Biology Reference
In-Depth Information
14000
12000
10000
8000
6000
4000
2000
0
Male
Female
FIGURE 18.11
Sex-linked difference in titer of wBruAus. Titers of wBruAus in adult males and females
were quantiÝed in terms of
wsp
gene copies per nanogram of total insect DNA. The difference was statistically
signiÝcant (Mann-Whitney
U
-test,
p
< 0.001). [From Kondo, N., Nikoh, N., Ijichi, N., Shimada, M., and
Fukatsu, T. (2002b).
Proc. Natl. Acad. Sci. U.S.A.
99:
14280Ï14285.]
some of the host insect. If this were true, female insects should contain twice as much titer of
wBruAus as male insects. Quantitative PCR analysis certainly demonstrated that females contained
about twice as much
wsp
copies per nanogram of total DNA as males (Figure 18.11), which
conÝrmed the idea of the X chromosome linkage.
DETECTION OF
WOLBACHIA
GENES IN CURED STRAIN
OF
C. CHINENSIS
Using speciÝc PCR and Southern blotting, we attempted to detect
Wolbachia
genes in the cured
strain jC
Aus
that contained only wBruAus. The following 17 genes were targeted: 16S rDNA,
atpD,
cytB, ftsZ, gltA, glyA, groE, gyrA, IswI, lipA, polA, rpoB, sdhA, sucD, thrA, tufA
, and
wsp
. While
all the genes were detected from the triple-infected strain jC, only three genes Ð
ftsZ, gyrA
, and
wsp
Ð were detected from the strain jC
Aus
(data not shown). These results indicated that the strain
jC
Aus
contained only a fraction of the
Wolbachia
gene repertoire, which agrees with the idea that
the wBruAus strain is a genome fragment of
Wolbachia
located on the host chromosome.
STRUCTURE OF
WOLBACHIA
GENOME FRAGMENTS OBTAINED
FROM CURED STRAIN OF
C. CHINENSIS
We cloned and sequenced the ampliÝed fragments of
wsp
,
ftsZ
, and
gyrA
genes from the total DNA
of the strain jC
Aus
. In addition, we successfully isolated large DNA fragments Þanking
wsp
and
ftsZ
genes using an inverse PCR technique. A 4141-bps fragment containing
wsp
(Figure 18.12A),
an
11,400-bps fragment containing
ftsZ
(Figure 18.12B), and a 373-bps fragment of
gyrA
were obtained.
These DNA fragments encoded bacterial genes that showed high sequence similarity to those of the
b-proteobacteria such as
Wolbachia
,
Rickettsia
, and
Brucella
. In the 4.1-kb fragment, four bacterial
open reading frames (ORFs) were identiÝed:
dnaX
,
wsp
, and two ORFs homologous to RP866 and
RP416 in the genome of
Rickettsia prowazekii.
In the 11.4-kb fragment, seven bacterial ORFs were
detected:
ftsZ
,
sdhB
,
pgpA
,
g3pdh
,
czcR
, an ORF homologous to RP741 in the genome of
R.
prowazekii,
and an ORF homologous to BMEI0172 in the genome of
B. melitensis
. One ORF showed
a partial weak homology to acetyltransferase genes from various bacteria, archaea, and plants.
NON-LTR RETROTRANSPOSON-LIKE SEQUENCE
IN THE
WOLBACHIA
GENOME FRAGMENT
Notably, a non-long terminal repeat (non-LTR) retrotransposon-like sequence, which showed a
signiÝcant similarity to
I
and
Yo u
elements from
Drosophila
species (Berezikov et al., 2000) and