Afut
1, a retrotransposon-like element from
Aspergillus fumigatus
Afut 1, a retrotransposon-like element from Aspergillus fumigatus
Cécile
Neuveglise
,
Jacqueline
Sarfati
,
Jean-Paul
Latge
and
Sophie
Paris*
Laboratoire des Aspergillus, Institut Pasteur, 25 rue du Dr Roux, F-75724
Paris
Cedex 15,
France
Received January 26, 1996;
Revised and Accepted February 28, 1996
GenBank accession nos L76085 and L76086
ABSTRACT
A repeated DNA sequence used for epidemiological studies of the human opportunistic pathogen
Aspergillus fumigatus
has been characterized. It is a retroelement of 6914 bp in length, bounded by long terminal repeats of 282 bp,
with sequence and features characteristic of retroviruses and retrotransposons.
A 5 bp duplication site was found at its borders. This element, designated
Afut
1, encodes amino acid sequences homologous to the reverse transcriptase, RNase H and endonuclease
encoded by the
pol
genes of retroelements. Comparison of the peptidic sequences with other putative polypeptides of fungal LTR retrotransposons showed that
Afut
1 is a member of the gypsy group. This is the first report of a transposable
element in
A.fumigatus
.
Afut
1 is a defective element: the putative coding domains contain multiple stop
codons due exclusively to transitions from C:G to T:A.
INTRODUCTION
Aspergillus fumigatus
is an opportunistic fungus causing several respiratory diseases, such as allergic bronchopulmonary aspergillosis,
aspergilloma and invasive aspergillosis. The latter is presently a major cause of death amongst immunocompromised patients in hospitals,
especially in transplant units. Understanding the infection steps in the
hospital environment has required the development of molecular methods for
fingerprinting
A.fumigatus
strains. Repeated sequences specific for
A.fumigatus
have been isolated that allow precise strain identification and that provide a
measure of strain relatedness which has been very helpful in molecular
epidemiological studies of invasive aspergillosis (
1
-
3
).
Southern blot hybridization with dispersed repeated DNA sequences has been used
in molecular typing studies amongst human and plant fungal pathogens (
4
-
9
). The nature of these sequences is seldom identified. In the human pathogen
Candida albicans
most of the numerous repeated sequences (CARE1, CARE2, Ca3, Ca7, RPSs, 27a,
Tca1, etc.) show no homology to any known nucleotide sequence (
10
-
12
), except for the Ca7 sequence, which is a 23 bp repeated sequence associated
with telomeres (
13
), and for the Tca1 sequence, which is a retrotransposon-like element (
14
). In plant pathogenic fungi most of the dispersed repetitive sequences were
characterized as transposable elements, in particular retrotransposons with
long terminal repeats (LTRs) (
6
,
7
,
15
). Since the fungal LTR retroelements are merely found by analyzing repetitive
sequences, their ability to transpose has rarely been proven. Maggy, which has been identified by transposon tagging,
is the only LTR retrotransposon which has been shown to transpose (
16
). The
Cf
T-1 element of
Cladosporium fulvum
is supposed to be active, since this element is transcribed into an RNA which is
packaged in virus-like particles that contain reverse transcriptase (
7
). In contrast, elements like
Foret
1 (
17
) that possess an accumulation of stop codons in putative coding regions are
clearly defective.
Since the nature of the repeated sequences of
A.fumigatus
was not known, we investigated the repetitive DNA sequence [lambda]3.9 (
1
) used as a probe for epidemiological studies of
A.fumigatus
(
2
,
3
).
MATERIALS AND METHODS
Fungal strains and culture media
The reference strain of
A.fumigatus
used in this study was originally isolated from a patient in 1971 and deposited
at the Centraalbureau voor Schimmelcultures in 1989 as CBS143-89. The strain was maintained on 2% malt agar. For DNA isolation CBS143-89 was grown overnight at 37oC in Sabouraud's medium [1% mycopeptone (Biocar, France), 2%
glucose].
Bacterial strains and plasmids
Escherichia coli
DH5[alpha] [
endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1
[Delta](
lacZYA-argF
)
U169 deoR
([Phi]
80dlac
[Delta](
lacZ
)
M15
)] and PAP105 [[Delta](
lac-pro
) F'(
lacI
q1
[Delta](
lacZ
)
M15
pro
+
Tn
10
)] were used for propagation and amplification of recombinant plasmids as
described by Sambrook
et al
. (
18
). Plasmid Bluescript
)
SK(+) (Stratagene, La Jolla, CA) was used in subcloning procedures.
Isolation and manipulation of DNA
Aspergillus fumigatus
DNA preparations and Southern blot hybridization were done as previously
described (
1
). Plasmid DNA manipulations, cloning techniques and DNA sequencing were
performed by standard procedures (
18
).
The [lambda]3.9 recombinant phage (
1
) was first digested with
Eco
RI. Four fragments of 1.0, 1.1, 1.7 and 5.0 kb showed strong intensity when probed with
32
P-labeled genomic DNA of the CBS143-89 strain. These fragments, containing portions of the repetitive sequence, were subcloned into pBluescript
)
SK(+), yielding pJS2, pJS1, pJS24 and pCN13 respectively. The
Hin
dIII and
Acc
I fragments contained in the insert of pCN13 were subcloned into pBluescript
)
SK(+), yielding pCN25H and pCN20A respectively. The repetitive sequence of [lambda]3.9 phage was sequenced on both strands using primers that hybridized with the vector sequence and using
appropriately designed primers.
Nucleotide sequence accession numbers
The nucleotide sequences of the 3.9 copy of
Afut
1 and of the 5' LTR of the 4.11 copy have been submitted to the GenBank database under
accession nos L76086 and L76085 respectively.
RESULTS AND DISCUSSION
Structure of repetitive DNA from
A.fumigatus
The recombinant [lambda] phage 3.9 contained a repetitive DNA sequence, which was divided into
four
Eco
RI restriction fragments subcloned into Bluescript
)
SK(+) (Fig.
1
), and that was bordered with single copy sequences (data not shown). Sequence data of the four subclones
revealed: (i) two direct repeats of 282 bp in pJS1 and pCN13; (ii) between
these two repeats a DNA sequence with significant homology to the
pol
genes of LTR retrotransposons; (iii) two short direct repeats of 5 bp (TCCTT) flanking the 3.9 repetitive
sequence. These data suggested that the 3.9 repetitive sequence is a retransposon with long terminal repeats (LTRs).
This element was therefore called
Afut
1 (
A.fu
migatus
t
ransposon).
The two 282 bp direct repeats corresponding to the LTRs, as well as the two 5 bp direct repeats characteristic of a target site duplication
generated during transposition, delimited the extremities of the repeated sequence. Nucleotide analysis of the element revealed that it
had a total length of 6914 bp and was A+T-rich (63%), while structural genes of
A.fumigatus
show ~47% A+T.
The 5' and 3' LTRs are not perfect direct repeats, since they share only 90% nt identity. There are 27 nt differences between
the two LTRs, corresponding exclusively to C:G -> T:A transitions (Fig.
2
). The 5' and 3' LTRs contain the 5'-terminal TG and the 3'-terminal CA respectively (see boxed
nucleotides in Fig.
2
) that are characteristics of retroviruses and of most retrotransposons. However, the other end of each LTR does not possess this property, because of a
C:G -> T:A base pair transition (Fig.
2
). Short direct repeats were identified in the LTR sequence. None of them
corresponds to sequences previously described in the LTR of other
retrotransposons.
[lambda]
4.11, another copy of
Afut
1
The [lambda]4.11 recombinant phage was isolated from the same library as the [lambda]3.9 phage and cross-hybridized with it. The repeated region of the [lambda]4.11 phage was cut into two
Bgl
II fragments which were subsequently subcloned. The restriction maps of these
two subclones revealed that the three
Eco
RI sites and most of the
Hin
dIII sites previously identified in the 3.9 copy are conserved, whereas the
Acc
I sites are absent. This implies that the repeated sequence of the two phage
corresponds to two different copies.
The 5' LTR of the 4.11 copy was sequenced and compared with the 5' LTR of the 3.9 sequence (Fig.
4
A). They are only 86.5% identical. All nucleotide mutations correspond to C:G -> T:A transitions. The insertion site of the 4.11 copy is ATAAT, which is
distinct from the 3.9 insertion site (TCCTT). Moreover, the sequences flanking
the elements do not show any homology. This confirms that 3.9 and 4.11 are two
distinct copies.
Distribution, conservation and copy number of
Afut
1 elements
To determine the copy number of the
Afut
1 elements per genome the DNA of strain CBS143-89 was digested with enzymes that do not cut the 3.9 copy of
Afut
1 (
Bam
HI,
Cla
I,
Eco
RV,
Pvu
II,
Sal
I and
Xho
I), transferred to nylon membrane and probed with internal subclones of
Afut
1 (pCN20A and pJS2). The hybridization patterns of the
Eco
RV and
Pvu
II digests show bands whose sizes are smaller than 7 kb, confirming that the
restriction sites of the different copies of
Afut
1 are not conserved. In the other digests (
Bam
HI,
Cla
I,
Sal
I and
Xho
I) the number of hybridizing bands (>7 kb) is at least 10, but with variable
intensity (data not shown).
CpG dinucleotides in genes or sequences of
A.fumigatus
Gene or sequence
Size (bp)
CpG
Gene accession no.*
O
E
O/E
Afut
1
6924
39
236
0.165
L76086
PEP
1540
71
104
0.683
X85092
RES
834
44
56
0.785
X58278
RAS
2590
126
146
0.863
L42299
HSP
360
17
28
0.607
S60074
RODA
1202
54
76
0.710
L25258
ALP
2163
112
147
0.762
Z11580
MEP
3068
193
222
0.869
Z30424
O, observed number of CpG in the sequence; E, expected number of CpG in the
sequence = (number of C residues in the sequence) * (number of G residues in the sequence) [divide] (total bases in the sequence), according to Kricker
et al
. (38).
*GenBank.
It is not yet certain whether this difference in intensity reflects a nucleotide
divergence (the 3.9 and 4.11 copies share only 92% identity in the RT and 86.5%
in the 5' LTR) or reveals a variable number of copies. Transposable elements such
as
Pogo
from
Neurospora crassa
(
26
) and
Ty1
from
Saccharomyces cerevisiae
(
27
) showed a similar heterogeneity. Since the restriction sites of
Afut
1 are poorly conserved and prevent an accurate count of the number of full-length copies, an approximate number of at least 10 copies can be
proposed. This number is consistent with the moderate copy number (25 copies)
of
Cf
T-1 from
C.fulvum
(
7
).
Conservation of
Afut
1 copies was more precisely studied by analyzing their restriction site
polymorphism within the CBS143-89 genome. There are three
Eco
RI, four
Hin
dIII and four
Acc
I restriction sites in the 3.9 copy of
Afut
1 (Fig.
1
).
Eco
RI-,
Hind
III- or
Acc
I-digested genomic DNA was subjected to Southern analysis by probing with
pJS2 (1 kb) and pJS24 (1.7 kb), pCN25H (1.3 kb) or pCN20A (1.4 kb) respectively
(Fig.
5
). The patterns revealed a ladder of bands of heterogeneous intensity, in addition to
the expected one. This confirms again that the positions of the restriction
sites mapped within the sequenced element are poorly conserved in the other
genomic copies of
Afut
1.
The presence of
Afut
1 LTR as solo elements was investigated in strain CBS143-89 by digesting genomic DNA with different enzymes that do not cut the 3.9
copy of
Afut
1 (
Bam
HI,
Cla
I,
Eco
RV,
Pvu
II,
Sal
I and
Xho
I) and by probing the Southern blot with two different fragments of
Afut
1: first, an amplified fragment of the 5' and 3' LTRs (Fig.
6
A) and, second, amplification products of the two internal regions of
Afut
1 close to the LTRs (Fig.
6
B). These two fragments, located 52 bp downstream of the 5' LTR and 218 bp upstream of the 3' LTR, are respectively 571 and 686 bp long. They were chosen in
order to minimize the occurrence of hybridizing fragments that do not
correspond to true solo LTRs. Comparison of the hybridization patterns revealed
the presence of additional bands when digested genomic DNA was hybridized with
the LTR fragment. This strongly suggests that there are several solo LTRs in
the genome, but cloning and sequencing of the fragments corresponding to the
additional bands should be done to confirm the presence or absence of solo
elements. LTRs as solo elements have not previously been identified in
filamentous fungi. However, in other eukaryotic genomes LTRs are often found as solo elements separated from
the transposon. In the dimorphic fungus
Yarrowia lipolytica
Schmid-Berger
et al
. (
28
) reported the existence of >30 copies of the solo [xi] element (the LTR of the composite element Ylt1). In
S.cerevisiae
LTRs of the
Ty
retrotransposons are often found as solo elements: [delta] elements for
Ty1
(
27
,
29
), [sigma] elements for
Ty3
(
30
) and
Ty5
LTR (
31
). In the maize species
Zea mays
Cin1 sequences are only known as solo LTR elements (
32
). According to Roeder and Fink (
33
), the solo LTRs arose by homologous recombination between the LTRs of the
retroelement followed by excision of the internal region. Chen and Fonzi (
14
) explained the existence of solo LTRs in the Tca1 element of
C.albicans
by this type of recombinational event.
Afut
1 is a defective retrotransposon
Several arguments show that
Afut
1 is a defective transposable element. First, the amino acids sequence
homologous to the polyprotein of retroviruses is interrupted by many stop
codons in both the 3.9 and 4.11 copies. Moreover, the overall conserved amino
acid boxes, such as YXDD in the RT domain, are mutated in the two
Afut
1 copies, whereas this box has been proposed to be part of the RT active site (
23
). The two LTRs of the 3.9 copy share only 90% identity. Accumulation of stop codons in putative coding regions and
divergence between the LTRs of the same copy have already been described. In
the fungal retroelement
Foret
1 the amino acid sequence corresponding to the protease, RT and RNase H domains
is interrupted by many stop codons. Similarly,
del
, the
Lilium henryi
retrotransposon (
34
) and
Ty5
from
S.cerevisiae
(
31
) contain several stop codons.
In the
Pogo
element of
N.crassa
the two copies of the LTR-like sequences, which are 89% identical, differ by 34 nt, including 33
transitions from C:G to T:A (
35
). In the 3.9 copy of
Afut
1 the 27 nt substitutions correspond exclusively to transitions. Similarly, nucleotide differences between the RT of the 3.9 and 4.11 copies are transitions
from C:G to T:A. Such a pattern of nucleotide variations would be consistent
with a process described for
Neurospora
repeated sequences (
36
). This process, referred to as RIP (repeat-induced point mutation) produces numerous C:G -> T:A mutations in both copies of duplicated sequences during the
period between fertilization and karyogamy. In
Neurospora
changes occurred primarily at sites where there was an adenine 3' of the changed cytosine (
37
). In order to determine if mutations occurred randomly the frequencies of
dinucleotides in
Afut
1 were analyzed. The
Afut
1 sequence contains no CpA depletions, but the frequency of CpG dinucleotides
was unusually low ( <= 0.165) when compared with the frequencies observed in
A.fumigatus
structural genes, which have a mean value of 0.68 (Table
1
). In other words, the substantial deficit of CpG clearly suggest that mutations
occurred preferentially in CpG dinucleotides, as in mammalian genomes (
38
). As cytosine methylation is typically associated with mutations in sequences
affected by RIP (
39
), it was of interest to determine whether the
Afut
1 copies were subjected to methylation. The genomic DNA of strain CBS143-89 was digested with isoschizomers of restriction endonucleases which
exhibit differential sensitivity to cytosine methylation:
Sau
3AI and
Nde
II or
Msp
I and
Hpa
II. In contrast to
Sau
3A,
Nde
II is blocked by methylation. When the external C in the sequence CCGG is
methylated neither
Msp
I nor
Hpa
II can cleave. However, unlike
Hpa
II,
Msp
I can cleave the sequence when the internal C residue is methylated. The
restriction patterns obtained after ethidium bromide staining of the digested
genomic DNA appeared identical with both couples of isoschizomers. Moreover,
the Southern blot probed with the [lambda]3.9 phage revealed identical patterns with the isoschizomers, which
implies the absence of methylated cytosine in
Afut
1 copies.
Two hypotheses are proposed to explain this accumulation of mutations. First,
the mechanism of CpG site mutation in
Afut
1 copies is a new phenomenon that does not accurately correspond to the RIP
process described in
N.crassa
since: (i)
A.fumigatus
sexual reproduction is unknown; (ii) no methylation was detected in
Afut
1 copies by DNA digestion analysis. The more attractive hypothesis is that the
strains studied are presently defective for RIP, but that
Afut
1 has been subjected to RIP at a time when
A.fumigatus
possessed a functional sexual cycle and an active DNA methylation process. Thus the 3.9 and 4.11 copies could be relics of RIP.
ACKNOWLEDGEMENTS
We thank Dr G. Faugeron for helpful discussions about the RIP mechanism,
suggestions and stimulating encouragement.We are grateful to Drs M. J. Daboussi
and T. Langin for useful comments about fungal transposable elements. We are
also thankful to Dr C. d'Enfert for critical reading of the manuscript. This
research was supported by CNAMTS-INSERM grant 3AM051 to J.P.L. and a post-doctoral fellowship from the Association Française de Lutte contre la Mucoviscidose (AFLM) to C.N.
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