Journal of Heredity 2004:95(1)
© 2004 The American Genetic Association 95:62-69
A Minisatellite with Fold-Back Structure is Included in the 5'-Flanking Region of the Adh Gene of Scaptodrosophila lebanonensis
From the Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. This work was supported by DGICYT grant PB96-0172 and BOS2000-0770 (to E.J.) and by CIRIT grant 1999SGR-25 (to M. Aguadé). We thank Serveis Cientificotècnics, Universitat de Barcelona for automated sequencing facilities.
Address correspondence to D. J. Orengo at the address above, or e-mail: dorcas{at}porthos.bio.ub.es.
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A tandem repetitive sequence with a repeat unit of 12 bp has been found 1.3 kb upstream of the Adh gene of Scaptodrosophila lebanonensis. This repetitive sequence extends over 4.3 kb and consists of two inverted arrays (a fold-back segment). The repeated unit with a consensus sequence GAATACAGAATA is highly conserved and the nucleotide substitutions are not distributed randomly among the 12 bp. In situ hybridization in S. lebanonensis polytene chromosomes revealed two signals, one at the 60A section, the Adh locus, and a second site in the same chromosome at the 60C section close to the telomere. This same pattern of hybridization is obtained in all the analyzed strains including the subspecies S. lebanonensis casteeli. The minisatellite sequence accounts for about 0.03–0.04% of the S. lebanonensis genome and showed intraspecific variability in tandem repeat numbers. Possible functions of this sequence are discussed.
A characteristic of all eukaryotic genomes is the presence of various repetitive DNA sequences. Simple tandem repetitive sequences have been classified according to the size of the repeat unit, the number of repeat units per array, and the genomic location of the tandem arrays (Li 1997). Satellites have a repeat unit size of two to hundreds of base pairs, an array size of more than 1000 repeat units, and are usually located in the centromeric heterochromatin. Minisatellite sequences have 9–100 bp per repeat, arrays of 0.5–30 kb long (Armour and Jeffreys 1992), and a subtelomeric or dispersed distribution. Finally, short tandem repeats, microsatellites, and Alu trail arrays have 3–5, 1–2, and 1–5 bp per repeat, respectively, from 10 to 100 repeats per array, and are dispersed in the genome. In several Drosophila species, satellite sequences have been identified and all of them mapped in heterochromatin (Gall et al. 1971; Lohe et al. 1993). Microsatellites also are abundantly distributed across the three major chromosomes of Drosophila melanogaster (Bachtrog et al. 1999). The minisatellites, as stated above, widely distributed in vertebrates, fungi, and plants (Charlesworth et al. 1994), have seldom been described in Drosophilidae. Jacobson et al. (1992) reported the minisatellite mD4.2 of Drosophila mauritiana that consists of 13 tandemly repeated monomers, 10 of which are 33 bp long. The tandem repeats of 457 bp, located in a subterminal region of chromosome 2L of D. melanogaster, were described as minisatellite (Walter et al. 1995), although Kurenova et al. (1998) referred to it as a satellite. Another repeated sequence of these structural characteristics (359 bp per repeat unit) has been described as satellite related (SR) in the euchromatic portion of the X chromosome, which in at least two cases is immediately adjacent to transcriptionally active genes (DiBartolomeis et al. 1992).
We describe here a type of moderately repeated sequence with a repeat unit of 12 bp, located in two euchromatic sites of Scaptodrosophila lebanonensis chromosome 4. This repeated sequence has the features described above for minisatellites and has been found during the characterization of the regulatory region of the alcohol dehydrogenase (Adh) gene (Juan et al. 1994). Furthermore, the characterized 4.3 kb sequence revealed that the repeats are organized in a fold-back structure (Finnegan 1982). It has been argued that different families of minisatellites in vertebrates, plants, and fungi may have different functions, although the biological meaning of this type of genomic redundancy is far from being understood. The characterization of this minisatellite is the first step in studying its function.
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Materials and Methods |
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Drosophilidae Strains
Strain 323G of S. lebanonensis (from Gandesa, Tarragona) was used as a source of genomic DNA. In the in situ hybridization experiments we used the following stocks: 323G (Gandesa, Tarragona), Freixenet2 (San Sadurní d'Anoia, Barcelona), four isofemale lines from the natural population 828 (Cordoba), and two isofemale lines from population 727 (Cheste, Valencia) of S. lebanonensis lebanonensis; S. lebanonensis casteeli (Bowling Green Center, Ohio); D. melanogaster (Canton S); Drosophila willistoni (LDS); Drosophila subobscura (chcu); Drosophila virilis (Tokyo); Drosophila funebris (Bilbao), and Drosophila immigrans (El Prat, Barcelona). Individuals from all the Scaptodrosophila strains were also used in the polymerase chain reaction (PCR) analysis.
Cloning and Sequencing
Two subclones with fragments HincII–HindIII of 5987 bp and StyI–XmnI of 2576 bp (Figure 1) were derived from a 9 kb clone isolated from a BSKSm plasmid partial HindIII genomic library of S. lebanonensis. To overcome the difficulty to obtain the correct sequence in this region, due to its repetitive nature, we used the Genome Priming System (New England Biolabs), which inserts a transposon (Transprimer) randomly into the target DNA. Unique priming sites on both ends of the Transprimer element allow DNA sequences to be obtained from both strands of the target DNA at the position of insertion. Clones were sequenced automatically on an ABIPRISM 377 automated DNA sequencer (Perkin-Elmer) with the Thermo Sequenase Dye Terminator Cycle Sequencing Kit v.2 (Amersham). Since this region is very A-T rich, 5 mmols of dATP or dTTP for each 20 µl reaction were added depending on the strand being sequenced. Overlapping sequences from 20 clones with transprimer insertions and 17 nested clones allowed us to determine the sequence of both strands. Each nucleotide was sequenced three times on average. The sequence of 5606 bp HincII–XbaI (Figure 1) is under the accession number AJ300179.
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In Situ Hybridization
Salivary gland polytene chromosomes from third instar larvae were prepared for in situ hybridization according to Montgomery et al. (1987). Mitotic chromosomes were prepared from brains of third instar larvae according to Gatti et al. (1994). The clone StyI–XmnI of 2576 bp (Figure 1) labeled with biotin-16-dUTP by nick translation was used as the probe. The prehybridization, hybridization, and detection conditions were as described by Montgomery et al. (1987) except that the hybridization temperature was 25°C when the chromosome preparations were from species different from S. lebanonensis. The chromosome map of S. lebanonensis casteeli constructed by Berendes and Thijssen (1971) was used to identify the hybridization sites.
Slot Blot
The amount of repetitive sequence in the genome was determined by blotting serial (1:2) dilutions of genomic DNA from 323G flies and plasmidic DNA from clone StyI–XmnI of 2576 bp on a nylon membrane (Hybond-N, Amersham) using a Bio-Dot SF Apparatus (BioRad). The probe p2 (Figure 1) was labeled by random priming (Boehringer Mannheim) using 50 ng of DNA and 3000 Ci/mmol of [
32P]dATP and [32P]dTTP (NEN Life Science Products) to a specific activity of 3 x 109 cpm/µg of DNA. Hybridizations were carried out in 15 ml of hybridization solution at 58°C (Sambrook et al. 1989). Radioisotopic imaging of the membrane was obtained with the Molecular Imager FX System (BioRad) in order to quantify the amount of hybridized probe.
PCR and Molecular Analysis
Genomic DNA of single flies was purified according to Ashburner (1989). The variable number of tandem repeats (VNTRs) were identified by agarose gel electrophoresis of amplified PCR products, obtained from primers that recognize regions flanking the repeat units: SAT1 primer 5' TAA GTA CCG AGA TTC TCC AAG G 3' and SAT2 primer 5' AGT TCA TCT TGG TGC TCC ATT C 3'. PCRs were performed in 50 µl reactions on a Gene Amp PCR System 2400 thermal cycler (Perkin-Elmer) using Expand Long polymerase (Roche) and the 10x buffer (22.5 mM MgCl2) supplied with the enzyme. Each reaction contained 2.5 U of enzyme, 0.2 mM dNTPs, 1 µM primers, and one-fourth of a single fly genomic DNA. The reaction was heated to 94°C for 5 min, then cycled 30 times at 92°C for 45 s, 57°C for 45 s, and 68°C for 4.5 min with a final extension at 68°C for 10 min. VNTRs were also detected by Southern analysis of genomic DNA. For this analysis, genomic DNA from single flies was digested with StyI that cuts outside the repetitive fragment, electrophoresed on 0.8% agarose gel, and transferred to a nylon membrane (Hybond-N, Amersham) by Southern blot (Sambrook et al. 1989). The hybridization probe p3 (BamHI–StyI; Figure 1) was a fragment of single-copy DNA included in the StyI fragment. PCRs with primers within the inverted repeats 5' TGG ATG AAC GCA AGT ATA TCAA 3' and 5' GCA AGG ATC CCA AAA ACT ATA A 3' were performed in the conditions described above.
Sequence Analysis
The sequence was analyzed using the software package of the Genetics Computer Group, version 8.UNIX (Devereux et al. 1984). The program DnaSp3.14 (Rozas and Rozas 1999) was used to calculate the nucleotide variation and its graphical display. The sliding window method was used to obtain a graphic representation of the pattern of change of nucleotide variation along the sequence (window length = 48, step size = 12).
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Results |
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The sequence of the fragment HincII–HindIII located 1.3 kb upstream of the distal start site of the Adh gene (Figure 1) revealed that 4356 bp of the 5987 bp consisted of a tandemly repeated DNA. The repeated unit is a dodecanucleotide, 75% A-T rich, whose consensus sequence is 5' G94A98A99T97A96C88A96G97A70A94T82A86 3'. This dodecanucleotide shows two GAATA blocks, which are internal repeats, separated by two nucleotides. This internal repeat is identical to the pentanucleotide AATAG (since the first nucleotide of a repeat is arbitrarily chosen), which is mainly present in the heterochromatic regions of chromosomes Y and 2 of D. melanogaster (Lohe et al. 1993).
The repetitive DNA in this region is divided into two arrays, which are inverted one with respect to the other, and separated by an XmnI site (Figure 1). These two arrays, which are capable of forming a fold-back structure, contain 211 and 154 repeats, respectively. Figure 2 shows, for each of these two inverted arrays, the frequency of the consensus and its variants. The dodecanucleotide sequence is highly conserved, since 40% of the repeats are identical to the consensus and 31% show changes in only one nucleotide (Figure 2). In addition, variations are mainly due to nucleotide substitutions, and only 3.28% of the repeats show nucleotide deletions. The more frequent variants in one array are also the more frequent in the other, and variants only found in one array are unique or very rare, showing not more than three repeats.
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The nucleotide variability within the repeats is not distributed randomly among the 12 bp. For example, the third nucleotide only changes once (0.27%), while the ninth nucleotide differs from the consensus in 30% of the repeats. When the 11th nucleotide changes to A, the 12th changes to G, and vice versa in 46 repeats (12.6%). Changes in only one of these two positions to A or G were observed in only three repeats near the ends of the repeated region where repeats are more degenerate.
The two internal repeated blocks (GAATA) also behave in a different way. The first block is more conserved than the second. Considering the 12 bp repeats from the two inverted arrays, we found 34 variable repeats with 43 nucleotide changes in the first block and 171 variable repeats with 235 nucleotide changes in the second block. In addition, qualitative differences are also observed in the variation of these two blocks. So in the first block, transitions are more frequent than transversions (25:18), while in the second block transversions are the more frequent changes (68:167). These differences between the two blocks are significant (
21df = 12.64; P =.00038). In the first block, the last A only changes to G (nine times) and when it changes alone produces a BsmI site. Nevertheless, in the second block the last A changes to G 47 times out of 49 and in 46 blocks changes simultaneously with the fourth nucleotide (T) to A as reported above. Another example of differences between the two blocks is the variation of the third nucleotide (A). This nucleotide only varies once in the first block, in one repeat located at the end of the repeated region, while in the second block it varies in 15 repeats.
The distribution of nucleotide variability in the whole region of 4356 bp with respect to a consensus sequence is not uniform. Figure 3 shows the nucleotide variation of the 4.4 kb sequence calculated with the DnaSP3.14.3 program (Rozas and Rozas 1999) versus a consensus sequence of the same length. The arrays StyI–XmnI and XmnI–BamHI show an average of 0.076 and 0.095 changes per nucleotide, respectively. Differences in nucleotide variability between the two inverted arrays were statistically significant (21df = 4.668; P =.031). The repeats at both ends are the most degenerate, as predicted by the unequal exchange model (Brutlag 1980). At both ends, after the last completely conserved repeat, we found 14 degenerate repeats (Figure 4); also at both ends, one of these degenerate repeats shows a deletion of five and seven nucleotides, respectively. However, degeneration is more marked at the BamHI end.
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Furthermore, we have identified in the regions flanking the 4.4 kb fragment some sequences identical to the A-box and T-box found in the scaffold attachment regions (SARs) (Gasser and Laemmli 1986). In the StyI end we have identified two A-boxes (AATAAATAAA), like those described upstream of the Adh distal promoter of D. melanogaster (Gasser and Laemmli 1986), separated by 12 nucleotides and located at 270 bp from the beginning of the repeated region. In the BamHI end we have identified a T-box (TTATTTTATA) (Figure 4) as that described in the SARs of histone genes, which is a part of the last two repeats. Strikingly, we found imperfect inverted repeats of 47 bp flanking the repeated region. One of the inverted repeats is located 84 bp before the beginning of the degenerated, but identifiable repeats (at the StyI end) and the other 56 bp after the end of identifiable repeats (at the BamHI end) (Figure 4). The two inverted repeats show 83% identity.
Suspecting a multiple location of this dodecanucleotide repetitive sequence, in situ hybridization was performed on polytene chromosomes of S. lebanonensis lebanonensis (Gandesa 323G). Only two hybridization signals on the polytene chromosome 4R were observed: 4R-60A (corresponding to the Adh site) and 4R-60C at the subtelomeric region (Figure 5a). Since repetitive DNA is commonly found in pericentromeric heterochromatin that is underreplicated in polytene chromosomes, in situ hybridization on mitotic chromosomes was also carried out. In the mitotic chromosomes, only one telomeric signal was observed on the small metacentric chromosome pair (Figure 5b), indicating that this repetitive DNA does not occur or is not detectable in the pericentromeric heterochromatic regions.
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In order to search for intraspecific variability in the distribution of this repetitive DNA, in situ hybridization was also carried out on polytene chromosomes of three additional populations of S. lebanonensis lebanonensis from different geographic origins and in one strain of the subspecies S. lebanonensis casteeli. All the strains showed the same pattern of hybridization described above.
The amount of this dodecanucleotide sequence was determined by a slot blot hybridization experiment with serial (1:2) dilutions of genomic DNA and plasmidic DNA from clone StyI–XmnI, probed with p2. The scanning of the hybridized slot blot membrane gave in the second (0.362 ng) and the third (0.181 ng) plasmidic slots, 14% and 4% more counts per square millimeter than the first (860 ng) and second (430 ng) genomic slots, respectively. From these results it has been estimated that the amount of this repetitive DNA is in the range of 0.03–0.04% of the genome.
The overall characteristics described above lead us to consider the dodecanucleotide repetitive sequence as a minisatellite like those described in vertebrates, plants, and fungi. It is a moderately repetitive sequence (0.03–0.04% of the genome) with a repeat unit size of 12 bp and located at two euchromatic sites. Minisatellites frequently show VNTRs among individuals (Jeffreys et al. 1995). This variability was tested by PCR amplification of the Adh tandem repeated region in single flies from different isofemale lines of S. lebanonensis, using as a control the amplification of the HincII–HindIII plasmid, which contains the whole repetitive region (Figure 6). Amplification product of the HincII–HindIII plasmid was composed of three bands: the one expected according to the known sequence (arrow 1 in Figure 6), plus two bands of higher mobility (arrows 2 and 3 in Figure 6). This pattern must be due to different conformational DNA structures of the fold-back repetitive sequence, since it also appears when the fragment StyI–BamHI from this plasmid undergoes the same temperature changes as PCRs, but without polymerase, primers, and nucleotides. The same pattern also appeared in the PCRs of individual genomic DNAs from lines 323, 828-13, and 828-22. A similar pattern, but with bands of different size, was observed in individuals of line 727-24. Other patterns appeared in the rest of the lines (F2, 727-18, 828-19, 828-21, and S. lebanonensis casteeli). Furthermore, variability among individuals was detected in line 727-18 (Figure 6). The variation observed by Southern analysis among several lines (data not shown) confirmed the PCR results on VNTRs.
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The fold-back structure of the 4.3 kb sequence with internal tandem repeats reminds us of the fold-back elements described in Drosophila (Bingham and Zachar 1989). On the other hand, the presence of inverted repeats flanking this sequence might indicate another kind of transposable element. To further investigate this possibility we searched for open reading frames (ORFs) between the 47 bp inverted repeats. We found 33 ORFs that include a start codon ATG and a stop codon. Nevertheless, none of these ORFs have associated either a Drosophila consensus translation initiation site (Cavener 1987) or a polyA signal. In addition, FASTA analysis revealed a very low similarity with any of the proteins in the data bank. To examine the possibility that the 47 bp inverted repeats might correspond to the inverted repeats of an unknown transposable element, we performed PCRs using primers within these 47 bp inverted repeats. The results obtained (data not shown) using the same genomic DNAs as for the VNTR analysis revealed that these inverted repeats only occur flanking the 4.3 kb tandem repeat sequence in the genome of S. lebanonensis.
The minisatellite described above has not been identified in other Drosophila species since the in situ hybridization performed on the polytene chromosomes of other six species, D. melanogaster, D. willistoni, and D. subobscura of the subgenus Sophophora, and D. virilis, D. funebris, and D. immigrans of the subgenus Drosophila, yield no hybridization signals.
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Discussion |
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The tandem repeated sequences located on chromosome arm 4R of S. lebanonensis have the characteristics of the minisatellites frequently described in vertebrates, fungi, and plants. Minisatellites are short, tandemly repeated, DNA sequences that are found in moderate numbers in the euchromatic regions of eukaryotes and are genetically unstable, since new length alleles arise by gain or loss of repeat units. The slot blot experiment indicated that the minisatellite described accounts for 0.03–0.04% of the entire genome of S. lebanonensis. Assuming a genome size of 170 Mb (as considered for D. melanogaster by Ashburner et al. [1999]), this minisatellite would expand over 50–70 kb. This amount must be mainly distributed between the two chromosomal sites detected by in situ hybridization (60A and 60C). The characterized 13.9 kb Adh genomic region (Figure 1) contains 4.3 kb of this minisatellite, but we cannot discard the presence of more repeats outside this region at the 60A site.
Polymerase chain reaction amplification of genomic DNA from different individuals showed variability in the number of tandem repeats. VNTRs were mainly observed among isofemale lines. The small number of individuals analyzed per line, plus the high consanguinity within lines (they are isofemale lines maintained in the laboratory for more than 10 years), could account for the low level of variability within lines.
Different processes can produce variations in the number of tandem repeats: unequal crossing over, replication slippage, and unequal sister-chromatid exchange (Li 1997). The analysis of the minisatellite at the Adh region suggests that the number of repeats expands by replication slippage. Some evidence supports this hypothesis. Repeat variants are more similar the closer they are. In addition, some contiguous segments of 60–100 nucleotides are identical. This is the case for two segments of 99 nucleotides (2686–2784/2798–2896) in the StyI–XmnI array that includes a deletion of seven nucleotides and other rare variants of the dodecanucleotide consensus, indicating that one segment must have originated from the other.
The hybridization signal on the mitotic chromosomes shows that the minisatellite is only found near one telomere of the small metacentric chromosome pair. The presence of this single signal is consistent with the detection of two distal signals on the polytene chromosome arm 4R, given the higher compacted structure of the mitotic chromosomes. Two aspects can be highlighted from this result. First, this repeated sequence is not detected in pericentromeric heterochromatin. Second, the small metacentric chromosome pair corresponds to the polytene chromosome 4, as inferred by Papaceit and Juan (1993) from the amount of constitutive heterochromatin and the relative size of each chromosome pair.
The two hybridization signals (60A and 60C) at the same distal section of polytene chromosomes, observed in all the strains of S. lebanonensis tested (including the two subspecies), indicate that this pattern appeared before the divergence of the two subspecies. This pattern can be explained by intrachromosomal transposition or a paracentric inversion from an initial single repetitive array. The fact that the sequenced region (at 60A) shows a fold-back structure is consistent with both hypotheses. The fold-back structure recalls that of Drosophila FB transposable elements (Bingham and Zachar 1989), and the 47 bp inverted repeats flanking the FB structure, are consistent with other kinds of transposable elements. However, the presence of highly degenerate repeats at both ends of the fold-back segment, the lack of ORFs, and the constant chromosomal distribution of the repetitive sequence in several strains indicate that if this sequence has ever been part of a transposable element, it was no longer active before divergence of the two subspecies.
The two inverted arrays at the Adh region must have been evolving independently since their inverted sequences prevent phenomena such as unequal crossing over or replication slippage between them. Although the difference in nucleotide variation between inverted arrays is statistically significant, the same kind of nucleotide substitutions took place at the same position of the repeats in both arrays (Figure 2). These similar kinds of changes might indicate that the repeated sequence has some unknown function that would allow only certain changes in certain positions. An alternative possibility is that an initial tandem repeat diverged to some degree before another event created the fold-back structure. Further divergence after this later event would obscure the identity between the two inverted sequences.
Cross-hybridization analysis did not detect this minisatellite in any of the Drosophila species analyzed. We also performed different in silico searches for the minisatellite in the D. melanogaster and D. pseudoobscura genomes (FlyBase Consortium 2003). Pattern Search using the monomers most frequently found in our minisatellite identified several matches in the D. melanogaster genome, as expected by random combination of the nucleotides. Near the Adh gene we found a single monomer GAATATAGAATA at 5425 bp upstream of the distal TATA. A FASTA search (Pearson 1990), with a sequence containing multiple consensus dodecanucleotide, found a single match of 12 repeats (TAATACAAAATA) with 81.2% identity within an A-T-rich intronic sequence of the heph gene, near the telomere of the chromosome arm 3R of D. melanogaster. We conclude that the GAATACAGAATA sequence is not present as a minisatellite in D. melanogaster or D. pseudoobscura. This result is not surprising since closely related species or even different chromosomes of the same species can have distinct repetitive sequences (Lohe et al. 1993). Furthermore, dot plot analyses of the 6 kb region upstream of the Adh distal TATA of D. melanogaster and D. pseudoobscura did not identify any minisatellite sequence of any type.
Different functions have been proposed for minisatellites. It has been suggested that subtelomeric minisatellites may be implicated in meiotic chromosome pairing and recombination (Gilson et al. 1993). Some minisatellites are known to be associated with proteins, which might locally influence chromatin structure, inhibiting or activating genes in the vicinity. This is the case of the minisatellite in the human diabetes-susceptibility locus IDDM2 that regulates insulin transcription (Kennedy et al. 1995).
The minisatellite described here is located only 1.3 kb upstream of the Adh distal promoter of S. lebanonensis. Minisatellite sequences have not been described in any Drosophila Adh gene upstream of the coding region. The proximity of this minisatellite in S. lebanonensis Adh might have some influence on the Adh expression of this species. A possibility is that this minisatellite might affect Adh gene expression acting as a SAR (Hart and Laemmli 1998). Three characteristics will account for this possibility: a very rich A-T sequence, its association with the regulatory region of the gene, and the presence of one T-box (Figure 4) identical to those described in D. melanogaster SARs.
In order to define the cis-regulatory region of the S. lebanonensis Adh gene, we have performed germ-line transformation experiments in D. melanogaster. Our results (manuscript submitted) indicate that the cis-regulatory region extends over 1.1 kb of the 3' end of the minisatellite. The identified sequence encompasses 69 repeats; 26 of them fit the consensus sequence and the rest have different nucleotide substitutions. The repeats become more degenerated as they reach the 3' end. This degeneration could give rise to new binding sites for transcription factors or proteins that keep the chromatin around the gene in a more open structure, increasing the transcriptional activity of the gene.
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Footnotes |
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Corresponding Editor: R. C. Woodruff
Received February 14, 2003
Accepted July 31, 2003
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  M. Papaceit, D. Orengo, and E. Juan Sequences Upstream of the Homologous cis-elements of the Adh Adult Enhancer of Drosophila Are Required for Maximal Levels of Adh Gene Transcription in Adults of Scaptodrosophila lebanonensis Genetics, May 1, 2004; 167(1): 289 - 299. [Abstract] [Full Text] [PDF]
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