Biochhnica et Biophysica Acta, 113(I(1992) 349-351
349
~ 1992 Elsevier Science Publishers B.V. All rights reserved (|167-4781/t)2/$1)5.00
BBAEXP 90338
Short Sequence-Paper
The nucleotide sequence of rat liver glycogen phosphorylase cDNA Katrin Schiebel, Elisabeth Pekel and Doris Mayer Deutsches Kreb,~Jrschm~gszentrton, Abteihmg Cytopathologie, Heidelberg ((;ermmzy) (Received 24 January 1992)
Key words: Glycogen phosphowlase, eDNA sequence; (Rat)
The nueleotide sequence of a eDNA coding l~r rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the eDNA arc sufficient to encode the total protein as dclcrmincd by comparison with the liver type of glycogen phosphorylasc of man. Human and rat liver glycogen phosphorylase showed 86t)f. hon'mlogy at the DNA level whcrcas the deduced amino acid sequence has 93.5c~ identity.
Glycogen phosphorylase (EC 2.4.1.1) catalyzes the first step in the intracellular degradation of glycogen. In mammals three different isozymes have been identified. They were named after the tissue of predominant appearance: muscle, brain and liver (for review see Ref. 1). A possible fourth type of glycogen phosphorylase occurring in a Morris hepatoma cell line and in a non-tumorigenic rat liver epithelial cell line was recently described [2]. The three well characterized glycogen phosphorylase isoenzymes from human tissue are encoded by three different genes localized on three diffcrent chromosomes [3]. For humans the complete cDNA sequences of brain a)ld liver type of glycogen phosphorylase, and the genomic nucleotide sequence and part of the eDNA sequence for the muscle type have been published [3-6]. The nueleotide sequence of rabbit muscle glycogen phosphorylase eDNA has also been determined [7], whereas the nucleotide sequences of rat glycogen phosphorylases were only partly available as shown in Fig. 1. Two pieces of the liver type cDNA, 1241 bp (base pairs) and 294 bp in length, respectively, were ;,nalysed [8,9]. For the brain and muscle isoenzymes the nucleotide sequence of eDNA pieces of only 484 bp and 589 bp length were determined, respectively [ 10,8].
in order to obtain a reference for the comparison of the tumour type glycogen phosphorylase isolated from a rat hepatoma cell line [2] with the liver type phosphorylase of rats, we have sequenced the full length eDNA of rat liver glycogen phosphorylase. RNA extracted from rat liver cells [ll] was reverse transcribed by oligo(dT)-priming [12] and amplified by the PCRmethod using primers (see Fig. 2) designed with the help of the nuclcotidc sequence of the already published part of rat liver glycogen phosphorylase eDNA [8]. After the amplification (30 cycles, I min 92 °C, ! min 48 °C, I rain 72 °C) a 492 bp PCR-fragment was obtained, which was used to screen a Agtll cDNA library prepared from adult rat liver [13].
ATG
., . . . .
,..
. . . . . .
..
,
,.:.
STOP ,
I
human muscle genomtc ]5J human muscle eDNA 161
ATG
STOP human brain eDNA [3]
j
L
ATG
STOP
I
I
ATG
STOP
~---
human liver eDNA [4]
]
rabbit muscle cDNA [71
ATG
.STOP
j
I STOP J
The sequence reported in this p a p e r has been submitted EMBL/Databank under the accession number X63515.
to
rat liver eDNA [this paper] rat liver cDNA [9,6J
the
Correspondence: D. Mayer, Abteilung Cytopathologie, Deulsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69(10Heidelberg, Germany.
rat brain eDNA [10l ra! muscle eDNA 16]
Fig. 1. Overview of the published g l y c o g e n pho~phorylas¢ sequences of mammals, v. inlnm of variable length.
350 hul~n • a~ • at
10 10 50 ~ CG C G G¢G A GC A G G C ~:J~CC~.J~kCC~-TC~2¢GAAG~ I ~ V ~ CCGACC?-GGAAAAGCCACGGC&Gk~AG C R A K P ~ T D Q E K R R Q I S 70
•mt ra~ human htl~ln eat. rm~ human h ~ rgt ra~ hUnCh haman • at rat h~q~m
hum4m ~ll~ rat hUIah hl~ tat r&h huee~ bin ~&lr. ~J~
110
90
ATC~CGGCA~-~'~3AGAACGTAGCGGAGCTGAAAAAGGG Z R G I V G v E N V A E L K K
G s
CTTt ~ATCG TCA C F N R H
130 ?.50 170 G C A • G CTGCACTTCA~GGACCGCAATGTGGCCACCCCCCGCGACTACTACTTCGCC ~ K F T L V g D R g V k T P R 0 Y Y F & T 190 210 230 G G G G CTTGCG~Jt~.ACAG'I~CGC~CeACCTGG'~GGGGCG CTGGAT~'~.~..ACACAGCAG"~,CTAC L A H T V R D H L V G R W I R T Q O H Y 250 270 2~0 C AA T&/~A~GGGTGTATTA~I~TCTCTGGAATTTTAC&TGGGCCGAACA Y D g C P K R V Y Y L s L E r Y N a R T t 310 330 350 C q'c G & T T G e TTA~AGAA~A~CA%~ATP~A~'~L~ACAGAATGC C T G ~ A ¢ ~ A G ~ A ~ A ~ G L Q H T N X H L G L Q H A C D It & I Y Q
370 3)0 410 T AT T & A A & CT~'f~keAT~T~A~TT~0aA~AT~¢T~a¢TT~CAAT~0T L G L D N | ~ L B B I e B 0 & O L ~ N r~ X 430 450 470 k T ¢ k GGTCT'L'~GGA~¢~"/q~%'~¢C'~.~"TCTI~GACTCCATGGC : ~ ¢GCTGGG00TTGe&o ~ 0 L G R L k k e f L 0 B g k T L G L A 490
hoalm ~a~ ~a~c hUl~n hul~n ~at humn~
510 530 T G G T T A TATOOATk~eAT~'~TT~TC~ATATO~kATCTTCAATC~GAAGATC(~AGAAGOG TGG Y G Y G I R Y E Y G 1 F H Q K ! R E G W 0 aS0 570 590 A & T AT T 0 C A
Q
V
E
~
K A R P s 650 e T A & & CA C G C G~ATTCATG~IT~CTGTGCATTTCTACGGAAGAGTAGACCACACCC&GGCAGGAACA~AG e P N L P v H F Y G R V E H T Q A G T g g 14 T 670 690 710 k T T & C C A T AG ¢ C e TGGGTCGACACCCAGGTGGTGCTGG CTTTGCCGTACGACA¢CCCCGTACC'T~GGTAT&TG t~ V 0 T Q V V L A L P Y O T P V P G Y N I E 610
human ~at • at huaan
h~an ta~ ra~ h~n human t'a~ rat hu~&n
humon t'a~ ra~ human human • eE t'at huu~
k
O
D
W L
R
H Y
G
N
P
W
E
630
730 7~0 770 T T C C T T G A C ~J~CRACACGGTC~ACACTATG CGCeT ~TCGTCGGCCCGAGC & C C C & A T G & C T T T ~ N N T v N T g g L ~ S k R & P N 0 F H L 790 810 830 &G T A C C C~%(:~CTTTJ~ATGTCGOAGACTACATTC&G GCTGTGCTG0&CCGG&ACCTGGCTG&GAAT Q 0 P ~ V G 0 Y | Q A V L ~ R N L & E N R aS0 070 890 C G C C T T C T A AT ATCTCC&OA~TGCTGT&COC¢~ACGATJ~ACTTrTTT~GGGA~GG&0CTG&0GCTGJ~G 1 8 g V L Y P H D N ~' ~" I; G K E L R L K 910
hu~ ~a~ ~at h~ma~ hul~ tit • i~; h~Mn h~an ~m~ :m~
h ~ rat ra~ h~n haman • at rm~ human
human ra~ rat h~n hu~n rat ra~ h~n ~uaan rmt ~m~ human h~an raE T'a~. human
930 950 & A T A & ¢ A ¢A~GT&CTT~GTGGTGGCT~CQ&CCCTGCAOGATGTeATCCr~CGITTC/~GG CCTC¢ Q ~ Y te V V A & T L Q O V Z R R F K A 5 I ~70 ggo 1010 T CCC'G CA T C C G G AAG~t~TAA~.~T~ GT~TAr.~AAC¢GTGITTr.~T~ ~-F;-~-~.AGATr.AGGT&G ~ K P 0 S K 0 G V G T V r D k F P D g V & T R Q 1030 1050 1070 T T e I~GG~ G T T &TCC&GCTC-AATG&CAC&¢AT~.'~.'GCAC/CGCCATCCCGGAGCTGATGAGGATCiTTGTG ~ Q L N D T R P A L A l P E L N R l F ~' 1090 1110 1130 'r C k C A ¢ C ¢,~TI'GC't%"~,GI'¢r.~GGCCTGG~GNr~ccAAGAAG~CCTI'TGCCT&¢ D ~ E K L P W ~ K A ~ £ 1 T K ~ T F & Y L N Q 1150 1170 1190 & A ¢ ACCAA~.At'GGTG~GGC~'Tr~AGCGCTGGCCAGTGGACCtGGTGGAG&A0 T H H T v L P E A L E R W P V D L V E K 1210 1230 1250 C T G k k T CT0 ~CCTCG&CACTTC;CAG~TC&TTT&TGAGATCAATCAGAAGC&TTTAGATA~TC ~ G P R H ~ Q 1 1 ¥ E I N Q K H L D R 1 E 1270 1290 1310 T TG G CT¢ & ~U~ G k GTGG~~GACATCGACCGCATC;C~;GCGGA~TCGAAGAGG~A v A L F P K O I D R N R R N S L 1 E E E V 0 L 1330 1350 1370 A T T C T C T T T e GGAGGCA&AAGGATCAA~TGGCCCACCT~TCGTGGGCTGCCACGCGGTGAACGGG G G I~ R I H Iq & H L C 1 V (3 C 14 A V N G S S 1300 1410 1430 G T A A TA GTAGCGAAG&TCCACTCGGACATCGTGAAGACCCAAGTATT~AAGGACTTCAGTGAGC~,A V A K ]r H 5 D ~ V I~ T Q V F K D F S E L K
human rat rat human
hunan rat rat human ~u~an human human Eat rat human human human human Ya~ human
human rat rat human human human huna~ ~at human
human rat tat human human rat ~at human human ra~ ca~ human
human ~at human hu~ hu.~4m hu~m
1450 1470 1490 T T T T A C GAACCAGAC&AGTTCC&GAATA~%AC CAACGGGATCACCCCGAGGCGCTGGCTCTTACTC E I:' D K F Q N K T N G I T P R R W L L L 1510 1530 1950 A T A GC C TGCAACCCAGGGCTC~CTGACTTGATAGCAGAC~qAAATTGGAGAAGACTATGTG AAAGAC C N P G L A D L I A E K I G E D Y V K D E 1570 1590 1610 C T T TG 0C CTGAGCCAGCTG&CGAAGCTCCACAGCTTCGTGGGCGACGACATCTTCCTCCGGG~ T & L S Q L T K L H S F V G 0 0 1 F [. R Z I L V L 1630 1fi50 1670 G G G G T T GC A GCCAAAGTGAAGCAGGAA~ATA&ACTG~TTCTCCCAGTTCCTGGAAAAGGAGTACAAG & K V K Q E g K L K ¥ S Q F L E K E ¥ K T 1690 1710 1730 C T C 0 & & T G GTGAAGATC&&CCCATCTTCCATGTTTG&CGTGCACGTGAAGCGGATCCACG>AC~ V K I N P S S N F D V ~ V K R T H E Y K Q 1750 1770 1990 CT T G C T CCTG~ATGTQ&TCACC&TGTACAAT~CATC~AGAAA~qCCCT R Q L L N C L H V I T H Y N R l K K D P 1810 1830 1850 k T C I' /~AGAAGTTCTTCGTGCCAAGGAC>CAT~TTGGTGGGAAAGCTGCCCCAGGATATC~¢ K K F F V P R T V Z I G G K A & P G Y H L 1870 |800 1010 & T A T T ATGG¢CAN~ATGATC&TA/tAGCTGGTC&CCTCCGTGGC&GAAGTGGTC.~ACAACGA¢C~ N A K N I I K L V T S V A £ V V N N D P X O 1910 1950 1970 A A ATGQTTGGCAGCA>TGAAA~TCATCTTCTTGO/tG,kAeTACAO& G ~ T ~ H V G $ g L K V Z F L £ 1t ¥ R V S L & E 1090 2010 2030 A T G T A C ~AGTCATTCCAGCCA'2GGACCTGTCAGIk&CAG&TCTCCACT0 eTGGCACGGAAGC~ K V l P A T 0 L S E Q 1 S T A G T E A S
2050 2070 2090 A T A T A T C GGG&CGGGCJ~ACATG~AGTTCATGCTGJU4CGGGGCCCTG&CC&TCGGGACTA~A~ G T G N H K F N L N G & L T I G T N D G 2110 2130 2150 & A & A T & G C GCCAATGTGG&GATGGCGG&GQAGGCCGG~~AGGAAAACCTGTTCATCTTTGGCATGAGG k N V E N A E £ A G E E N L F Z F G N R 2170 21g0 2210 A T T C & C A GTAG&TGATGTGGCCGCT¢TGGACAAGAAAGGGTATG&GG CCAAAGAATATTATGAGG CC V O D V A & L 0 g K G Y E & K E Y ¥ E A X 2230 2250 2270 G T TT G C T T ¢ C & G A A ~ G CTGGTC&TTG&CCAAATTGACAATGGCTTC~-r~-~r CCC~TCAG L P E L K L V X D Q 1 D N 0 F F S P N Q K |290 2330 2330 T T ¢ G CCAG&CCTCTTCAN~GACATCATCAACATGTTATTTTATCATG&CAGATTTAAAGTCTTT P O L F K O X 1 N g L F Y H D R F ~ Y F
human
human rat
;1350 2370 2390 T G C C GCAGAeTACOAAGCCT&TG%~.AAG~GTCAGTCAG CTGTATATOAATCAA & D Y E & ¥ V K e Q E K V S Q L ¥ ll N Q 0 P 2410 3430 2450 G & A & AAAGCC'IT,GAAeA~ATGGTTCTCAGAAAPJ~TAGCTGCCTCGGGGAAGTTCTCCAGTGAC K A H g T N V L R H 1 & A S O K F S S 0 K 3470 2490 2510 T~ & ¢.'J~ G A A A T LT,AAGqAT~q~OAGTATG CCAA~ACATCI~GAACATGGAGCCTTCCGATCTGAAGATC R T 1 R E Y & K D Z W N N E P 8 D L g I K Q N V 2530 2550 2570 T ¢ T A T A A "'T G& T" e ¢T CA& GTCTCTJ~ Tt~¢T&TCTAAGOAGTCCAGCRATGGGGTC/UtC~C~ATC~GAAGTAAATGCTAAAATAT 5 L 8 K E 5 S N G V N k N G K g K G 2590 2E10 3630 G/~J~¢ AGent CTTACTGA T ACATTT T&CAAC TTCAC GGT TTTGT T G ATTCTTATTCAATAA~ACTGGAt~TGAGT&CTCTTAG&GCTTCCCTGAGTCTGTT
human rat
2650 2570 2690 &GeT&AT eTA A GT AG & C GGG AT GCG GGGAA TTATATGTAA TTGTT&TTG ~A~'~;~.TAGTA~ATGTATTTCTGTATTAGAGCTRAA/~TA~AATGTCAA~
human ra~
~710 T~, CT~AA T T¢GAGTTGTC&~A A
human rat h~a~ hdllt~h
h~mah human human Eat human
Fig. 2. Nucleotide sequence and deduced amino acid sequence of the rat liver glycogen phosphorylase cDNA in comparison with the human liver glycogen phosphorylase cDNA. For the human enzyme only bases and amino acids which differ between rat and man are indicated. Nt 1013 to 1305 correspond to the sequence of the cDNA piece published by Glaser et al. [9], nt 1340 to 2578 to those sequenced by Osawa et al. [8]. Bases which were found different from the already published parts of the rat sequence are marked in bold face. The bases used for primer design are underlined.
351 A 2.75 kbp insert of a positive clone was subcloned in Bluescript SK vectors and DNA sequences were determined by the dideoxy sequencing method of Sanger et ai. [14]. By comparing with the nueleotide sequence of human liver glycogen phosphorylase and translating the nueleotide sequence into amino acid sequence, the transcription start codon ATG could be located at nt 16-18 and the stop codon TAA at nt 2565-2567. The difference in length between the liver type isoenzymes from man and rat (847 compared to 850 amino acids) is due to three additional amino acids at the C-terminal end of the rat protein. 794 amino acids (93.5%) are identical and 29 (3.5%) additional amino acids are similar. Three amino acids which are different in man and rat are located in functional domains, if the X-ray crystal structure of the rabbit muscle phosphorylase is taken as reference [l]. In the dimer contact region amino acid 187 determined as Tyr in man was found to be His in rat, Ser-194 in man was replaced by Ala in rat. The Glu-402 involved in the glycogen storage site in man was replaced by the similar amino acid Gin in rat. The nucleotide sequence and the deduced amino acid sequence of the full length cDNA of rat liver glycogen phosphorylase is shown in Fig. 2. The bases which are different from the published sequences are marked in bold face. The tissue specific codon usage of the eDNA encoding human liver glycogen phosphorylase reported by Newgard et al. [4] is also obvious for the rat eDNA. The protein encoding region contains 52% G + C and thus lies within the average of 51 _+ 6% G + C usually
found in liver [4]. An average G + C content at the third codon position of 59 + 12% G + C described for liver proteins [4], was also found for rat liver phosphorylase, namely 68.5% G + C. References ! Newgard, C.B., Hwang, P.K. and Fletterick, R.J. (1989) CR( Crit. Rev. Biochem. Mol. Biol. 24, 69-99. 2 Mayer, D., Seelmann-Eggebert, G. and Letseh, I. (1992) Biochem. J. 282, in press. 3 Newgard, C.B., Littman, D.R., Van Genderen, C., Smith, M. and Fletterick, R.J. (1988)J. Biol. Chem. 263. 3850-3857. 4 Newgard, C.B., Nakano, K., Hwang, P,K. and Fletterick, R.J. (1986) Proe. Natl. Acad. Sci. USA 83, 8132-8136. 5 Burke, J., Hwang, P., Anderson, L., Lebo. R., Gorin. F. and Fletterick R.J. (1987) Prof. Struct. Funct. Genet. 2, 177-187. 6 Gautron, S., Daegelen, D., Mennecier, F., Dubocq, D.. Kaha, A, and Dreyfus, J.C. (1987)J. Clin. Invest. 79, 275-28L 7 Nakano, K., Hwang, P.K. and Fletterick, R.J. (198h) FEBS Lctl. 204, 283-287. 8 0 s a w a , S., Chiu, R.II., McDonough. A., Miller. T,B. and Johnson, G.L. (1986) FEBS Left. 2{12, 282-288. 9 Glaser, T., Matthews, K.E., Hudson, J.W., Seth, P., Itousman. D.E. and Crerar, M.M. (1989) Genomics 5, 510-521. 111 Crerar, M.M., Hudson, J.W.. Matthews, K.E., David, E.S. and Golding, G.B. (1988) Genome 30, 582-590. 11 Chirgwin, J.M., Prz'ybyla. A.E., MacDonald. R.J. and Ruttcr. W.J. (1979) Biochemistry, 18, 5294-5299. 12 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning; A laboratory manual, Cold Spring Harhor Laboratory Press, Cold Spring Harbor. 13 Frain, M, Swart, G., Monaci, P., Nicosia, A., St~.impfli, S.. Frank. R. and Cortese, R. (1989) Cell 59, 145-157. 14 Sanger, F., Nicklen, S. and Coulson. A.R. (1977) Proc. Nail. Acad. Sci. USA 74, 5463-5467.
349
~ 1992 Elsevier Science Publishers B.V. All rights reserved (|167-4781/t)2/$1)5.00
BBAEXP 90338
Short Sequence-Paper
The nucleotide sequence of rat liver glycogen phosphorylase cDNA Katrin Schiebel, Elisabeth Pekel and Doris Mayer Deutsches Kreb,~Jrschm~gszentrton, Abteihmg Cytopathologie, Heidelberg ((;ermmzy) (Received 24 January 1992)
Key words: Glycogen phosphowlase, eDNA sequence; (Rat)
The nueleotide sequence of a eDNA coding l~r rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the eDNA arc sufficient to encode the total protein as dclcrmincd by comparison with the liver type of glycogen phosphorylasc of man. Human and rat liver glycogen phosphorylase showed 86t)f. hon'mlogy at the DNA level whcrcas the deduced amino acid sequence has 93.5c~ identity.
Glycogen phosphorylase (EC 2.4.1.1) catalyzes the first step in the intracellular degradation of glycogen. In mammals three different isozymes have been identified. They were named after the tissue of predominant appearance: muscle, brain and liver (for review see Ref. 1). A possible fourth type of glycogen phosphorylase occurring in a Morris hepatoma cell line and in a non-tumorigenic rat liver epithelial cell line was recently described [2]. The three well characterized glycogen phosphorylase isoenzymes from human tissue are encoded by three different genes localized on three diffcrent chromosomes [3]. For humans the complete cDNA sequences of brain a)ld liver type of glycogen phosphorylase, and the genomic nucleotide sequence and part of the eDNA sequence for the muscle type have been published [3-6]. The nueleotide sequence of rabbit muscle glycogen phosphorylase eDNA has also been determined [7], whereas the nucleotide sequences of rat glycogen phosphorylases were only partly available as shown in Fig. 1. Two pieces of the liver type cDNA, 1241 bp (base pairs) and 294 bp in length, respectively, were ;,nalysed [8,9]. For the brain and muscle isoenzymes the nucleotide sequence of eDNA pieces of only 484 bp and 589 bp length were determined, respectively [ 10,8].
in order to obtain a reference for the comparison of the tumour type glycogen phosphorylase isolated from a rat hepatoma cell line [2] with the liver type phosphorylase of rats, we have sequenced the full length eDNA of rat liver glycogen phosphorylase. RNA extracted from rat liver cells [ll] was reverse transcribed by oligo(dT)-priming [12] and amplified by the PCRmethod using primers (see Fig. 2) designed with the help of the nuclcotidc sequence of the already published part of rat liver glycogen phosphorylase eDNA [8]. After the amplification (30 cycles, I min 92 °C, ! min 48 °C, I rain 72 °C) a 492 bp PCR-fragment was obtained, which was used to screen a Agtll cDNA library prepared from adult rat liver [13].
ATG
., . . . .
,..
. . . . . .
..
,
,.:.
STOP ,
I
human muscle genomtc ]5J human muscle eDNA 161
ATG
STOP human brain eDNA [3]
j
L
ATG
STOP
I
I
ATG
STOP
~---
human liver eDNA [4]
]
rabbit muscle cDNA [71
ATG
.STOP
j
I STOP J
The sequence reported in this p a p e r has been submitted EMBL/Databank under the accession number X63515.
to
rat liver eDNA [this paper] rat liver cDNA [9,6J
the
Correspondence: D. Mayer, Abteilung Cytopathologie, Deulsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69(10Heidelberg, Germany.
rat brain eDNA [10l ra! muscle eDNA 16]
Fig. 1. Overview of the published g l y c o g e n pho~phorylas¢ sequences of mammals, v. inlnm of variable length.
350 hul~n • a~ • at
10 10 50 ~ CG C G G¢G A GC A G G C ~:J~CC~.J~kCC~-TC~2¢GAAG~ I ~ V ~ CCGACC?-GGAAAAGCCACGGC&Gk~AG C R A K P ~ T D Q E K R R Q I S 70
•mt ra~ human htl~ln eat. rm~ human h ~ rgt ra~ hUnCh haman • at rat h~q~m
hum4m ~ll~ rat hUIah hl~ tat r&h huee~ bin ~&lr. ~J~
110
90
ATC~CGGCA~-~'~3AGAACGTAGCGGAGCTGAAAAAGGG Z R G I V G v E N V A E L K K
G s
CTTt ~ATCG TCA C F N R H
130 ?.50 170 G C A • G CTGCACTTCA~GGACCGCAATGTGGCCACCCCCCGCGACTACTACTTCGCC ~ K F T L V g D R g V k T P R 0 Y Y F & T 190 210 230 G G G G CTTGCG~Jt~.ACAG'I~CGC~CeACCTGG'~GGGGCG CTGGAT~'~.~..ACACAGCAG"~,CTAC L A H T V R D H L V G R W I R T Q O H Y 250 270 2~0 C AA T&/~A~GGGTGTATTA~I~TCTCTGGAATTTTAC&TGGGCCGAACA Y D g C P K R V Y Y L s L E r Y N a R T t 310 330 350 C q'c G & T T G e TTA~AGAA~A~CA%~ATP~A~'~L~ACAGAATGC C T G ~ A ¢ ~ A G ~ A ~ A ~ G L Q H T N X H L G L Q H A C D It & I Y Q
370 3)0 410 T AT T & A A & CT~'f~keAT~T~A~TT~0aA~AT~¢T~a¢TT~CAAT~0T L G L D N | ~ L B B I e B 0 & O L ~ N r~ X 430 450 470 k T ¢ k GGTCT'L'~GGA~¢~"/q~%'~¢C'~.~"TCTI~GACTCCATGGC : ~ ¢GCTGGG00TTGe&o ~ 0 L G R L k k e f L 0 B g k T L G L A 490
hoalm ~a~ ~a~c hUl~n hul~n ~at humn~
510 530 T G G T T A TATOOATk~eAT~'~TT~TC~ATATO~kATCTTCAATC~GAAGATC(~AGAAGOG TGG Y G Y G I R Y E Y G 1 F H Q K ! R E G W 0 aS0 570 590 A & T AT T 0 C A
Q
V
E
~
K A R P s 650 e T A & & CA C G C G~ATTCATG~IT~CTGTGCATTTCTACGGAAGAGTAGACCACACCC&GGCAGGAACA~AG e P N L P v H F Y G R V E H T Q A G T g g 14 T 670 690 710 k T T & C C A T AG ¢ C e TGGGTCGACACCCAGGTGGTGCTGG CTTTGCCGTACGACA¢CCCCGTACC'T~GGTAT&TG t~ V 0 T Q V V L A L P Y O T P V P G Y N I E 610
human ~at • at huaan
h~an ta~ ra~ h~n human t'a~ rat hu~&n
humon t'a~ ra~ human human • eE t'at huu~
k
O
D
W L
R
H Y
G
N
P
W
E
630
730 7~0 770 T T C C T T G A C ~J~CRACACGGTC~ACACTATG CGCeT ~TCGTCGGCCCGAGC & C C C & A T G & C T T T ~ N N T v N T g g L ~ S k R & P N 0 F H L 790 810 830 &G T A C C C~%(:~CTTTJ~ATGTCGOAGACTACATTC&G GCTGTGCTG0&CCGG&ACCTGGCTG&GAAT Q 0 P ~ V G 0 Y | Q A V L ~ R N L & E N R aS0 070 890 C G C C T T C T A AT ATCTCC&OA~TGCTGT&COC¢~ACGATJ~ACTTrTTT~GGGA~GG&0CTG&0GCTGJ~G 1 8 g V L Y P H D N ~' ~" I; G K E L R L K 910
hu~ ~a~ ~at h~ma~ hul~ tit • i~; h~Mn h~an ~m~ :m~
h ~ rat ra~ h~n haman • at rm~ human
human ra~ rat h~n hu~n rat ra~ h~n ~uaan rmt ~m~ human h~an raE T'a~. human
930 950 & A T A & ¢ A ¢A~GT&CTT~GTGGTGGCT~CQ&CCCTGCAOGATGTeATCCr~CGITTC/~GG CCTC¢ Q ~ Y te V V A & T L Q O V Z R R F K A 5 I ~70 ggo 1010 T CCC'G CA T C C G G AAG~t~TAA~.~T~ GT~TAr.~AAC¢GTGITTr.~T~ ~-F;-~-~.AGATr.AGGT&G ~ K P 0 S K 0 G V G T V r D k F P D g V & T R Q 1030 1050 1070 T T e I~GG~ G T T &TCC&GCTC-AATG&CAC&¢AT~.'~.'GCAC/CGCCATCCCGGAGCTGATGAGGATCiTTGTG ~ Q L N D T R P A L A l P E L N R l F ~' 1090 1110 1130 'r C k C A ¢ C ¢,~TI'GC't%"~,GI'¢r.~GGCCTGG~GNr~ccAAGAAG~CCTI'TGCCT&¢ D ~ E K L P W ~ K A ~ £ 1 T K ~ T F & Y L N Q 1150 1170 1190 & A ¢ ACCAA~.At'GGTG~GGC~'Tr~AGCGCTGGCCAGTGGACCtGGTGGAG&A0 T H H T v L P E A L E R W P V D L V E K 1210 1230 1250 C T G k k T CT0 ~CCTCG&CACTTC;CAG~TC&TTT&TGAGATCAATCAGAAGC&TTTAGATA~TC ~ G P R H ~ Q 1 1 ¥ E I N Q K H L D R 1 E 1270 1290 1310 T TG G CT¢ & ~U~ G k GTGG~~GACATCGACCGCATC;C~;GCGGA~TCGAAGAGG~A v A L F P K O I D R N R R N S L 1 E E E V 0 L 1330 1350 1370 A T T C T C T T T e GGAGGCA&AAGGATCAA~TGGCCCACCT~TCGTGGGCTGCCACGCGGTGAACGGG G G I~ R I H Iq & H L C 1 V (3 C 14 A V N G S S 1300 1410 1430 G T A A TA GTAGCGAAG&TCCACTCGGACATCGTGAAGACCCAAGTATT~AAGGACTTCAGTGAGC~,A V A K ]r H 5 D ~ V I~ T Q V F K D F S E L K
human rat rat human
hunan rat rat human ~u~an human human Eat rat human human human human Ya~ human
human rat rat human human human huna~ ~at human
human rat tat human human rat ~at human human ra~ ca~ human
human ~at human hu~ hu.~4m hu~m
1450 1470 1490 T T T T A C GAACCAGAC&AGTTCC&GAATA~%AC CAACGGGATCACCCCGAGGCGCTGGCTCTTACTC E I:' D K F Q N K T N G I T P R R W L L L 1510 1530 1950 A T A GC C TGCAACCCAGGGCTC~CTGACTTGATAGCAGAC~qAAATTGGAGAAGACTATGTG AAAGAC C N P G L A D L I A E K I G E D Y V K D E 1570 1590 1610 C T T TG 0C CTGAGCCAGCTG&CGAAGCTCCACAGCTTCGTGGGCGACGACATCTTCCTCCGGG~ T & L S Q L T K L H S F V G 0 0 1 F [. R Z I L V L 1630 1fi50 1670 G G G G T T GC A GCCAAAGTGAAGCAGGAA~ATA&ACTG~TTCTCCCAGTTCCTGGAAAAGGAGTACAAG & K V K Q E g K L K ¥ S Q F L E K E ¥ K T 1690 1710 1730 C T C 0 & & T G GTGAAGATC&&CCCATCTTCCATGTTTG&CGTGCACGTGAAGCGGATCCACG>AC~ V K I N P S S N F D V ~ V K R T H E Y K Q 1750 1770 1990 CT T G C T CCTG~ATGTQ&TCACC&TGTACAAT~CATC~AGAAA~qCCCT R Q L L N C L H V I T H Y N R l K K D P 1810 1830 1850 k T C I' /~AGAAGTTCTTCGTGCCAAGGAC>CAT~TTGGTGGGAAAGCTGCCCCAGGATATC~¢ K K F F V P R T V Z I G G K A & P G Y H L 1870 |800 1010 & T A T T ATGG¢CAN~ATGATC&TA/tAGCTGGTC&CCTCCGTGGC&GAAGTGGTC.~ACAACGA¢C~ N A K N I I K L V T S V A £ V V N N D P X O 1910 1950 1970 A A ATGQTTGGCAGCA>TGAAA~TCATCTTCTTGO/tG,kAeTACAO& G ~ T ~ H V G $ g L K V Z F L £ 1t ¥ R V S L & E 1090 2010 2030 A T G T A C ~AGTCATTCCAGCCA'2GGACCTGTCAGIk&CAG&TCTCCACT0 eTGGCACGGAAGC~ K V l P A T 0 L S E Q 1 S T A G T E A S
2050 2070 2090 A T A T A T C GGG&CGGGCJ~ACATG~AGTTCATGCTGJU4CGGGGCCCTG&CC&TCGGGACTA~A~ G T G N H K F N L N G & L T I G T N D G 2110 2130 2150 & A & A T & G C GCCAATGTGG&GATGGCGG&GQAGGCCGG~~AGGAAAACCTGTTCATCTTTGGCATGAGG k N V E N A E £ A G E E N L F Z F G N R 2170 21g0 2210 A T T C & C A GTAG&TGATGTGGCCGCT¢TGGACAAGAAAGGGTATG&GG CCAAAGAATATTATGAGG CC V O D V A & L 0 g K G Y E & K E Y ¥ E A X 2230 2250 2270 G T TT G C T T ¢ C & G A A ~ G CTGGTC&TTG&CCAAATTGACAATGGCTTC~-r~-~r CCC~TCAG L P E L K L V X D Q 1 D N 0 F F S P N Q K |290 2330 2330 T T ¢ G CCAG&CCTCTTCAN~GACATCATCAACATGTTATTTTATCATG&CAGATTTAAAGTCTTT P O L F K O X 1 N g L F Y H D R F ~ Y F
human
human rat
;1350 2370 2390 T G C C GCAGAeTACOAAGCCT&TG%~.AAG~GTCAGTCAG CTGTATATOAATCAA & D Y E & ¥ V K e Q E K V S Q L ¥ ll N Q 0 P 2410 3430 2450 G & A & AAAGCC'IT,GAAeA~ATGGTTCTCAGAAAPJ~TAGCTGCCTCGGGGAAGTTCTCCAGTGAC K A H g T N V L R H 1 & A S O K F S S 0 K 3470 2490 2510 T~ & ¢.'J~ G A A A T LT,AAGqAT~q~OAGTATG CCAA~ACATCI~GAACATGGAGCCTTCCGATCTGAAGATC R T 1 R E Y & K D Z W N N E P 8 D L g I K Q N V 2530 2550 2570 T ¢ T A T A A "'T G& T" e ¢T CA& GTCTCTJ~ Tt~¢T&TCTAAGOAGTCCAGCRATGGGGTC/UtC~C~ATC~GAAGTAAATGCTAAAATAT 5 L 8 K E 5 S N G V N k N G K g K G 2590 2E10 3630 G/~J~¢ AGent CTTACTGA T ACATTT T&CAAC TTCAC GGT TTTGT T G ATTCTTATTCAATAA~ACTGGAt~TGAGT&CTCTTAG&GCTTCCCTGAGTCTGTT
human rat
2650 2570 2690 &GeT&AT eTA A GT AG & C GGG AT GCG GGGAA TTATATGTAA TTGTT&TTG ~A~'~;~.TAGTA~ATGTATTTCTGTATTAGAGCTRAA/~TA~AATGTCAA~
human ra~
~710 T~, CT~AA T T¢GAGTTGTC&~A A
human rat h~a~ hdllt~h
h~mah human human Eat human
Fig. 2. Nucleotide sequence and deduced amino acid sequence of the rat liver glycogen phosphorylase cDNA in comparison with the human liver glycogen phosphorylase cDNA. For the human enzyme only bases and amino acids which differ between rat and man are indicated. Nt 1013 to 1305 correspond to the sequence of the cDNA piece published by Glaser et al. [9], nt 1340 to 2578 to those sequenced by Osawa et al. [8]. Bases which were found different from the already published parts of the rat sequence are marked in bold face. The bases used for primer design are underlined.
351 A 2.75 kbp insert of a positive clone was subcloned in Bluescript SK vectors and DNA sequences were determined by the dideoxy sequencing method of Sanger et ai. [14]. By comparing with the nueleotide sequence of human liver glycogen phosphorylase and translating the nueleotide sequence into amino acid sequence, the transcription start codon ATG could be located at nt 16-18 and the stop codon TAA at nt 2565-2567. The difference in length between the liver type isoenzymes from man and rat (847 compared to 850 amino acids) is due to three additional amino acids at the C-terminal end of the rat protein. 794 amino acids (93.5%) are identical and 29 (3.5%) additional amino acids are similar. Three amino acids which are different in man and rat are located in functional domains, if the X-ray crystal structure of the rabbit muscle phosphorylase is taken as reference [l]. In the dimer contact region amino acid 187 determined as Tyr in man was found to be His in rat, Ser-194 in man was replaced by Ala in rat. The Glu-402 involved in the glycogen storage site in man was replaced by the similar amino acid Gin in rat. The nucleotide sequence and the deduced amino acid sequence of the full length cDNA of rat liver glycogen phosphorylase is shown in Fig. 2. The bases which are different from the published sequences are marked in bold face. The tissue specific codon usage of the eDNA encoding human liver glycogen phosphorylase reported by Newgard et al. [4] is also obvious for the rat eDNA. The protein encoding region contains 52% G + C and thus lies within the average of 51 _+ 6% G + C usually
found in liver [4]. An average G + C content at the third codon position of 59 + 12% G + C described for liver proteins [4], was also found for rat liver phosphorylase, namely 68.5% G + C. References ! Newgard, C.B., Hwang, P.K. and Fletterick, R.J. (1989) CR( Crit. Rev. Biochem. Mol. Biol. 24, 69-99. 2 Mayer, D., Seelmann-Eggebert, G. and Letseh, I. (1992) Biochem. J. 282, in press. 3 Newgard, C.B., Littman, D.R., Van Genderen, C., Smith, M. and Fletterick, R.J. (1988)J. Biol. Chem. 263. 3850-3857. 4 Newgard, C.B., Nakano, K., Hwang, P,K. and Fletterick, R.J. (1986) Proe. Natl. Acad. Sci. USA 83, 8132-8136. 5 Burke, J., Hwang, P., Anderson, L., Lebo. R., Gorin. F. and Fletterick R.J. (1987) Prof. Struct. Funct. Genet. 2, 177-187. 6 Gautron, S., Daegelen, D., Mennecier, F., Dubocq, D.. Kaha, A, and Dreyfus, J.C. (1987)J. Clin. Invest. 79, 275-28L 7 Nakano, K., Hwang, P.K. and Fletterick, R.J. (198h) FEBS Lctl. 204, 283-287. 8 0 s a w a , S., Chiu, R.II., McDonough. A., Miller. T,B. and Johnson, G.L. (1986) FEBS Left. 2{12, 282-288. 9 Glaser, T., Matthews, K.E., Hudson, J.W., Seth, P., Itousman. D.E. and Crerar, M.M. (1989) Genomics 5, 510-521. 111 Crerar, M.M., Hudson, J.W.. Matthews, K.E., David, E.S. and Golding, G.B. (1988) Genome 30, 582-590. 11 Chirgwin, J.M., Prz'ybyla. A.E., MacDonald. R.J. and Ruttcr. W.J. (1979) Biochemistry, 18, 5294-5299. 12 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning; A laboratory manual, Cold Spring Harhor Laboratory Press, Cold Spring Harbor. 13 Frain, M, Swart, G., Monaci, P., Nicosia, A., St~.impfli, S.. Frank. R. and Cortese, R. (1989) Cell 59, 145-157. 14 Sanger, F., Nicklen, S. and Coulson. A.R. (1977) Proc. Nail. Acad. Sci. USA 74, 5463-5467.
