phytopathology 85 339-347, the Hawaiian isolate of 
BBTV was purified from infected banana cultivar Williams. Three 
single-stranded DNA (ssDNA) components were cloned and sequenced; they 
were named component 1, 3 and 4 respectively. Component 1 is 1,110 
nucleotides in length and shares 98% nucleotide sequence identity with the 
BBTV DNA component 1 of the Australian isolate as described In Harding et 
al. (1993) above. This component contains two open reading frames (ORF) 
capable of encoding a protein of 33.5 kDa, which may function as a 
replicase, and a protein about 15.2 kDa, with unknown functions. Component 
3 is 1,057 nucleotides in length and does not contain any ORFs larger than 
10 kDa. Component 4 is 1,017 nucleotides in length and potentially encodes 
a protein of 18.9 kDa. All three ssDNA components have a same stem-loop 
sequence and have a conserved non-coding region. The sequence of each of 
these-three components is different from that of BBTV DNA components of 
two Taiwanese isolates. BETV-specific clones were used in dot-blot 
hybridisation assays for detection of BBTV in plants using radioactive and 
non-radioactive probes. A polymerase chain reaction (PCR) assay was 
developed for detection of BBTV in banana samples and single aphids. 
Dot-blot hybridisation assays were as sensitive as enzyme-linked 
immunosorbent assay (ELISA) while PCR was 1,000 times more sensitive than 
dot-blot and ELISA assays for detection of BBTV in bananas. 
Although so called intergenic regions of genomic components 1, 3 and 4 have 
been detected in Harding et al., 1993 supra; and Xie and Hu, 1995, supra, 
the only charcteristic of such intergenic regions has been a stem-loop 
structure in component 1 as disclosed in Harding et al., 1993, supra. 
In an earlier application, (i.e. International Application PCT/AU95/00311) 
reference is made to BBTV components 3, 4 and 6 which are claimed per se. 
Regions outside the ORF regions (i.e. the so called "intergenic regions") 
have also been disclosed in this earlier application. Genomic components 
1, 3 and 4 disclosed in Xie and Hu, 1995, supra correspond to components 
1, 2 and 5 referred to in the earlier application PCT/AU95/00311. 
SUMMARY OF THE INVENTION 
Surprisingly, it has now been discovered that intergenic regions of BBTV 
genomic components 1-6, or parts thereof, have sequences derived therefrom 
capable of promoting, enhancing, regulating or modifying transcription of 
non-BBTV genes. 
The invention therefore includes within its scope, the aforementioned 
intergenic regions when used for promoting, enhancing, regulating or 
modifying transcription of a non-BBTV gene as well as parts thereof, 
sequences derived therefrom and mutants thereof per se. 
The invention also includes within its scope a DNA molecule which is a 
partial fragment of an intergenic region of a BBTV component or 
alternatively which DNA molecule is derived from said intergenic region 
whereby the DNA molecule is capable of promoting, enhancing, regulating or 
modifying transcription of a non-BBTV gene. 
The partial fragment is defined in this specification as being a sequence 
less than the full sequence of the intergenic region of a BBTV component 
but equal to or greater than a sequence of approximately 172 base pairs 
from a BBTV component. The sequence of said DNA molecule may be identical 
to the complementary sequence of the partial fragment of an intergenic 
region of a BBTV component. 
The intergenic region is used in this specification to define the 
non-coding region of a BBTV component or the region outside the ORF in a 
BBTV component. 
The sequence of the DNA molecule may be substantially identical or 
substantially complementary to the partial fragment of the intergenic 
region of BBTV components 1-6. Substantially is used in this specification 
to refer to sequences having variations up to 20%. The amount of sequence 
variation can be determined by standard hybridisation procedures or 
sequence comparison. The percentage of 20% is the variation shown with the 
region outside of the ORF of component 1 between different geographical 
isolates (Karan et al., 1994, Journal of General Virology, 75, 3541-3546; 
and U.S. patent application Ser. No. 08/202186). Both of these documents 
are herein incorporated by reference to support the claim of 20% variation 
of all components of BBTV. The variation determined for component 1 of 
different geographical isolates is representative of the variation between 
each component from different geographical isolates. Thus variation up to 
20% also applies to the sequences of components 2 through to 6 discussed 
below. 
The term derived defines any sequence that has been changed, altered or 
modified by whatever procedure including mutagenesis from a fragment of 
the intergenic region of a BBTV component. 
In particular, the sequence of the DNA molecule may be the BBTV intergenic 
region derived inserts of pBT6.1 (approximately 623 base pair fragment), 
pBT6.2 (approximately 351 base pair fragment), pBT6.3 (approximately 239 
base pair fragment), and pBT6.4 (approximately 172 base pair fragment) 
from component 6; pBT1.1 (approximately 214 base pair fragment), pBT1.INT 
(approximately 980 base pair fragment) from component 1; pBT2.1 
(approximately 855 base pair fragment) from component 2; pBT3.1 
(approximately 526 base pair fragment) from component 3; pBT4.1 
(approximately 659 base pair fragment) from component 4; and pBT5.1 
(approximately 454 base pair fragment) from component 5. The DNA molecule 
may be 275 base pair region which includes the CR-M that is present in the 
insert of pBT6.1 but not in the insert of pBT6.2. The 275 base pair region 
may be a regulatory region. The DNA molecule may comprise the region 
between or including the CRSL region and the ATG of the open reading frame 
of any one of the BBTV components 1 to 6. The inserts of pBT1.1, pBT2.1, 
pBT3.1, pBT4.1, pBT5.1 and pBT6.1 are shown in FIG. 11. FIG. 12 shows the 
sequence of the insert of pBT1.INT. FIG. 13 shows the DNA sequence of the 
inserts of (a) pBT6.1; (b) pBT6.2; (c) pBT6.3; and (d) pBT6.4. 
The non-BBTV gene may be any suitable gene such as GUS, NPTII, insecticide 
resistance gene, herbacide resistance gene or a growth promoting gene. 
The DNA molecule may transcribe the gene in any suitable prokaryote or 
eukaryote host. Preferably, the DNA molecule may transcribe the gene in a 
monocotyledon plant cell such as plant cells from Musa spp (banana), wheat 
cells. The DNA molecule may transcribe the gene in a monocotyledon plant 
such as banana and wheat. Preferably the DNA molecule may transcribe the 
gene in a dicotyledon plant cell such as tobacco and zucchini cells. The 
DNA molecule may transcribe the gene in a dicotyledon plant such as 
tobacco and zucchini. The DNA molecule preferably transcribes the gene in 
cells of undifferentiated tissue in a dicotyledon plant. 
In a second aspect, the invention is a DNA chimaeric vector or cassette 
having a DNA molecule as described above upstream of a gene of interest to 
enable the promoting, enhancing, regulating or modifying of transcription 
of the gene. 
The chimaeric vector may be derived from pBI101.3. The vector may be any 
suitable construct mentioned below. The cassette may be any suitable 
construct mentioned below. The gene of interest may be any suitable gene 
as mentioned above. The chimaeric vector or cassette may be introduced 
into any suitable host including monocotyledon plant cells and dicotyledon 
plant cells for expression in the host. 
The invention in a third aspect is a plant cell having a DNA molecule as 
described above. 
The invention in a fourth aspect is a plant with the plant cells as 
described above. 
The invention in a fifth aspect provides a method of expressing a non-BBTV 
gene in a plant cell using the DNA molecule as described above. 
The invention will now be described with reference to preferred 
embodiments. However, these preferred embodiments are given by way of 
example. Example 1 describes the discovery and identification of the 
further three components of BBTV and is incorporated in the specification 
for convenience to allow a man skilled in the art to isolate these BBTV 
components. This information has essentially been published in Burns et 
al., 1995, Journal of General Virology, 76, 1471-1482).