Vol. 173, No. 6
JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 2125-2127
0021-9193/91/062125-03$02.00/0
A Unique Repetitive DNA Sequence in the Myxococcus xanthus Genome SHOJI FUJITANI,1t TERUYA KOMANO,1
AND
SUMIKO INOUYE2*
Department of Biology, Tokyo Metropolitan University, Fukazawa, Setagaya-ku, Tokyo 158, Japan,1 and Department of Biochemistry, Robert Wood Johnson Medical School at Rutgers, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 088542 Received 4 October 1990/Accepted 10 January 1991
We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.
sequence
In prokaryotic genomes a variety of repetitive DNA sequences are found (for a review, see reference 6). First are duplicated genes, such as the seven rrn genes for rRNA present in the Escherichia coli genome, and many closely related genes, presumably derived from common ancestral genes. Second are insertion sequences and transposons. For
example, 7 copies of IS], 12 copies of IS2, and 6 copies of IS3 are found in the genome of E. coli K-12 W3110 (9). Third are recognition sequences bound by various sequence-specific DNA-binding proteins and other repetitive sequences with unknown functions. Repetitive extragenic palindromic sequences compose up to 0.5% of the whole genome of E. coli. Myxococcus xanthus is a unique gram-negative bacterium which undergoes multicellular development (for a review, see reference 7). When nutrients are depleted in a solid medium, cells aggregate to form mounds which eventually convert to fruiting bodies. Rod-shaped cells differentiate to round or ovoid spores. During the development of M. xanthus, a large amount of an extracellular protein, protein S, is produced (1). The tps gene coding for protein S was cloned and its DNA sequence was determined (3). A highly homologous gene, ops, which encodes an intraspore protein, protein S-1, is also found 1.4 kb upstream of the tps gene. Therefore, the tps and ops genes are examples of repetitive DNA sequences in M. xanthus. We used the 2.4-kb HindlIl-EcoRI DNA probe (probe a [see Fig. 2]), which contains the entire ops gene and most of the tps gene together with the 1.4-kb spacer region, to hybridize with restriction fragments of M. xanthus genomic DNA. In addition to the fragments corresponding to the probe DNA region, many weakly hybridizing bands were detected (Fig. 1A), suggesting the presence of a repetitive sequence(s) in the M. xanthus genome. We used various subfragments from the 2.4-kb HindlIl-EcoRI fragment as probes to locate the repetitive sequence. When the 239-bp BstEII-BamHI fragment (probe b [see Fig. 2]) was used as a probe, many restriction fragments hybridized rather strongly (Fig. 1B), indicating the presence of the repetitive sequence in the intergenic region between the ops and tps genes of M.
xanthus. This repetitive DNA sequence was tentatively designated RMX (repetitive DNA sequence of M. xanthus), and the one between ops and tps was tentatively designated RMX1. The same BstEII-BamHI fragment was used to screen a M. xanthus genomic library (5) for the other clones containing an RMX sequence. The library was constructed on pHC79 (4) as cosmids, and each clone contains a 30- to 40-kb DNA insert. About 3% of the clones in the library hybridized with the RMX1 probe. The results suggest that M. xanthus genome carries at least six copies of RMX sequences, since the genome size of M. xanthus was reported to be 5,690 kb (11). However, Southern blot analysis of the XhoI-digested chromosomal DNA with RMX1 as the probe revealed that at least 15 bands hybridized with the probe (Fig. 1B, lane 4). The analyses using the other chromosomal DNA fragments indicate that some RMX sequences are present as clusters on the M. xanthus genome, as will be described below. Three clones were further analyzed (Fig. 2). The locations
A
23.1
-
9.4
1 2 3 4 5
B
3
4
5
6
7
23.14 9 *
6.6
-
66-
4.4
-
4.4-
2.32.0-
2
-i
*
s
_
i
2.3
2.0 -
FIG. 1. Southern blot hybridization of the genomic DNA from M. xanthus DZF1. (A) 32P-labeled 2.4-kb HindIII-EcoRI fragment (probe a [see Fig. 2]) was used as the probe. DNA samples were digested with EcoRI (lane 1), HindlIl (lane 2), BamHI (lane 3), PstI (lane 4), and Sall (lane 5). (B) 32P-labeled 239-bp BstEII-BamHI fragment (probe b [see Fig. 2]) was used as the probe. DNA samples were digested with PstI (lane 1), Sall (lane 2), BstEII (lane 3), XhoI (lane 4), EcoRI (lane 5), HindIII (lane 6), and BamHI (lane 7). Molecular lengths (in kilobases) are indicated on the left.
* Corresponding author. t Present address: Life Science Laboratories, Ajinomoto Co.,
Yokohama 244, Japan. 2125
2126
NOTES
J. BACTERIOL. probe b probe a
E
HPH
H
P
P
1-
PP
B
H
P
P
H
P P P
RMX2
RMX3
17
RMX4
RMX5
117
113
116
95
\163
128
131
114
\38
RMX6
tps
P
E
P
RMX4
RMX5
RMXI RMX2
89
RMX3
88
91
1 54
114
RMX4
88
92
92151
91
RMX5
88
87
92
91
94
RMX6
77
84
82
80
B 2 kb
rpoD
RMXI
E eB
RMXI I~~~~~~~~~~
-
RMX3 P BB
B
P$s
RMX2
I I
E
B
RMX6
FIG. 2. Locations of the repetitive DNA sequences of M. xanthus (RMX sequences) on the restriction maps. Above the top restriction map, the regions of probe a and b are indicated. B, BamHI; E, EcoRI; H, HindIll; P, PstI. Large arrowheads (not to scale) represent the location and orientation of RMX sequences, and small arrows indicate the ops, tps and rpoD genes.
of RMX sequences on the three clones were determined by Southern hybridization analysis, and sequence orientation was deduced from the DNA sequence described below. One repeat, RMX2 was located 14.5 kb upstream of RMX1. The second clone contained three repeats, RMX3, RMX4, and RMX5. The three RMX sequences were separated by 2 to 3 kb and were situated in the same orientation. In the third clone, RMX6 was located downstream of the rpoD gene coding for the major sigma factor (2). The orientation of RMX6 is opposite to the direction of the rpoD gene. DNA sequences of RMX2 through RMX6 were determined by the dideoxy-chain termination method (8). Figure 3 aligns the DNA sequences of all six RMX sequences. The length and level of homology for each pair of RMX sequences compared in Fig. 4 was calculated from the results of Fig. 3. The following general features about RMX sequences are derived from this comparison. (i) The length of
80
FIG. 4. Comparison of the six RMX sequences. The top right half shows the lengths (in base pairs) of the homologous sequences, and the bottom left half shows the level (percent) of homology. All values were calculated from the results of Fig. 3.
homologous DNA differs from sequence to sequence. Every RMX sequence consists of a 87-bp core sequence (box in solid lines, Fig. 3), and each RMX contains sequences of extra homology in both 3' and 5' regions (box in broken lines, Fig. 3). RMX3 and RMX4 share the longest homology (358 bp). (ii) The level of homology differs from sequence to sequence. The homology between RMX2 and RMX4, between RMX3 and RMX4, and between RMX3 and RMX5 is highest (92%), while that between RMX1 and RMX6 is lowest (77%). (iii) There are deletions or insertions among homologous sequences, suggesting the absence of a proteincoding region within RMX sequences. The type of repetitive sequence described above is unique, since in most cases, the lengths of other known repetitive sequences, such as insertion sequences and transposons, are
RMX1 RMX2 RMX3 RMX4 RMX5
CCCACTCGGTGACCCCATTCACGCTCACGGCCGAGCTAGTGCCCTGAI CCCAGACGCCAGGCCTGGGCCGCAGGGAGATGGCGGGCGTGGCCTGG GCTTGAGGCAGCGGCGAGGAAGGAGCGGGTGAAGAAGAGGACACCGCI CGCCAATCCAGACGTGTGCGTGTTCCGGGTAAGTCTTAGCCGTGCCC
G5ECGCGCICGC
RMX6
CGTAACTGGGCAGGCGTCATGCTCCGTCAACACGGGGCGCCCGCGCC
rCGACGTATC
"CGAGGTGT
100
CGC 100
'CGAGGTGT iGC 100 'ACATA TCGT 100
CTCGGGCGCTGAGCCGTTTCCGCCTCCGTGCTGCGTTGCCCCATCG
:TGSGGTGTKCGC G
100 100
RMX1 RMX2
GZ
GCCCACGGCATG 198
RMX3 RMX4
CT
GCCCACGGGATG 199
RMX5 RMX6
GT
................ .....
RMX1
RMX2 RMX3 RHX4
RMX5 RMX6 RMX1 RMX2
RMX3 RMX4 RMX5 RMX6
RMXI RMX2
GCCCACGGGATG 200
GT GT GT
'GCCCACGGGATG 200 GCCCACGGGATG 198 ;TAATGCTGGCCC 199 0
s 00 *0000000000 @00
@
00
0
*-.DO 0 0 0-
'AGCCCA 298
'TGGCGA 'GAAGGG GAAGGG ;TAGTTG
300 297 298 298 ;TGGAGA 299
GGTTGAGCCCGAGGGCGAGGTTCCACGGTCGGTTTCCGGTGACTGGCATCGGCGGCCCTCCTCCCTGGGAGCAACGATGCGCACGCCGCTCGGACAGGGA 398 CATCCCGGATGGTGACG_CCCGAGTACTTCCGCGACGAGCTGCGCTCGTTCAGA_GGGGCGG_TCCTCCCCAGTGTCGCCACGTTCCTC 400 AGCGCGGTCTGGGCAGGCGTGTACCCAAAGGGGGCTCAAGCGGCCTGTCCACTGGACGGGAGCACCACTCGGGCGGCCCCGTCAGCGGTCCTCCTCGGCC 397
AGCGCGGTCTGGGCTGGCGTGTACCCCAA_GGGG-CTCAAGCGGCCTGTCCACTGGACAGGAGCATCACTCGGGC_GC_CCC_TCAGCGGTCCTCCTCGGCC GCGGGTCTTTTCACTGTCGAC TTCCCGAGGCGCCATGCGGGGTTAAGAGGAGTGGGCCCTCCTGGATTCGAACCAGGGACCAATCGGTTATGAGCCGACAGCTCTAACCGCTGAGCTAAGG CAGGCACACGGAGGGGCCCAACTCCGGCTCAGGTGGTCAAGAGTGACGGAGGGCTCTGGCGCGTTGAGAGATGAGCCCCCGCGCGAAGGCGCGCCGTCGC
397 319 399
498
_ _ CGGGCGCAAGCGCGGAGCGAAGACGTC_ 427 CCTCTGCACCTTTCCTCCCCCCTCAACAGGGCCCCCATCCCTCTGATGCG, KGGGGCAACGCTAGGGAGTTCACA 9 RMX4 CCTCTGCACCCTCCCTCCCCACTCAACAGGGCCCCCATCCCTATTATGCGTA JTCCGCTGATGTATGGCTCTCAGCACTTCTCGCTGCACGCGCAGCTGA 497 RMX5 RMX6 GCCCTCATGCGCCAACCAACCGCGTGACGGATTGCATATCGCCGGGTACGCAGTCCTGGCAAGGGGCGTCGTTTTACCCGGCTTCAGCGCTGCCGCTTCG 499 FIG. 3. Alignment of DNA sequences of RMX1 through RMX6. The absence of a nucleotide (-) and conserved nucleotides (0) are shown. The region of 87-bp core sequence is boxed with solid lines, and the region with extra homology is boxed with broken lines. DNA sequence of RMX1 was taken from Inouye et al. (3) (corresponds to positions 1122 to 1619). RMX3
NOTES
VOL. 173, 1991
constant. A similar type of repetitive DNA sequence has been reported in Mycoplasma pneumoniae (10). Two types
of repetitive DNA sequences, RepMP1 and RepMP2, 300 and 150 bp long, respectively, were found at least 10 and 8 times, respectively, in the M. pneumoniae genome. In some clones of RepMP1 and RepMP2, additional homology was found upstream and downstream of the repetitive sequence. The functions of RMX sequences are not clear at present. RMX may have played an important role in rearrangement and, thereby, evolution of the M. xanthus genome. We are grateful to Bert Lampson for critically reading the manuscript.
REFERENCES 1. 2.
3.
4.
Inouye, M., S. Inouye, and D. R. Zusman. 1979. Gene expression during development of Myxococcus xanthus; pattern of protein synthesis. Dev. Biol. 68:579-591. Inouye, S. 1990. Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus. J. Bacteriol. 172:80-85. Inouye, S., T. Franceschini, and M. Inouye. 1983. Structural similarities between the development-specific protein S from a Gram-negative bacterium, Myxococcus xanthus, and calmodulin. Proc. Natl. Acad. Sci. USA 80:6829-6833. Hohn, B., and J. Collins. 1980. A small cosmid for efficient
2127
cloning of large DNA fragments. Gene 11:291-298. 5. Komano, T., S. Inouye, and M. Inouye. 1985. Physical mapping of a 330 x 103-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination. J. Bacteriol. 162:124-130. 6. Riley, M., and S. Krawiec. 1987. Genome organization, p. 967-981. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 7. Rosenberg, E. 1984. Myxobacteria: development and cell interaction. Springer-Verlag, New York. 8. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 9. Umeda, M., and E. Ohtsubo. 1989. Mapping of insertion elements IS], IS2 and IS3 on the Escherichia coli K-12 chromosome: role of the insertion elements in formation of Hfrs and F' factors in rearrangement of bacterial chromosomes. 1. Mol. Biol. 208:601-614. 10. Wenzel, R., and R. Herrmann. 1988. Repetitive DNA sequence in Mycoplasma pneumoniae. Nucleic Acids Res. 16:8337-8350. 11. Yee, T., and M. Inouye. 1982. Two-dimensional DNA electrophoresis applied to the study of DNA methylation and the analysis of genome size Myxococcus xanthus. J. Mol. Biol. 154:181-196.
JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 2125-2127
0021-9193/91/062125-03$02.00/0
A Unique Repetitive DNA Sequence in the Myxococcus xanthus Genome SHOJI FUJITANI,1t TERUYA KOMANO,1
AND
SUMIKO INOUYE2*
Department of Biology, Tokyo Metropolitan University, Fukazawa, Setagaya-ku, Tokyo 158, Japan,1 and Department of Biochemistry, Robert Wood Johnson Medical School at Rutgers, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 088542 Received 4 October 1990/Accepted 10 January 1991
We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.
sequence
In prokaryotic genomes a variety of repetitive DNA sequences are found (for a review, see reference 6). First are duplicated genes, such as the seven rrn genes for rRNA present in the Escherichia coli genome, and many closely related genes, presumably derived from common ancestral genes. Second are insertion sequences and transposons. For
example, 7 copies of IS], 12 copies of IS2, and 6 copies of IS3 are found in the genome of E. coli K-12 W3110 (9). Third are recognition sequences bound by various sequence-specific DNA-binding proteins and other repetitive sequences with unknown functions. Repetitive extragenic palindromic sequences compose up to 0.5% of the whole genome of E. coli. Myxococcus xanthus is a unique gram-negative bacterium which undergoes multicellular development (for a review, see reference 7). When nutrients are depleted in a solid medium, cells aggregate to form mounds which eventually convert to fruiting bodies. Rod-shaped cells differentiate to round or ovoid spores. During the development of M. xanthus, a large amount of an extracellular protein, protein S, is produced (1). The tps gene coding for protein S was cloned and its DNA sequence was determined (3). A highly homologous gene, ops, which encodes an intraspore protein, protein S-1, is also found 1.4 kb upstream of the tps gene. Therefore, the tps and ops genes are examples of repetitive DNA sequences in M. xanthus. We used the 2.4-kb HindlIl-EcoRI DNA probe (probe a [see Fig. 2]), which contains the entire ops gene and most of the tps gene together with the 1.4-kb spacer region, to hybridize with restriction fragments of M. xanthus genomic DNA. In addition to the fragments corresponding to the probe DNA region, many weakly hybridizing bands were detected (Fig. 1A), suggesting the presence of a repetitive sequence(s) in the M. xanthus genome. We used various subfragments from the 2.4-kb HindlIl-EcoRI fragment as probes to locate the repetitive sequence. When the 239-bp BstEII-BamHI fragment (probe b [see Fig. 2]) was used as a probe, many restriction fragments hybridized rather strongly (Fig. 1B), indicating the presence of the repetitive sequence in the intergenic region between the ops and tps genes of M.
xanthus. This repetitive DNA sequence was tentatively designated RMX (repetitive DNA sequence of M. xanthus), and the one between ops and tps was tentatively designated RMX1. The same BstEII-BamHI fragment was used to screen a M. xanthus genomic library (5) for the other clones containing an RMX sequence. The library was constructed on pHC79 (4) as cosmids, and each clone contains a 30- to 40-kb DNA insert. About 3% of the clones in the library hybridized with the RMX1 probe. The results suggest that M. xanthus genome carries at least six copies of RMX sequences, since the genome size of M. xanthus was reported to be 5,690 kb (11). However, Southern blot analysis of the XhoI-digested chromosomal DNA with RMX1 as the probe revealed that at least 15 bands hybridized with the probe (Fig. 1B, lane 4). The analyses using the other chromosomal DNA fragments indicate that some RMX sequences are present as clusters on the M. xanthus genome, as will be described below. Three clones were further analyzed (Fig. 2). The locations
A
23.1
-
9.4
1 2 3 4 5
B
3
4
5
6
7
23.14 9 *
6.6
-
66-
4.4
-
4.4-
2.32.0-
2
-i
*
s
_
i
2.3
2.0 -
FIG. 1. Southern blot hybridization of the genomic DNA from M. xanthus DZF1. (A) 32P-labeled 2.4-kb HindIII-EcoRI fragment (probe a [see Fig. 2]) was used as the probe. DNA samples were digested with EcoRI (lane 1), HindlIl (lane 2), BamHI (lane 3), PstI (lane 4), and Sall (lane 5). (B) 32P-labeled 239-bp BstEII-BamHI fragment (probe b [see Fig. 2]) was used as the probe. DNA samples were digested with PstI (lane 1), Sall (lane 2), BstEII (lane 3), XhoI (lane 4), EcoRI (lane 5), HindIII (lane 6), and BamHI (lane 7). Molecular lengths (in kilobases) are indicated on the left.
* Corresponding author. t Present address: Life Science Laboratories, Ajinomoto Co.,
Yokohama 244, Japan. 2125
2126
NOTES
J. BACTERIOL. probe b probe a
E
HPH
H
P
P
1-
PP
B
H
P
P
H
P P P
RMX2
RMX3
17
RMX4
RMX5
117
113
116
95
\163
128
131
114
\38
RMX6
tps
P
E
P
RMX4
RMX5
RMXI RMX2
89
RMX3
88
91
1 54
114
RMX4
88
92
92151
91
RMX5
88
87
92
91
94
RMX6
77
84
82
80
B 2 kb
rpoD
RMXI
E eB
RMXI I~~~~~~~~~~
-
RMX3 P BB
B
P$s
RMX2
I I
E
B
RMX6
FIG. 2. Locations of the repetitive DNA sequences of M. xanthus (RMX sequences) on the restriction maps. Above the top restriction map, the regions of probe a and b are indicated. B, BamHI; E, EcoRI; H, HindIll; P, PstI. Large arrowheads (not to scale) represent the location and orientation of RMX sequences, and small arrows indicate the ops, tps and rpoD genes.
of RMX sequences on the three clones were determined by Southern hybridization analysis, and sequence orientation was deduced from the DNA sequence described below. One repeat, RMX2 was located 14.5 kb upstream of RMX1. The second clone contained three repeats, RMX3, RMX4, and RMX5. The three RMX sequences were separated by 2 to 3 kb and were situated in the same orientation. In the third clone, RMX6 was located downstream of the rpoD gene coding for the major sigma factor (2). The orientation of RMX6 is opposite to the direction of the rpoD gene. DNA sequences of RMX2 through RMX6 were determined by the dideoxy-chain termination method (8). Figure 3 aligns the DNA sequences of all six RMX sequences. The length and level of homology for each pair of RMX sequences compared in Fig. 4 was calculated from the results of Fig. 3. The following general features about RMX sequences are derived from this comparison. (i) The length of
80
FIG. 4. Comparison of the six RMX sequences. The top right half shows the lengths (in base pairs) of the homologous sequences, and the bottom left half shows the level (percent) of homology. All values were calculated from the results of Fig. 3.
homologous DNA differs from sequence to sequence. Every RMX sequence consists of a 87-bp core sequence (box in solid lines, Fig. 3), and each RMX contains sequences of extra homology in both 3' and 5' regions (box in broken lines, Fig. 3). RMX3 and RMX4 share the longest homology (358 bp). (ii) The level of homology differs from sequence to sequence. The homology between RMX2 and RMX4, between RMX3 and RMX4, and between RMX3 and RMX5 is highest (92%), while that between RMX1 and RMX6 is lowest (77%). (iii) There are deletions or insertions among homologous sequences, suggesting the absence of a proteincoding region within RMX sequences. The type of repetitive sequence described above is unique, since in most cases, the lengths of other known repetitive sequences, such as insertion sequences and transposons, are
RMX1 RMX2 RMX3 RMX4 RMX5
CCCACTCGGTGACCCCATTCACGCTCACGGCCGAGCTAGTGCCCTGAI CCCAGACGCCAGGCCTGGGCCGCAGGGAGATGGCGGGCGTGGCCTGG GCTTGAGGCAGCGGCGAGGAAGGAGCGGGTGAAGAAGAGGACACCGCI CGCCAATCCAGACGTGTGCGTGTTCCGGGTAAGTCTTAGCCGTGCCC
G5ECGCGCICGC
RMX6
CGTAACTGGGCAGGCGTCATGCTCCGTCAACACGGGGCGCCCGCGCC
rCGACGTATC
"CGAGGTGT
100
CGC 100
'CGAGGTGT iGC 100 'ACATA TCGT 100
CTCGGGCGCTGAGCCGTTTCCGCCTCCGTGCTGCGTTGCCCCATCG
:TGSGGTGTKCGC G
100 100
RMX1 RMX2
GZ
GCCCACGGCATG 198
RMX3 RMX4
CT
GCCCACGGGATG 199
RMX5 RMX6
GT
................ .....
RMX1
RMX2 RMX3 RHX4
RMX5 RMX6 RMX1 RMX2
RMX3 RMX4 RMX5 RMX6
RMXI RMX2
GCCCACGGGATG 200
GT GT GT
'GCCCACGGGATG 200 GCCCACGGGATG 198 ;TAATGCTGGCCC 199 0
s 00 *0000000000 @00
@
00
0
*-.DO 0 0 0-
'AGCCCA 298
'TGGCGA 'GAAGGG GAAGGG ;TAGTTG
300 297 298 298 ;TGGAGA 299
GGTTGAGCCCGAGGGCGAGGTTCCACGGTCGGTTTCCGGTGACTGGCATCGGCGGCCCTCCTCCCTGGGAGCAACGATGCGCACGCCGCTCGGACAGGGA 398 CATCCCGGATGGTGACG_CCCGAGTACTTCCGCGACGAGCTGCGCTCGTTCAGA_GGGGCGG_TCCTCCCCAGTGTCGCCACGTTCCTC 400 AGCGCGGTCTGGGCAGGCGTGTACCCAAAGGGGGCTCAAGCGGCCTGTCCACTGGACGGGAGCACCACTCGGGCGGCCCCGTCAGCGGTCCTCCTCGGCC 397
AGCGCGGTCTGGGCTGGCGTGTACCCCAA_GGGG-CTCAAGCGGCCTGTCCACTGGACAGGAGCATCACTCGGGC_GC_CCC_TCAGCGGTCCTCCTCGGCC GCGGGTCTTTTCACTGTCGAC TTCCCGAGGCGCCATGCGGGGTTAAGAGGAGTGGGCCCTCCTGGATTCGAACCAGGGACCAATCGGTTATGAGCCGACAGCTCTAACCGCTGAGCTAAGG CAGGCACACGGAGGGGCCCAACTCCGGCTCAGGTGGTCAAGAGTGACGGAGGGCTCTGGCGCGTTGAGAGATGAGCCCCCGCGCGAAGGCGCGCCGTCGC
397 319 399
498
_ _ CGGGCGCAAGCGCGGAGCGAAGACGTC_ 427 CCTCTGCACCTTTCCTCCCCCCTCAACAGGGCCCCCATCCCTCTGATGCG, KGGGGCAACGCTAGGGAGTTCACA 9 RMX4 CCTCTGCACCCTCCCTCCCCACTCAACAGGGCCCCCATCCCTATTATGCGTA JTCCGCTGATGTATGGCTCTCAGCACTTCTCGCTGCACGCGCAGCTGA 497 RMX5 RMX6 GCCCTCATGCGCCAACCAACCGCGTGACGGATTGCATATCGCCGGGTACGCAGTCCTGGCAAGGGGCGTCGTTTTACCCGGCTTCAGCGCTGCCGCTTCG 499 FIG. 3. Alignment of DNA sequences of RMX1 through RMX6. The absence of a nucleotide (-) and conserved nucleotides (0) are shown. The region of 87-bp core sequence is boxed with solid lines, and the region with extra homology is boxed with broken lines. DNA sequence of RMX1 was taken from Inouye et al. (3) (corresponds to positions 1122 to 1619). RMX3
NOTES
VOL. 173, 1991
constant. A similar type of repetitive DNA sequence has been reported in Mycoplasma pneumoniae (10). Two types
of repetitive DNA sequences, RepMP1 and RepMP2, 300 and 150 bp long, respectively, were found at least 10 and 8 times, respectively, in the M. pneumoniae genome. In some clones of RepMP1 and RepMP2, additional homology was found upstream and downstream of the repetitive sequence. The functions of RMX sequences are not clear at present. RMX may have played an important role in rearrangement and, thereby, evolution of the M. xanthus genome. We are grateful to Bert Lampson for critically reading the manuscript.
REFERENCES 1. 2.
3.
4.
Inouye, M., S. Inouye, and D. R. Zusman. 1979. Gene expression during development of Myxococcus xanthus; pattern of protein synthesis. Dev. Biol. 68:579-591. Inouye, S. 1990. Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus. J. Bacteriol. 172:80-85. Inouye, S., T. Franceschini, and M. Inouye. 1983. Structural similarities between the development-specific protein S from a Gram-negative bacterium, Myxococcus xanthus, and calmodulin. Proc. Natl. Acad. Sci. USA 80:6829-6833. Hohn, B., and J. Collins. 1980. A small cosmid for efficient
2127
cloning of large DNA fragments. Gene 11:291-298. 5. Komano, T., S. Inouye, and M. Inouye. 1985. Physical mapping of a 330 x 103-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination. J. Bacteriol. 162:124-130. 6. Riley, M., and S. Krawiec. 1987. Genome organization, p. 967-981. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 7. Rosenberg, E. 1984. Myxobacteria: development and cell interaction. Springer-Verlag, New York. 8. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 9. Umeda, M., and E. Ohtsubo. 1989. Mapping of insertion elements IS], IS2 and IS3 on the Escherichia coli K-12 chromosome: role of the insertion elements in formation of Hfrs and F' factors in rearrangement of bacterial chromosomes. 1. Mol. Biol. 208:601-614. 10. Wenzel, R., and R. Herrmann. 1988. Repetitive DNA sequence in Mycoplasma pneumoniae. Nucleic Acids Res. 16:8337-8350. 11. Yee, T., and M. Inouye. 1982. Two-dimensional DNA electrophoresis applied to the study of DNA methylation and the analysis of genome size Myxococcus xanthus. J. Mol. Biol. 154:181-196.
