©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcription of the Rat -Adrenergic Receptor Gene
CHARACTERIZATION OF THE TRANSCRIPT AND IDENTIFICATION OF IMPORTANT SEQUENCES (*)

(Received for publication, July 20, 1994; and in revised form, October 10, 1994)

Robert P. Searles Clare N. Midson Valerie J. Nipper (§) Curtis A. Machida (1)(¶)

From the Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006 Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have characterized the 5` and 3` ends of the rat beta(1)-adrenergic receptor transcript using RNase protection assays and have used transient transfection analysis to identify regions of the beta(1)-adrenergic gene 5`-flanking sequences which are important for expression. The transcript has multiple start sites, occurring primarily in two clusters at bases -250 and -280, relative to the first base of the initiation codon. Two potential polyadenylation signals at +2450 and +2732 are both functional, although the site at +2732 is preferred both in C6 glioma cells and in heart tissue. Characterization of the gene by transient transfection analysis has identified a region between bases -389 and -325 which is necessary for expression. The specific deletion of a potentially functional inverted CCAAT sequence within this region does not significantly alter activity. In addition to the region from -389 and -325, deletion of the bases between -1 and -159 and between -186 and -211 significantly alters expression. Both of these regions are downstream from the beta(1)-adrenergic receptor gene start sites and may function either through regulation of transcription or through alteration of the transcript structure.


INTRODUCTION

Physiological response to the catecholamines epinephrine and norepinephrine is initiated by agonist interaction with the beta-adrenergic receptors (beta-ARs)(^1)(1) . Two major beta-AR subtypes, the beta(1)- and beta(2)-adrenergic receptors (beta(1)- and beta(2)-ARs, respectively), account for most of the mammalian beta-AR complement. These two receptors are the products of separate genes (2) and can be distinguished by their relative affinities for their endogenous ligands epinephrine and norepinephrine(3) . In addition to these two subtypes, the gene for a third subtype, the beta(3)-AR, has been isolated recently from several species (4, 5, 6) . Northern analysis and in situ hybridization have determined that the beta(1)-AR is the primary or exclusive beta-AR in the cerebral cortex(7, 8) , the pineal gland(7) , the ventral thalamic nuclear complex(8) , and the heart (7) and that beta(2)-AR is the primary or exclusive beta-AR in the prostate (7) , the cerebellar cortex(8) , and the centrolateral and centromedial thalamic nuclei(8) . By contrast, the beta(3)-AR mRNA is detected exclusively in brown and white adipose tissue, where it is the predominant beta-AR(6) .

Multiple transcriptional start sites for the beta(2)-AR mRNA have been identified by S1 nuclease digestion and primer extension analysis(9) . Based upon these data, it has been suggested that the beta(2)-AR gene contains a promoter that utilizes an inverted CCAAT box upstream from the multiple start sites(9) . The beta(1)-AR gene has not been assigned a promoter, although potential elements have been suggested. These elements include an inverted AGCCAAT sequence at -353 (^2)to -359 in the rat gene (10) and at the corresponding location in the rhesus macaque gene (10) and an inverted CCAAT box at -1001 in the human beta(1)-AR gene(11) . Start sites for the mouse brain beta(1)-AR transcript occur at the equivalent of rat beta(1)-AR gene bases -416, -421, and -492(12) . Using ribonuclease (RNase) protection assays, we have identified multiple start sites for the rat beta(1)-AR mRNA, occurring primarily in two clusters surrounding bases -250 and -280. Using the same protocol, we have identified two polyadenylation sites, both of which are used in C6 cells and in cardiac tissue. Using transient transfection assays we have identified the region between bases -389 and -325 as necessary for expression. We have also determined that the bases between -1 and -156 and between bases -186 and -211 contain repressor and enhancer activities, respectively, which affect the expression of the rat beta(1)-AR gene.


EXPERIMENTAL PROCEDURES

Materials

TriReagent was purchased from Molecular Research, Inc. Sequenase® was purchased from U. S. Biochemical Corp. P-Labeled nucleotides were from DuPont NEN. Restriction enzymes were from New England Biolabs, Life Technologies, Inc., and Boehringer Mannheim. Luciferin and ribonuclease T(1) were from Sigma. Ribonuclease A was from Boehringer Mannheim. G418 (Geneticin) was from Life Technologies, Inc. The cloning vector pGEM3Zf(+), RNA polymerases T7 and SP6, HindIII linkers, and RNasin were from Promega. The bacterial beta-galactosidase expression vector ptkbeta was from Clontech. The cloning vector pBluescript II SK was from Stratagene. The GalactoLight Kit was from Tropix, Inc. All other materials were of the highest available quality.

Luciferase expression vectors pXP1 and pT109luc (13) were used with the kind permission of Dr. Steven Nordeen, University of Colorado. Luciferase and beta-galactosidase activities were measured using an EG & G Berthold AutoLumat LB 953 luminometer. RNA annealing incubations were performed using an MJ Research PTC-100 programmable thermal controller. Rat C6 glioma cells were from Dr. Kim Neve, Veterans Administration Hospital, Portland, OR. Rat L6 myoblast cells were from ATCC.

RNase Protection Assays

The TriReagent protocol for cultured cells was used to isolate total RNA from rat C6 glioma and rat L6 myoblast cells grown to confluence in 100-mm dishes. Total RNA from 1.1 g of cardiac tissue was prepared using the TriReagent protocol for whole tissue. The integrity of the recovered RNA was verified by electrophoresis using a formaldehyde gel. Total RNA was divided into 20- or 100-µg aliquots and stored as an ethanol precipitate at -20 °C until needed. RNA was tested for contaminating chromosomal DNA by RNase protection assay using a beta(1)-AR gene sense transcript.

RNase protection assays were based on the method of Gilman(14) . Template-containing plasmids were linearized and then stored in 1-µg aliquots in 2 µl of water at -20 °C. Antisense transcripts were generated from 1 µg of template in a total of 20 µl of reaction mix (40 mM TrisbulletHCl, pH 7.9, 6 mM MgCl(2), 2 mM spermidine, 10 mM dithiothreitol, 0.5 mM rNTPs (rATP, rGTP, rUTP), 12 µM rCTP, 20 units of RNasin, 20 units of T7 or SP6 RNA polymerase, and 50 µCi of 800 Ci/mmol [alpha-P]rCTP). The reaction was incubated at 37 °C for 1 h. 25 µg of tRNA was added, and the RNA was precipitated with ethanol. The pellet was resuspended in 5 µl of 50% formamide with xylene cyanol and bromphenol blue and applied to a 6% acrylamide, 8.3 M urea gel to separate transcript from template and unincorporated label. The transcript was visualized by autoradiography, excised, and then eluted into 140 µl of 10 mM TrisbulletHCl, pH 7.5, 400 mM NaCl, and 1 mM EDTA for 1-3 h.

Antisense transcript (10^6 cpm) was combined with either 20 or 100 µg of total RNA and recovered by ethanol precipitation. The RNA was resuspended in 30 µl of hybridization solution (40 mM PIPES, pH 6.8, 400 mM NaCl, 1 mM EDTA, 80% formamide) and then denatured by incubating the mix at 95 °C for 30 min. Annealing conditions were transcript-dependent and are described in the figure legends. Following the annealing step, 29 µl of the hybridization mix was added to an RNase mixture (final volume 400 µl; final composition: 10 mM TrisbulletHCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 5.4% formamide, with RNases A and T(1) as indicated in the figure legends). Incubation conditions are indicated in the figure legends. The remaining 1 µl of the hybridization solution was used as an undigested control. RNase digestion was terminated by the addition of 10 µl of 20% sodium dodecyl sulfate and 25 µl of 2 mg/ml proteinase K. The mix was incubated first at room temperature for 10 min and then at 37 °C for 15 min. The solution was extracted with phenol/chloroform and ethanol precipitated. The recovered sample was resuspended in formamide/dye solution, and the products were separated by electrophoresis on a 6% acrylamide, 8.3 M urea gel. A DNA sequencing ladder using pGEM3Zf(+) as the template was prepared to size the protected fragments. To correct for any differences that might occur between the migration of RNA and DNA, RNA standards of known size were compared with the DNA ladder.

Construction of Deletion Mutants and Luciferase Plasmids

Expression was analyzed using the promoterless luciferase-expressing plasmid pXP1(13) . A promoterless luciferase-expressing plasmid with a multicloning site oriented opposite that of pXP1 was constructed using the plasmid pT109luc(13) . The plasmid pT109luc contains a luciferase gene under the control of a herpes simplex virus thymidine kinase promoter. The thymidine kinase promoter of pT109luc was removed by an XhoI/BglII double digest, followed by T4 DNA polymerase blunting of the ends and blunt end ligation. The XhoI and BglII sites were regenerated by the final ligation. The multicloning site of this plasmid, pd09, was verified by sequencing using a primer specific for the luciferase gene. The plasmids pXP1 and pd09 differ only in the orientation of the multicloning site, and pd09 is equivalent to pXP2 (13) .

The beta(1)-AR gene PstI fragment from base -484 to +270 was subcloned into the PstI site of pGEM3Zf(+). Bases +1 to +270 were then removed from this fragment by exonuclease III deletion(15) . A HindIII linker was inserted immediately 3` of base -1, and the 5`-flanking sequence of the beta(1)-AR gene was reconstructed using this fragment as the 3` end. Deletion mutants of the rat beta(1)-AR 5`-flanking sequences were then generated by one of two methods. For the first method, deletion mutants were generated by using endogenous restriction sites and available sites in the vector multicloning sites. For the second method, when no conveniently located restriction sites were available, unidirectional overlapping deletions were generated using exonuclease III(15) . Deletion mutants were first screened by size and then characterized by sequencing. Selected deletion fragments generated by either protocol were subcloned into the multicloning site of pXP1 or pd09. All constructs were verified by dideoxy sequencing and restriction digests. The nomenclature of the beta(1)-AR gene constructs is [5` end, 3` end] to indicate the 5` and 3` limits of a segment of DNA.

Transfections

Plasmids were introduced into rat C6 glioma cells by calcium phosphate transfection using HEPES-buffered saline (16) . C6 cells were seeded at 6 times 10^5 cell/60-mm dish. After 24-48 h, each dish was cotransfected with 2.5 µg of the beta(1)-AR construct and 2.5 µg of the plasmid ptkbeta (Clontech), which expresses the gene for bacterial beta-galactosidase under control of the thymidine kinase promoter. Cells were harvested after incubation for 16-24 h following the removal of the precipitate and assayed for luciferase activity, beta-galactosidase activity, and protein. Parallel dishes transfected with pT109luc and ptkbeta were prepared for each experiment, and all values were expressed as a percentage of the expression of pT109luc after normalizing for ptkbeta expression. Because both pT109luc and ptkbeta utilize the thymidine kinase promoter, control experiments were performed to verify that the thymidine kinase promoter has a linear response following the transfection of as much as 20 µg of pT109luc. Mock transfected cells were prepared for each experiment to determine the contribution of endogenous beta-galactosidase activity. Each experiment included three dishes/construct, and each construct was measured in two or three separate experiments (n = 6 or n = 9). The values for all experiments were combined and subjected to analysis of variance. Significance was determined by the Tukey Compromise post hoc analysis (p = 0.05).

Assays

Luciferase assays were performed as described(17) , with the lysis buffer modified to use 0.1% Triton X-100. beta-Galactosidase was assayed using the GalactoLight Kit. Protein was measured using the method of Dulley and Grieve(18) , which is designed to eliminate interference from detergent.


RESULTS

The Rat beta(1)-AR Gene Utilizes Multiple Transcriptional Start Sites

Transcriptional start sites for the mouse beta(1)-AR gene were determined recently by primer extension analysis of total RNA(12) . We attempted primer extension analysis of the rat beta(1)-AR transcript with two different primers and found that the primers gave different start sites. In addition, the same primers yielded products from rat L6 total RNA control assays, despite the absence of beta(1)-AR transcript in L6 cells(19) . To resolve these problems, we analyzed the 5` end of the rat beta(1)-AR transcript in rat C6 glioma cells by RNase protection assay. To circumvent problems encountered when transcribing long probes from the high GC region of the beta(1)-AR DNA, we developed a series of short overlapping probes which allowed us to ``walk'' along the beta(1)-AR transcript. These three probes are [-216, -1], [-299, -81], and [-484, -159] (Fig. 1A).


Figure 1: Transcription start sites of the rat beta(1)-AR gene. Panel A, probes used to determine the start site of beta(1)-AR gene transcription by RNase protection assay. The open bar represents the beta(1)-AR transcript. The shaded bars represent the RNase protection assay probes. Panel B, RNase protection assay products of antisense RNA probe [-299, -81] hybridized to total RNA. Lane 1, RNA standards; lanes 2-5, pGEM3Zf(+) sequence using the reverse primer; left to right, the lanes are A, C, G, T; lane 6, rat C6 glioma total RNA; lane 7, rat L6 myoblast total RNA. The sizes of the RNA standards are indicated by the numbers on the left. The approximate locations of the protected start site clusters are indicated by the numbers on the right. Probe was hybridized to 100 µg of total RNA. The RNA/probe mix was denatured at 95 °C for 30 min and then cooled to 85 °C. After 2 h at 85 °C, the incubation temperature was reduced in 5 °C increments with a 2-h incubation at each increment. The final incubation was at 55 °C. Hybridized RNAs were digested with 140 µg/ml RNase A and 7 µg/ml of RNase T(1) at room temperature for 2 h. After the RNases were inactivated, the RNA was recovered and the products separated on a 6% acrylamide, 8.3 M urea gel. Variation between RNA standard and DNA migration is less than 2%. Panel C, RNase protection assay products of antisense RNA probe [-479, -159] hybridized to total RNA. Panels B and C were prepared from the same autoradiogram. Lane 1, rat L6 myoblast total RNA; lane 2, rat C6 glioma total RNA; lane 3, RNA standard; lanes 4-7, pGEM3Zf(+) sequence using the reverse primer; left to right, the lanes are A, C, G, T. The approximate locations of the start site clusters are indicated by the numbers on the left. In addition to the start site clusters at -250 and -280, lane 2 has additional bands that correspond to start sites at -295, -304, and -309. The length of the RNA standard is indicated by the number on the right. Panel D, the sequence of the rat beta(1)-AR gene indicating the start sites (bullet) identified by RNase protection assay. The sequence is from Searles et al.(10) .



RNase protection assays using probe [-216, -1] annealed to 20 µg of total RNA showed only a single band about 40 bases shorter than intact probe (data not shown). The 40-base difference is equivalent to the pGEM3Zf(+)-derived sequence in the probe and indicates that the entire length of the beta(1)-AR antisense probe was protected. RNase digestion of probe [-299, -81] annealed to 20 µg of total C6 RNA produced three faint bands. Two of these bands were diffuse and somewhat smaller than the full-length probe; the third appeared to be equivalent to probe minus vector sequence. To increase the signal from these multiple start sites, we increased the amount of total C6 RNA 5-fold to 100 µg and increased the amount of RNase A and T(1) 4-fold to 140 µg/ml and 7 µg/ml, respectively, to accommodate the increased amount of RNA. The increased amounts of RNase for this assay did not change the pattern of the digestion, but the increased signal allowed us to identify two clusters of start sites at approximately -250 and -280 (Fig. 1B, lane 6). In addition, the third band represented RNA initiated close to or upstream of -299, indicating that all beta(1)-AR start sites are not within the -250 and -280 clusters. Control experiments using 100 µg of C6 cell RNA and probe [-299, -81] digested at 15 °C for 2 h with 3.5 µg/ml RNase T(1) verified that the clusters are not the result of RNA ``breathing'' (data not shown).

To identify the start sites with greater resolution and to identify additional start sites upstream of base -299, RNase protection assays were performed with probe [-484, -159]. This probe identified the same clusters of start sites as probe [-299, -81] (Fig. 1C, lane 2). In addition to the -280 and -250 clusters, digests using probe [-484, -159] contained several additional bands (Fig. 1C, lane 2). These are derived from transcriptional start sites at -295, -304, and -309. These additional sites contribute to the band at -299 for probe [-299, -81] in lane 6 of Fig. 1B. In total, 18 sites are identified by probe [-484, -159] (Fig. 1D). The darkest band in the -280 cluster (Fig. 1C, lane 2) corresponds to base -281. The probe has a cytosine at the base complementary to base -281. This cytosine is 3` to 2 consecutive adenosines. Since neither RNase A nor T(1) digests 3` of adenosines, the signal at -281 may represent the signal from start sites at -279, -280, and -281. Alternatively, base -281 may be the primary transcription start site. The digest of probe [-484, -159] also has a product which indicates intact probe minus vector sequence (data not shown). This suggests that there are more start sites upstream of base -484, which is consistent with the transfection analysis of the beta(1)-AR 5`-flanking sequences (below). The control experiments with each of these probes using L6 RNA did not produce any protected fragments.

The Rat beta(1)-AR Gene Utilizes Two Polyadenylation Sites in C6 Cells and in Cardiac Tissue

To complement the identification of the transcriptional start sites for this gene, and thereby delineate the 5` and 3` extent of the beta(1)-AR transcript, we have used RNase protection assays to characterize the use of the two potential polyadenylation signals found at +2450 and +2732 in the rat beta(1)-AR gene. RNase protection assays using antisense probe [+2084, +2901] hybridized to C6 glioma total RNA gave four bands (Fig. 2A, lane 3). Two bands correspond to the use of each of the potential polyadenylation signals. The 650-base band corresponds to mRNAs that have used the polyadenylation signal at +2732. Based upon relative signal intensities, this signal is the preferred site. The 370-base band corresponds to use of the polyadenylation signal at +2450. We believe that the third band at 817 bases is the product of beta(1)-AR transcript that has not been polyadenylated. Such transcript would be eliminated by the isolation of mRNA using oligo(dT) but is present in the total RNA that we have used for this experiment. The fourth band is undigested probe, the recovery of which is variable, even when the conditions of the hybridization and digestion are unchanged. RNase protection assays of total RNA from cardiac tissue (Fig. 2B, lane 2) produce the same pattern of products in the same proportions, but in this autoradiogram it lacks the band from undigested probe. There is a minor amount of variable background banding in these experiments which has been minimized by the digest conditions. This background results from the high AT content of the probe.


Figure 2: Polyadenylation sites of the rat beta(1)-AR gene. Panel A, RNase protection assay of beta(1)-AR transcript using probe [+2084, +2901] with total RNA from rat C6 glioma and L6 myoblast cells. Lane 1, undigested probe; lane 2, rat L6 myoblast total RNA; lane 3, rat C6 glioma total RNA; lane 4, RNA standards. The location of the polyadenylation site corresponding to the product is indicated by the numbers on the left. 2901 corresponds to the product derived from unprocessed transcript. The sizes of the RNA standards, which include the undigested probe, are indicated by the numbers on the right. The probe and RNA were denatured at 95 °C for 30 min and then cooled to 45 °C for an overnight incubation. The annealed RNA/probe mix was digested with 3.5 µg/ml RNase T(1) for 2 h at 15 °C. After the RNase was inactivated, the RNA was recovered, and the products were separated on a 6% acrylamide, 8.3 M urea gel. Panel B, RNase protection assay of beta(1)-AR transcript using probe [+2084, +2901] with C6 cell and cardiac tissue total RNA. Lane 1, undigested probe; lane 2, cardiac tissue total RNA; lane 3, C6 glioma total RNA; lane 4, RNA standards. The location of the polyadenylation site corresponding to a given product is indicated by the numbers on the left. 2901 corresponds to the product from unprocessed transcript. The sizes of the RNA standards, which include the undigested probe, are indicated by the numbers on the right. The parameters for the RNase protection assay are described in the legend for panel A.



Transfection Analysis Identifies Three Elements between Bases -484 and -1 Which Are Important for Expression of the Rat beta(1)-AR Gene

To identify regions of importance in the control of rat beta(1)-AR expression, deletion mutants of the 5`-flanking sequences were constructed and then linked to the expression of the gene for firefly luciferase. Deletion of the beta(1)-AR 5`-flanking sequences from the 5` side showed a moderate increase in activity as the deletion progressed from -3354 to -484 (Fig. 3). Several of these deletions involved large sections of DNA (e.g. the deletion of the bases between -3354 and -1804). Because the transfections are done with a constant 2.5 µg of construct/dish, it is likely that most of the differences in expression are the result of significant changes in the molar amounts of construct as the constructs become smaller. Therefore, no significant elements are identified by this series of deletions. However, the much lower level of expression of the construct [-1253, -479] compared to the expression of construct [-1253, -1] is significant. There is a 76% decline in activity when the segment between bases -484 and -1 is removed. This indicates that important elements of expression are present between bases -484 and -1. The start site data from the RNase protection assays localize the primary start sites to this section of the gene, and the high levels of expression obtained with [-484, -1] indicate that promoter activity is also present in this region. The residual activity of [-1253, -479] is consistent with the RNase protection assay data, which indicate that a portion of the transcript population initiates upstream of -484.


Figure 3: Expression analysis of the 5`-flanking sequences of the rat beta(1)-AR gene. The left side is a schematic diagram of the rat beta(1)-AR 5`-flanking sequences subcloned into the luciferase expression vector and transfected into rat C6 glioma cells. The right side represents the expression of the beta(1)-AR constructs relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three transfections. Each transfection utilized triplicate samples.



To examine further the region between -484 and -1, two series of overlapping deletions were generated and used to promote luciferase expression. Unlike the deletions performed above, these deletions do not result in a significant change in the size of the overall plasmid, and therefore interpretation of changes in expression is not complicated by the change in molar ratios of the transfected DNAs. The first set of deletions progressed from the 5` side of [-484, -1] (Fig. 4). Overall, there is a 78% difference in activity between [-484, -1] and [-160, -1]. Deletion of the bases between -484 and -389 does not significantly change expression, but when the 18 bases between -389 and -370 are deleted, there is a 35% decline in expression. Expression declines by 61% when the bases between -370 and -299 are deleted. Continued deletion from -299 to -160 does not affect expression. The expression of the last three constructs ([-299, -1], [-244, -1], [-160, -1]) differs significantly from the expression of pXP1 (p = 0.05). This may result from cryptic promoters present in the high GC regions of the remaining DNA.


Figure 4: Expression analysis of fragment [-479, -1] using 5` deletions. The left side is a schematic diagram of the rat beta(1)-AR 5`-flanking sequences subcloned into the luciferase expression vector and transfected into rat C6 glioma cells. The right side represents the expression of the beta(1)-AR constructs relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three transfections. Each transfection utilized triplicate samples.



A second set of recombinants was prepared by deletion of [-484, -1] from the 3` side. Luciferase activity varied in response to the deletion of three specific regions of the gene (Fig. 5). First, there is a 35% increase in signal when the region between bases -1 and -159 is deleted. Second, deletion of the region between bases -159 and -258 results in a 62% decline in activity. The majority of this decline occurs with the deletion of the region between -186 and -211, removal of which results in a 52% loss of activity. Interestingly, activity remains essentially level when the bases between -211 and -325 are removed, even though all of the start sites are deleted. Deletion of bases between bases -325 and -367 results in a loss of 81% of the remaining activity. Expression from the plasmid [-484, -367] is not significantly different from that of pXP1 (p = 0.05). These data imply that the region between -325 and -367 is necessary and sufficient for activity, even in the absence of the endogenous start sites.


Figure 5: Expression analysis of fragment [-479, -1] using 3` deletions. The left side is a schematic diagram of the rat beta(1)-AR 5`-flanking sequences subcloned into the luciferase expression vector and transfected into rat C6 glioma cells. The right side represents the relative expression of the beta(1)-AR constructs relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three transfections. Each transfection utilized triplicate samples.



The Inverted AGCCAAT Sequence between Bases -353 and -359 Is Not a Significant Factor in the Expression of the Rat beta(1)-AR Gene

The region between bases -389 and -325 is implicated by both the 5` and 3` deletions as containing elements important for beta(1)-AR gene expression. There is an inverted AGCCAAT box at base -353 to base -359, and the deletion of this element may contribute to the decline in activity which occurs when the region between -389 and -299 is deleted. To examine this, a linker was used to ligate two deletion clones to produce a construct without the inverted AGCCAAT sequence (Fig. 6A). The resultant internal deletion lacked the endogenous bases between bases -348 and -360. Expression of luciferase linked to this internal deletion was not significantly different than expression from the intact fragment [-484, -1] (p = 0.05) (Fig. 6B). This indicates that the inverted AGCCAAT is not a significant factor in the expression of the beta(1)-AR gene.


Figure 6: Expression analysis of the internal deletion of the AGCCAAT element. Panel A, the beta(1)-AR gene fragment [-479, -1] was reconstructed as described under ``Experimental Procedures'' to replace the sequence from -348 to -359 with an 8-base linker sequence. This deleted the inverted AGCCAAT sequence at -353 to -359. The resultant recombinant was subcloned in the luciferase expression plasmid pXP1 and transfected as described under ``Experimental Procedures.'' Panel B, expression from the recombinant expression plasmid(-)CCAAT is compared with the expression from intact [-479, -1]. Expression from both plasmids is presented as a percentage of the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three transfections. Each transfection utilized triplicate samples.




DISCUSSION

We have identified multiple cap sites for the rat beta(1)-AR mRNA by RNase protection assay. The use of multiple transcription start sites is common among adrenergic receptor genes(9, 20) . Of particular interest, the rat alpha-adrenergic receptor gene has been reported to use five start sites controlled by three different promoters(21) . In contrast to this, the human alpha-adrenergic receptor transcript was shown by RNase protection assay and primer extension analysis to possess a cluster of start sites about 177-193 bases upstream from the translation initiation codon(22) . Similarly, the rat and the mouse beta(1)-AR genes both use multiple start sites, but there are no start sites common to the two genes. This may represent species variations, or it may represent tissue variation: the mouse transcript was isolated from brain, and the rat transcript was isolated from a cultured glioma cell line. Overall, the use of multiple start sites may reflect the high GC content commonly found in the genes for the adrenergic receptors, since multiple start sites are common in genes with high GC promoters(23, 24, 25) .

In addition to the heterogeneity of the 5` end of the transcript, our data also identify two functional polyadenylation sites for the beta(1)-AR gene. Multiple sites of polyadenylation for single genes are known, and they can be part of the complex regulation of transcript processing(26) . For example, multiple polyadenylation sites are present in the adenovirus major transcriptional unit, and selection of specific polyadenylation sites varies over the course of infection (27) . Functionally, the switching of polyadenylation signals may have an effect on the stability of a transcript by altering the secondary structure of the 3`-untranslated sequence(28) . Therefore, regulation of beta(1)-AR gene expression may be a complex function involving promoter/enhancer elements modulating transcription levels while changes in polyadenylation site alter mRNA stability. Variation in the use of the polyadenylation sites is not apparent in the comparison of C6 cell and cardiac tissue. Whether this is the case for beta(1)-AR mRNAs obtained from other tissue is under investigation. Also, whether there is a shift in the selection of polyadenylation sites under conditions that have an effect on receptor number (e.g.in vitro during agonist-induced down-regulation; (31) ) and in vivo during conditions such as congestive heart failure, which exhibits a marked decline in the levels of beta(1)-AR expression (29) is also currently under investigation.

Transfection analyses of specific upstream flanking sequences of the rat beta(1)-AR gene inserted into luciferase expression vectors show that there is no change in expression when the primary start sites at -280 and -250 are deleted. However, sequences both upstream and downstream from the start sites appear to have a significant effect on expression. First, the combination of 5` and 3` deletion constructs identifies the region between bases -389 and -325 as important for expression. There may be multiple elements within this region, since the decline in activity with progressive 5` deletion has a notably smooth appearance, suggesting the progressive deletion of additive elements. A linker mutant removing the consensus AGCCAAT sequence in this region demonstrates that it is not functional. We are currently examining this region more closely to characterize specific functional elements.

Two more important regions of the beta(1)-AR gene flanking sequences, -1 to -186 and -186 to -211, are identified by the 3` deletion constructs. Both exist downstream of the start sites of transcription, so deletions of these regions must be considered in the context of both transcriptional regulation and transcript structure. The increase in activity accompanying the 3` deletions from -1 to -211 may represent destabilization of the DNA. Just as we have encountered difficulty in the in vitro transcription of this gene, the high GC content may impede transcription of the gene in vivo, and removal of this portion of the gene may facilitate the progress of the transcription complex. The dramatic decline in activity with the deletion of the 25 bases between -186 and -211 may result from the loss of two overlapping AP-4 (30) sites (bases -204 to -196 and bases -201 to -193). However, as indicated above, these variations in expression may also result from changes in transcript structure.

In conclusion, we have identified multiple transcription start sites for the rat beta(1)-AR gene. These sites occur primarily in two clusters, located at about bases -280 and -250. Our data indicate that deletion of the sites does not affect expression. Sequences located between -389 and -325 appear to be critical for expression, presumably through the presence of a promoter or an enhancer. An inverted AGCCAAT sequence within this region is not functional. The regions between -1 and -159 and between -186 and -211 also appear to have a significant effect upon the levels of expression. Because these regions are downstream from the transcription start sites, it is not clear whether their effect is transcriptional or if it results from an alteration in the structure of the mRNA. We have also identified two functional polyadenylation signals in the rat beta(1)-AR gene. Further analysis is currently under way to characterize elements necessary for the expression of the beta(1)-AR gene.


FOOTNOTES

*
This work was supported in part by National Institutes of Health National Research Service Award Postdoctoral Fellowship HL08189 (to R. P. S.), by National Institutes of Health Grant HL42358 and Murdock Charitable Trust and Research Corporation Grant HS-185 (to C. A. M.), and by National Institutes of Health Grants RR0163 and P30HD18185 to the Oregon Regional Primate Research Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, OR 97201.

American Heart Association Established Investigator. To whom correspondence should be addressed. Tel.: 503-690-5509; Fax: 503-690-5384.

(^1)
The abbreviations used are: beta-AR, beta-adrenergic receptor; alpha-AR, alpha-adrenergic receptor; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
All numbers are relative to the first base of the initiation codon.


ACKNOWLEDGEMENTS

We thank Kirsten Pilcher and David Spackman for expert technical assistance; Drs. Michael Melner, Kenneth Low, Steven Kohama, Gregory Dissen, Eliot Spindel, and Srinivasa Nagalla for helpful comments and discussions; and Dr. Spindel for the use of the EG & G Berthold luminometer.


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