Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-23
2003-10-14
Smith, Lynette R. F. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S023200, C536S024500, C536S023700, C435S024000, C435S317100, C435S346000, C435S418000, C435S419000, C435S069500, C435S253200, C435S253600
Reexamination Certificate
active
06632935
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to genomic DNA and plasmid DNA of aphids Buchnera sp.
BACKGROUND OF THE INVENTION
Buchnera sp. APS is a bacterial symbiont harbored by aphids. The host aphids are insects belonging to the suborder homoptero of the order hemiptera. Nearly 10,000 species of them are known throughout the world. Aphids have extremely strong fertility based on diploid parthenogenesis, and are one of the most serious agricultural insect pests on the earth. Aphids harbor many bacteria called Buchnera sp. in specialized cells, called bacteriocyte. The mutualism between Buchnera and aphids is so obligate that the symbiont Buchnera cannot survive outside the host aphid and aphids lacking Buchnera lose their fertility in addition to decreased growth.
Hence, noticing the host-symbiont relationship of the aphid and Buchnera is useful to obtain information to destroy aphids.
SUMMARY OF THE INVENTION
The present invention is to provide genomic DNA and plasmid DNA of Buchnera sp.
The present inventors have succeeded in determining a whole nucleotide sequence of genome of Buchnera, which is a bacterial symbiont harbored by
Acyrthosiphon pisum
and in identifying 619 genes (including plasmids) contained in the genome as a result of diligent research on the above problems.
That is, the present invention provides genes derived from Buchnera sp., comprising DNA of (a) or (b) as follows.
(a) a DNA selected from a group consisting of a DNA having a nucleotide sequence ranging from a start point to an end point as shown in Table 1 in a nucleotide sequence represented by SEQ ID NO:1, or a DNA complementary thereto, and
(b) a DNA hybridizing to said DNA of (a) under stringent conditions and encoding a protein having a function same as that of the product expressed by the DNA.
Here, the term “the product expressed by said DNA” means one of (a substance encoded by a sequence ranging from a start point to an end point) substances described in “Substance Name” column of Table 1.
Further, the present invention provides a recombinant vector containing the above gene or a transformant containing the vector.
Furthermore, the present invention provides genomic DNA of Buchnera sp. having a nucleotide sequence represented by SEQ ID NO:1.
Furthermore, the present invention provides a plasmid derived from Buchnera sp., comprising DNA of the (c) or (d) as follows.
(c) a DNA having a nucleotide sequence represented by SEQ ID NO:2 or 3, and
(d) a plasmid, capable of hybridizing to the DNA having a nucleotide sequence represented by SEQ ID NO:2 or 3 under stringent conditions, and self-replicating.
Further, the present invention provides a method of producing the above-mentioned protein, comprising the steps of culturing the transformant and collecting the protein expressed by a target gene from the resulting culture product.
Hereinafter, a more detailed explanation of this invention will be given. The present specification includes the contents of the specifications and/or drawings of the Japanese Patent Applications No. 2000-107160 based on which the present application claims priority.
The present invention relates to genomic DNA with a length of approximately 640 kb of Buchnera sp. (hereinafter also referred to as Buchnera) and two plasmid DNAs present in Buchnera sp.
1. Cloning of Buchnera genomic DNA and plasmids
Buchnera can be obtained by the following techniques. For example, the host aphids harboring Buchnera are dissected, and huge cells (called bacteriocyte) in which Buchnera is living are isolated. The bacteriocytes are crushed and filtered through a 5 &mgr;m pore size filter, thereby isolating Buchnera. Buchnera can also be isolated by homogenizing aphids and filtering the homogenates through 20, 10, and 5 &mgr;m pore size filters in order. Moreover, Buchnera can be isolated by density gradient centrifugation using sucrose or percoll (Pharmacia).
An example of aphids is
Acyrthosiphon pisum
(Harris).
Next, genomic DNA is prepared from Buchnera. The genomic DNA can be prepared by known methods including a phenol/chloroform protocol.
Thus obtained DNA can be analyzed by the whole genome shotgun sequencing in this invention. The whole genome shotgun sequencing is to provide information on a whole genomic sequence, comprising the steps of fragmenting and sequencing randomly the whole genome in large quantities, and searching fragment ends overlapping to each other using a computer to join them together. That is, this method involves sequencing each DNA fragment treated with restriction enzymes or each DNA fragment fragmented at a random site using HydroShear (GeneMachines) and the like, comparing the sequences to each other to find overlapping portions, and then connecting the overlapping ends of the fragments, whereby determining the whole sequence.
This technique is basically the same as that of Fleischmann R. D. et al (Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 469-512, 1995). In order to avoid chimera formation in preparing shotgun sequence libraries, some methods (for example, Partial Fill-in method) can be adapted. In the partial fill-in method, bases of overhang ends are partially polymerized.
The nucleotide sequences of the above DNA fragments can be determined by known techniques including Sanger method (Molecular Cloning, vol. 2, 13.3, 1989) and methods based on PCR. Normally, nucleotide sequences are determined by performing sequencing reactions with PRISM sequencing kit and the like containing fluorescent dideoxy terminator (Perkin Elmer), and using an autosequencer (model ABI 377, Applied Biosystem).
SEQ ID NO:1 represents the whole sequence of the genomic DNA of this invention. In addition, Table 1 shows all the genes (608 genes excluding plasmids) contained in the nucleotide sequence of the chromosome represented by SEQ ID NO:1. 572 genes encoding proteins contained in the above genes can be isolated by, for example, PCR method. In Table 1, “F” represents + chain and “R” represents − chain in the data in “Orientation” column. “Type” represents the sequence type of a gene. For example, CDS represents translation regions for proteins, tRNA transfer RNA, rRNA ribosomal RNA, and PS pseudogenes. Pseudogenes (PS) contain frameshift mutation or a stop codon inserted in the middle. When a direction is “F,” data in “Start point” column represents an initiation point for translation of a substance to be encoded by the gene, and data in “End point” column represents a termination point for the translation. For example, in Table 1, a second (BU002) gene (gene name: atpB) corresponds to a nucleotide sequence from 2278
th
to 3102
nd
bases and encodes ATP-synthetase A-chain. When a direction is “R,” translation proceeds in the direction opposite to that of the complementary strand from an initiation to an end point. For example, in Table 1, a 10
th
(BU010) gene (gene name: gyrB) represents a complementary strand of a nucleotide sequence from 8911
th
to 11322
nd
bases of a nucleotide sequence of SEQ ID NO:1. Translation proceeds in the direction from 11322
nd
to 8911
th
base based on the sequence position in SEQ ID NO:1. The remainder genes also encode substances (proteins, enzymes nucleic acids and the like) described in “Substance name” column according to nucleotide sequences between “Start point” and “End point” described in Table 1 or their complementary sequences.
TABLE 1
ID
gene name
type
a
orientation
start (bp)
end (bp)
description
BU001
gidA
CDS
F
197
2083
glucose inhibited division protein A
BU002
atpB
CDS
F
2278
3102
ATP synthase A chain
BU003
atpE
CDS
F
3139
3378
ATP synthase C chain
BU004
atpF
CDS
F
3497
3982
ATP synthase B chain
BU005
atpH
CDS
F
3982
4515
ATP synthase delta chain
BU006
atpA
CDS
F
4530
6068
ATP synthase alpha chain
BU007
atpG
CDS
F
6101
6973
ATP synthase gamma chain
BU008
atpD
CDS
F
6997
8394
ATP synthase beta chain
BU009
atpC
CDS
F
8421
8837
ATP synthase epsilon chain
BU010
gyrB
CDS
R
8911
11322
DNA gyrase subunit B
BU011
dnaN
CDS
R
11449
12549
DNA polymerase III beta chain
BU012
Hattori Masahira
Ishikawa Hajime
Sakaki Yoshiyuki
Shigenobu Shuji
Watanabe Hidemi
Baskar Padmavathi
Foley & Lardner
Riken
Smith Lynette R. F.
LandOfFree
Genome DNA of bacterial symbiont of aphids does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Genome DNA of bacterial symbiont of aphids, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Genome DNA of bacterial symbiont of aphids will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3127158