Muscle-specific Transcriptional Regulation of theslowpoke Ca2+-activated K+Channel Gene
2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês
10.1074/jbc.275.6.3991
ISSN1083-351X
AutoresWhei-meih Chang, Rudi A. Bohm, Jeffrey C. Strauss, Tao Kwan, Tarita O. Thomas, Roshani B. Cowmeadow, Nigel S. Atkinson,
Tópico(s)Insect and Pesticide Research
ResumoTranscriptional regulation of theDrosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified, which are responsible for slowpokeexpression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, we introduced small deletions into the slowpoke transcriptional control region. Using transformed flies, we asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion was required for promoter activity. We identified 55-, 214-, and 20-nucleotide sequences that control promoter activity. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells. Transcriptional regulation of theDrosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified, which are responsible for slowpokeexpression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, we introduced small deletions into the slowpoke transcriptional control region. Using transformed flies, we asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion was required for promoter activity. We identified 55-, 214-, and 20-nucleotide sequences that control promoter activity. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells. base pair(s) transcription start site dorsal longitudinal muscle dorso-ventral muscle direct control muscles, specifically the basalare and pterale I and II tergotrochanter muscle polymerase chain reaction 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside To acquire the appropriate electrical character, a neuron or muscle must express the correct subset of ion channels in the proper amounts (1.Baro D.J. Levini R.M. Kim M.T. Willms A.R. Lanning C.C. Rodriguez H.E. Harris-Warrick R.M. J. Neurosci. 1997; 17: 6597-6610Crossref PubMed Google Scholar). This is not a simple problem since even invertebrates have the capacity to produce more than a thousand different ion channel proteins (2.Atkinson N.S. Brenner R. Bohm R.A., Yu, J.Y. Wilbur J.L. Ann. N. Y. Acad. Sci. 1998; 860: 296-305Crossref PubMed Scopus (17) Google Scholar). Obviously, one should expect the expression of channel genes to be heavily regulated. Potassium channels belong to a large superfamily of genes. Particularly interesting are the calcium-activated potassium channels. These respond to changes in both calcium and membrane potential. The coupling of local calcium concentrations to a hyperpolarizing potassium current enables the cell to produce local circuits, which can rapidly and dynamically modulate both membrane potential and calcium influx (3.Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1173) Google Scholar,4.Sah P. Trends Neurosci. 1996; 19: 150-154Abstract Full Text PDF PubMed Scopus (802) Google Scholar). These channels participate in shaping the firing patterns of neurons and skeletal muscles, moderating synaptic efficacy, controlling smooth muscle tone, generating cyclical calcium waves during fertilization, active transport, controlling osmotic pressure, and demarcating the binding of ligands to receptors (5.Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar, 6.Rudy B. Neuroscience. 1988; 25: 729-749Crossref PubMed Scopus (1069) Google Scholar, 7.Elkins T. Ganetzky B. J. Neurosci. 1988; 8: 428-434Crossref PubMed Google Scholar, 8.Navaratnam D.S. Bell T.J. Tu T.D. Cohen E.L. Oberholtzer J.C. Neuron. 1997; 19: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9.Rosenblatt K.P. Sun Z.P. Heller S. Hudspeth A.J. Neuron. 1997; 19: 1061-1075Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10.Fay F.S. Science. 1995; 270: 588-589Crossref PubMed Scopus (36) Google Scholar, 11.Sheppard D.N. Giraldez F. J. Membr. Biol. 1988; 105: 65-75Crossref PubMed Scopus (37) Google Scholar, 12.Turnheim K. Constantin J. Chan S. Schultz S.G. J. Membr. Biol. 1989; 112: 247-254Crossref PubMed Scopus (26) Google Scholar, 13.Lewis R.S. Cahalan M.D. Annu. Rev. Immunol. 1995; 13: 623-653Crossref PubMed Scopus (445) Google Scholar). The maxi-K-type calcium-activated potassium channels have conductances ranging upward of 200 picosiemens (14.Blatz A.L. Magleby K.L. Trends Neurosci. 1987; 10: 463-467Abstract Full Text PDF Scopus (299) Google Scholar). Therefore, the activation of even a small number of such channels can effect the membrane potential of the cell and, as a result, the activity of voltage-gated ion channels in the membrane. The slowpoke gene encodes a maxi-K-type calcium-activated potassium channel that shows strong evolutionary sequence conservation and is expressed in a similar suite of tissues in vertebrates and invertebrates (15.Becker M.N. Brenner R. Atkinson N.S. J. Neurosci. 1995; 15: 6250-6259Crossref PubMed Google Scholar, 16.Tseng-Crank J. Foster C.D. Krause J.D. Mertz R. Godinot N. DiChiara T.J. Reinhart P.H. Neuron. 1994; 13: 1315-1330Abstract Full Text PDF PubMed Scopus (382) Google Scholar). An independent metric of the similarity between invertebrate and vertebrate channels is the demonstration that theDrosophila slowpoke calcium sensor can activate the pore-forming domain of the mouse slowpoke protein (17.Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (227) Google Scholar). We are using the Drosophila gene as a model to study how ion channel gene expression is regulated. The slowpoketranscriptional control region is extremely complex. To date, five tissue-specific promoters have been identified (2.Atkinson N.S. Brenner R. Bohm R.A., Yu, J.Y. Wilbur J.L. Ann. N. Y. Acad. Sci. 1998; 860: 296-305Crossref PubMed Scopus (17) Google Scholar). These promoters are distributed over 7 kilobases of DNA and drive expression in the nervous system, larval midgut, muscle fibers, and tracheal cells (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar, 19.Brenner R. Atkinson N. Dev. Biol. 1996; 177: 536-543Crossref PubMed Scopus (19) Google Scholar). Here, we focus on a slowpoke promoter active in muscle and tracheal cells (Promoter C2). We use evolutionary sequence conservation coupled with deletion analysis to ask what cis-acting sequences activate the promoter in these cells and whether the promoter is regulated differently in distinct cell types. Promoter studies are usually performed in vivo in tissue culture lines; here, deletion analysis of the slowpoke transcriptional control region is performed in situ. We use animals stably and uniformly transformed with reporter genes. An advantage to this tack is that expression of a wild type or mutated transgene can be assayed in many tissues all situated in their native environment. A 414-base pair (bp)1 BamHI/ApaI fragment from the Drosophila melanogaster slowpoke cDNA Z54 (15.Becker M.N. Brenner R. Atkinson N.S. J. Neurosci. 1995; 15: 6250-6259Crossref PubMed Google Scholar), which contained exon C1 and C3, was used to probe a D. hydeigenomic library (20.O'Neil M.T. Belote J.M. Genetics. 1992; 131: 113-128Crossref PubMed Google Scholar) generously provided by Dr. John Belote (Syracuse University) under reduced stringency (hybridization: 20% (v/v) formamide (Ambion), 6× SSPE, 10× Denhardt's solution, 0.2% SDS, and 200 μg/ml salmon sperm DNA at 42 °C; wash: 2× SSPE, 0.1% SDS at 65 °C). DNA fragments from D. hydei were subcloned into pBluescriptII. Exonuclease Bal31-generated nested deletions (21.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) were sequenced by the dideoxy chain termination method (22.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52250) Google Scholar). Accession numbers for the D. melanogaster and D. hydeisequences are U40221 and AF208226, respectively. The construction of P6 and P7 has been described (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar). The construction of all other reporter genes has been described in detail by Chang (23.Chang W.-M. Identification of Transcriptional Regulatory Elements in Muscle Promoter of Ca2 + -activated Potassium Channel, slowpoke.in: in Drosophila. Ph.D. thesis. University of Texas, Austin1998Google Scholar) and is summarized below. Deletion constructs BR17 and EX remove sequences 5′ of Promoter C2. To produce BR17, the P6 reporter gene was digested with BamHI and EcoRI (partial) to release the 574-bp fragment between −975 and −401. Overhanging ends were converted to blunt ends and ligated to one another. Deletion EX was built by deleting the material between theEcoRI and XbaI sites (−400 to −62).EcoRI is not unique in P6, so deletion EX is built by a three-step cloning process. The XhoI-NotI fragment from P614 was inserted into pBluescript to produce plasmid J10. The EcoRI-XbaI fragment was deleted from J10, the sticky ends converted to blunt ends using Klenow enzyme, and the plasmid ligated shut to produce the plasmid J10EX. This recreated an EcoRI site but destroyed the XbaI site. The J10EX insert was excised using a XhoI and NotI double digestion and used to replace the XhoI toNotI fragment from P614. The GAL4BII reporter gene carries the entire transcriptional control region and includes Promoters C0, C1, C1b, C1c, and C2. This DNA fragment has been shown to reproduce the slowpoke expression pattern (15.Becker M.N. Brenner R. Atkinson N.S. J. Neurosci. 1995; 15: 6250-6259Crossref PubMed Google Scholar). In GAL4BII, the GAL4 gene has been inserted into a uniqueBglII site within exon C2, such that transcription from Promoter C2 expresses the GAL4 transcription factor. The translation start site is provided by the consensus start site withinslowpoke exon C2. The Gal4 gene was derived from the promoter-less pGaTB plasmid kindly provided by Andrea Brand (24.Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) and includes a hsp70 termination site. The GAL4B2.1 transgene is identical to Gal4BII, except that the C2/C3 intronic region (theBglII-ApaI fragment; Fig. 1 A) has been deleted. Blast searches confirmed that the newly created junction fragments for BR17, EX, and Gal4BII did not themselves represent known transcription factor binding sites. The EX deletion removed three evolutionarily conserved regions called the 55 box, the 4E region, and the 20 box. Each was added back into the EX deletion and then tested for activity. To make the construct 55/EX, the 55 box was produced by PCR. The primers 55 upper (5′-GTCTGATCACTCTGCCTTTTAATT-3′) and 55 lower (5′-TATGGATCCGACCGCGAAAAGTGTCAG-3′) were used to amplify the 55 box from P6. The PCR fragment was gel-purified and cloned into the vector PCRblunt (Invitrogen) to produce plasmid 55/pblunt. AnEcoRI digestion was used to release the 55 box from 55/pblunt. The purified fragment was ligated into the uniqueEcoRI site of J10EX to produce 55/J10EX. Sequence analysis confirmed that the construct contained one copy of the 55 box and that it was in the positive orientation. This places the 55 box almost in its original position. The 55/J10EX insert was excised withXhoI and NotI and ligated intoXhoI-NotI-digested P614 construct to produce plasmid 55/EX. The 4E/EX reporter gene was built by adding the 214-bp 4E region back into the EX deletion construct. The 4E region was PCR-amplified from P6 DNA using the 4E upper (5′-TTCAGATCTTAGCCAAATGCCCGTATA-3′) and 4E lower primers (5′-ACCGGATCCACCGCACAACTGGCG-3′). The product was blunt-end-cloned into the vector PCRblunt (Invitrogen) to produce 4E/pblunt. In this vector, EcoRI flanks the insert. The 4E insert from 4E/pblunt was excised with EcoRI and ligated into the recreated EcoRI site of J10EX produce 4E/J10EX. Sequence analysis confirmed that 4E/J10EX contained one copy of the 4E region in the positive orientation. The 4E/J10EX insert was then excised with XhoI and NotI and then ligated into a XhoI-NotI-digested P614 construct. The resulting transformation construct was called 4E/EX. In the construct 20/EX, the 20 box has been inserted into plasmid EX at the site of the original deletion. A double-stranded oligomer representing the 20 box was prepared by annealing oligomer 20A upper (5′-AATTCGCGGCCGCTTCGCTCGGTGCCTCCTTTTG-3′) to oligomer 20A lower (5′-AATTCAAAAGGAGGCACCGAGCGAAGCGGCCGCG-3′). This produces a double-stranded oligomer that anneals to the 5′ overhanging ends produced by EcoRI. An additional NotI site has been introduced to help identify the appropriate ligation product. The 20 oligomer was phosphorylated (polynucleotide kinase) and ligated directly into the EcoRI site of plasmid J10EX. This product is called 20/EX. The insertion was sequenced to confirm the number of copies of the 20 box and their orientation. Both one and two copies of the 20 box were obtained and are referred to as 1 × 20/J10EX and 2 × 20/J10EX, respectively. The 1 × 20/J10EX and 2 × 20/J10EX inserts were excised from the vector using XhoI andNotI and ligated intoXhoI-NotI-digested P614 construct. These products are called 1 × 20/EX and the 2 × 20/EX, respectively. Both produced identical expression patterns in transformed flies; therefore, the transformants are collectively referred to as 20/EX. P-element transformations were carried out largely as described by Spradling et al. (25.Spradling A.C. Roberts D.B. Drosophila: A Practical Approach. IRL Press, Oxford1986: 175-196Google Scholar). Potential transformants were crossed to w 1118;Sco/CyO; MKRS/TM6Tb. The presence of the w + gene (orange to red eyes) was used to identify transformants. Larvae and adults were stained for β-galactosidase activity as described by Brenner et al. (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar). Relative expression levels were quantified by staining all transformants in the same dish at the same time and by monitoring the appearance of the blue reaction product throughout the staining period. The previously described P6 transgene (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar, 19.Brenner R. Atkinson N. Dev. Biol. 1996; 177: 536-543Crossref PubMed Scopus (19) Google Scholar) was used as a positive control. Because the expression pattern of transgenes can be influenced by chromosomal position, the expression results were a consensus of no less than three independent P-element insertions. In each case all exhibited the same expression pattern. Homozygous transformants were used where possible, however, some transgene insertions were homozygous lethal and therefore were assayed as heterozygotes. In this case all animals in the comparison group were heterozygous. Animals carrying transgenes employing the Gal4 transcription factor as a reporter were first crossed to animals carrying the Gal4 responsive UAS-lacZ reporter (24.Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar). To determine the level and pattern of expression, control and experimental animals were stained together on the same slide or in the same dish. Our understanding of the slowpoke transcriptional control region results from 1) the physical mapping of promoter transcription start sites by 5′ rapid amplification of cDNA ends and 2) the assignment of promoter tissue specificity by deletion mapping (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar, 19.Brenner R. Atkinson N. Dev. Biol. 1996; 177: 536-543Crossref PubMed Scopus (19) Google Scholar, 26.Thomas T. Wang B. Brenner R. Atkinson N.S. Invert. Neurosci. 1997; 2: 283-291Crossref PubMed Google Scholar). The slowpoke gene has been shown to have five transcriptional promoters (2.Atkinson N.S. Brenner R. Bohm R.A., Yu, J.Y. Wilbur J.L. Ann. N. Y. Acad. Sci. 1998; 860: 296-305Crossref PubMed Scopus (17) Google Scholar). From 5′ to 3′, they are Promoters C0, C1, C1b, C1c, and C2 (Fig.1 A). Deletion analysis indicated that Promoters C0 and C1 are active in the nervous system, that the DNA fragment containing Promoters C1b and C1c is required for expression in two bands in the larval midgut, and finally that deletion of a fragment containing Promoter C2 causes a loss of expression in muscle fibers and tracheal cells (2.Atkinson N.S. Brenner R. Bohm R.A., Yu, J.Y. Wilbur J.L. Ann. N. Y. Acad. Sci. 1998; 860: 296-305Crossref PubMed Scopus (17) Google Scholar, 18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar). Transcription from each of the slowpoke promoters begins with a unique 5′ exon, which is subsequently spliced to exons common to all slowpoketranscripts. Each of these unique 5′ exons is named after its promoter. Thus, exon C2 is a product specifically produced by transcription from Promoter C2. Following transcription, exon C2 is spliced to exon C3, which is an exon common to all slowpoke transcripts. We would like to identify sequences that regulate the activity of slowpoke Promoter C2. Promoter C2 is responsible for muscle and tracheal expression ofslowpoke. While this promoter is also active in a small region of the larval brain (Ref. 18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar and Fig. 5 B), our focus here is on its regulation in muscle and tracheal cells. With respect to Promoter C2, transgenes that contain nucleotides −1902 to +1472, as in construct P6, reliably reproduce the slowpoke expression pattern in trachea and muscles (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar, 19.Brenner R. Atkinson N. Dev. Biol. 1996; 177: 536-543Crossref PubMed Scopus (19) Google Scholar) and therefore are predicted to contain all elements required for normal activity of Promoter C2. Promoter C2 includes a single strong transcription start site followed by a number of minor start sites distributed within exon C2. In this document, nucleotides are numbered with respect to the Promoter C2 major transcription start site (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar). Random deletion analysis is an inefficient approach for identifying small control elements in such a large transcriptional control region. Therefore, we have chosen to use evolutionary conservation to guide our search for DNA elements important for controlling transcription fromslowpoke Promoter C2. Toward this end, we have cloned and sequenced genomic DNA from D. hydei homologous to the Promoter C2 control region of D. melanogaster. These species diverged from a common ancestor approximately 60 million years ago (27.Patterson J.T. Stone W.S. Evolution in the Genus Drosophila. Macmillan Co., New York1952Google Scholar). The program MACAW was used to identify and organize the sequences into blocks of homology (28.Schuler G. Multiple Alignment Construction and.in: Analysis (MACAW), Version 2.0 (5) MAC 68K. National Center for Biotechnology, National Library of Medicine, Bethesda, MD1995Google Scholar). Eleven boxes of homology were identified (Figs. 1 C and 2). All of these blocks were conserved in both sequence and relative position with respect to one another and to theslowpoke exons. As expected, the most striking conservation was within the coding region of exons C2 and C3. However, three other relatively large homology blocks were identified. They are the 55 box, located upstream of the Promoter C2 transcription start site (tss) and the 36 and 60 boxes (Figs. 1 and 2) found within the 5′-untranslated region of exon C2. Smaller blocks of homology (10–20 nucleotides) were also considered significant if they were conserved in both sequence and position (Figs. 1 and 2). Transcription start sites are difficult to identify by inspection of DNA sequence. The D. melanogaster Promoter C2 tss was previously mapped by 5′ rapid amplification of cDNA ends and confirmed by RNase protection assays and deletion analysis (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar). This site is flanked by the evolutionarily conserved 10B and 36 boxes. In the absence of physical mapping data, we assume that the D. hydei Promoter C2 tss is in the same relative position between these two conserved boxes. We also searched the sequence for known transcription factor binding motifs. Three mef2 and 20 E box motifs were identified. The mef2 and myoD family of transcription factors recognize these motifs and are key regulators of myogenesis (29.Molkentin J.D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (372) Google Scholar). A single zfh1 motif was found 5′ of exon C2 in the D. melanogasterand D. hydei sequences. Transcription factors that bind this site have been shown to be important in silencing muscle-specific genes in non-muscle tissue (30.Fortini M.E. Lai Z.C. Rubin G.M. Mech. Dev. 1991; 34: 113-122Crossref PubMed Scopus (132) Google Scholar, 31.Postigo A.A. Ward E. Skeath J.B. Dean D.C. Mol. Biol. Cell. 1999; 19: 7255-7263Crossref Google Scholar). In a previous study (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar), it was shown that the P7 deletion, which removed sequences 5′ of Promoter C2 up to nucleotide −975 (the BamHI site in Fig. 1 and 2), was capable of reproducing the Promoter C2 expression pattern. This indicated that all essential, positively acting elements are 3′ of this BamHI site. However, P7 was noted to be sensitive to chromosome position effects, which sometimes resulted in ectopic expression (23.Chang W.-M. Identification of Transcriptional Regulatory Elements in Muscle Promoter of Ca2 + -activated Potassium Channel, slowpoke.in: in Drosophila. Ph.D. thesis. University of Texas, Austin1998Google Scholar). This deletion removes a conserved zfh1 site. This suggests that this site may be a negative regulator of Promoter C2 expression, which serves to prevent expression in inappropriate tissues. Nevertheless, this study has focused on elements between nucleotides −975 and +1472, the fragment of DNA carried in the P7 construct. The conserved blocks were tested for functional importance by deletion analysis. The P6 transgene was used as the starting material, since it reproduces theslowpoke muscle and tracheal cell expression pattern and is not sensitive to chromosome position effects (18.Brenner R. Thomas T.O. Becker M.N. Atkinson N.S. J. Neurosci. 1996; 16: 1827-1835Crossref PubMed Google Scholar, 19.Brenner R. Atkinson N. Dev. Biol. 1996; 177: 536-543Crossref PubMed Scopus (19) Google Scholar). Transgenic flies were used to compare the expression pattern of the deleted and intact versions of P6. Animals being compared were sectioned or dissected together and stained on the same slide or in the same dish. TableI provides a summary of the data discussed below.Table ISummary of expression patternsA.Indirect, asynchronousIndirect, synchronousDirect, synchronousDLMDVMTTPSBasalarePterale IPterale IILegP6++++++++++++++++++++++++++++EX−−−+++++20/EX−−−+++++4E/EX+++++++++++++++++++++++++55/EX−−−+++++Gal4BII++++++++−++++++++++++++++++++Gal4B2.1−−−+++++++++++B.554E20Intronic regionAdult asynchronous muscleNot requiredSufficientNot requiredRequiredAdult synchronous muscleNot requiredNot requiredNot requiredNot requiredLarval body wall muscleSufficientSufficientNot requiredNot requiredLarval tracheal cellsSufficientNot requiredSufficientNot requiredTable gives a summary of the muscle expression pattern ofslowpoke transgenes. In part A, the number of pluses represents a visual estimation of the relative expression level in stained animals. A minus indicates a lack of expression. Muscle subtypes are grouped as indirect asynchronous, indirect synchronous, and direct synchronous flight muscle. Abbreviations are as defined in Fig. 5. In part B, dependence of muscle subtypes and tracheal cells on different conserved regions is shown for expression from Promoter C2. Open table in a new tab Table gives a summary of the muscle expression pattern ofslowpoke transgenes. In part A, the number of pluses represents a visual estimation of the relative expression level in stained animals. A minus indicates a lack of expression. Muscle subtypes are grouped as indirect asynchronous, indirect synchronous, and direct synchronous flight muscle. Abbreviations are as defined in Fig. 5. In part B, dependence of muscle subtypes and tracheal cells on different conserved regions is shown for expression from Promoter C2. The BR17 derivative of P6 has suffered a deletion that removes the nucleotides flanked by the BamHI and EcoRI sites of P6 (nucleotides −975 to −401) eliminating the 10A box (Fig.3). In transformant lines, BR17 expressed β-galactosidase in the same pattern and with the same relative intensity as the P6 reporter gene in both larval and adult muscles and in tracheal cells (data not shown). It appears that the BR17 deletion causes no alteration in the pattern or the intensity of the muscle expression in larvae or adult. Clearly, the conserved 10A and E box removed by this deletion are not essential for normal activity in muscles. The EX deletion removes nucleotides −400 to −62 and eliminates the 55 box, the 4E region, and the 20 box. In D. melanogaster, the 214-nucleotide 4E region includes four E boxes. In larvae, this deletion caused the loss of all muscle expression (Fig.4 A). The effect of the EX deletion in the adult can be summarized as a loss of expression in the fibrillar power muscles and a reduction in expression in other adult muscles (Fig. 4 B). The muscle groups showing a loss of expression were the dorsal longitudinal muscles (DLM), the dorso-ventral muscles (DVM), and the tergotrochanter muscle (TT). The DLM and DVM provide the mechanical power for the wing beat, while the TT generates the jump used to initiate flight (32.Dickinson M.H. Tu M.S. Comp. Biochem. Physiol. A. Physiol. 1997; 116A: 223-238Crossref Scopus (166) Google Scholar). Reduced expression was observed in the pterale direct control muscles (steering muscles), and in an indirect flight control muscle, the pleurosternal I muscle. The pterale muscles are involved in directly controlling wing kinematics while the pleurosternal muscles are thought to modulate wing beat frequency. A reduced but readily detectable level of expression was also observed in the prothoracic, mesothoracic, and metathoracic leg muscles (data not shown). The expression in these muscles indicates that the promoter is still functional and that the deletion has merely affected its tissue specificity. The 339-nucleotide EX deletion removes the conserved 55 and 20 boxes and the 214-bp 4E region. To determine the relative contribution of these sequences to Promoter C2 activity, each was individually added back to the EX deletion and the modified reporter gene assayed for expression in transformed flies. The EX deletion has an EcoRI site at the site of the deletion. Oligonucleotides representing the 55 box, the 4E region, and the 20 box were individually prepared and inserted with their original polarity into this site. The products are called 55/EX, 4E/EX, and 20/EX to designate which oligonucleotide they contain. To be able to compare expression levels of the constructs, different genotypes were stained for the same length of time in the same dish or microscope slide. In larvae, the insertion of the 55 box restored larval body wall muscle expression to a level indistinguishable from P6 (Fig. 4 A). The 4E region, however, only partially restored larval muscle expression, and, finally, the insertion of one copy or two copies of the 20 box back into the EX deletion was unable to activate Promoter C2 in larval muscle (Fig. 4 A). The expression levels of these reporter gene constructs in the adult are substantially different than that observed in the larvae. In adults, the 55/EX construct is expressed at extremely low levels (Fig.4 B). Thus, the 55 box is not sufficient in the absence of the 20 box and the 4E region to properly activate Promoter C2 in adults. However, the reinsertion of the 4E region alone restored expression in muscles of the thorax to near normal levels (Fig.4 B). Expression in muscles in the head and legs was also augmented (data not shown). The reinsertion of the 20 box had no effect on adult muscle expression and sections from these animals expre
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