Gene, 109 (1991) 265-267 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50
265
GENE 06187
Nucleotide sequence of a cDNA encoding rat brain carbonic anhydrase II and its deduced amino acid sequence * (Recombinant DNA; molecular cloning; restriction enzyme site; amino acid conservation; catalysis; isozymes)
Catherine A. Stoile, Michelle H. McGowan, Ruth A. Heim, Monica Varia and Judith A. Neubauer Department of Medicine, Universityof Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903 (U.S.A.) Tel. (908)937-7840
Received by S.T. Case: 7 May 1991 Revised/Accepted: 29 July/7 August 1991 Received at publishers: 23 September 1991
SUMMARY A carbonic anhydrase II (CAll)-encoding cDNA clone was isolated from a rat brain 2gtl 1 library. The 1459-bp cDNA codes for 260 amino acids with sequence similarity to mouse and human CAll and hybridizes to a single 1.7-kb mRNA.
INTRODUCTION
EXPERIMENTAL AND DISCUSSION
Carbonic anhydrase (CA; EC 4.2.1.1; carbonate dehydratase) is a monomeric zinc metalloenzyme that catalyses the reversible hydration of carbon dioxide (Tashian, 1989). In animals, this highly efficient enzyme is encoded by a muitigene family that yields at least seven isozymes that exhibit characteristic properties and tissue distributions. The CAll is the most widely distributed isozyme of this family.
(a) Ooning the CAH eDNA To facilitate studies on the function and synthesis of CAll in neuronal cells, we isolated and sequenced a eDNA clone encoding rat CAll. Approx. 105 recombinant phage plaques from a rat brain 2gtll cDNA library (Clontech) were screened by hybridization with a nick-translated human CAH cDNA probe (Murakami et al., 1987). A 1.4-kb cDNA insert from one of five hybridization positive clones was excised from the bacteriophage vector by EcoRI, subcloned into a double-stranded plasmid, and sequenced. The sequencing strategy and a restriction enzyme map of the clone are presented in Fig. 1.
Correspondenceto: Dr. C.A. Stolle, Depar:ment of Medicine, UMDNJRobert Wood Johnson Medical School~675 Hoes La., Piscataway, NJ 08854 (U.S.A.) "lei. (908)463-4053; Fax (908)463-4288. * On request, the authors will~upplydetailed experimentalevidence for the sequencing results in this Short Communication.
Abbreviations: aa, an~inoacid(s); bp, base pair(s); CA, carbonic anhydrase; CA, gene (DNA) encoding CA; cDNA, DNA complementaryto RNA; kb, kilobase(s) or 1000bp; nt, nucleotide(s); oligo,oligodeoxyribonucleotide; UTR, untranslated region.
(b) Sequence of the C A H eDNA The eDNA sequence consists of 8 bp of 5'-flanking sequence, the complete protein-coding region, and a 668-bp 3'-UTR containing a translation stop codon and two polyadenylation signals (Fig. 2). Comparison of the nt sequence of the translated region of the rat clone to other mammalian CAH eDNA sequences revealed 93 % similarity to mouse (Venta etal., 1985) and 81% similarity to human
266 3' UTR
CAH
5'
A.
( M u r a k a m i et al., 1987) CAH. B y contrast, c o m p a r i s o n o f
this s e q u e n c e to the c o d i n g region o f t h e m o u s e C A I ( F r a s e r
I
a n d Curtis, 1986) and CAI!! (Tweedie a n d E d w a r d s , 1989) c D N A s r e v e a l e d o n l y 6 4 % a n d 6 3 % similarity, respec-
4
q
4
B.
,
q
,
>
4
( 4
t
,
k
tively, s u g g e s t i n g that the rat brain c l o n e e n c o d e s the CAll
= C.
,,o,
D-
I
~.
I
I
I
~
5(
I
'
0
I
0.5
'
'
isozyme. ,
'
,
I
I
1.0
1.5 kb
. Fig. I. Schematic diagram of the rat CAH eDNA clone indicating: (A) relative sizes of the Y-flanking region (5', thin line), the translated region (open box), and the Y-UTR (3' UTR, thin line); (B) overlapping sequence obtained with synthetic oligo primers; (C) restriction enzyme map ofcaH eDNA; and (D) scale in kb.
1
I 61 19
GCGTGACTATGTCCCACCACTGGGGATACAGCAAGAGCAACGGACCAGAGAACTGGCACA M ;~ H H W G ~ S K S H G P E N W H K AGGAGTTCCCCATTGCCAATGGAGACCGACAGTCCCCTGTGGACATTGACACCGGGACTG E
F
I:) I
A
N
G
D
R
Q
~
P
V
D
I
D
T
G
T
A
121 39
CCCAGCATOACCCTTCCCTACAGCCTCTGCTCATATGTTACGATAAGGTTGCTTCCAAGA Q H D P S L Q P L L I C Y D K V A S K S
181 s,
GCATTGTC~,ACAATGGCCATTCCTTCAACGTTGAGTTTGATGACTCCCAGGACTTTG CAG , v ® ® Q ® ® ® ® v ® v D D s Q D F ^ v
241 ~9
TGC"rGAAAGAGGGACCCCTCAGTGGCTCCTACAGATTGATCCAGTTTCACTTTCACTGGG L ,~ E ~ P ,. s c s Y ,~ , . ® ® F ® F ® w G
301 99
G C T C A T C T G A T G G C C A G G G C T C T G A G C A C A C C G T G A A C A A A A ~ T A T G C T G CAGAGC s s . c o G s ® ® T v . K . . Y A ,,®,.
361 i~9
TT__CACTTGG_TTCACTGGAACACCAAATATGGGGATTTTGGAAAAGCTGTGCAGCACCCAG ® T . ® . . . T ~ Y G D ~C K a V O " P D
421 139
A T G G A C T G G C T ~ T T T T G G G T A T T T T ~ G A A G A T T G G A C C T G CCTCACAAGGCCTTCAGA G @ A @ L @ I F L R I G P A S Q G L Q K
481
AAATCACTGAAGCACTGCATTCCATTAAAACAAAGGGGAAACGTGCAGCCTTTGCTAACT l T E k L H S I K T K G K R A A F k . F
159 541
.9
TTGATeCTTGCTCCCTTCTTCCTGGAAACTTGGACTACTGGACATATCCTGGCTCTCTGA D ,, c s ,~ ,. ,, a . L D Y ® . ® , , ~ s®®
661
GCAGTGAGCAGATGTCTCATTTCCGTAAACTGAACTTCAATTCGGAGGGGGAGGCTGAAG
(c) Analysis of the nt and aa sequences Analysis of the deduced aa sequence supports the identity of the rat brain clone as a CAH eDNA. The translated region of rat brain eDNA specifies 260 aa with a calculated Mr of 29 081, consistent with previously characterized CAll isozymes from other species (Deutsch, 1987). In addition, the aa sequence exhibits conservation of those residues in CA isozymes thought to be involved in active site formation or Zn 2+ binding (Fig. 2; Tashian, 1989). Futhermore, a comparison between the aa sequence deduced from rat brain CAH eDNA and aa that are distinct for a particular CA isozyme (Hewett-Emmett et al., 1985) reveals that it is most similar to the CAll isozyme (Table I). Northern-blot analysis of total RNA from rat brain hybridized to nick-translated rat CAH eDNA revealed a single 1.7-kb mRNA (Fig. 3), similar in size to human call mRNA (Murakami et al., 1987) and human CAIII mRNA
M
B
O!: 219
S
E
Q
H
S
H
F
R
K
L
N
F
N
S
E
G
E
A
E
E
721 239
AACTGATGGTGGACAAACTGC~CGTCeAGCTCAGCCGCTGAAOAACAGAAAGATCAAGGeGT L M V D (~ W ~ P A Q e L K N R K I K A S
781
CCTTTAAATAAAATGACCCTGCAGCTGGGGTCCAAAAAGCACAAGTGTGG CTGCCTCTCT F K *
259
841
GTAGCTAAGCACAGTTCCGCCTTGGTGATTCAGATeCCGACTTTGCATCTGATATTGTAG
901
GCCTTTTTACCTCTCACCCATTGTGCTTACT~TGTGAAAAGGAAGACCCAGGTG
961
TCTCATGTGGTGGTAGCATGGTGGCAGGCTGGTGGTTGACTTAGGGCATCCTTTCTCAGC
1021
CACAACAATGCAATGCAAAGAACAGATATGGCCTCTTGCTTCTCCACAGCCATAGAATAA
1081
TGAGTACTCAGGCCTGTTTATTAAAATOCTATTTTTAAAACCATATAAGGTAGAATGATT
1141
GTTTACAAATCCACATCATGAGACAAACTGAGG CAATTTAGGCAAATCAGGTAAAACAGT
1201
CATAGTTTTATGGTTATTAATTAGATGAATGTTCACTATTCCAAGATCTTATATTAAAGA
1261
AAAACTTTTAAAAAGCTTATATATTTGTAGCAAAGTTATTCTTAAATATGAATTATGTTG
kb ..
9.5 7.S
---
4.4
--
2.4
--
1.4
--
0.24
--
,
1323. TAACTTAGTGACTTTTGATTTCTAGAGGTGTAAATGAAGATGTAAAAATTGATATAGTTG 1381 1441
TGATACAGAGTATATTTCCCTTCAGATAACTTACCATAACCTAATGGATAATGTATTTTA GATATATTCTCTAATAAAA - 1459
Fig The nt sequence of rat CAll cDNA and deduced aa sequence. Douale-stranded plasmid DNA containing the rat CAll eDNA was alkali denatured (Chen and Seeburg, 1985) and sequenced by the dideoxy method (Sanger et al., 1977) using [~SS]dATP and Sequenase (U.S. Biochemicals, Cleveland, OH). Synthetic oligos were generated to a newly derived sequence with an Applied Biosystems 381A DNA synthesizer and used to prime overlapping sequences. The sequence ofboth DNA strands was confirmed by multiple reactions. The deduced aa sequence is indicated below the corresponding codons. The aa thought to be involved in active site formation or Zn 2 + binding (Tashian, 1989) are circled. The translation stop codon is marked with an asterisk; polyadenylation signals are underfined. EMBL accession No. is X58294.
Fig. 3. Northern-blot analysis of CAH mRNA in rat brain. Total RNA was isolated from rat brain by the method ofChirgwin et al. (1979). RNA (15 #g) was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde, transferred by blotting to a nitro-cellulose filter, and hybridized (Thomas, 1980) using a nick-translated rat CAH eDNA probe. RNA molecular size markers (Bethesda Research Laboratories) electrophoresed according to the manufacturer's instructions were visualized by hybridization of the nitrocellulose filter to nick-translated phage ;t DNA. M, RNA molecular size markers; B, rat brain total RNA.
267 TABLE I
H L 3 3 9 3 8 a n d A R 4 0 5 5 6 ) a n d t h e C a r d i o v a s c u l a r Institute
Comparison of aa residues in rat CAll to invariant residues in CA isozymes from several species
of UMDNJ.
CA isozyme
CAI CAll CAIll
Number of invariant residues a
18 15 40
Number of rat residues identical with invariant residues of other CA isozymes ! 14b 3
a From Hewett-Emmett et al. (1985). b These are at positions 3, 4, 27, 56, 67, 69, 112, 125, 128, 129, 137, 165, 229 and 230 in Fig. 2.
(Lloyd et al., 1986). Since the rat CAll cDNA is only 1.46 kb, it apparently lacks approx. 240 nt of the mRNA. Nevertheless, this cDNA clone represents the complete coding region of rat CAll, provides sequence information not previously available for this protein, and will be a useful reagent for analyzing the regulated expression of the CAll gene in neuronal cell subpopulations.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr. William Sly for the human CAH cDNA probe, Ms. Hyeon-Gyu Shin for excellent technical assistance, and Ms. Marcella Spioch for preparation of the manuscript. This research was supported by grants from the National Institutes of Health (HL 16022,
REFERENCES Chen, E.Y. and Seeburg, P.H.: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4 (1985) 165-170. Chirgwin, J.M., Przybyla, A.E., MacDonald, RJ. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18 (1979) 5294-5301. Deutsch, H.F.: Carbonic anhydrases. Int. J. Biochem. 19 (1987) 101-113. Fraser, P.J. and Curtis, P.J.: Molecular evolution of the carbonic anhydrase genes: calculation of divergence time for mouse carbonic anhydrase I and II. J. Mol. Evol. 23 (1986) 294-299. Hewett-Emmett, D., Hopkins, PJ., Tashian, R.E. and Czelusniak, J.: Origins and molecular evolution of the carbonic anhydrase isozymes. Ann. NY Acad. Sci. 429 (1985) 338-358. Lloyd, J., McMillan, S., Hopkinson, D. and Edwards, Y.H.: Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase. Gene 41 (1986) 233-239. Murakami, H., Marelich, G.P., Grubb, J.H., Kyle, J.W. and Sly, W.S.: Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II. Genomics ! (1987) 159-166, Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Tashian, R.E.: The carbonic anhydrases: widening perspectives on their evolution, expression and function. BioEssays 10 (1989) 186-192. Thomas, P.S.: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77 (1980) 5201-5205. Tweedie, S. and Edwards, Y.: Mouse carbonic anhydrase Ill: nucleotide sequence and expression studies. Biochem. Genet. 27 (1989) 17-30. Venta, P.J., Montgomery, J.C., Hewett-Emmett, D., Wiebauer, K. and Tashian, R.E.: Structure and exon to protein domain relationships of the mouse carbonic anhydrase I1 gene. J. Biol. Chem. 260 (1985) 12130-12135.
265
GENE 06187
Nucleotide sequence of a cDNA encoding rat brain carbonic anhydrase II and its deduced amino acid sequence * (Recombinant DNA; molecular cloning; restriction enzyme site; amino acid conservation; catalysis; isozymes)
Catherine A. Stoile, Michelle H. McGowan, Ruth A. Heim, Monica Varia and Judith A. Neubauer Department of Medicine, Universityof Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903 (U.S.A.) Tel. (908)937-7840
Received by S.T. Case: 7 May 1991 Revised/Accepted: 29 July/7 August 1991 Received at publishers: 23 September 1991
SUMMARY A carbonic anhydrase II (CAll)-encoding cDNA clone was isolated from a rat brain 2gtl 1 library. The 1459-bp cDNA codes for 260 amino acids with sequence similarity to mouse and human CAll and hybridizes to a single 1.7-kb mRNA.
INTRODUCTION
EXPERIMENTAL AND DISCUSSION
Carbonic anhydrase (CA; EC 4.2.1.1; carbonate dehydratase) is a monomeric zinc metalloenzyme that catalyses the reversible hydration of carbon dioxide (Tashian, 1989). In animals, this highly efficient enzyme is encoded by a muitigene family that yields at least seven isozymes that exhibit characteristic properties and tissue distributions. The CAll is the most widely distributed isozyme of this family.
(a) Ooning the CAH eDNA To facilitate studies on the function and synthesis of CAll in neuronal cells, we isolated and sequenced a eDNA clone encoding rat CAll. Approx. 105 recombinant phage plaques from a rat brain 2gtll cDNA library (Clontech) were screened by hybridization with a nick-translated human CAH cDNA probe (Murakami et al., 1987). A 1.4-kb cDNA insert from one of five hybridization positive clones was excised from the bacteriophage vector by EcoRI, subcloned into a double-stranded plasmid, and sequenced. The sequencing strategy and a restriction enzyme map of the clone are presented in Fig. 1.
Correspondenceto: Dr. C.A. Stolle, Depar:ment of Medicine, UMDNJRobert Wood Johnson Medical School~675 Hoes La., Piscataway, NJ 08854 (U.S.A.) "lei. (908)463-4053; Fax (908)463-4288. * On request, the authors will~upplydetailed experimentalevidence for the sequencing results in this Short Communication.
Abbreviations: aa, an~inoacid(s); bp, base pair(s); CA, carbonic anhydrase; CA, gene (DNA) encoding CA; cDNA, DNA complementaryto RNA; kb, kilobase(s) or 1000bp; nt, nucleotide(s); oligo,oligodeoxyribonucleotide; UTR, untranslated region.
(b) Sequence of the C A H eDNA The eDNA sequence consists of 8 bp of 5'-flanking sequence, the complete protein-coding region, and a 668-bp 3'-UTR containing a translation stop codon and two polyadenylation signals (Fig. 2). Comparison of the nt sequence of the translated region of the rat clone to other mammalian CAH eDNA sequences revealed 93 % similarity to mouse (Venta etal., 1985) and 81% similarity to human
266 3' UTR
CAH
5'
A.
( M u r a k a m i et al., 1987) CAH. B y contrast, c o m p a r i s o n o f
this s e q u e n c e to the c o d i n g region o f t h e m o u s e C A I ( F r a s e r
I
a n d Curtis, 1986) and CAI!! (Tweedie a n d E d w a r d s , 1989) c D N A s r e v e a l e d o n l y 6 4 % a n d 6 3 % similarity, respec-
4
q
4
B.
,
q
,
>
4
( 4
t
,
k
tively, s u g g e s t i n g that the rat brain c l o n e e n c o d e s the CAll
= C.
,,o,
D-
I
~.
I
I
I
~
5(
I
'
0
I
0.5
'
'
isozyme. ,
'
,
I
I
1.0
1.5 kb
. Fig. I. Schematic diagram of the rat CAH eDNA clone indicating: (A) relative sizes of the Y-flanking region (5', thin line), the translated region (open box), and the Y-UTR (3' UTR, thin line); (B) overlapping sequence obtained with synthetic oligo primers; (C) restriction enzyme map ofcaH eDNA; and (D) scale in kb.
1
I 61 19
GCGTGACTATGTCCCACCACTGGGGATACAGCAAGAGCAACGGACCAGAGAACTGGCACA M ;~ H H W G ~ S K S H G P E N W H K AGGAGTTCCCCATTGCCAATGGAGACCGACAGTCCCCTGTGGACATTGACACCGGGACTG E
F
I:) I
A
N
G
D
R
Q
~
P
V
D
I
D
T
G
T
A
121 39
CCCAGCATOACCCTTCCCTACAGCCTCTGCTCATATGTTACGATAAGGTTGCTTCCAAGA Q H D P S L Q P L L I C Y D K V A S K S
181 s,
GCATTGTC~,ACAATGGCCATTCCTTCAACGTTGAGTTTGATGACTCCCAGGACTTTG CAG , v ® ® Q ® ® ® ® v ® v D D s Q D F ^ v
241 ~9
TGC"rGAAAGAGGGACCCCTCAGTGGCTCCTACAGATTGATCCAGTTTCACTTTCACTGGG L ,~ E ~ P ,. s c s Y ,~ , . ® ® F ® F ® w G
301 99
G C T C A T C T G A T G G C C A G G G C T C T G A G C A C A C C G T G A A C A A A A ~ T A T G C T G CAGAGC s s . c o G s ® ® T v . K . . Y A ,,®,.
361 i~9
TT__CACTTGG_TTCACTGGAACACCAAATATGGGGATTTTGGAAAAGCTGTGCAGCACCCAG ® T . ® . . . T ~ Y G D ~C K a V O " P D
421 139
A T G G A C T G G C T ~ T T T T G G G T A T T T T ~ G A A G A T T G G A C C T G CCTCACAAGGCCTTCAGA G @ A @ L @ I F L R I G P A S Q G L Q K
481
AAATCACTGAAGCACTGCATTCCATTAAAACAAAGGGGAAACGTGCAGCCTTTGCTAACT l T E k L H S I K T K G K R A A F k . F
159 541
.9
TTGATeCTTGCTCCCTTCTTCCTGGAAACTTGGACTACTGGACATATCCTGGCTCTCTGA D ,, c s ,~ ,. ,, a . L D Y ® . ® , , ~ s®®
661
GCAGTGAGCAGATGTCTCATTTCCGTAAACTGAACTTCAATTCGGAGGGGGAGGCTGAAG
(c) Analysis of the nt and aa sequences Analysis of the deduced aa sequence supports the identity of the rat brain clone as a CAH eDNA. The translated region of rat brain eDNA specifies 260 aa with a calculated Mr of 29 081, consistent with previously characterized CAll isozymes from other species (Deutsch, 1987). In addition, the aa sequence exhibits conservation of those residues in CA isozymes thought to be involved in active site formation or Zn 2+ binding (Fig. 2; Tashian, 1989). Futhermore, a comparison between the aa sequence deduced from rat brain CAH eDNA and aa that are distinct for a particular CA isozyme (Hewett-Emmett et al., 1985) reveals that it is most similar to the CAll isozyme (Table I). Northern-blot analysis of total RNA from rat brain hybridized to nick-translated rat CAH eDNA revealed a single 1.7-kb mRNA (Fig. 3), similar in size to human call mRNA (Murakami et al., 1987) and human CAIII mRNA
M
B
O!: 219
S
E
Q
H
S
H
F
R
K
L
N
F
N
S
E
G
E
A
E
E
721 239
AACTGATGGTGGACAAACTGC~CGTCeAGCTCAGCCGCTGAAOAACAGAAAGATCAAGGeGT L M V D (~ W ~ P A Q e L K N R K I K A S
781
CCTTTAAATAAAATGACCCTGCAGCTGGGGTCCAAAAAGCACAAGTGTGG CTGCCTCTCT F K *
259
841
GTAGCTAAGCACAGTTCCGCCTTGGTGATTCAGATeCCGACTTTGCATCTGATATTGTAG
901
GCCTTTTTACCTCTCACCCATTGTGCTTACT~TGTGAAAAGGAAGACCCAGGTG
961
TCTCATGTGGTGGTAGCATGGTGGCAGGCTGGTGGTTGACTTAGGGCATCCTTTCTCAGC
1021
CACAACAATGCAATGCAAAGAACAGATATGGCCTCTTGCTTCTCCACAGCCATAGAATAA
1081
TGAGTACTCAGGCCTGTTTATTAAAATOCTATTTTTAAAACCATATAAGGTAGAATGATT
1141
GTTTACAAATCCACATCATGAGACAAACTGAGG CAATTTAGGCAAATCAGGTAAAACAGT
1201
CATAGTTTTATGGTTATTAATTAGATGAATGTTCACTATTCCAAGATCTTATATTAAAGA
1261
AAAACTTTTAAAAAGCTTATATATTTGTAGCAAAGTTATTCTTAAATATGAATTATGTTG
kb ..
9.5 7.S
---
4.4
--
2.4
--
1.4
--
0.24
--
,
1323. TAACTTAGTGACTTTTGATTTCTAGAGGTGTAAATGAAGATGTAAAAATTGATATAGTTG 1381 1441
TGATACAGAGTATATTTCCCTTCAGATAACTTACCATAACCTAATGGATAATGTATTTTA GATATATTCTCTAATAAAA - 1459
Fig The nt sequence of rat CAll cDNA and deduced aa sequence. Douale-stranded plasmid DNA containing the rat CAll eDNA was alkali denatured (Chen and Seeburg, 1985) and sequenced by the dideoxy method (Sanger et al., 1977) using [~SS]dATP and Sequenase (U.S. Biochemicals, Cleveland, OH). Synthetic oligos were generated to a newly derived sequence with an Applied Biosystems 381A DNA synthesizer and used to prime overlapping sequences. The sequence ofboth DNA strands was confirmed by multiple reactions. The deduced aa sequence is indicated below the corresponding codons. The aa thought to be involved in active site formation or Zn 2 + binding (Tashian, 1989) are circled. The translation stop codon is marked with an asterisk; polyadenylation signals are underfined. EMBL accession No. is X58294.
Fig. 3. Northern-blot analysis of CAH mRNA in rat brain. Total RNA was isolated from rat brain by the method ofChirgwin et al. (1979). RNA (15 #g) was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde, transferred by blotting to a nitro-cellulose filter, and hybridized (Thomas, 1980) using a nick-translated rat CAH eDNA probe. RNA molecular size markers (Bethesda Research Laboratories) electrophoresed according to the manufacturer's instructions were visualized by hybridization of the nitrocellulose filter to nick-translated phage ;t DNA. M, RNA molecular size markers; B, rat brain total RNA.
267 TABLE I
H L 3 3 9 3 8 a n d A R 4 0 5 5 6 ) a n d t h e C a r d i o v a s c u l a r Institute
Comparison of aa residues in rat CAll to invariant residues in CA isozymes from several species
of UMDNJ.
CA isozyme
CAI CAll CAIll
Number of invariant residues a
18 15 40
Number of rat residues identical with invariant residues of other CA isozymes ! 14b 3
a From Hewett-Emmett et al. (1985). b These are at positions 3, 4, 27, 56, 67, 69, 112, 125, 128, 129, 137, 165, 229 and 230 in Fig. 2.
(Lloyd et al., 1986). Since the rat CAll cDNA is only 1.46 kb, it apparently lacks approx. 240 nt of the mRNA. Nevertheless, this cDNA clone represents the complete coding region of rat CAll, provides sequence information not previously available for this protein, and will be a useful reagent for analyzing the regulated expression of the CAll gene in neuronal cell subpopulations.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr. William Sly for the human CAH cDNA probe, Ms. Hyeon-Gyu Shin for excellent technical assistance, and Ms. Marcella Spioch for preparation of the manuscript. This research was supported by grants from the National Institutes of Health (HL 16022,
REFERENCES Chen, E.Y. and Seeburg, P.H.: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4 (1985) 165-170. Chirgwin, J.M., Przybyla, A.E., MacDonald, RJ. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18 (1979) 5294-5301. Deutsch, H.F.: Carbonic anhydrases. Int. J. Biochem. 19 (1987) 101-113. Fraser, P.J. and Curtis, P.J.: Molecular evolution of the carbonic anhydrase genes: calculation of divergence time for mouse carbonic anhydrase I and II. J. Mol. Evol. 23 (1986) 294-299. Hewett-Emmett, D., Hopkins, PJ., Tashian, R.E. and Czelusniak, J.: Origins and molecular evolution of the carbonic anhydrase isozymes. Ann. NY Acad. Sci. 429 (1985) 338-358. Lloyd, J., McMillan, S., Hopkinson, D. and Edwards, Y.H.: Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase. Gene 41 (1986) 233-239. Murakami, H., Marelich, G.P., Grubb, J.H., Kyle, J.W. and Sly, W.S.: Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II. Genomics ! (1987) 159-166, Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Tashian, R.E.: The carbonic anhydrases: widening perspectives on their evolution, expression and function. BioEssays 10 (1989) 186-192. Thomas, P.S.: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77 (1980) 5201-5205. Tweedie, S. and Edwards, Y.: Mouse carbonic anhydrase Ill: nucleotide sequence and expression studies. Biochem. Genet. 27 (1989) 17-30. Venta, P.J., Montgomery, J.C., Hewett-Emmett, D., Wiebauer, K. and Tashian, R.E.: Structure and exon to protein domain relationships of the mouse carbonic anhydrase I1 gene. J. Biol. Chem. 260 (1985) 12130-12135.
