DEVELOPMENTAL GENETICS 12:431436 (1991)

Distinct Developmental Regulatory Mechanisms for Two Members of the Aldolase Gene Family ANN B. MAINE AND ELENA CIEJEK-BAEZ Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York the adult tissue [Schapira et al., 19751.This pattern of fetal isozyme expression reoccurs during hepatocarcinogenesis where some hepatomas have high levels of aldolase A and C and decreased levels of aldolase B [Schapira et al., 1963;Matsushima et al., 1968;Schapira, 19811.The cDNAs of aldolase A and B have been isolated from rat [Simon et al., 19831 and the aldolase B gene has been cloned from rat [Tsutsumi et al., 19851, human [Rottman et al., 19841,chicken [Burgess and Penhoet, 19851,and mouse [Maine et al., 19911.The availability of these clones has led to the finding that changes in aldolase protein levels during liver development [Numazaki et al., 19841 and carcinogenesis [Daimon et al., 19841 are due to changes in the steady state levels of mRNA. To undcrstand better aldolase mRNA expression, we have investigated its expression and regulation in the liver during normal gestation and in the adult liver and kidney. Here we show that although the aldolase genes encode functionally similar products, the regulation of expression of the A and B genes differs. Aldolase A expression in the liver is regulated a t a posttranscriptional level resulting in decreasing levels of aldolase A mRNA as liver development proceeds. HowKey words: Regulatory mechanisms, mRNA, alever, aldolase B expression is regulated at the trandolase, post-transcriptional control scriptional level and is induced during fetal liver development. In addition, there are tissue-specific controls of the genes in which a similar level of initiaINTRODUCTION tion of transcription of aldolase B in the kidney and The aldolase isozyme family is a n excellent system liver results in the kidney having %fold more steady for the study of tissue-specific factors involved in gene state mRNA, while the same level of initiation of tranexpression during differentiation and development. scription of aldolase A in the kidney and liver results in Fructose-l,6-bisphosphate aldolase (aldolase, EC the kidney having 40-fold more steady state aldolase A 4.1.2.13)is a ubiquitous glycolytic enzyme that cata- mRNA. lyzes the cleavage of fructose-1,6 bisphosphate. There are three aldolase isozymes, A, B, and C [Penhoet et al., 19661. These isozymes are expressed in a tissuerestricted manner. In the adult animal the A form is found in every tissue except the liver, while the B isozyme is found in the liver, kidney and the small intestine. The brain is the only adult tissue that contains aldolase C [Lebherz and Rutter, 19691.Many tissues change their expression of these isoforms in a Received for publication December 4, 1991; accepted December 20, 1991. characteristic manner during development. For example, during gestation, the fetal liver initially expresses Ann B. Maine is now a t the Department of Dental Research, Univerthe A and C forms, in addition to the B form, while the sity of Rochester School of Medicine and Dentistry, Rochester, NY expression of the A and C forms is low to negligible in 14642. Address reprint requests there. The aldolase isozyme family is ABSTRACT composed of three members, A, B, and C, which are encoded by separate genes. The proteins are expressed in a tissue-restricted manner during development and in the adult. To elucidate the regulation of aldolase mRNA in the mouse liver, we analyzed its expression by a number of methods including Northern blot, RNA dot blot, and nuclear run-on assays. Our experiments demonstrate that the expression of aldolase A in the liver is primarily regulated by post-transcriptional control. In contrast, we found that changes in the level of aldolase B mRNA are due to changes in the rate of initiation of transcription. In addition, we examined the regulation of aldolase expression in the adult kidney. We found that although the kidney has eight times more aldolase B than the liver, the rate of initiation of transcription is similar in both tissues. Also, the rate of initiation of transcription of aldolase A is the same in the adult kidney and liver although there is 40 times more steady state aldolase A mRNA in the kidney than in the liver. o 1992 WiIey-Liss, Inc.

0 1992 WILEY-LISS, INC.

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MATERIALS AND METHODS RNA Isolation Total RNA was isolated from the tissues of a n outbred strain of Swiss Webster mice from Charles River Laboratories, Crl: CFW (SWIBR at days 15,17,19, and 20 of gestation (vaginal plug considered a s day 1 in a 20-21 day gestation period). Newborn mice were less than 12 hours old. Adult animals were a t least 6 weeks old. The method used was a modification of Chirgwin et al.’s procedure [1979]. Briefly, fresh tissue or tissue frozen in liquid nitrogen was homogenized in a l o x volume (v/w) of 4 M guanidine isothyiocyanayte with a Virtis tissue homogenizer. The solution (25 ml) was layered onto a n 8 ml CsCl cushion (5.7 M CsC1/2 mM Na-EDTA/25 mM NaOAc pH 5.2) and centrifuged for 24 hours at 26,000 rpm in a n SW 27 rotor. The resulting pellet was resuspended in 7.5 M guanidine hydrochloride/5 mM Na-EDTN0.025 volume NaCit and precipitated with HAc.

Isolation of Nuclei Nuclei were prepared from fresh tissue by a combination of the methods of Tata and Baumbach [Tata, 1974; Baumbach et al., 19871 with the following modifications. The homogenization buffer contained 1 mM HEPES pH 6.8 and 0.1% Triton X-100. The tissue was homogenized in 4 volumes (v/w) of buffer with 4-5 strokes in a Kontes ground glass homogenizer. To decrease loss of nuclei, the homogenate was not filtered. The nuclei were pelleted a t 4°C by a 5 minute centrifugation at 800 rpm using a Sorval RCdB. The nuclei pellet was resuspended in 4 volumes of 2.1 M sucrose buffer (2.1 M sucrose/l mM MgC1,/1 mM HEPES) and layered onto a n equal volume of the same buffer and then centrifuged at 4°C for 1 hour at 30,000 rpm using a n SW60 rotor. The final nuclei pellet was resuspended in 0.1-0.2 volumes of cold resuspension buffer (25% glyceroli30 mM Tris-HC1, pH 7.8/50 mM NH,S0,/5 mM MgCl,/l mM MnC1,). The nuclei were either used immediately or stored for up to 3 days at -70°C.

Transcription of Isolated Nuclei Northern Blot Analysis of RNA Nuclei were quantified by counting samples in a heRNA (20 pg) was loaded in each lane and fractionated on a 1.3% agarose-formaldehyde gel [Lehrach et mocytometer. A typical reaction contained 16-25 x al., 19771. The RNA was transferred overnight to lo6 nuclei in 500 yl. Transcription reactions were perSchleicher & Schuell (S&S) nitrocellulose [Thomas, formed according to Flint et al. [1984] with the follow19801 and the filter was baked at 80°C for 2 hours. The ing modifications. The transcription buffer contained filters were prehybridized for a t least 8 hours at 45°C in 25% glcyeroli30 mM Tris-HC1 pH 7.9/50 mM Northern buffer which is 50% formamide/5x SSC/ (NH,),S0,/5 mM MgC1,/1 mM MnC1,/10 mM p-mer2.5 x Denhardt’s/0.2% sodium dodecyl sulfate (SDS)/ captoethanolil mM ATP/0.25 mM GTP/0.25 mM CTP 0.25 mg per ml yeast RNA (1x SSC is 150 mM NaCl, 15 and 30-40 pl (w3’P) UTP (3,000 Ciimmole, AmermM sodium citrate; 5 0 Denhardt’s ~ is 1% each of sham). The reaction was terminated after 20 minutes ficoll, bovine serum albumin, and polyvinylpyrroli- a t 25°C. To measure the efficiency of the hybridization reacdone). Hybridization was performed overnight at 45°C with radiolabeled cDNA inserts [Feinberg and Vo- tions the vector pBS+ was used as a positive control. gelstein, 19831 of p l l (mouse aldolase B) or pFL (mouse The plasmid was linearized with Xmn I. RNA tranaldolase A) at a final concentration of at least 1million scripts were produced with the manufacturer’s instruccounts per ml. The specific activity of the probes was tions and reagents (Stratagene). The bacteriophage T3 P) approximately lo8 cpm/pg DNA. The filters were RNA polymerase was used with 5 p1 of ( C X - ~ ~CTP washed 4 times for 5 minutes in 2 x SSC/O.l%SDS at (3,000 Ci/mmole). Unincorporated nucleotides were removed by etha25°C and then twice a t 45°C for 30 minutes in nol precipitation. The amount of labeled transcript was 0.1 x SSC/O.l% SDS. The filters were exposed to Kodak XAR film overnight with an intensifying screen at determined by trichloroacetic acid precipitation of 2 pl -70°C. The molecular weights were determined from samples. The precipitations were done in duplicate followed by filtration on nitrocellulose. The filters were migration of RNA standards on the same gel. dissolved in Ecolume scintillant fluid and counted in a liquid scintillation counter. RNA Dot Blots DNA Dot Blots for Nuclear Run-on Assays Total cellular RNA (30-40 yg) was denatured for 15 Genomic DNA samples from mouse liver were denaminutes a t 68°C in a solution of 37% formaldehyde and 6 x SSC (final volume 200 pl). The solution was cooled tured by a 10 minute incubation in a boiling water bath on ice and diluted with 1 5 SSC ~ to the desired con- and then chilled on ice for 10 minutes. DNA (5 pg) was centration. Samples of 150-200 yl were applied to ni- spotted onto S & S nitrocellulose, air dried and then trocellulose using a n S&Sdot blot manifold. Each well baked a t 80°C for 2 hours. Preliminary experiments was washed twice with 15 x SSC. The filter was dried determined that 5 yg of DNA is in excess under the and treated as a Northern blot for baking, prehybrid- hybridization conditions used. The dots were cut out ization, hybridization, and washing a s described above. and prehybridized for at least 1 hour at 68°C in 6~

ALDOLASE GENE EXPRESSION SSC/2 x Denhardt’s. The RNA transcripts (either from in vitro nuclear transcription or from the in vitro transcription of the plasmid DNA pBS+ were resuspended in water and denatured by a 10 minute incubation at 68°C. The solution was cooled on ice and then a known amount of precipitable counts (10,000 or 15,000) were added to the prehybridization solution containing the DNA dot blots. Hybridization was for 2-3 days at 68°C. After hybridization, the filters were washed with 2 x SSC for 2.5 hours a t 68°C. The filters were counted in 5 ml of Ecolume scintillation fluid. Each experiment was done in triplicate a t 2 different concentrations of labeled probe.

RESULTS Expression of Aldolase in the Adult Tissues We examined the level of aldolase mRNA expression in the adult mouse brain, kidney, and liver. As seen in Figure 1, aldolase A is expressed in the brain and kidney while aldolase B is expressed in both the kidney and liver. The A transcript is 1.55 kb long, indicating use of the housekeeping promoter [Stauffer et al., 19901. The existence of two aldolase B mRNAs of 1.6 and 2.0 kb is due to the presence of alternative polyadenylation signal sequences [Maine et al., 19911. RNA dot blot analyses (Fig. 2) show that there is 40 times more aldolase B mRNA than A in the liver. In the kidney, there is 8 times more aldolase B than A present. A comparison of the two tissues shows that the kidney has 40-fold more aldolase A and %fold more aldolase B than the liver.

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Distinct developmental regulatory mechanisms for two members of the aldolase gene family.

The aldolase isozyme family is composed of three members, A, B, and C, which are encoded by separate genes. The proteins are expressed in a tissue-res...
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