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Human brain glycogen phosphorylase: characterization of fetal cDNA and genomic sequences
Molecular Brain Research, 6 (1989) 177-185 Elsevier
177
BRESM 70157
Human brain glycogen phosphorylase" characterization of fetal cDNA and genomic sequences Richard P. Gelinas 2, Byron E. Froman 2, Fred McElroy 1, Robert C.
Zait 1
and
F r e d r i c A. G o r i n 1 Departments of 1Neurology and 2Genetics, University of California, Davis, Davis, CA 95616 (U.S.A.) (Accepted 30 May 1989)
Key words: Glycogenphosphorylase; Brain isozyme;Transcription; Human tissue
Glycogen phosphorylase (a-l,4-D-glucan:orthophosphate D-glucosyltransferase, EC 2.4.1.1) is the rate-determining enzyme catalyzing glycogen degradation. Human brain has been demonstrated previously to express genes of both the liver and muscle isozymes of glycogen phosphorylase. In this report, a human fetal brain cDNA and genomic DNA corresponding to the brain isozyme of glycogenphosphorylase were isolated and characterized. Transcripts corresponding to this isozyme are present in human adult and fetal brain, and at low levels in other human fetal tissues. The predicted C-terminal sequence of the protein encoded by this cDNA and gene differ from that encoded by a phosphorylase cDNA isolated from a human astrocytoma cell line.
INTRODUCTION Glycogen stores within the central nervous system provide the immediate source of energy during acute changes in regional metabolism 34. The hydrolysis of glycogen within the central nervous system is activated during such pathological conditions as ischemia 22, hypoxia 19, epilepsy 9, and spreading cortical depression 6. Additionally, glycogenolysis is rapidly activated by changes in K ÷ flux 13, adenosine 17, and several neurotransmitters 31 and neuropeptides TM. Recent positron emission tomography data indicate that, under certain normal physiological conditions, transient increases in regional brain metabolism preferentially utilize glycolysis and glycogen metabolism rather than oxidative metabolism 1°. Glycogen phosphorylase (EC 2.4.1.1) is the ratedetermining enzyme catalyzing glycogenolysis. Three forms of glycogen phosphorylase, muscle (M), liver (L), and brain (B), have been identified in mammalian tissues using immunologic and electrophoretic techniques 7'35. Previous work in this laboratory has determined that transcripts corresponding
to the M- and L-isozymes are present in human adult and fetal brain 11. We now report the isolation and sequence characterization of human fetal brain cDNA and human genomic D N A encoding the B-isozyme of glycogen phosphorylase. These sequences differ significantly at the 3" end from that of a glycogen phosphorylase c D N A isolated from a human astrocytoma cell line 26. MATERIALS AND METHODS
cDNA library screening Approximately 8 x 105 plaques from a 2gtll human fetal brain cDNA library 24 were screened by nucleic acid hybridization using a radiolabeled 8 rabbit M-phosphorylase cDNA fragment encoding amino acids 1-304 and approximately 100 bp of the 5" untranslated region 23. Hybridizations were performed at moderately low stringency (55 °C for 16 h in 6x SSC, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate, 200/~g/ml salmon sperm DNA, 25 mM sodium phosphate, pH 7.0). Denhardt's solution is 0.02% polyvinylpyrrolidone, 0.02% Ficoll,
Correspondence: F.A. Gorin, Section of Neurosciences, Surge III Room 1161, University of California, Davis, Davis, CA 95616, U.S.A. 0169-328X/89/$03.50 © 1989 Elsevier Science Putglishers B.V. (Biomedical Division)
178 0.02% bovine serum albumin, and I x SSC is 0.15 M NaCI, 0.015 M sodium citrate.
Genomic library construction and screening High molecular weight D N A was isolated from human placental tissue and partially digested with Sau3A. Genomic fragments of 15-22 kb were ligated to the BamHl site of Charon 3516 and the library was screened using a 662 bp Smal fragment of brain phosphorylase c D N A beginning at amino acid 841 and extending 660 nucleotides into the 3" untranslated region. Hybridizations were carried out at high stringency (42 °C for 16 h in 50% (v/v) deionized formamide, 3x SSC, 5x D e n h a r d r s solution, 200 ug/ml salmon sperm D N A , 50 mM sodium phosphate, pH 7.0). A 6 kb EcoRI fragment corresponding to a 3" portion of the B-phosphorylase gene was partially sequenced. DNA sequencing D N A fragments were mapped with restriction enzymes and cloned into M13 vectors for sequence determination by the chain termination method 33. Unidirectional deletions of restriction fragments were generated using exonuclease Ill 12. Both strands were sequenced for 85% of the D N A . In regions where the fetal brain c D N A sequences differed with the astrocytoma phosphorylase c D N A sequence2~'~ c D N A sequence results were verified by comparison with genomic nucleotide sequence data. Sequence analysis was performed using the B I O N E T electronic network. R N A preparation R N A was isolated from human and macaque tissue as previously described tl. Poly(A) + R N A was isolated by chromatography over an oligo(dT)cellulose column ~. R N A from skeletal and cardiac muscle tissue used for S1 analysis was prepared by the method of Seedorf 3°. R N A was quantitated by spectroscopy. Northern blot analysis R N A was size-fractionated by electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde 15 and transferred to a Nytran membrane (Schleicher and Schuell). A 336 bp BamH1 fragment (B14) of human B-phosphorylase encoding
amino acids 458-570 was radiolabeled ~ and hybridized at high stringency (genomic library, vide supra). This fragment's small size and 74% sequence identity with L- and M-phosphorylase permitted isozyme-specific hybridization at this stringency (see Results, Fig. 3). Sizes of R N A on the Northern blot were determined using a 1 kb D N A ladder (Bethesda Research Labs).
S1 nuclease analysis SI nuclease analysis was performed as described by Burke ~. A 218 bp SmaI/BamH1 fragment of B-phosphorylase encoding amino acids 497-570 and a 157 bp Pvull/SmaI fragment encoding amino acids 789-84(I were separately cloned into M13mpl8 and used to prepare radiolabeled single-stranded D N A . The single-stranded probe was hybridized with 2840 Bg of total R N A and subsequently digested with S1 nuclease. One-fifth of the sample was run on a denaturing polyacrylamide gel and autoradiography performed at room temperature using Kodak X A R 5 film. RESULTS
Brain phosphorylase cDNA A human fetal brain c D N A library was screened using a c D N A probe encoding the N-terminus of rabbit M-phosphorylase 2~. D N A sequence analysis
BEP i"
P I
B ,
BM ,,
B ,
P I
PM
I i
I
~ m
-~
I III
L ,a,,-,,~, m
S I
P PBM
A I
q
i m
500
'
PPM
'
~
P PBM
S
I
IIll •
m
•
Fig. 1. Human B-glycogen phosphorylase cDNA restriction map. The open reading frame (ORF) is indicated by a thick line and the sequenced regions are indicated by arrows. The C-terminal and 3" non-coding region of the gene are shown for comparison, with an intron of undetermined size indicated by an open box. Scale is indicated at left in base pairs. Restriction sites shown include BamHl, B: EcoRI, E: PstI. P; SmaI, M: and Sphl, S. The location of the poly-A tail on the cDNA is indicated by A. The 4 DNA fragments used in hybridization studies are indicated by Roman numerals I-IV.
179 -30 -20 -I0 CCTCCATCTCTTTTCCTCCGCCTCCGCCGGCGCG I 10 20 30 40 M G E P L T D S E K R K Q I S V R G L A G L G D V A E V R K S F N R H L H F T L V K D R N V A T AT•••cGAACCGCTGAcGGACAGCGAGAAGCGGAAGCAGATCAGcGTGCGC•GCcTGGcGGGGCTAGGCGACGTGGCCGAGGTGcGGAAGAGCT•cAA•cGGCACTTGcACTTCAcGcT•GTCAAGGACCGCAATGTG•CCAcG
144
50 60 70 80 90 P R D Y F F A L A H T V R D H L V G R W I R T Q Q H Y Y E R D P K R I Y Y L S L E F Y M G R T L CCCCGCGACTACTTCTTCGCGCTGGCGCACACG~TGCGCGACCACCTCGTGGGCC~CTGGATCCGCACGCAGCAGCACTACTACGA~CGCGACCCCAAGcGCATTTATTATCTTTCCCTGGAATTCTACATGGGTCGCACGCTG
288
100 110 120 130 140 Q N T M V N L G L Q N A C D E A I Y Q L G L D L E E L E E I E E D A G L G N G G L G R L A A C F CAGAACACGATGGTGAACCTGGGCCTTCAGAAT~CCTGCGATGAAGCCATCTATCAG~TGGGGTTAGACTT~GAGGAACTCGAGGAGA~AGAAGAAGATGCTGGcCTTGGGAATGGAG~CC~GGGGAGGCT~GCAGCGTGTTTC
432
150 160 170 180 190 L D S M A T L G L A A Y G Y G I R Y E F G I F N Q K I V N G W Q V E E A D D W L R Y G N P W E K CTTGACTCAATGGCTACCT•GGGCCTGGCAGcATACGGc•ATGGAA•CCGCTATGAATTTGGGATTTTTAACCAGAAGATTGTCAA•GG•TGGCAGGTAGAGGAGGCCGATGACTGGCTGC•CTACGGCAACCCCTGGGA•AAA
576
200 210 220 230 A R P E Y M L P V H F Y G R V E H T P D G V K W L D T Q V V L A M P Y D T P V P G Y K N N T V GCGCGGCCTGAGTATATGCTTCCcGTGCAC•TCTACGGACGCGTGGAGCACA•CCCCGAcGGcGTGAAGTGGCTGGACACACAGGTGGTGCTGGCCATGCCCTACGACACCCCAGTGCcCGGCTACAAGAACAACACCGTCAAC
720
N
240 250 260 270 280 T M R L W S A K A P N D F K L Q D F N V G D Y I E A V L D R N L A E N I S R V L Y P N D N F F E ACCATGCGGC•GTGGTCCGCCAAGG•TCCCAACGACTTCAAGCTGCAGGACTTCAAcGTGGGAGACTACATCGAGGCG•TCCTGGACCGGAACTTGGCTGAGAACATCTCCAGGGTCCTGTATCCAAATGATAACTTCTTTGAG
864
290 300 310 320 330 G K E L R L K Q E Y F V V A A T L Q D I I R R F K S S K F G C R D P V R T C F E T F P D K V A I GGGAAGGAGCTGCGGCTGAAGCAGGAG~ACTTCGTGGTGGCCGCCACGCTCCAG~ACATCATCCGcCGCTTCAAGTcGTCCAAGTTCGGCTGCCGGGACCCTG~GAGAACCTGTTTCGAGACGTTCCCAGACAAGGTGGCCATC
1008
340
350
360
370
380
Q L N D T H P A L S I P E L M R I L V D V E K V D W D K A W E I T K K T C A Y T N H T V L P E A CAGcTGAACGACAcCCACCCCGCCC~CTCCATCCCTGAGCTCATGCGGATCCTGG~GGACGTGGAGAAGG~GGACTG~GACAAG~CCTGGGAAATCAcGAAGAAGACCT~TGCATACACCAACCACACTGTGCTGCC]~GAGGCC
1152
390 400 410 420 430 L E R W P V S M F E K L L P R H L E I I Y A I N Q R H L D H V A A L F P G D V D R L R R M S V I TTGGAGCGCTGGCCC~TGTCCATGTTTGAGAAGCTGCTGCCGcG~CACCTGGAGATAATCTATGCCATCAACCAGCGGCACCTGGACCACG~GGCCGCGCT~TTCCCGGCGATGTGGACCGCCTGC~CAGGATGTCTGTGATC
1296
440 450 460 470 E E G D C K R I N M A H L C V I G S H A V N G V A R I H S £ I V K Q S V F K D F Y E L E P E K F GAGGA~G~GGACTGCAAGCGGATCAACATGGCCCACCTGTGTGTGATTGGG~CCCATGCTGTCAATGGTGTGGCGAGGATCCACTCGGAGATCG~GAAACA~TCGGTCTTTAAGGATTT~TATGAACTGGAGCCAGAGAAI~TTC
1440
480 490 500 510 520 Q N K T N G I T P R R W L L L C N P G L A D T I V E K I G E E F L T D L S Q L K K L L P L V S D CAGAATAA~ACCAATGGCATCACCCCCCGCCGGTGGCTGCTGC~GTGCAACCcGGGGCTGGCCGATACCATCGTGGAGAAAATTGGG~AGGAGTTCCTGAcTGACCTGAGCCAGCTGAAGAAGCTGCTGCCGC~GGTCAGTGAC
1584
530 540 550 560 570 E V F I R D V A K V K Q E N K L K F S A F L E K £ Y K V K I N P S S M F D V H V K R I H E Y K R GAGGTGTTCATCA~GACGTGGCCAAGGTCAAACAG~AGAACAA~CTCAAGTTCTCGGCCTTCCTGGAGAAGGA~TACAAGGTGAAGATCAACCcCTCCTCCATGTTCGATGTGCAT~TGAAGAGGATCCAcGAGTACAAGcGG
1728
580 590 600 610 620 Q L L N C L H V V T L Y N R I K R D P A K A F V P R T V M I G G K A A P G Y H M A K L I I K L V CAGCTGCTCAACTGCCTGCACG•CGTCACCCTGTAcAATCGAATCAAGAGAGACCCGGCCAA•GCTTTTGTGCCCAGGACTGTTAT•ATTGGG•GCAAG•CA•CGCCCGGTTACCACATGGCCAA•CTGATCATCAAGTTGGTC
1872
630 640 650 660 670 T S I G D V V N H D P V V G D R L K V I F L E N Y R V S L A E K V I P A A D L S Q Q I S T A G T ACCTCCATCGGCGACGTCGTCAATCATGACCCAGTTGTGGGTGACAGGTTGAAAGTGATCTTC CTGGAGAACTACCGTGTGTCCTTGGCTGAGAAAGTGATCCCGGCCGCTGATCTGTCGCAGCAGATCTCCACTGCAGGCACC
2016
680 690 700 710 E A S G T G N M K F M L N G A L T I G T M D G A N V E M A E E A G A E N L F I F G L R V E D V E GAGGCCTCAGGCACAGGCAACATGAAGTTCATGCTCAACGGGGCCCTCACCATCGGCACCATGGACGGCGCC AACGTGGAGATGGCCGAGGAGGCCGGGGCCGAGAACCTCTTCATCTTCGGCCTGCGGGTGGAGGATGTCGAG
2160
720 730 740 750 760 A L D R K G Y N A R E Y Y D H L P E L K Q A V D Q I S S G F F S P K E P D C F K D I V N M L M H GCCTTGGACCGGAAAGGGTACAATGCCAGGGAGTACTACGACCACCTGCCCGAGCTGAAGCAGGCCGTGGAC CAGATCAGCAGTGGCTTTTTTTCTC CCAAGGAGCCAGACTGCTTCAAGGACATCGTGAACATGCTGATGCAC
2304
770 780 790 800 H D R F K V F A D Y E A Y M Q C Q A Q V D Q L Y R N P K E W T K K V
CATGACAGGTTCAAGGTGTTTGCAGACTATGAAGCCTACATGCAGTGCCAGGCACAGGTGGACCAGCTGTAC
I
R N I
810 A C S G K F S S D R
CGGAACCCCAAGGAGTGGACCAAGAAGGTCATCAGGAACATCGCCTGCTCGGGCAAGTTCTCCAGTGACCGG
820 830 840 T I T E Y A R E I W G V E P S D L Q I P P P N I P R D * ACCATCACGGAGTATGCACGGGAGATCTGGGGTGTGGAGCCCTCCGACCTGCAGATCCCGCCCCCCAACATC CCCCGGGACTAGGCACACCCTGCCTTGGCGGGACCAGCGGGCATTTGTTTTCTTGCTGACTTTGCACCTCCT
2448
2592
TTTTTCCCCAAACACT~TGCCAGCCACTGGTGGTCCCTGCTTTTCTGAGTACCATGTTTCCAGGAGG~GCCATGGGGGTCAGGGTGGTTTTGAGAGAGCAGGGTAAG~AAGGAA~TGCTAGAA~TGCTCCTAGTTTCTTGTAA (*)
2736
AGGAAGCCAGAGTTGACAGTACAAAG•GTCG••GCCAGCCCTGCAGCTTCAGCACCTGCCccACCCAGAGTGGGAGTCAGGTGGAGCCAcCTGCTGGGCTCCCCCAGAACTTTGCACACATCTTGCTATGTATTAGCCGATGTC
2880
TTTAGTGTTGAGcCTCT•GATTCTG•GGTCTGGGCCAG•GGCCATAGTGAAGCCTGGGAATGAGTGTTACTGCAGCATCTGGGCTGCCAGCCACAGGGAAGG•CCAA•CCCCAT•TAGCcCCAGTCATCCTGCCCAGCCCT•CC
3024
TCCTGGCcATGCCGGGA•GGGTCGGATCCTC•AGGCATCGCCTTCACAGCCCCCTGCCCCCTGCCCTCTGTCCT•GCTCTGCACCTGGTATATGGGTCATGGACcAGATGGGGCTTTCCCTTTGTAGCCATCCAA•GGGCATTG
3168
•G•GGGTGCTTGGAACCCGGGA•GACTGAGGGGGA•ACTGGAG•GGGTGCTTGTGTCTGCTGTCTCAGAGGCC••GG•CAGGATGAAG•TG•CTG•CACAGCTTAGCT••G•T•TGCT•A•TCAAAAGAGAAAATAAC•ACACA
3312
TGGAAATGAAACTAGCTGAAGCCTTT•CTT•TTTTAGCAAC•GAAAAT•GTACTTGGTCACTTTTGTGCTT•AGGAG•CCCATTTTCTGCCTGGCAG•GGCA•GTCT•TGCcCTCCCGCTTGACTCCTGCTGT•TCCTGAGGTG
3456
CATTTCCTGTTT•TTACACACAA•GGcCAG•CTCCATTCTCCCTCCC••TCCACCAGTGCCACAGC•TCGTCTGGAAAAAGGACCAGGGGTCCCGGA•GAACCCATTTGT••TCTGCTTGGACA•CAGGCCTGG•ACTGGGAGG
3600
TG•GG•T•AGCCCCTCACAGCC•TGCCCCTcCCCAAG•CTCGAACCTGCCTCCCATT•CCCAAGAGAGAGG•CAGG•AACAGGCTACTGTCCTTCCCTGTG•AATTGCCGAGAAATCTAGCACCTT•CATGCTG•ATCT•GGCT
3744
GCGGGGAGG•TCTTTTTCTCCCTGGCCTCCAGTGCCCACCAGGAG•ATCTGCGCACGGTGCACAGCCCACCAGAGCAC•ACAGCCTTTTATTGAGTGGGGCAAGTGCTGGGCTGTGGTCGTGcCCTGAcAGCATCTTCCCCAGG
3888
CAGCGGCTCTGTGGAGGAGGCCATACTCCCCTAGTTGGC~ACTGGGG~CACCACCCTGACCACCACTGTGCCCCTcATT~TTACTGCCTTGTGAGATAAAAACT~ATTAAACCTTTGTGGCTGTGGTTGGCTGA~AAAAAAAAA
4176
Fig. 2. Nucleotide sequence of the Bophosphorylase c D N A . The nucleotide and deduced amino acid sequences are shown, with amino acids indicated by a one-letter code, with the termination codon for the fetal brain c D N A O R F indicated by an asterisk, *. The 280 bp region absent from the phosphorylase c D N A isolated from astrocytoma cell line U25126 is underlined. Deletion of the underlined region results in an extended phosphorylase O R E shown in Table I, with a termination codon indicated by a bracketed asterisk, [*]. The sequence of the B-phosphorylase gene was identical to that of the fetal brain c D N A from the termination point of an intron of undetermined size at amino acid 793 to the polyadenylation site.
180 TABLE I Coding region difJorenees between brain and U251 glycogen phosphoo, lase cDNA sequences
Nucleotide sequence position and amino acid residue numbering are according to Fig. 2. Nucleotide differences are indicated in boldface. Brain cDNA
U251 cDNA z°
Base no.
Nucleotide
Amino acid
Nucleotide
Amino acid
741 743 906 ll61
GCC AAG GCC CGC
A-246 K-247 A-301 R-386
GCA AGG GCG CGG
A-246 R-247 G-301 R-386
2503-2783 IPPPNIPRD*
of a clone isolated from this library (Figs.l and 2) revealed 78% and 74% nucleotide identity with muscle 4 and liver 27 c D N A s , respectively. The deoxyguanosine plus deoxycytosine ( G + C ) content of the coding region of this isozyme (58%) is more similar to that of muscle (57%) than to liver (49%,). This difference in codon usage is p r o n o u n c e d at the third position, where B-phosphorylase contains 84% G + C , M-phosphorylase 77%, G + C and L-phosphorylase only 60% G + C . Examination of the predicted amino acid sequence of the protein encoded by the fetal Bphosphorylase c D N A (Fig. 2) revealed 84% and 78% amino acid identity with the human muscle and liver isozymes, respectively. The protein sequence deduced from the fetal B-phosphorylase c D N A is 842 amino acids in length, while the muscle and liver proteins are 841 and 846 amino acids long, respectively. This variability in length is derived solely from the addition of amino acids at the C-terminus (Table I). Based on nucleotide sequence and predicted amino acid sequence comparisons, the B- and M-isozymes a p p e a r more closely related to each other than either is to the L-isozyme. This conclusion is supported by similarities of the M- and B-isozymes in allosteric response to A M P 7"14, and the ability of these two isozymes to form functional h e t e r o d i m e r s 2°. Comparison
o f B-phosphorylase
with the astrocy-
toma-derived c D N A
There are significant nucleotide differences be-
280 bp insertion/deletion Predicted protein difference is: LQH LPHPEWESGGATCWAPPELCTHLAMY*
tween the human B-phosphorylase c D N A in this report and the c D N A previously isolated from a human astrocytoma cell line, U25126. Most of the single base coding region discrepancies do not affect the predicted protein sequence (Table 1). The most prominent difference is an a p p a r e n t 280 bp deletion within the c D N A o b t a i n e d from astrocytoma cell line U251. This deletion affects amino acids 834-842 and a portion of the 3" untranslated region (Fig. 2). The shortened c D N A obtained from cell line U251
a
b
c
d
6.15.14.13.12.01 ,O--
Fig. 3. Northern blot analysis of B-phosphorylase transcripts. RNA blot containing poly(A)+ RNA purified from adult tissues was probed at high stringency using a 336 bp fragment of human fetal B-phosphorylase cDNA. Lane a, 5.0/~g human brain RNA, lane b, 3.5 ~g macaque heart RNA, lane c, 5.0 ~g human liver RNA, and lane d, ().7 ~tg human skeletal muscle RNA. Size in kb is indicated at the left.
181 encodes a predicted protein of 862 amino acids rather than the 842 amino acids obtained for the fetal B-phosphorylase c D N A .
3" Genomic B-phosphorylase sequence To verify that the discrepancies between the B-phosphorylase c D N A s in human fetal brain and an astrocytoma cell line were not the result of cloning artifacts in the fetal brain c D N A library, a portion of the genomic clone for human B-phosphorylase was isolated and sequenced. The genomic nucleotide sequence was identical to the c D N A in this report from amino acid 793 (the 3" border of an intron) through the polyadenylation site (Figs. 1 and 2). A n exact match of the fetal brain c D N A and genomic D N A sequences verified the fetal c D N A sequence, and indicated there were no additional introns 3" to amino acid 793. This organization of exons corresponds exactly to that present in the human M-phosphorylase gene 4, further emphasizing the close evolutionary distance between these isozymes. Southern blot analysis was performed to verify the presence of only one gene encoding B-phosphorylase. Chromosomal D N A purified from a single human placenta was digested separately with 6 restriction enzymes, then 10/~g samples were subjected to electrophoresis on an 0.8% agarose gel and transferred to nylon membrane. Duplicate blots were prepared and independently hybridized with either the 280 bp Pstl fragment present only in the fetal brain c D N A (fragment I, Fig. 1), or with the adjacent 173 bp PstI fragment c o m m o n to both the fetal brain and astrocytoma c D N A sequences (fragment II, Fig. 1). All bands detected with the probe c o m m o n to both c D N A sequences (fragment II) were also detected with the probe unique to the fetal brain c D N A (fragment I) (data not shown). These results suggest that only one gene encoding Bphosphorylase is present in human tissues.
Tissue-specific expression of B-phosphorylase The level of B-phosphorylase transcript in different tissues was characterized by Northern blot analysis using poly(A) ÷ R N A from human adult brain, liver, and muscle, and from macaque adult heart tissues (Fig. 3). The B-isozyme-specific c D N A probe, B14, identified 4.2 kb transcripts in human adult brain and adult macaque heart.
To examine low levels of B-phosphorylase transcripts in human fetal tissues, we used the more sensitive technique of solution hybridization followed by S1 nuclease digestion 3. A n antisense, 218 bp BamHI/SmaI fragment of B-phosphorylase c D N A was used as the single stranded probe (fragment III, Fig. 1). As shown in Fig. 4, a 218 base
a bcde
f gh
290 -
218-
Fig. 4, S1 nuclease analysis of B-phosphorylase transcripts. A radiolabeled 290 base single-stranded probe, of which 218 bases was complementary to B-phosphorylase cDNA (fragment Ill, Fig. 1), was annealed to RNA from different sources, then subjected to S1 nuclease digestion. The S1resistant fragments were examined by electrophoresis on a 6% polyacrylamide sequencing gel. The 290 base probe in the absence, lane a, and presence, lane b, of S1 nuclease treatment. RNA was from: lane c, human adult brain; lane d, human adult liver; lane e, macaque adult heart; lane f, human fetal brain; lane g, human fetal liver; and lane h, human fetal muscle. Lanes a-f were exposed for 24 h, and lanes g and h for 72 h. The chain termination sequencing reaction at the left was used for size determination.
182 protected fragment was detected in human adult brain and macaque adult heart. Transcripts were also detected in human fetal brain and in much lower amounts in human fetal liver and muscle. Transcripts were not detected in human adult liver (Fig. 4d) or muscle (Fig. 5f), even with 10-fold longer exposure times (data not shown). Thus, both Northern blots and S1 nuclease ex-
a b c de f
periments demonstrate that B-phosphorylase transcripts are present in human adult brain and adult macaque heart, but not human adult liver or muscle. This tissue specific pattern of expression is consistent with the protein data of Proux and Dreyfus 2s, but differs from RNA data of Fletterick and cow o r k e r s 25,26.
The 4.2 kb apparent size of the brain transcript is considerably larger than the 3.2-3.4 kb transcript sizes corresponding to L- and M-phosphorylases n'27. The larger size is, in part, the result of the 1.5 kb long 3" untranslated region found in the brain phosphorylase transcript, in contrast, human M- and L-phosphorylase transcript 3" untranslated sequences are only 263 and 170 bp long, respectively.
Alternative processing of B-phosphorylase in human tissues The B-phosphorylase m R N A in the astrocytoma cell line U251 was found to be smaller than the B-phosphorylase m R N A in control tissues 26. Comparison of the nucleotide sequence of the fetal B-phosphorylase cDNA with that reported for the astrocytoma cDNA raised the possibility that alternative processing 2 of the B-phosphorylase gene in the astrocytoma gave rise to the deletion in the U251 cDNA. As shown in Fig. 2, the cDNA and genomic sequences at the borders of the region deleted in the cell line cDNA contain the sequences C T G C A G / ATC and CTGCAG/CT, which resemble a consensus splice site 2~ with two notable exceptions: (1) the
Fig. 5. 3" structural determination of B-phosphorylase transcripts. A 263 base radiolabeled single-stranded D N A fragment with 157 bases identical to the fetal B-phosphorylase cDNA, but only 132 bases identical to the astrocytoma phosphorylase cDNA 26 (fragment IV, Fig. l), was annealed with RNA, then subjected to digestion with S1 nuclease. Protected fragments were examined by electrophoresis on a 6% polyacrylamide sequencing gel. A protected fragment of 157 bases indicates perfect homology with the fetal brain cDNA and the absence of processing at the putative splice site, while a fragment of 132 bases indicates homology with the astrocytoma cDNA and suggests the presence of a processed transcript. Lane a shows the fragments present in the absence of S1 nuclease, and lanes b - f show the fragments generated following SI nuclease treatment. The D N A fragment was annealed with R N A purified from: lane b, no RNA; lane c, human fetal brain; lane d, human adult brain (grade IV astrocytoma); lane e, human adult liver; and lane f, human adult muscle. The chain termination sequencing reaction at the left was used for size determination.
183 'invariant' G of the 5" donor is an A and (2) the dinucleotide AG is found at position -9 of the 3" splice site. Although this dinucleotide has not been previously reported in the -5 to -15 region, it is not known if its presence prohibits proper splicing. S1 nuclease protection studies were used to examine human tissues for alternative processing involving this region of the cDNA. A single-stranded DNA probe containing a region common to both B-phosphorylase cDNAs and the region present only in the fetal B-phosphorylase cDNA and gene (fragment IV, Fig. 1) was annealed to mRNA from various human tissues prior to S1 nuclease digestion. Recognition of and processing at the potential splice site should result in protection of a 132 base fragment, while absence of splicing at this site should generate a 157 base protected fragment. The 157 base protected fragment was observed with RNA from human fetal brain and a naturally occurring grade IV human astrocytoma (Fig. 5, lanes c and d). No evidence of alternative processing corresponding to the truncated transcript reported in the human astrocytoma cell line U251 was detected in any adult human tissue (Fig. 5, additional data not shown). DISCUSSION In this report we describe the cloning, sequencing, and transcript levels of human B-phosphorylase genomic DNA and cDNA. The results presented demonstrate that this gene is expressed in adult and fetal brain and heart, and in low levels in other human fetal tissues. Previous work has demonstrated that transcripts of M- and L-phosphorylase are present in human adult brain, and that Lphosphorylase mRNA predominates in human fetal brain and other human fetal tissues 11. In contrast to previous reports concerning the expression of Bphosphorylase 25"26, the tissue-specific expression of B-phosphorylase transcripts presented here is in good agreement with the protein data of Proux and Dreyfus 2s. It has recently been proposed that conformational changes of amino acids 839-841 play a significant role in the transition of a dephosphorylated, catalytically inactive T state to the phosphorylated, active R state 32. These C-terminal residues are ordered in M-phosphorylase b and participate in
intersubunit hydrogen bonding, but become disordered with covalent phosphorylation of the Nterminus 32. The C-terminal amino acid sequence of B-phosphorylase predicted from the fetal brain cDNA and human genomic DNA favors the maintenance of a structurally disordered C-terminus, with 4 chain-breaking prolines at residues 835,836, 837, and 840. In the M-isozyme, aspartate-838 forms an intersubunit ion-pair bond that further stabilizes the ordered C-terminus found in phosphorylase b. The fetal brain cDNA B-phosphorylase, like L-phosphorylase, contains an asparagine at residue 838, which would be unable to participate in this intersubunit interaction. This should contribute further to disordering of the C-terminus. Thus, one would predict that the disordered C-terminus of B-phosphorylase should favor an activated, R state enzyme 7. The cDNA and gene sequences for human Bphosphorylase presented here differ from the sequence of a cDNA obtained from a human astrocytoma cell line 26. Overall, the sequences are nearly identical, except for a 280 bp deletion present in the astrocytoma-derived cDNA. The deletion in the astrocytoma-derived cDNA significantly alters and extends the predicted C-terminus of the protein by 20 amino acids, beginning at amino acid 834 (Table I). The C-terminal differences in the protein sequences predicted from these two cDNAs may be significant regarding the function of B-phosphorylase. The predicted B-phosphorylase sequence reported for the cDNA isolated from astrocytoma cell line U251 differs significantly from the other reported phosphorylase isozymes beginning at residue 835, and it is difficult a priori to predict its effect on R---~T state transitions. The significance of the variant characterized by Newgard et al. 26 remains uncertain. The B-phosphorylase transcript detected in the U251 astrocytoma cell line was smaller than the transcript observed in normal tissues 26, suggesting that the deletion present in the cDNA is representative of the phosphorylase transcripts present in the astrocytoma cell line, and is not merely a cloning or sequencing artifact. If the smaller transcript is a result of a chromosomal deletion, then cell line U251 must be homozygous for this deletion, since only a single transcript size was detected. A deletion arising on
184 one c h r o m o s o m e could have become homozygous as a result of aneuploidy, for example, perhaps arising from non-disjunction 5. Alternatively, the astrocytoma cell line might recognize a splice site and excise the 280 bp region from the m R N A . As previously discussed, a nearconsensus splice signal is present at the site of the deletion, suggesting the potential for alternative splicing. A l t h o u g h S1 nuclease analysis failed to detect alternative processing involving this site in normal human tissues and in a naturally occurring grade IV astrocytoma, this alternative processing might be present in specific transformed cell lines. In this regard, it is interesting that Sato et al., using electrophoretic and immunologic techniques, have identified a novel 'fetal-type' phosphorylase isozyme in specific rat ascites h e p a t o m a cell lines 29. Further analysis of the phosphorylase genes in cell line U251 will be necessary to ascertain the potential biological significance of the U251 phosphorylase c D N A variant. The identification of the c D N A for human brain REFERENCES 1 Aviv, H. and Leder, P., Purification of biologically active globin messenger RNA by chromatography or oligothymidylic acid-cellulose, Proc. Natl. Acad. Sci. U.S.A., 69 11972) 1408-1412. 2 Breitbart, R.E., Andreadis, A. and Nadal-Ginard, B., Alternative splicing: a ubiquitous mechanism for the generation of multiple isoforms from single genes, Annu. Rev. Biochem., 56 (1987) 467-495. 3 Burke, J.E, High-sensitivity SI mapping with singlestranded [32p]DNA probes synthesized from bacteriophage M13mp templates, Gene, 30 (1984) 63-68. 4 Burke, J., Hwang, P., Anderson, L., Lebo, R., Gorin, E and Fletterick, R., Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase, Proteins Struct. Funct. Genet., 2 (1987) 177-187, 5 Cavenee, W.K., Dryja, T.P., Phillips, R.A., Benedict, W.E, Godbout, R., Gallie, B.L., Murphee, A.L., Strong, L.C. and White, R.L., Expression of recessive alleles by chromosomal mechanisms in retinoblastoma, Nature (Lond.), 305 (1983) 779-784. 6 Csiba, L., Paschen, W. and Mies, G., Regional changes in tissue pH and glucose content during cortical spreading depression in rat brain, Brain Res., 336 (1985) 167-170. 7 Davis, C.H., Schliselfeld, L.H., Wolf, D.P., Leavitt, C.A. and Krebs, E.G., Interrelationships among glycogen phosphorylase isozymes, J. Biol. Chem., 242 (1967) 4824-4833. 8 Feinberg, A.P. and Vogelstein. B., A technique for radiolabeling DNA restriction endonuclease fragments, Anal. Biochem., 132 11983) 6-13. 9 Ferendelli, J. and McDougal, D., The effects of audiogenic seizures on regional energy reserves, glycolyis and citric
phosphorylase enables future characterization of the structural and biochemical p r o p e r t i e s of this enzyme. We have p r e p a r e d antibodies against the C-terminal sequence predicted by the fetal B-phosphorylase c D N A , and are currently cytologically localizing expression of this isozyme of glycogen phosphorylase within the central nervous system.
ACKNOWLEDGEMENTS We would like to thank Dr. R o b e r t Fletterick's lab for the rabbit muscle phosphorylase c D N A probe. We thank Dr. R. Teplitz, Ms. M. Pierce and the U C D M C Pathology D e p a r t m e n t Tissue Bank for providing human tissues. This work was supp o r t e d in part by grants from the California Affiliate of the A m e r i c a n H e a r t Association, the U.S. Public Health Service, and the U.S. D e p a r t m e n t of Education ( U S D E / N I D R R H133 B80016). R . G . was the recipient of an N I H training fellowship and a University of California graduate research award. acid cycle flux, J. Neurochem., 17 (1971) 1207-1220. 10 Fox, P.T., Raichle, M.E., Mintun, M.A. and Dence, C., Nonoxidative glucose consumption during focal physiologic neural activity, Science, 241 11988) 462-464. 11 Gorin, EA., Mullinax, R.L., Ignacio, P.C., Neve, R.L. and Kurnit, D.M., McArdle's and Hers' diseases: glycogen phosphorylase transcriptional expression in human tissues, J. Neurogenet., 4 (1987) 293-308. 12 Henikoff~ S.. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, 28 (1984) 351-359. 13 Hof, R., Pascale, E. and Magistretti, J., K + at concentrations reached in the extracellular space during neuronal activity promotes a Ca + dependent glycogen hydrolysis in the mouse cerebral cortex, J. Neurosci., 8 11988) 19221928. 14 Kamogawa, A. and Fukui, T., Purification and properties of glycogen phosphorylase from bovine spleen, Biochim. Biophys. Acta, 242 (1971) 55-58. 15 Lehrach, H., Diamond, D., Wozney, J.M. and Boedtke, H., RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 16 (1977) 4743-4751. 16 Loenen, W.A.M. and Blanner, ER., Lambda Charon vectors (Ch32,33,34 and 35) adapted for DNA cloning in recombinant-deficient hosts, Gene, 26 (1983) 171-179. 17 Magistreni, P.J., Hof, P.R. and Martin, J.-L., Adenosine stimulates glycogenolysis in mouse cerebral cortex: a possible coupling mechanism between neuronal activity and energy metabolism, J. Neurosci., 6 (1986) 2558-2562. 18 Magistretti, P.J.. Morrison, J.H., Shoemaker, W.J., Sapin, V. and Bloom, EE., Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices. A possible
185
19
20
21 22
23
24
25
26
regulatory mechanism for the local control of energy metabolism, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6535-6539. McCandless, D.W., Feussner, G.K., Lust, W.D. and Passoneau, J.V., Metabolite levels in brain following experimental seizures: the effects of maximal electroshock and phenytoin in cerebellar layer, J. Neurochem., 32 {1979) 743-753. Miranda, A.F., Nette, E.G., Hartlage, P.L. and Di Mauro, S., Phosphorylase isoenzymes in normal heart and myophosphorylase deficient human heart, Neurology, 29 (1979) 1538-1541. Mount, S.M., A catalogue of splice junction sequences, Nucleic Acids Res., 10 (1982) 459-472. Mrsulja, B.B., Lust, W.D., Mrsulja, B.J., Passonneau, J.V. and Klatzo, I., Brain glycogen following experimental cerebral ischemia in gerbils (Meriones unguiculatus), Experientia, 32 (1976) 732-733. Nakano, K., Hwang, P.K. and Fletterick, R.J., Complete cDNA sequence for rabbit muscle glycogen phosphorylase, FEBS Lett., 204 (1986) 283-287. Neve, R.L., Harris, P., Kosik, K.S., Kurnit, D.M. and Donlon, T.A., Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubuleassociated protein 2, Mol. Brain Res., 1 (1986) 271-280. Newgard, C.B., Hwang, P.K. and Fletterick, R.J., The family of glycogen phosphorylases: structure & function, CRC Crit. Rev. Biochem. Mol. Biol., 24(1) (1989) 69-99. Newgard, C.B., Littman, D.R., van Genderen, C., Smith, M. and Fletterick, R.J., Human brain glycogen phosphory-
lase, J. Biol. Chem., 263 (1988) 3850-3857. 27 Newgard, C.B., Nakano, K., Hwang, P.K. and Fletterick, R.J., Sequence analysis of the cDNA encoding human liver glycogen phosphorylase reveals tissue-specific codon-usage, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8132-8136. 28 Proux, D. and Dreyfus, J.-C., Phosphorylase isoenzymes in tissues: prevalence of the liver type in man, Clin. Chim. Acta, 48 (1973) 167-172. 29 Sato, K., Satoh, K., Sato, T., Imai, F. and Morris, H.P., Isozyme patterns of glycogen phosphorylase in rat tissues and transplantable hepatomas, Cancer Res., 36 (1976) 487-495. 30 Seedorf, U., Leberer, E., Kirschbaum, B.J. and Pette, D., Neural control of gene expression in skeletal muscle, Biochem. J., 239 (1986) 115-120. 31 Siesjo, B.K., Brain Energy Metabolism, Wiley, New York, 1978, and references within. 32 Sprang, S.R., Acharya, K.R., Goldsmith, E.J., Stuart, D.I., Varvill, K., Fletterick, R.J., Madsen, N.B. and Johnson, L.N., Structural changes in glycogen phosphorylase induced by phosphorylation, Nature (Lond.), 336 (1988) 215-221. 33 Tabor, S. and Richardson, C.C., DNA sequence analysis with a modified bacteriophage T7 DNA polymerase, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 4767-4771. 34 Watanabe, H. and Passonneau, J.V., Factors affecting the turnover of cerebral glycogen and limit dextrin in vivo, J. Neurochem., 20 (1973) 1543-1554. 35 Yonezawa, S. and Hori, S.H., Electrophoretic studies on the phosphorylase isozymes, J. Histochem. Cytochem., 23 (1975) 745-751.
177
BRESM 70157
Human brain glycogen phosphorylase" characterization of fetal cDNA and genomic sequences Richard P. Gelinas 2, Byron E. Froman 2, Fred McElroy 1, Robert C.
Zait 1
and
F r e d r i c A. G o r i n 1 Departments of 1Neurology and 2Genetics, University of California, Davis, Davis, CA 95616 (U.S.A.) (Accepted 30 May 1989)
Key words: Glycogenphosphorylase; Brain isozyme;Transcription; Human tissue
Glycogen phosphorylase (a-l,4-D-glucan:orthophosphate D-glucosyltransferase, EC 2.4.1.1) is the rate-determining enzyme catalyzing glycogen degradation. Human brain has been demonstrated previously to express genes of both the liver and muscle isozymes of glycogen phosphorylase. In this report, a human fetal brain cDNA and genomic DNA corresponding to the brain isozyme of glycogenphosphorylase were isolated and characterized. Transcripts corresponding to this isozyme are present in human adult and fetal brain, and at low levels in other human fetal tissues. The predicted C-terminal sequence of the protein encoded by this cDNA and gene differ from that encoded by a phosphorylase cDNA isolated from a human astrocytoma cell line.
INTRODUCTION Glycogen stores within the central nervous system provide the immediate source of energy during acute changes in regional metabolism 34. The hydrolysis of glycogen within the central nervous system is activated during such pathological conditions as ischemia 22, hypoxia 19, epilepsy 9, and spreading cortical depression 6. Additionally, glycogenolysis is rapidly activated by changes in K ÷ flux 13, adenosine 17, and several neurotransmitters 31 and neuropeptides TM. Recent positron emission tomography data indicate that, under certain normal physiological conditions, transient increases in regional brain metabolism preferentially utilize glycolysis and glycogen metabolism rather than oxidative metabolism 1°. Glycogen phosphorylase (EC 2.4.1.1) is the ratedetermining enzyme catalyzing glycogenolysis. Three forms of glycogen phosphorylase, muscle (M), liver (L), and brain (B), have been identified in mammalian tissues using immunologic and electrophoretic techniques 7'35. Previous work in this laboratory has determined that transcripts corresponding
to the M- and L-isozymes are present in human adult and fetal brain 11. We now report the isolation and sequence characterization of human fetal brain cDNA and human genomic D N A encoding the B-isozyme of glycogen phosphorylase. These sequences differ significantly at the 3" end from that of a glycogen phosphorylase c D N A isolated from a human astrocytoma cell line 26. MATERIALS AND METHODS
cDNA library screening Approximately 8 x 105 plaques from a 2gtll human fetal brain cDNA library 24 were screened by nucleic acid hybridization using a radiolabeled 8 rabbit M-phosphorylase cDNA fragment encoding amino acids 1-304 and approximately 100 bp of the 5" untranslated region 23. Hybridizations were performed at moderately low stringency (55 °C for 16 h in 6x SSC, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate, 200/~g/ml salmon sperm DNA, 25 mM sodium phosphate, pH 7.0). Denhardt's solution is 0.02% polyvinylpyrrolidone, 0.02% Ficoll,
Correspondence: F.A. Gorin, Section of Neurosciences, Surge III Room 1161, University of California, Davis, Davis, CA 95616, U.S.A. 0169-328X/89/$03.50 © 1989 Elsevier Science Putglishers B.V. (Biomedical Division)
178 0.02% bovine serum albumin, and I x SSC is 0.15 M NaCI, 0.015 M sodium citrate.
Genomic library construction and screening High molecular weight D N A was isolated from human placental tissue and partially digested with Sau3A. Genomic fragments of 15-22 kb were ligated to the BamHl site of Charon 3516 and the library was screened using a 662 bp Smal fragment of brain phosphorylase c D N A beginning at amino acid 841 and extending 660 nucleotides into the 3" untranslated region. Hybridizations were carried out at high stringency (42 °C for 16 h in 50% (v/v) deionized formamide, 3x SSC, 5x D e n h a r d r s solution, 200 ug/ml salmon sperm D N A , 50 mM sodium phosphate, pH 7.0). A 6 kb EcoRI fragment corresponding to a 3" portion of the B-phosphorylase gene was partially sequenced. DNA sequencing D N A fragments were mapped with restriction enzymes and cloned into M13 vectors for sequence determination by the chain termination method 33. Unidirectional deletions of restriction fragments were generated using exonuclease Ill 12. Both strands were sequenced for 85% of the D N A . In regions where the fetal brain c D N A sequences differed with the astrocytoma phosphorylase c D N A sequence2~'~ c D N A sequence results were verified by comparison with genomic nucleotide sequence data. Sequence analysis was performed using the B I O N E T electronic network. R N A preparation R N A was isolated from human and macaque tissue as previously described tl. Poly(A) + R N A was isolated by chromatography over an oligo(dT)cellulose column ~. R N A from skeletal and cardiac muscle tissue used for S1 analysis was prepared by the method of Seedorf 3°. R N A was quantitated by spectroscopy. Northern blot analysis R N A was size-fractionated by electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde 15 and transferred to a Nytran membrane (Schleicher and Schuell). A 336 bp BamH1 fragment (B14) of human B-phosphorylase encoding
amino acids 458-570 was radiolabeled ~ and hybridized at high stringency (genomic library, vide supra). This fragment's small size and 74% sequence identity with L- and M-phosphorylase permitted isozyme-specific hybridization at this stringency (see Results, Fig. 3). Sizes of R N A on the Northern blot were determined using a 1 kb D N A ladder (Bethesda Research Labs).
S1 nuclease analysis SI nuclease analysis was performed as described by Burke ~. A 218 bp SmaI/BamH1 fragment of B-phosphorylase encoding amino acids 497-570 and a 157 bp Pvull/SmaI fragment encoding amino acids 789-84(I were separately cloned into M13mpl8 and used to prepare radiolabeled single-stranded D N A . The single-stranded probe was hybridized with 2840 Bg of total R N A and subsequently digested with S1 nuclease. One-fifth of the sample was run on a denaturing polyacrylamide gel and autoradiography performed at room temperature using Kodak X A R 5 film. RESULTS
Brain phosphorylase cDNA A human fetal brain c D N A library was screened using a c D N A probe encoding the N-terminus of rabbit M-phosphorylase 2~. D N A sequence analysis
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Fig. 1. Human B-glycogen phosphorylase cDNA restriction map. The open reading frame (ORF) is indicated by a thick line and the sequenced regions are indicated by arrows. The C-terminal and 3" non-coding region of the gene are shown for comparison, with an intron of undetermined size indicated by an open box. Scale is indicated at left in base pairs. Restriction sites shown include BamHl, B: EcoRI, E: PstI. P; SmaI, M: and Sphl, S. The location of the poly-A tail on the cDNA is indicated by A. The 4 DNA fragments used in hybridization studies are indicated by Roman numerals I-IV.
179 -30 -20 -I0 CCTCCATCTCTTTTCCTCCGCCTCCGCCGGCGCG I 10 20 30 40 M G E P L T D S E K R K Q I S V R G L A G L G D V A E V R K S F N R H L H F T L V K D R N V A T AT•••cGAACCGCTGAcGGACAGCGAGAAGCGGAAGCAGATCAGcGTGCGC•GCcTGGcGGGGCTAGGCGACGTGGCCGAGGTGcGGAAGAGCT•cAA•cGGCACTTGcACTTCAcGcT•GTCAAGGACCGCAATGTG•CCAcG
144
50 60 70 80 90 P R D Y F F A L A H T V R D H L V G R W I R T Q Q H Y Y E R D P K R I Y Y L S L E F Y M G R T L CCCCGCGACTACTTCTTCGCGCTGGCGCACACG~TGCGCGACCACCTCGTGGGCC~CTGGATCCGCACGCAGCAGCACTACTACGA~CGCGACCCCAAGcGCATTTATTATCTTTCCCTGGAATTCTACATGGGTCGCACGCTG
288
100 110 120 130 140 Q N T M V N L G L Q N A C D E A I Y Q L G L D L E E L E E I E E D A G L G N G G L G R L A A C F CAGAACACGATGGTGAACCTGGGCCTTCAGAAT~CCTGCGATGAAGCCATCTATCAG~TGGGGTTAGACTT~GAGGAACTCGAGGAGA~AGAAGAAGATGCTGGcCTTGGGAATGGAG~CC~GGGGAGGCT~GCAGCGTGTTTC
432
150 160 170 180 190 L D S M A T L G L A A Y G Y G I R Y E F G I F N Q K I V N G W Q V E E A D D W L R Y G N P W E K CTTGACTCAATGGCTACCT•GGGCCTGGCAGcATACGGc•ATGGAA•CCGCTATGAATTTGGGATTTTTAACCAGAAGATTGTCAA•GG•TGGCAGGTAGAGGAGGCCGATGACTGGCTGC•CTACGGCAACCCCTGGGA•AAA
576
200 210 220 230 A R P E Y M L P V H F Y G R V E H T P D G V K W L D T Q V V L A M P Y D T P V P G Y K N N T V GCGCGGCCTGAGTATATGCTTCCcGTGCAC•TCTACGGACGCGTGGAGCACA•CCCCGAcGGcGTGAAGTGGCTGGACACACAGGTGGTGCTGGCCATGCCCTACGACACCCCAGTGCcCGGCTACAAGAACAACACCGTCAAC
720
N
240 250 260 270 280 T M R L W S A K A P N D F K L Q D F N V G D Y I E A V L D R N L A E N I S R V L Y P N D N F F E ACCATGCGGC•GTGGTCCGCCAAGG•TCCCAACGACTTCAAGCTGCAGGACTTCAAcGTGGGAGACTACATCGAGGCG•TCCTGGACCGGAACTTGGCTGAGAACATCTCCAGGGTCCTGTATCCAAATGATAACTTCTTTGAG
864
290 300 310 320 330 G K E L R L K Q E Y F V V A A T L Q D I I R R F K S S K F G C R D P V R T C F E T F P D K V A I GGGAAGGAGCTGCGGCTGAAGCAGGAG~ACTTCGTGGTGGCCGCCACGCTCCAG~ACATCATCCGcCGCTTCAAGTcGTCCAAGTTCGGCTGCCGGGACCCTG~GAGAACCTGTTTCGAGACGTTCCCAGACAAGGTGGCCATC
1008
340
350
360
370
380
Q L N D T H P A L S I P E L M R I L V D V E K V D W D K A W E I T K K T C A Y T N H T V L P E A CAGcTGAACGACAcCCACCCCGCCC~CTCCATCCCTGAGCTCATGCGGATCCTGG~GGACGTGGAGAAGG~GGACTG~GACAAG~CCTGGGAAATCAcGAAGAAGACCT~TGCATACACCAACCACACTGTGCTGCC]~GAGGCC
1152
390 400 410 420 430 L E R W P V S M F E K L L P R H L E I I Y A I N Q R H L D H V A A L F P G D V D R L R R M S V I TTGGAGCGCTGGCCC~TGTCCATGTTTGAGAAGCTGCTGCCGcG~CACCTGGAGATAATCTATGCCATCAACCAGCGGCACCTGGACCACG~GGCCGCGCT~TTCCCGGCGATGTGGACCGCCTGC~CAGGATGTCTGTGATC
1296
440 450 460 470 E E G D C K R I N M A H L C V I G S H A V N G V A R I H S £ I V K Q S V F K D F Y E L E P E K F GAGGA~G~GGACTGCAAGCGGATCAACATGGCCCACCTGTGTGTGATTGGG~CCCATGCTGTCAATGGTGTGGCGAGGATCCACTCGGAGATCG~GAAACA~TCGGTCTTTAAGGATTT~TATGAACTGGAGCCAGAGAAI~TTC
1440
480 490 500 510 520 Q N K T N G I T P R R W L L L C N P G L A D T I V E K I G E E F L T D L S Q L K K L L P L V S D CAGAATAA~ACCAATGGCATCACCCCCCGCCGGTGGCTGCTGC~GTGCAACCcGGGGCTGGCCGATACCATCGTGGAGAAAATTGGG~AGGAGTTCCTGAcTGACCTGAGCCAGCTGAAGAAGCTGCTGCCGC~GGTCAGTGAC
1584
530 540 550 560 570 E V F I R D V A K V K Q E N K L K F S A F L E K £ Y K V K I N P S S M F D V H V K R I H E Y K R GAGGTGTTCATCA~GACGTGGCCAAGGTCAAACAG~AGAACAA~CTCAAGTTCTCGGCCTTCCTGGAGAAGGA~TACAAGGTGAAGATCAACCcCTCCTCCATGTTCGATGTGCAT~TGAAGAGGATCCAcGAGTACAAGcGG
1728
580 590 600 610 620 Q L L N C L H V V T L Y N R I K R D P A K A F V P R T V M I G G K A A P G Y H M A K L I I K L V CAGCTGCTCAACTGCCTGCACG•CGTCACCCTGTAcAATCGAATCAAGAGAGACCCGGCCAA•GCTTTTGTGCCCAGGACTGTTAT•ATTGGG•GCAAG•CA•CGCCCGGTTACCACATGGCCAA•CTGATCATCAAGTTGGTC
1872
630 640 650 660 670 T S I G D V V N H D P V V G D R L K V I F L E N Y R V S L A E K V I P A A D L S Q Q I S T A G T ACCTCCATCGGCGACGTCGTCAATCATGACCCAGTTGTGGGTGACAGGTTGAAAGTGATCTTC CTGGAGAACTACCGTGTGTCCTTGGCTGAGAAAGTGATCCCGGCCGCTGATCTGTCGCAGCAGATCTCCACTGCAGGCACC
2016
680 690 700 710 E A S G T G N M K F M L N G A L T I G T M D G A N V E M A E E A G A E N L F I F G L R V E D V E GAGGCCTCAGGCACAGGCAACATGAAGTTCATGCTCAACGGGGCCCTCACCATCGGCACCATGGACGGCGCC AACGTGGAGATGGCCGAGGAGGCCGGGGCCGAGAACCTCTTCATCTTCGGCCTGCGGGTGGAGGATGTCGAG
2160
720 730 740 750 760 A L D R K G Y N A R E Y Y D H L P E L K Q A V D Q I S S G F F S P K E P D C F K D I V N M L M H GCCTTGGACCGGAAAGGGTACAATGCCAGGGAGTACTACGACCACCTGCCCGAGCTGAAGCAGGCCGTGGAC CAGATCAGCAGTGGCTTTTTTTCTC CCAAGGAGCCAGACTGCTTCAAGGACATCGTGAACATGCTGATGCAC
2304
770 780 790 800 H D R F K V F A D Y E A Y M Q C Q A Q V D Q L Y R N P K E W T K K V
CATGACAGGTTCAAGGTGTTTGCAGACTATGAAGCCTACATGCAGTGCCAGGCACAGGTGGACCAGCTGTAC
I
R N I
810 A C S G K F S S D R
CGGAACCCCAAGGAGTGGACCAAGAAGGTCATCAGGAACATCGCCTGCTCGGGCAAGTTCTCCAGTGACCGG
820 830 840 T I T E Y A R E I W G V E P S D L Q I P P P N I P R D * ACCATCACGGAGTATGCACGGGAGATCTGGGGTGTGGAGCCCTCCGACCTGCAGATCCCGCCCCCCAACATC CCCCGGGACTAGGCACACCCTGCCTTGGCGGGACCAGCGGGCATTTGTTTTCTTGCTGACTTTGCACCTCCT
2448
2592
TTTTTCCCCAAACACT~TGCCAGCCACTGGTGGTCCCTGCTTTTCTGAGTACCATGTTTCCAGGAGG~GCCATGGGGGTCAGGGTGGTTTTGAGAGAGCAGGGTAAG~AAGGAA~TGCTAGAA~TGCTCCTAGTTTCTTGTAA (*)
2736
AGGAAGCCAGAGTTGACAGTACAAAG•GTCG••GCCAGCCCTGCAGCTTCAGCACCTGCCccACCCAGAGTGGGAGTCAGGTGGAGCCAcCTGCTGGGCTCCCCCAGAACTTTGCACACATCTTGCTATGTATTAGCCGATGTC
2880
TTTAGTGTTGAGcCTCT•GATTCTG•GGTCTGGGCCAG•GGCCATAGTGAAGCCTGGGAATGAGTGTTACTGCAGCATCTGGGCTGCCAGCCACAGGGAAGG•CCAA•CCCCAT•TAGCcCCAGTCATCCTGCCCAGCCCT•CC
3024
TCCTGGCcATGCCGGGA•GGGTCGGATCCTC•AGGCATCGCCTTCACAGCCCCCTGCCCCCTGCCCTCTGTCCT•GCTCTGCACCTGGTATATGGGTCATGGACcAGATGGGGCTTTCCCTTTGTAGCCATCCAA•GGGCATTG
3168
•G•GGGTGCTTGGAACCCGGGA•GACTGAGGGGGA•ACTGGAG•GGGTGCTTGTGTCTGCTGTCTCAGAGGCC••GG•CAGGATGAAG•TG•CTG•CACAGCTTAGCT••G•T•TGCT•A•TCAAAAGAGAAAATAAC•ACACA
3312
TGGAAATGAAACTAGCTGAAGCCTTT•CTT•TTTTAGCAAC•GAAAAT•GTACTTGGTCACTTTTGTGCTT•AGGAG•CCCATTTTCTGCCTGGCAG•GGCA•GTCT•TGCcCTCCCGCTTGACTCCTGCTGT•TCCTGAGGTG
3456
CATTTCCTGTTT•TTACACACAA•GGcCAG•CTCCATTCTCCCTCCC••TCCACCAGTGCCACAGC•TCGTCTGGAAAAAGGACCAGGGGTCCCGGA•GAACCCATTTGT••TCTGCTTGGACA•CAGGCCTGG•ACTGGGAGG
3600
TG•GG•T•AGCCCCTCACAGCC•TGCCCCTcCCCAAG•CTCGAACCTGCCTCCCATT•CCCAAGAGAGAGG•CAGG•AACAGGCTACTGTCCTTCCCTGTG•AATTGCCGAGAAATCTAGCACCTT•CATGCTG•ATCT•GGCT
3744
GCGGGGAGG•TCTTTTTCTCCCTGGCCTCCAGTGCCCACCAGGAG•ATCTGCGCACGGTGCACAGCCCACCAGAGCAC•ACAGCCTTTTATTGAGTGGGGCAAGTGCTGGGCTGTGGTCGTGcCCTGAcAGCATCTTCCCCAGG
3888
CAGCGGCTCTGTGGAGGAGGCCATACTCCCCTAGTTGGC~ACTGGGG~CACCACCCTGACCACCACTGTGCCCCTcATT~TTACTGCCTTGTGAGATAAAAACT~ATTAAACCTTTGTGGCTGTGGTTGGCTGA~AAAAAAAAA
4176
Fig. 2. Nucleotide sequence of the Bophosphorylase c D N A . The nucleotide and deduced amino acid sequences are shown, with amino acids indicated by a one-letter code, with the termination codon for the fetal brain c D N A O R F indicated by an asterisk, *. The 280 bp region absent from the phosphorylase c D N A isolated from astrocytoma cell line U25126 is underlined. Deletion of the underlined region results in an extended phosphorylase O R E shown in Table I, with a termination codon indicated by a bracketed asterisk, [*]. The sequence of the B-phosphorylase gene was identical to that of the fetal brain c D N A from the termination point of an intron of undetermined size at amino acid 793 to the polyadenylation site.
180 TABLE I Coding region difJorenees between brain and U251 glycogen phosphoo, lase cDNA sequences
Nucleotide sequence position and amino acid residue numbering are according to Fig. 2. Nucleotide differences are indicated in boldface. Brain cDNA
U251 cDNA z°
Base no.
Nucleotide
Amino acid
Nucleotide
Amino acid
741 743 906 ll61
GCC AAG GCC CGC
A-246 K-247 A-301 R-386
GCA AGG GCG CGG
A-246 R-247 G-301 R-386
2503-2783 IPPPNIPRD*
of a clone isolated from this library (Figs.l and 2) revealed 78% and 74% nucleotide identity with muscle 4 and liver 27 c D N A s , respectively. The deoxyguanosine plus deoxycytosine ( G + C ) content of the coding region of this isozyme (58%) is more similar to that of muscle (57%) than to liver (49%,). This difference in codon usage is p r o n o u n c e d at the third position, where B-phosphorylase contains 84% G + C , M-phosphorylase 77%, G + C and L-phosphorylase only 60% G + C . Examination of the predicted amino acid sequence of the protein encoded by the fetal Bphosphorylase c D N A (Fig. 2) revealed 84% and 78% amino acid identity with the human muscle and liver isozymes, respectively. The protein sequence deduced from the fetal B-phosphorylase c D N A is 842 amino acids in length, while the muscle and liver proteins are 841 and 846 amino acids long, respectively. This variability in length is derived solely from the addition of amino acids at the C-terminus (Table I). Based on nucleotide sequence and predicted amino acid sequence comparisons, the B- and M-isozymes a p p e a r more closely related to each other than either is to the L-isozyme. This conclusion is supported by similarities of the M- and B-isozymes in allosteric response to A M P 7"14, and the ability of these two isozymes to form functional h e t e r o d i m e r s 2°. Comparison
o f B-phosphorylase
with the astrocy-
toma-derived c D N A
There are significant nucleotide differences be-
280 bp insertion/deletion Predicted protein difference is: LQH LPHPEWESGGATCWAPPELCTHLAMY*
tween the human B-phosphorylase c D N A in this report and the c D N A previously isolated from a human astrocytoma cell line, U25126. Most of the single base coding region discrepancies do not affect the predicted protein sequence (Table 1). The most prominent difference is an a p p a r e n t 280 bp deletion within the c D N A o b t a i n e d from astrocytoma cell line U251. This deletion affects amino acids 834-842 and a portion of the 3" untranslated region (Fig. 2). The shortened c D N A obtained from cell line U251
a
b
c
d
6.15.14.13.12.01 ,O--
Fig. 3. Northern blot analysis of B-phosphorylase transcripts. RNA blot containing poly(A)+ RNA purified from adult tissues was probed at high stringency using a 336 bp fragment of human fetal B-phosphorylase cDNA. Lane a, 5.0/~g human brain RNA, lane b, 3.5 ~g macaque heart RNA, lane c, 5.0 ~g human liver RNA, and lane d, ().7 ~tg human skeletal muscle RNA. Size in kb is indicated at the left.
181 encodes a predicted protein of 862 amino acids rather than the 842 amino acids obtained for the fetal B-phosphorylase c D N A .
3" Genomic B-phosphorylase sequence To verify that the discrepancies between the B-phosphorylase c D N A s in human fetal brain and an astrocytoma cell line were not the result of cloning artifacts in the fetal brain c D N A library, a portion of the genomic clone for human B-phosphorylase was isolated and sequenced. The genomic nucleotide sequence was identical to the c D N A in this report from amino acid 793 (the 3" border of an intron) through the polyadenylation site (Figs. 1 and 2). A n exact match of the fetal brain c D N A and genomic D N A sequences verified the fetal c D N A sequence, and indicated there were no additional introns 3" to amino acid 793. This organization of exons corresponds exactly to that present in the human M-phosphorylase gene 4, further emphasizing the close evolutionary distance between these isozymes. Southern blot analysis was performed to verify the presence of only one gene encoding B-phosphorylase. Chromosomal D N A purified from a single human placenta was digested separately with 6 restriction enzymes, then 10/~g samples were subjected to electrophoresis on an 0.8% agarose gel and transferred to nylon membrane. Duplicate blots were prepared and independently hybridized with either the 280 bp Pstl fragment present only in the fetal brain c D N A (fragment I, Fig. 1), or with the adjacent 173 bp PstI fragment c o m m o n to both the fetal brain and astrocytoma c D N A sequences (fragment II, Fig. 1). All bands detected with the probe c o m m o n to both c D N A sequences (fragment II) were also detected with the probe unique to the fetal brain c D N A (fragment I) (data not shown). These results suggest that only one gene encoding Bphosphorylase is present in human tissues.
Tissue-specific expression of B-phosphorylase The level of B-phosphorylase transcript in different tissues was characterized by Northern blot analysis using poly(A) ÷ R N A from human adult brain, liver, and muscle, and from macaque adult heart tissues (Fig. 3). The B-isozyme-specific c D N A probe, B14, identified 4.2 kb transcripts in human adult brain and adult macaque heart.
To examine low levels of B-phosphorylase transcripts in human fetal tissues, we used the more sensitive technique of solution hybridization followed by S1 nuclease digestion 3. A n antisense, 218 bp BamHI/SmaI fragment of B-phosphorylase c D N A was used as the single stranded probe (fragment III, Fig. 1). As shown in Fig. 4, a 218 base
a bcde
f gh
290 -
218-
Fig. 4, S1 nuclease analysis of B-phosphorylase transcripts. A radiolabeled 290 base single-stranded probe, of which 218 bases was complementary to B-phosphorylase cDNA (fragment Ill, Fig. 1), was annealed to RNA from different sources, then subjected to S1 nuclease digestion. The S1resistant fragments were examined by electrophoresis on a 6% polyacrylamide sequencing gel. The 290 base probe in the absence, lane a, and presence, lane b, of S1 nuclease treatment. RNA was from: lane c, human adult brain; lane d, human adult liver; lane e, macaque adult heart; lane f, human fetal brain; lane g, human fetal liver; and lane h, human fetal muscle. Lanes a-f were exposed for 24 h, and lanes g and h for 72 h. The chain termination sequencing reaction at the left was used for size determination.
182 protected fragment was detected in human adult brain and macaque adult heart. Transcripts were also detected in human fetal brain and in much lower amounts in human fetal liver and muscle. Transcripts were not detected in human adult liver (Fig. 4d) or muscle (Fig. 5f), even with 10-fold longer exposure times (data not shown). Thus, both Northern blots and S1 nuclease ex-
a b c de f
periments demonstrate that B-phosphorylase transcripts are present in human adult brain and adult macaque heart, but not human adult liver or muscle. This tissue specific pattern of expression is consistent with the protein data of Proux and Dreyfus 2s, but differs from RNA data of Fletterick and cow o r k e r s 25,26.
The 4.2 kb apparent size of the brain transcript is considerably larger than the 3.2-3.4 kb transcript sizes corresponding to L- and M-phosphorylases n'27. The larger size is, in part, the result of the 1.5 kb long 3" untranslated region found in the brain phosphorylase transcript, in contrast, human M- and L-phosphorylase transcript 3" untranslated sequences are only 263 and 170 bp long, respectively.
Alternative processing of B-phosphorylase in human tissues The B-phosphorylase m R N A in the astrocytoma cell line U251 was found to be smaller than the B-phosphorylase m R N A in control tissues 26. Comparison of the nucleotide sequence of the fetal B-phosphorylase cDNA with that reported for the astrocytoma cDNA raised the possibility that alternative processing 2 of the B-phosphorylase gene in the astrocytoma gave rise to the deletion in the U251 cDNA. As shown in Fig. 2, the cDNA and genomic sequences at the borders of the region deleted in the cell line cDNA contain the sequences C T G C A G / ATC and CTGCAG/CT, which resemble a consensus splice site 2~ with two notable exceptions: (1) the
Fig. 5. 3" structural determination of B-phosphorylase transcripts. A 263 base radiolabeled single-stranded D N A fragment with 157 bases identical to the fetal B-phosphorylase cDNA, but only 132 bases identical to the astrocytoma phosphorylase cDNA 26 (fragment IV, Fig. l), was annealed with RNA, then subjected to digestion with S1 nuclease. Protected fragments were examined by electrophoresis on a 6% polyacrylamide sequencing gel. A protected fragment of 157 bases indicates perfect homology with the fetal brain cDNA and the absence of processing at the putative splice site, while a fragment of 132 bases indicates homology with the astrocytoma cDNA and suggests the presence of a processed transcript. Lane a shows the fragments present in the absence of S1 nuclease, and lanes b - f show the fragments generated following SI nuclease treatment. The D N A fragment was annealed with R N A purified from: lane b, no RNA; lane c, human fetal brain; lane d, human adult brain (grade IV astrocytoma); lane e, human adult liver; and lane f, human adult muscle. The chain termination sequencing reaction at the left was used for size determination.
183 'invariant' G of the 5" donor is an A and (2) the dinucleotide AG is found at position -9 of the 3" splice site. Although this dinucleotide has not been previously reported in the -5 to -15 region, it is not known if its presence prohibits proper splicing. S1 nuclease protection studies were used to examine human tissues for alternative processing involving this region of the cDNA. A single-stranded DNA probe containing a region common to both B-phosphorylase cDNAs and the region present only in the fetal B-phosphorylase cDNA and gene (fragment IV, Fig. 1) was annealed to mRNA from various human tissues prior to S1 nuclease digestion. Recognition of and processing at the potential splice site should result in protection of a 132 base fragment, while absence of splicing at this site should generate a 157 base protected fragment. The 157 base protected fragment was observed with RNA from human fetal brain and a naturally occurring grade IV human astrocytoma (Fig. 5, lanes c and d). No evidence of alternative processing corresponding to the truncated transcript reported in the human astrocytoma cell line U251 was detected in any adult human tissue (Fig. 5, additional data not shown). DISCUSSION In this report we describe the cloning, sequencing, and transcript levels of human B-phosphorylase genomic DNA and cDNA. The results presented demonstrate that this gene is expressed in adult and fetal brain and heart, and in low levels in other human fetal tissues. Previous work has demonstrated that transcripts of M- and L-phosphorylase are present in human adult brain, and that Lphosphorylase mRNA predominates in human fetal brain and other human fetal tissues 11. In contrast to previous reports concerning the expression of Bphosphorylase 25"26, the tissue-specific expression of B-phosphorylase transcripts presented here is in good agreement with the protein data of Proux and Dreyfus 2s. It has recently been proposed that conformational changes of amino acids 839-841 play a significant role in the transition of a dephosphorylated, catalytically inactive T state to the phosphorylated, active R state 32. These C-terminal residues are ordered in M-phosphorylase b and participate in
intersubunit hydrogen bonding, but become disordered with covalent phosphorylation of the Nterminus 32. The C-terminal amino acid sequence of B-phosphorylase predicted from the fetal brain cDNA and human genomic DNA favors the maintenance of a structurally disordered C-terminus, with 4 chain-breaking prolines at residues 835,836, 837, and 840. In the M-isozyme, aspartate-838 forms an intersubunit ion-pair bond that further stabilizes the ordered C-terminus found in phosphorylase b. The fetal brain cDNA B-phosphorylase, like L-phosphorylase, contains an asparagine at residue 838, which would be unable to participate in this intersubunit interaction. This should contribute further to disordering of the C-terminus. Thus, one would predict that the disordered C-terminus of B-phosphorylase should favor an activated, R state enzyme 7. The cDNA and gene sequences for human Bphosphorylase presented here differ from the sequence of a cDNA obtained from a human astrocytoma cell line 26. Overall, the sequences are nearly identical, except for a 280 bp deletion present in the astrocytoma-derived cDNA. The deletion in the astrocytoma-derived cDNA significantly alters and extends the predicted C-terminus of the protein by 20 amino acids, beginning at amino acid 834 (Table I). The C-terminal differences in the protein sequences predicted from these two cDNAs may be significant regarding the function of B-phosphorylase. The predicted B-phosphorylase sequence reported for the cDNA isolated from astrocytoma cell line U251 differs significantly from the other reported phosphorylase isozymes beginning at residue 835, and it is difficult a priori to predict its effect on R---~T state transitions. The significance of the variant characterized by Newgard et al. 26 remains uncertain. The B-phosphorylase transcript detected in the U251 astrocytoma cell line was smaller than the transcript observed in normal tissues 26, suggesting that the deletion present in the cDNA is representative of the phosphorylase transcripts present in the astrocytoma cell line, and is not merely a cloning or sequencing artifact. If the smaller transcript is a result of a chromosomal deletion, then cell line U251 must be homozygous for this deletion, since only a single transcript size was detected. A deletion arising on
184 one c h r o m o s o m e could have become homozygous as a result of aneuploidy, for example, perhaps arising from non-disjunction 5. Alternatively, the astrocytoma cell line might recognize a splice site and excise the 280 bp region from the m R N A . As previously discussed, a nearconsensus splice signal is present at the site of the deletion, suggesting the potential for alternative splicing. A l t h o u g h S1 nuclease analysis failed to detect alternative processing involving this site in normal human tissues and in a naturally occurring grade IV astrocytoma, this alternative processing might be present in specific transformed cell lines. In this regard, it is interesting that Sato et al., using electrophoretic and immunologic techniques, have identified a novel 'fetal-type' phosphorylase isozyme in specific rat ascites h e p a t o m a cell lines 29. Further analysis of the phosphorylase genes in cell line U251 will be necessary to ascertain the potential biological significance of the U251 phosphorylase c D N A variant. The identification of the c D N A for human brain REFERENCES 1 Aviv, H. and Leder, P., Purification of biologically active globin messenger RNA by chromatography or oligothymidylic acid-cellulose, Proc. Natl. Acad. Sci. U.S.A., 69 11972) 1408-1412. 2 Breitbart, R.E., Andreadis, A. and Nadal-Ginard, B., Alternative splicing: a ubiquitous mechanism for the generation of multiple isoforms from single genes, Annu. Rev. Biochem., 56 (1987) 467-495. 3 Burke, J.E, High-sensitivity SI mapping with singlestranded [32p]DNA probes synthesized from bacteriophage M13mp templates, Gene, 30 (1984) 63-68. 4 Burke, J., Hwang, P., Anderson, L., Lebo, R., Gorin, E and Fletterick, R., Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase, Proteins Struct. Funct. Genet., 2 (1987) 177-187, 5 Cavenee, W.K., Dryja, T.P., Phillips, R.A., Benedict, W.E, Godbout, R., Gallie, B.L., Murphee, A.L., Strong, L.C. and White, R.L., Expression of recessive alleles by chromosomal mechanisms in retinoblastoma, Nature (Lond.), 305 (1983) 779-784. 6 Csiba, L., Paschen, W. and Mies, G., Regional changes in tissue pH and glucose content during cortical spreading depression in rat brain, Brain Res., 336 (1985) 167-170. 7 Davis, C.H., Schliselfeld, L.H., Wolf, D.P., Leavitt, C.A. and Krebs, E.G., Interrelationships among glycogen phosphorylase isozymes, J. Biol. Chem., 242 (1967) 4824-4833. 8 Feinberg, A.P. and Vogelstein. B., A technique for radiolabeling DNA restriction endonuclease fragments, Anal. Biochem., 132 11983) 6-13. 9 Ferendelli, J. and McDougal, D., The effects of audiogenic seizures on regional energy reserves, glycolyis and citric
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ACKNOWLEDGEMENTS We would like to thank Dr. R o b e r t Fletterick's lab for the rabbit muscle phosphorylase c D N A probe. We thank Dr. R. Teplitz, Ms. M. Pierce and the U C D M C Pathology D e p a r t m e n t Tissue Bank for providing human tissues. This work was supp o r t e d in part by grants from the California Affiliate of the A m e r i c a n H e a r t Association, the U.S. Public Health Service, and the U.S. D e p a r t m e n t of Education ( U S D E / N I D R R H133 B80016). R . G . was the recipient of an N I H training fellowship and a University of California graduate research award. acid cycle flux, J. Neurochem., 17 (1971) 1207-1220. 10 Fox, P.T., Raichle, M.E., Mintun, M.A. and Dence, C., Nonoxidative glucose consumption during focal physiologic neural activity, Science, 241 11988) 462-464. 11 Gorin, EA., Mullinax, R.L., Ignacio, P.C., Neve, R.L. and Kurnit, D.M., McArdle's and Hers' diseases: glycogen phosphorylase transcriptional expression in human tissues, J. Neurogenet., 4 (1987) 293-308. 12 Henikoff~ S.. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, 28 (1984) 351-359. 13 Hof, R., Pascale, E. and Magistretti, J., K + at concentrations reached in the extracellular space during neuronal activity promotes a Ca + dependent glycogen hydrolysis in the mouse cerebral cortex, J. Neurosci., 8 11988) 19221928. 14 Kamogawa, A. and Fukui, T., Purification and properties of glycogen phosphorylase from bovine spleen, Biochim. Biophys. Acta, 242 (1971) 55-58. 15 Lehrach, H., Diamond, D., Wozney, J.M. and Boedtke, H., RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 16 (1977) 4743-4751. 16 Loenen, W.A.M. and Blanner, ER., Lambda Charon vectors (Ch32,33,34 and 35) adapted for DNA cloning in recombinant-deficient hosts, Gene, 26 (1983) 171-179. 17 Magistreni, P.J., Hof, P.R. and Martin, J.-L., Adenosine stimulates glycogenolysis in mouse cerebral cortex: a possible coupling mechanism between neuronal activity and energy metabolism, J. Neurosci., 6 (1986) 2558-2562. 18 Magistretti, P.J.. Morrison, J.H., Shoemaker, W.J., Sapin, V. and Bloom, EE., Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices. A possible
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