FRANcISco FIERRO*, JOSE Luis BARREDOt, BRUNO DfEZt, SANrIAGoGUTIERREZ*, FRANcISco J. FERNANDEZ*,. AND JUAN F. MARTIN*:. *Section of ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6200-6204, June 1995 Microbiology
The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences FRANcISco FIERRO*, JOSE Luis BARREDOt, BRUNO DfEZt, SANrIAGo GUTIERREZ*, FRANcISco J. FERNANDEZ*, AND JUAN F. MARTIN*: *Section of Microbiology and Institute of Biotechnology (INBIOTEC), University of Le6n, Faculty of Biology, 24071 Le6n, Spain; and tAntibi6ticos, S.A., Avenida de Antibi6ticos, 56, Le6n, Spain
Communicated by Arnold L. Demain, Massachusetts Institute of Technology, Cambridge, MA, February 13, 1995 (received for review, September 26, 1994)
The penicillin biosynthetic genes (pcbAB, ABSTRACT pcbC,penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum El, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npelO showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in El. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.
Strains. P. chrysogenum NRRL 1951 (wild-type strain) and Penicillium notatum ATCC 9478, the initial isolate of Fleming, were used. P. chrysogenum npelO is a deletion mutant that lacks the entire penicillin gene cluster (15). P. chrysogenum AS-P-78 is an early high penicillin producing strain provided by Antibioticos, S.A. P. chrysogenum P2, an initial strain of the PanLabs series (16), was donated by J. Lein (Panlabs, Deer Harbor, WA). P. chrysogenum El, a high penicillin producer, was developed at Antibioticos, S.A. Gene Libraries, Southern Blot Analysis, Hybridizations, and Sequencing. A genomic DNA library of P. chrysogenum AS-P-78 was made in AEMBL3, and libraries of genomic DNA of the wild-type P. chrysogenum NRRL 1951 and the high penicillin producer P. chrysogenum El were constructed in AGEM-12. Southern blot analysis, hybridizations, and sequencing were carried out by standard procedures (17).
Amplification of DNA sequences occurs in a variety of eukaryotic organisms (1-4). In some cases, repeated genes are a normal component of the genome (e.g., ribosomal RNA genes), whereas in others amplifications are due to mutations that increase expression of genes conferring resistance to metals (5, 6) or to antibiotics (7). Very few examples of gene amplification have been studied in filamentous fungi (8, 9). The penicillin biosynthetic genes (pcbAB,pcbC, and penDE) are clustered in a region of Penicillium chrysogenum DNA extending for 15 kb (10, 11). A large amplification of a DNA region of at least 35 kb was found in three high producing strains of P. chrysogenum by two groups (12, 13). The genes for the penicillin cluster were found to be amplified between 6 and 16 copies in different strains. However, it was impossible to conclude whether the amplification was tandemly repeated or dispersed throughout one or more chromosomes. The penicillin gene cluster of P. chrysogenum was located in the largest chromosome (chromosome I, 10.4 megabases in the wild type). No hybridization was observed in the other chromosomes ofP. chrysogenum AS-P-78 with probes internal to the pcbAB gene (14), suggesting that the amplification occurred within a single chromosome. Chromosomes of the high penicillin producing strains have undergone considerable changes in size (14), but it is unknown whether these chromosomal reorganizations affect penicillin biosynthesis. We report in this article that the amplified region (hereafter named AR) in AS-P-78 consists of tandem repeats of a unit of 106.5 kb. An industrial strain P. chrysogenum El contained 12-14 tandemly repeated copies of a 57.9-kb unit that overlapped with the 106.5-kb unit of AS-P-78.
RESULTS Cloning of the Right End of the AR in Strain AS-P-78. Based on the different intensity of hybridization of restriction fragments in strains AS-P-78 and P2 obtained with probes internal or external to the AR, we searched for the right end of the AR. Estimation by densitometry after hybridization of serial dilutions of total DNA of AS-P-78 with probes internal to the pebAB gene indicated that strain AS-P-78 contained five or six copies of the penicillin biosynthetic genes. By chromosome walking from phage F6A and F16A that contained the penicillin gene cluster, we cloned the region downstream of the penDE gene (phage F56) (Fig. 1). By using as probe a 1.4-kb Sal I-BamHI fragment corresponding to the distal end (with regard to the pen cluster) of phage F56, we observed that the DNA fragment was already out of the AR. To define precisely the junction between the amplified and the nonamplified DNA, several small probes corresponding to DNA fragments of phage F56 were made. One of these probes (probe C, a 1.1-kb Sal I-EcoRI fragment, Fig. 2) showed a high-copy-number hybridization pattern with strains AS-P-78 and P2 (Fig. 3), indicating that this fragment was located inside the AR. A different probe (probe D, a 0.88-kb Sal I-HindIII fragment, Fig. 2) hybridized with a DNA fragment located outside of the AR. In this way, a 1.42-kb Sal I band was defined that contained the right end border (REB) of the AR (downstream from the penDE gene). This fragment was mapped in detail and sequenced. Cloning of the Union Fragment Between Tandem Repeats in Strain AS-P-78. If the five or six copies of the repeated unit in AS-P-78 are organized in tandem, probe C (that is internal to the right end of the AR, Fig. 2) should hybridize also with the
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Abbreviations: AR, amplified region; REB, right end border; TRU, tandem repeats union; LEB, left end border; SF, shift fragment. *To whom reprint requests should be addressed.
MATERIALS AND METHODS
-
6200
Ii~ .
Proc. Natl. Acad. Sci. USA 92 (1995)
Microbiology: Fierro et aL LEB
TRU
_
P76
F76
N43
TRU
L-i
TRU
TRU
iL-7i
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H
F56 Ni
N32
_
F83
N5
F6
F64
N40
N61
F16A F6A
.20
pcbAB
AS-P-78
~ ~~H ~
F56 pcbC penDE
IREBI E
~
s s s s ~ss SsS s Is Si$Bs WiS.I54-1255 nPe1O Pll RBfBm i T Bn b tBB B~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ E E j E E R BE E E EM ! . E B E ,}4**5Es E' =;.SSX2 j~I'
S S S~~~~~~~~~~~~~~~~~~~~~~S
B
ii
I
S
S B
'~ ~ ~~~~s
'I~ ~ ~ ~~~i El
54.8Kb
~~~~~AL64
AL1O5 D Kb
AL92
th ahdln nbakt ~~~ ntelwrpato Wis. 54-1255 npel1- h P11iue ,SlI
by~~~ ~
REB l-
H
F36
N57
boxes).
6201
,BmI
pcbC penDE!i ~~~~~~~~~~~pcbAB AL99
~~~~~~
AL70 ,EcRI
B4
AL85
~~~~~~~~~~~~~~~~~~~~~~ Pi11
FIG. 1. Restriction map of the 106.5-kb unit that is amplified in P. chrysogenum AS-P-78 and the 57.9-kb unit of P. chrysogenum El (stippled boxes). The LEB and REB fragments are indicated by solid boxes. The arrangement of the five TRUs in AS-P-78 is indicated in the upper part of the figure. DNA fragments cloned in the different phage are indicated by thin lines. Phage of the F and N series correspond to DNA fragments of AS-P-78 and NRRL 1951, respectively. Phage of the AL series contain DNA fragments of the penicillin-overproducing El strain. Phage P11 contains DNA from the nonproducer mutant npelO. The DNA region deleted in mutant npelO (which coincides with the AR in strain El) is indicated by the dashed line in brackets in the lower part of the figure. S, Sal I; B, BamHl; E, EcoRI.
end of repeated units near the border between tandem repeats. As expected, several phage were obtained from the AS-P-78 library (e.g., phage F36, Fig. 1) by hybridization with probe C that showed a restriction map identical to that of F56 up to a point located inside the REB but different thereafter. In this way, a 1.47-kb Sal I fragment was identified that contained the end of the identical sequence of phage F56 and F36 (Fig. 2) and corresponds to the tandem repeats union (TRU). As shown in S
A
Sa
S
B
'B'SS
1j4 probe A
H
2.4
H
E
I.3
EV EV EV /S EV
S
probe B Sa i.S
3503R2
REB
E
H
B
fj I -,,l -W
2.4
left end
Z2 tandem repeat union 6.55
3,
C
probe C
B
3'5
Sa
B
S
probe B
TRU
S
\
EV
EV
>~~~-=
LEB
probe C
H
EV
5._63
2.58
S H Sa C B E B ..i~j Q
Sa
EV
H
B
47
C
EV
S
-right end 6.4
probe D
1-'GCTCGAGGGTGTAGCC C-C CCGGGCTC CtI'TTACAICCATCAA1GC'TA'I'GTC TlGC-A'CACCCCTCCA TCTCCATGGTTGTGATATGTTGGTCMAAT TTACACCATCAATCTATGTCTGGAT'CCCCTCCA TCTCCATGGTTGTCATATGTTGTCAAG TTTACAIACTAGAATATCGGAACCTGTGGGATTGA
LEB TRU REB
FIG. 2. (A) Detailed restriction maps of the LEB, TRU, and REB of the AR in P. chrysogenum AS-P-78 (stippled boxes). S, Sal I; Sa, Sac I; E, EcoRI; EV, EcoRV; B, BamHI; C, Cla I; and H, HindIII. Probes A, B, C, and D, used for hybridization studies to determine the ends of the AR, are shown by arrows. A double line indicates AR DNA and a thin line indicates the non-AR DNA. The junction points inside the LEB, TRU, and REB are indicated by vertical bars. (B) Nucleotide sequences of the LEB, TRU, and REB. Note the conservation of the hexanucleotide TTTACA in the borders and the junction zone.
Fig. 2, the region labeled as probe C was identical to that found at the REB of the AR, up to the Sac I site inside the TRU. Cloning of the Left End Border (LEB) of the AR in Strain AS-P-78. If the five or six repeats are organized in tandem without stretches of single-copy DNA between them, the left end of the AR should show the same restriction map as the sequence at the left end of the repeats. Indeed, when a 2.73-kb Sal I fragment (probe B, Fig. 2) of phage F36 contiguous to the TRU was used as probe, the pattern of hybridization of strains AS-P-78 and P2 showed that this fragment is located within the AR (Fig. 3, probe B). Several phage (e.g., F76) selected by hybridization with this probe showed a restriction map identical to that of F36 up to a point inside the TRU sequence, but different thereafter. The fragment enclosing the end of the AR distal from the pen cluster (a 1.5-kb Sal I fragment) has been named the LEB (Fig. 2). To confirm that this region corresponds to the left end of the AR, the DNA of the different strains was digested and hybridized with probe A (1.4-kb Sal I fragment) contiguous but external to the LEB. As shown in Fig. 3, probe A, only the nonamplified 6.9-kb EcoRI fragment was observed in all strains including AS-P-78 and P2. The Wild-Type P. chrysogenum NRRL 1951 Contains a Single Copy of the Pen Region. Phage carrying the pen region from a genomic library of the wild-type were selected by hybridization with probes corresponding to the REB and LEB fragments. None of the phage cloned from strain NRRL 1951 corresponded to the TRU region found in strain AS-P-78, indicating that the wild type contains a single copy of the pen region. Mapping of a Single Unit of the AR. A complete unit of the AR of strain AS-P-78 was cloned and mapped by "chromosome walking" from the LEB (phage N43) toward the right and from phage F83 toward the left. Phage from the AS-P-78 and NRRL 1951 strains were used for this purpose. The restriction maps of phage from one or the other P. chrysogenum strains were identical. The unit, which is repeated in tandem, extended for 106.5 kb between the LEB and the REB. The restriction map of the pen region was identical in NRRL 1951 (single copy) and in AS-P-78 (five or six units) as shown by
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probe A 1 2 3 4 5 6
6.9
am_m____ m a
probe C 1 2 3 4 5 6 6.3-
Proc. Natl. Acad. Sci. USA 92
Microbiology: Fierro et aL
0
prolbe B 1 2 3 4 5 6
Om
_D
5
4m
6.9 6.3
prolbe D 1 2 3 4 5 6
o
4.35
3.4
___ -- 2.1
0.8 _ 0.52 FIG. 3. Hybridizations of total DNA of P. chryssogenum Wis54-1255 (lane 1), P2 (lane 2), npelO (lane 3), NRRL 195:1 wild type (lane 4), and AS-P-78 (lane 5) and P. notatum ATCC 9478 (lane 6) with probes A, B, C, and D as indicated (see Fig. 2). DNAs were digested with EcoRI for probes A-C and with BamHI for probe D. A common band appears in all the strains (except in the deletion mutant npelO when hybridized with probes C and D, which are inter nal to the deletion). A second thick band corresponding to the AR (uni is seen in strains P2 and AS-P-78 when hybridize with probes B and C (internal to the AR) but not with probes A an Ld D (external to the AR). Note that the hybridizing bands with probes C and D in strains Wis54-1255 and P2 (lanes 1 and 2) are different fr*om those of the wild type, AS-P-78, and P. notatum (lanes 4-6) diue to the different orientation of the SF.
di
digestion with several restriction enzymes and hybridization with DNA of phage N40, N57, N61, F20, and F83 as probes. Hybridization ofP. chrysogenum P2 DNA 1with probes A and B (of the LEB) and with probes C and I) (REB) (Fig. 3) showed that the AR of strain P2 is almost i dentical to that of AS-P-78 extending also for 106.5 kb. A smiall difference was observed, however, between different P. chtrysogenum strains near the right end of the AR. As shown in Fiig. 3, probes C and D, hybridization results indicated that the wild-type NRRL 1951 and AS-P-78 strains showed an identic al organization of the right end region, but it was different frc)m that existing in the Wis54-1255 and P2 strain. P.notatum (IFleming's isolate) showed the same restriction fragments in th[e right end region as the wild-type P. chrysogenum NRRL 1 951 and AS-P-78 strains. Nucleotide Sequence of the Borders of the AR and the Union Between Repeats. A region of -0.8 kb of REB, LEB, and TRU was sequenced in both strands. A 6-nt sequeince TTTACA was found in the junctions of the REB and LEB with the non-AR (Fig. 2) and in the regions correspondinig to the junction between repeats. This suggests that a recomiibination phenomenon resulting in tandem repeats has occuirred between the REB and LEB of the wild type. The crossi ng-over may have occurred between any two nucleotides of t}his sequence.
(1995)
Industrial Strains Show a More Complex Amplification Pattern. The organization of the AR was also studied in a very high penicillin producing strain P. chrysogenum El. This strain was shown to contain 12-14 copies of the penicillin gene cluster. By using the same strategy described for P. chrysogenum AS-P-78, we found that the REB in El was close to that found in AS-P-78 although not at the same point (Figs. 1 and 4). In contrast, the LEB of the industrial El strain was located 57.9 kb from the REB, inside the 106.5-kb unit of the wild type; i.e., the repeated unit in strain El is only 57.9 kb long, corresponding to the right part of the 106.5-kb unit of the P2 and AS-P-78 strains. A detailed map of the ends of the 57.9-kb unit and the TRU of El is shown in Fig. 4. An internal 3.4-kb fragment adjacent to the REB has shifted its orientation in some of the copies of the AR of strain El. This fragment, named shift fragment (SF), is present in strain AS-P-78 (and in the wild type) adjacent to the REB but outside of the AR (Fig. 1); i.e., the AR in P. chrysogenum El extends 3.4 kb from the REB of strain AS-P-78, corresponding exactly to the SF. The orientation of the SF existing in the wild-type and AS-P-78 strains occurs only in a minority (1 or 2 copies) of the 57.9-kB units of the high producer strain El, whereas most units of this strain (10-12 copies) contain the SF in orientation opposite to that of the wild type. Comparative Analysis of the Junctions in the Different Strains. The different junctions between the units (TRU) and the SF are shown schematically in Fig. 5. The sequence TTTACA found initially in AS-P-78 was also observed in the junctions EG' and EF (as expected) of strain El. It is interesting that the junctions CD and F'H and the TRU sequence (F'D) of the high producer El contain the sequence TGTAA(A), which is inverse complementary to the previously found TTTACA, suggesting that the borders in the high producer strain have been made de novo by using the same TTTACA sequence that occurs (in the opposite strand) 3.4 kb downstream of the amplified unit in the wild type. The Deletion in Mutant npelO Coincides with the Borders of the AR in the High Producer El. The deletion in mutant npelO was delimited by hybridizations with a set of probes of B
S
C
\
EV
E H H EV H \ H X C / B
EV
4.6
left end Sa
E
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.
\
C
B
\
S
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I
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8.0
tandem union 1 (1-2 copies) E B C Sa C E B Sa E H H H Sa S EV B H S H C B I'
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(10-12 copies)
Sa
H
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C
Sa
Sa C
E EV B
H
B Sa
S
B EV
S
3.49
7t, 7i 7- |~~...-~:!.........__ 8A2
right end FIG. 4. Restriction map of the left end, tandem union type I, tandem union type II, and right end of the high producing strain El. Symbols are as in Fig. 2. SF, stippled box. The junction points are indicated by the vertical dashed line.
Proc. Natl. Acad. Sci. USA 92 (1995)
Microbiology: Fierro et aL
6203
SHIFT FRAGMENT
P. chrysogenum NRRL 1951
AB
C,D
E,F GIH
P. chrysogenum Wis 54-1255
A,B
C,D
EjG' F:H
P. chrysogenum
AB
C
! ~
npel0
~~~
-- -- --
H ----
JUNCTIONS
GAGTTr-TAAAT GCTCC~
left end P. chrysogenum L AS-P-78 B A ARB
left end P. chrysogenum P2
EB EB tandem repeat EF G1H rightend
cH
CCATC
TCAAdf?qZCATC
EB
TCAA CTAG
EF
GAGT7AAACG
CD
GACCAg~AACG TCAAQX3CTAG
ED
TCAA(P3rC CG
FGD
EB
EB
W tandem repeats
EG' F' H right end
CD
A,B
left end
D EF GD ttandem repeat T4
(1-2 copies) D EG'FD '-tandem repeat II
P. chrysogenum _ El
(10-12 copies) EG,
FIG. 5. (Left) Comparative organization of the ARs in strains AS-P-78, P2, and El relative to the wild-type NRRL 1951 (single copy) and to the low-producer Wis54-1255 (single copy but with the fragment FG shifted to G'F'). Note the different orientation of the SF (FG and G'F', solid boxes) in the type I and type II tandem repeats of strain El. (Right) Nucleotide sequence (boxed) at each junction site.
F
F'H
right end
the AR. The two sides of the deletion corresponded to the nonamplified DNA fragments flanking the 57.9-kb AR of El. A 4.9-kb Sal I fragment of strain npelO including the junction at the deletion site was cloned from phage Pll (Fig. 1) and 0.3 kb was sequenced. The nucleotide sequence (Fig. 6) at the deletion site showed the sequence TGTAAT that seems to have originated by recombination between the two identical sequences located at the borders of the 57.9-kb unit, after mutation with nitrosoguanidine (see Discussion).
AS-P-78 CD AB EF GH GCTCcffCATC TCAALOMMACTAG GCTCC=EKCATC CD
ARB
AR GCTCC
TT
CD CATC
A4,
GH
EF
TQAA%CTAG
CD EB Ev CT 14 TCAAL CTC TCAAMTVM%CTAG
DISCUSSION Wild-type strains of P. chrysogenum are believed to be haploid since recessive auxotrophic mutants are easily obtained (18). Similarly, mutants blocked in penicillin production are readily obtained from the wild-type or early strains of the genealogical tree of improved penicillin producers (15, 19). By cloning and mapping the entire 106.5-kb region containing the penicillin gene cluster, we observed that this region is present in a single copy per genome in the wild-type strain NRRL 1951. Many of the improved penicillin producers contain several copies of the region carrying the penicillin gene cluster (12, 13). As shown here, the AR in AS-P-78 corresponds to a stretch of 106.5 kb of contiguous DNA that is amplified 5- or 6-fold in tandem repeats. The AR is not identical in the different high producing strains tested, although the mechaS /
B
M1
C
EV / EV EV
ji(S
H
AB
CD GAGT
EG'
AACG
FH
CTAGTAAAT
ARBCAGT~T A.B
CD
GAGT
EG'7
TIM91AACG
B
Wis. 54-1255 npelO AB
CD
GAGTT~
AB
FH
CTAGTME
EG'
MAAAT
FH
CTAG1MTAAAT
kACG
CD GAGTTITggACG CTAG
EV
FH
EG' ACG
AAAT F'H
3.5
Wss.54-1255 npelO
a
5.02
ATTTACCTTTCTGCGTGCCCTTTTTGAGTT TA
EG'
CTAGTAATAAAT
FYD
CTAG10LU
S
H
ACG
CD
TAAATTTCTGTTCAACGCAGCAAATATT
FIG. 6. Restriction map and nucleotide sequence of the regions on both sides of the deletion (vertical bar) of mutant npelO. Enzymes are as in Fig. 2. The sequence TGTAAT (boxed) at which the deletion has occurred corresponds exactly to the sequences at junctions CD and F'H of P. chrysogenum El (Fig. 5).
G' E
AB
4,
C.H
FIG. 7. Proposed model for recombination events that originate tandem repeats in P. chrysogenum AS-P-78 and P. chrysogenum El. Symbols are as in Fig. 5. Model for the deletion in P. chrysogenum npelO by crossing-over at the TGTAA sequences.
6204
Microbiology: Fierro et at
nism of amplification is probably similar. Thus, the high penicillin producer El contains an AR of 57.9 kb that overlaps (except for a 3.4-kb fragment) with the AR of AS-P-78. This 3.4-kb SF is present in both orientations in the AR of strain El. The mechanism of gene amplification is intriguing. Industrial strains have been subject to strong mutagenic treatments (16). Chromosome instability resulting in chromosomal duplication (9) or gene amplification (20, 21) was reported in Aspergillus nidulans; gene duplications have been also observed in Neurosporoa crassa (22). However, the genetic mechanism underlying gene amplification in all these cases remains unknown. Gene amplification in P. chrysogenum occurs within the same chromosome I, differing from the transposition phenomenon described in A. nidulans where a large terminal segment of chromosome IR was duplicated and attached terminally to chromosome IIR (9). The tandem amplification observed in P. chrysogenum is similar to that reported in a few examples in Saccharomyces cerevisiae. Industrial yeast strains resistant to copper contain a tandem amplification of the CUP-4 region (6, 7). Resistance to antimycin A in S. cerevisiae takes place by 50-fold amplification of the nuclear geneADH4 as linear extrachromosomal molecules 42 kb long that can be separated from chromosomal DNA (7). In P. chrysogenum, the AR remains chromosomal (chromosome I). A possible model to explain the formation of tandem repeats is recombination at the conserved TTTACA sequences located at the borders of the single-copy 106.5-kb unit in the wild type after mutation. As shown in Fig. 7, misaligned pairing between homologous chromosomes or between sister chromatids may occur (6). A reciprocal crossover in this paired but misaligned region generates a tandem repeat. The mechanism of formation of tandem repeats by recombination at the TTTACA sequences is strongly supported by the loss of the 57.9-kb unit in the deletion mutant npelO. Apparently this region has looped out by recombination between homologous sequences at its ends. The TTTACA sequence and its inverse complement TGTAAA may be hot spots for site-specific recombination after mutation with nitrosoguanidine, since our data suggest that crossing-over for amplification or deletions always occurs within this motif. The recombination process might be part of a fungal SOS system similar to that existing in Escherichia coli for repairing damaged fragments of DNA that is likely to be induced after mutagenic treatments. E. coli responds to DNA damage with the expression of a set of genes regulated by the RecA and LexA proteins (the SOS response) (23). Similar excision repair and postreplication repair mechanisms appear to occur in yeasts and filamentous fungi (24).
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