- Email: [email protected]
Cloning of the rat and human mitochondrial branched chain aminotransferases (BCATm)1
Biochimica et Biophysica Acta 1339 Ž1997. 9–13
Short sequence-paper
Cloning of the rat and human mitochondrial branched chain aminotransferases ž BCATm / 1 Randy K. Bledsoe a , Paul A. Dawson
b,c
, Susan M. Hutson
a,)
a
Department of Biochemistry, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA Department of Internal Medicine, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA Department of ComparatiÕe Medicine, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA b
c
Received 6 December 1996; revised 27 February 1997; accepted 28 February 1997
Abstract The rat and human mitochondrial branched chain aminotransferase ŽBCATm . cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCATm is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCATm gene resides on chromosome 19 and the human cytosolic enzyme ŽBCATc . gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed. Keywords: Branched chain amino acid; Aminotransferase; cDNA; Chromosome 19; Chromosome 12; ŽRat.; ŽHuman.
Branched chain aminotransferases ŽEC 2.6.1.42. catalyze reversible transamination of the branched chain amino acids, leucine, isoleucine, valine, to their respective a-keto acids. In mammals there are two isoenzymes-mitochondrial ŽBCATm . and cytosolic ŽBCATc .. In the rat BCATm is the predominant isoenzyme, and it is found in almost all tissues while expression of the cytosolic form is restricted to brain, ovary and placenta w1,2x. Because of the importance of the mitochondrial isoenzyme in amino acid metabolism and whole body nitrogen shuttling, rat )
Corresponding author. Fax: q1 Ž910. 7167671. E-mail: [email protected] 1 The protein sequences reported in this paper have been submitted to the GenBank tm rEMBL Data Bank with the following accession numbers: rat BCATm , U68417; human BCATm , U68418; murine BCATm , U68526; porcine BCATm , U68527.
BCATm has been purified and characterized w3x. It has also been suggested that rat BCATm may have a second function w4x, and recently the yeast BCATm homolog has been identified as a suppressor of a temperature-sensitive mutation in the yeast mitochondrial ABC transporter w5x. In addition to their physiological significance, the bacterial BCAT and recently cloned rat BCATc appear to belong to a unique folding class of pyridoxal phosphate-dependent enzymes w6,7x. Therefore, to extend our study of the structure and function of these proteins, we have cloned the rat and human BCATm cDNAs and determined the chromosomal location of the human isoenzymes. Amino acid sequencing of purified rat heart BCATm was performed as described w7x. This allowed for determination of the first 32 amino acids of the mature protein and the sequence of six internal pep-
0167-4838r97r$17.00 Copyright q 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 4 4 - 7
10
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
tides. PCR was used to obtain a rat BCATm cDNA probe which was used to screen a rat heart lgt10 cDNA library ŽClontech, Palo Alto, CA.. Seven positive clones were isolated, but none extended to the initiator methionine. Therefore, the full-length BCATm cDNA was obtained using the inverse PCR procedure described by Zeiner and Gehring w8x. The
entire clone is 1548 base pairs in length with 5X and 3X untranslated regions of 7 and 362 base pairs, respectively. The nucleotide sequence of the BCATm cDNA and the deduced amino acid sequence are shown in Fig. 1. Northern blot analysis performed with polyŽA.q RNA from adult rat heart revealed a single mRNA transcript of approximately 1.7 kilo-
Fig. 1. Nucleotide and predicted amino acid sequence of rat BCATm . The nucleotides and amino acids are numbered on the right of the sequence. Amino acid 1 Žboxed. is the first residue of the mature protein. The peptide sequences determined by direct amino terminal sequencing or sequencing of internal peptides obtained after Lys-C digestion of the purified protein are underlined. An asterisk denotes the stop codon, and the polyadenylation signal is underlined twice.
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
bases, therefore the cDNA is almost full-length. The predicted initiator methionine was the only methionine residue upstream of the N-terminal valine of the mature protein that was determined by direct sequencing of the purified enzyme. The open reading frame encodes a mature protein of 366 amino acids that is preceded by a presequence of 27 amino acids. This presequence displays features that are characteristic of other mitochondrial targeting sequences w9x; cleavage occurs between a cysteine and valine residue and is preceded by an arginine at the y2 position. The calculated molecular mass of the mature protein is 41 227 Da, a value that is in excellent agreement with the molecular mass of 41.5 kDa determined by SDS-PAGE w4x. To demonstrate that the rat BCATm cDNA encodes a protein that is proteolytically processed to a functionally active enzyme, the complete cDNA was ligated into a pCMV5 expression vector ŽpBCATm . and transfected into COS-1 cells w7x. As shown in the immunoblot in Fig. 2, a protein of 41.2 kDa, which co-migrated with BCATm in a rat heart mitochondrial extract, was readily detected in the pBCATm transfected COS-1 cells. This indicated that proteolytic processing of the N-terminal targeting sequence had occurred. The calculated molecular mass of the unprocessed rat BCATm protein is 44.3 kDa. The COS-1 cell extract exhibited BCAT activity which was 6-fold higher than in the rat heart mitochondrial extract and 56-fold higher than the mock Žp b Gal. transfected cells Žsee Fig. 2.. Therefore, the data indicate that BCATm is expressed and proteolytically processed in COS-1 cells. Using the rat BCATm nucleotide sequence to search the available protein and nucleic acid data bases, the human, murine and porcine homologues were identified as expressed sequence tags Žsee Fig. 3.. The human cDNA Ž accession number R13964. was obtained and sequenced on both strands. The putative N-terminus of the mature proteins was deduced from alignment with the known rat BCATm sequence Ž see Fig. 3.. The mature human BCATm protein consists of 365 amino acids with a calculated molecular mass of 41 318 Da. Another human BCATm amino acid sequence has been reported w10x that differs from the sequence in Fig. 3 in that it lacks the presequence and first 13 amino acids of the mature polypeptide. There are also 11 amino acid changes in the remainder of
11
Fig. 2. Expression of rat BCATm in transfected COS-1 cells. Cell extracts Ž5 m g. from pCMV-b-galactosidase-transfected COS-1 cells ŽpbGal., pBCATm transfected COS-1 cells ŽpBCATm ., and a rat heart mitochondrial extract Ž20 m g. ŽHM. were subjected to SDS-PAGE analysis and immunoblotted as described in w7x. BCAT activity was measured in the extracts. Activity is displayed as munitsrmg of protein and represents the means and standard deviations, ns 3. A milliunit is defined as one nmol of valine formed per min at 378C.
the protein sequence including 4 nonconservative changes. These changes deviate from the general consensus sequence of the mammalian BCATm and may represent sequencing errors or polymorphisms. All the mammalian BCATm contained a conserved presequence of 27 amino acids. The mature rat sequence was 82% and 95% identical with the human and murine BCATm , but only 45% identical to the lower eukaryotic BCATm sequence from yeast which also has a smaller targeting sequence of 16 amino acids w5,7x. The yeast BCATm and BCATc isoenzymes are also more similar to each other Ž77% identity. than the mammalian isoenzyme pairs Ž58– 59% identical.. Since it is now possible to identify the eukaryotic isoenzymes as cytosolic or mitochondrial, use of the more descriptive designations BCATc and BCATm for these proteins instead of Bat1, Bat2 w5x, Eca39, Eca40 w10x would avoid confusion in the literature. The BCAT w7x are members of a unique folding class of pyridoxal phosphate-dependent enzymes w6x that includes 4-amino-4-deoxychorismate lyase and D-amino acid aminotransferase ŽD-AAT.. Primary sequence comparisons of mammalian BCATm and these enzymes revealed 24–26% sequence identity with D-AAT from B. subtilis and 18–19% identity with the lyase from Escherichia coli. In spite of the low sequence identity, all six residues ŽD-AAT Arg-
12
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
50, Arg-138, K-145, E-177, N-182, F-184. conserved in the Fold type IV enzymes and shown to be involved in cofactor binding in D-AAT w11x are found in the BCATm sequence alignment Žsee Fig. 3.. Analysis of somatic cell hybrids made by fusion of a Chinese hamster cell line lacking BCAT activity with several human cell lines originally established the presence of two human genes for the BCAT which were tentatively assigned to chromosomes 12 and 19 w12x. Indeed, fibroblasts we obtained from a patient with Pallister Killian syndrome containing qiŽ12p. mosiacism w13x exhibited significantly higher BCAT activity than that found in normal fibroblasts, 85.1 " 15.4 versus 29.8 " 4.1 milliunitsrmg protein, respectively. Availability of the human BCATm and BCATc Žaccession number U21551. sequences now
permits definitive assignment of the chromosomal location of each BCAT isoenzyme gene. Southern blot analysis of genomic DNA from human-rodent somatic cell hybrids was performed using the human BCATm cDNA. Three bands of ; 3.9, 3.4 and 1.5 kb were detected in the human genomic DNA Žsee Fig. 4, lanes 2 and 16. but not in the hamster Ž lanes 1 and 15. or murine Žlane 13. DNA. The human-specific BCATm hybridization pattern was observed in six of the human-rodent somatic cell hybrids Žlanes 8, 10, 11, 20, 24, 27.. Correlation between the hybridization pattern and human chromosome content revealed discordance for every chromosome except 19. The pattern is consistent with a single BCATm gene on chromosome 19. The same analysis using the human BCATc cDNA was consistent with assignment of this
Fig. 3. Alignment of the complete rat, human, and partial murine and porcine BCATm proteins. The predicted cleavage site of the mitochondrial targeting sequence is indicated by an arrow Ž≠.. Amino acids conserved in the Fold type IV class of PLP-dependent enzymes w6x are boxed. The putative pyridoxal phosphate binding lysine residue is shown with a star Žw..
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
13
model for this BCAT isoenzyme and determination of its role in amino acid metabolism. The authors would like to thank Ann Craddock for assistance with the COS-1 cell transfections, and Dr. Debra Saxe for providing the fibroblast cell lines. This work was supported by Grant DK-34738 from the National Institutes of Health ŽS.M.H.. and in part by USDA Grant 95-37200-1651 Ž S.M.H.. . P.A.D. is an American Gastroenterology AssociationrJanssen Pharmaceutical Research Scholar.
References
Fig. 4. Southern blot analysis of rodent-human somatic cell hybrids. A filter containing HindIII-digested somatic cell hybrid DNA ŽBIOS Somatic Cell Hybrid Southern blot a2645. was hybridized with a 1252 bp w 32 Px-labeled random hexamer-primed human BCATm probe for 16 h in aqueous BIOS speed hybridization solution I. The blot was washed in 0.5=SSC containing 0.5% SDS and exposed to Kodak Biomax MR film.
gene to chromosome 12. In accordance with the HUGOrGDB Nomenclature Committee’s recommendations, these genes have been designated BCAT1 Žcytosolic. and BCAT2 Žmitochondrial.. In summary, cloning of the rat and human BCATm provides the tools for development of a molecular
w1x S.M. Hutson, J. Nutr. 118 Ž1988. 1475–1481. w2x A. Ichihara, in: P. Christen, D.E. Metzler ŽEds.., Transaminases, Vol. 2, John Wiley and Sons, New York, 1985, pp. 430–438. w3x R. Wallin, T.R. Hall, S.M. Hutson, J. Biol. Chem. 265 Ž1990. 6019–6024. w4x S.M. Hutson, T.R. Hall, J. Biol. Chem. 268 Ž1993. 3084– 3091. w5x G. Kispal, H. Steiner, D.A. Court, B. Rolinski, R. Lill, J. Biol. Chem. 271 Ž1996. 24458–24464. w6x N.V. Grishin, M.A. Phillips, E.J. Goldsmith, Protein Sci. 4 Ž1995. 1291–1304. w7x S.M. Hutson, R.K. Bledsoe, T.R. Hall, P.A. Dawson, J. Biol. Chem. 270 Ž1995. 30344–30352. w8x M. Zeiner, U. Gehring, Biotechniques 17 Ž1994. 1050–1052. w9x F.U. Hartl, N. Pfanner, D.W. Nicholson, W. Neupert, Biochim. Biophys. Acta 988 Ž1989. 1–45. w10x A. Eden, G. Simchen, N. Benvenisty, J. Biol. Chem. 271 Ž1996. 20242–20245. w11x S. Sugio, G.A. Petsko, J.M. Manning, K. Soda, D. Ringe, Biochemistry 34 Ž1995. 9661–9669. w12x S.L. Naylor, T.B. Shows, Somat. Cell Genet. 6 Ž1980. 641–652. w13x J.H. Priest, J.M. Rust, P.M. Fernhoff, Am. J. Med. Genet. 42 Ž1992. 820–824.
Short sequence-paper
Cloning of the rat and human mitochondrial branched chain aminotransferases ž BCATm / 1 Randy K. Bledsoe a , Paul A. Dawson
b,c
, Susan M. Hutson
a,)
a
Department of Biochemistry, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA Department of Internal Medicine, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA Department of ComparatiÕe Medicine, Wake Forest UniÕersity Medical Center, Winston-Salem, NC 27157, USA b
c
Received 6 December 1996; revised 27 February 1997; accepted 28 February 1997
Abstract The rat and human mitochondrial branched chain aminotransferase ŽBCATm . cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCATm is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCATm gene resides on chromosome 19 and the human cytosolic enzyme ŽBCATc . gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed. Keywords: Branched chain amino acid; Aminotransferase; cDNA; Chromosome 19; Chromosome 12; ŽRat.; ŽHuman.
Branched chain aminotransferases ŽEC 2.6.1.42. catalyze reversible transamination of the branched chain amino acids, leucine, isoleucine, valine, to their respective a-keto acids. In mammals there are two isoenzymes-mitochondrial ŽBCATm . and cytosolic ŽBCATc .. In the rat BCATm is the predominant isoenzyme, and it is found in almost all tissues while expression of the cytosolic form is restricted to brain, ovary and placenta w1,2x. Because of the importance of the mitochondrial isoenzyme in amino acid metabolism and whole body nitrogen shuttling, rat )
Corresponding author. Fax: q1 Ž910. 7167671. E-mail: [email protected] 1 The protein sequences reported in this paper have been submitted to the GenBank tm rEMBL Data Bank with the following accession numbers: rat BCATm , U68417; human BCATm , U68418; murine BCATm , U68526; porcine BCATm , U68527.
BCATm has been purified and characterized w3x. It has also been suggested that rat BCATm may have a second function w4x, and recently the yeast BCATm homolog has been identified as a suppressor of a temperature-sensitive mutation in the yeast mitochondrial ABC transporter w5x. In addition to their physiological significance, the bacterial BCAT and recently cloned rat BCATc appear to belong to a unique folding class of pyridoxal phosphate-dependent enzymes w6,7x. Therefore, to extend our study of the structure and function of these proteins, we have cloned the rat and human BCATm cDNAs and determined the chromosomal location of the human isoenzymes. Amino acid sequencing of purified rat heart BCATm was performed as described w7x. This allowed for determination of the first 32 amino acids of the mature protein and the sequence of six internal pep-
0167-4838r97r$17.00 Copyright q 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 4 4 - 7
10
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
tides. PCR was used to obtain a rat BCATm cDNA probe which was used to screen a rat heart lgt10 cDNA library ŽClontech, Palo Alto, CA.. Seven positive clones were isolated, but none extended to the initiator methionine. Therefore, the full-length BCATm cDNA was obtained using the inverse PCR procedure described by Zeiner and Gehring w8x. The
entire clone is 1548 base pairs in length with 5X and 3X untranslated regions of 7 and 362 base pairs, respectively. The nucleotide sequence of the BCATm cDNA and the deduced amino acid sequence are shown in Fig. 1. Northern blot analysis performed with polyŽA.q RNA from adult rat heart revealed a single mRNA transcript of approximately 1.7 kilo-
Fig. 1. Nucleotide and predicted amino acid sequence of rat BCATm . The nucleotides and amino acids are numbered on the right of the sequence. Amino acid 1 Žboxed. is the first residue of the mature protein. The peptide sequences determined by direct amino terminal sequencing or sequencing of internal peptides obtained after Lys-C digestion of the purified protein are underlined. An asterisk denotes the stop codon, and the polyadenylation signal is underlined twice.
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
bases, therefore the cDNA is almost full-length. The predicted initiator methionine was the only methionine residue upstream of the N-terminal valine of the mature protein that was determined by direct sequencing of the purified enzyme. The open reading frame encodes a mature protein of 366 amino acids that is preceded by a presequence of 27 amino acids. This presequence displays features that are characteristic of other mitochondrial targeting sequences w9x; cleavage occurs between a cysteine and valine residue and is preceded by an arginine at the y2 position. The calculated molecular mass of the mature protein is 41 227 Da, a value that is in excellent agreement with the molecular mass of 41.5 kDa determined by SDS-PAGE w4x. To demonstrate that the rat BCATm cDNA encodes a protein that is proteolytically processed to a functionally active enzyme, the complete cDNA was ligated into a pCMV5 expression vector ŽpBCATm . and transfected into COS-1 cells w7x. As shown in the immunoblot in Fig. 2, a protein of 41.2 kDa, which co-migrated with BCATm in a rat heart mitochondrial extract, was readily detected in the pBCATm transfected COS-1 cells. This indicated that proteolytic processing of the N-terminal targeting sequence had occurred. The calculated molecular mass of the unprocessed rat BCATm protein is 44.3 kDa. The COS-1 cell extract exhibited BCAT activity which was 6-fold higher than in the rat heart mitochondrial extract and 56-fold higher than the mock Žp b Gal. transfected cells Žsee Fig. 2.. Therefore, the data indicate that BCATm is expressed and proteolytically processed in COS-1 cells. Using the rat BCATm nucleotide sequence to search the available protein and nucleic acid data bases, the human, murine and porcine homologues were identified as expressed sequence tags Žsee Fig. 3.. The human cDNA Ž accession number R13964. was obtained and sequenced on both strands. The putative N-terminus of the mature proteins was deduced from alignment with the known rat BCATm sequence Ž see Fig. 3.. The mature human BCATm protein consists of 365 amino acids with a calculated molecular mass of 41 318 Da. Another human BCATm amino acid sequence has been reported w10x that differs from the sequence in Fig. 3 in that it lacks the presequence and first 13 amino acids of the mature polypeptide. There are also 11 amino acid changes in the remainder of
11
Fig. 2. Expression of rat BCATm in transfected COS-1 cells. Cell extracts Ž5 m g. from pCMV-b-galactosidase-transfected COS-1 cells ŽpbGal., pBCATm transfected COS-1 cells ŽpBCATm ., and a rat heart mitochondrial extract Ž20 m g. ŽHM. were subjected to SDS-PAGE analysis and immunoblotted as described in w7x. BCAT activity was measured in the extracts. Activity is displayed as munitsrmg of protein and represents the means and standard deviations, ns 3. A milliunit is defined as one nmol of valine formed per min at 378C.
the protein sequence including 4 nonconservative changes. These changes deviate from the general consensus sequence of the mammalian BCATm and may represent sequencing errors or polymorphisms. All the mammalian BCATm contained a conserved presequence of 27 amino acids. The mature rat sequence was 82% and 95% identical with the human and murine BCATm , but only 45% identical to the lower eukaryotic BCATm sequence from yeast which also has a smaller targeting sequence of 16 amino acids w5,7x. The yeast BCATm and BCATc isoenzymes are also more similar to each other Ž77% identity. than the mammalian isoenzyme pairs Ž58– 59% identical.. Since it is now possible to identify the eukaryotic isoenzymes as cytosolic or mitochondrial, use of the more descriptive designations BCATc and BCATm for these proteins instead of Bat1, Bat2 w5x, Eca39, Eca40 w10x would avoid confusion in the literature. The BCAT w7x are members of a unique folding class of pyridoxal phosphate-dependent enzymes w6x that includes 4-amino-4-deoxychorismate lyase and D-amino acid aminotransferase ŽD-AAT.. Primary sequence comparisons of mammalian BCATm and these enzymes revealed 24–26% sequence identity with D-AAT from B. subtilis and 18–19% identity with the lyase from Escherichia coli. In spite of the low sequence identity, all six residues ŽD-AAT Arg-
12
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
50, Arg-138, K-145, E-177, N-182, F-184. conserved in the Fold type IV enzymes and shown to be involved in cofactor binding in D-AAT w11x are found in the BCATm sequence alignment Žsee Fig. 3.. Analysis of somatic cell hybrids made by fusion of a Chinese hamster cell line lacking BCAT activity with several human cell lines originally established the presence of two human genes for the BCAT which were tentatively assigned to chromosomes 12 and 19 w12x. Indeed, fibroblasts we obtained from a patient with Pallister Killian syndrome containing qiŽ12p. mosiacism w13x exhibited significantly higher BCAT activity than that found in normal fibroblasts, 85.1 " 15.4 versus 29.8 " 4.1 milliunitsrmg protein, respectively. Availability of the human BCATm and BCATc Žaccession number U21551. sequences now
permits definitive assignment of the chromosomal location of each BCAT isoenzyme gene. Southern blot analysis of genomic DNA from human-rodent somatic cell hybrids was performed using the human BCATm cDNA. Three bands of ; 3.9, 3.4 and 1.5 kb were detected in the human genomic DNA Žsee Fig. 4, lanes 2 and 16. but not in the hamster Ž lanes 1 and 15. or murine Žlane 13. DNA. The human-specific BCATm hybridization pattern was observed in six of the human-rodent somatic cell hybrids Žlanes 8, 10, 11, 20, 24, 27.. Correlation between the hybridization pattern and human chromosome content revealed discordance for every chromosome except 19. The pattern is consistent with a single BCATm gene on chromosome 19. The same analysis using the human BCATc cDNA was consistent with assignment of this
Fig. 3. Alignment of the complete rat, human, and partial murine and porcine BCATm proteins. The predicted cleavage site of the mitochondrial targeting sequence is indicated by an arrow Ž≠.. Amino acids conserved in the Fold type IV class of PLP-dependent enzymes w6x are boxed. The putative pyridoxal phosphate binding lysine residue is shown with a star Žw..
R.K. Bledsoe et al.r Biochimica et Biophysica Acta 1339 (1997) 9–13
13
model for this BCAT isoenzyme and determination of its role in amino acid metabolism. The authors would like to thank Ann Craddock for assistance with the COS-1 cell transfections, and Dr. Debra Saxe for providing the fibroblast cell lines. This work was supported by Grant DK-34738 from the National Institutes of Health ŽS.M.H.. and in part by USDA Grant 95-37200-1651 Ž S.M.H.. . P.A.D. is an American Gastroenterology AssociationrJanssen Pharmaceutical Research Scholar.
References
Fig. 4. Southern blot analysis of rodent-human somatic cell hybrids. A filter containing HindIII-digested somatic cell hybrid DNA ŽBIOS Somatic Cell Hybrid Southern blot a2645. was hybridized with a 1252 bp w 32 Px-labeled random hexamer-primed human BCATm probe for 16 h in aqueous BIOS speed hybridization solution I. The blot was washed in 0.5=SSC containing 0.5% SDS and exposed to Kodak Biomax MR film.
gene to chromosome 12. In accordance with the HUGOrGDB Nomenclature Committee’s recommendations, these genes have been designated BCAT1 Žcytosolic. and BCAT2 Žmitochondrial.. In summary, cloning of the rat and human BCATm provides the tools for development of a molecular
w1x S.M. Hutson, J. Nutr. 118 Ž1988. 1475–1481. w2x A. Ichihara, in: P. Christen, D.E. Metzler ŽEds.., Transaminases, Vol. 2, John Wiley and Sons, New York, 1985, pp. 430–438. w3x R. Wallin, T.R. Hall, S.M. Hutson, J. Biol. Chem. 265 Ž1990. 6019–6024. w4x S.M. Hutson, T.R. Hall, J. Biol. Chem. 268 Ž1993. 3084– 3091. w5x G. Kispal, H. Steiner, D.A. Court, B. Rolinski, R. Lill, J. Biol. Chem. 271 Ž1996. 24458–24464. w6x N.V. Grishin, M.A. Phillips, E.J. Goldsmith, Protein Sci. 4 Ž1995. 1291–1304. w7x S.M. Hutson, R.K. Bledsoe, T.R. Hall, P.A. Dawson, J. Biol. Chem. 270 Ž1995. 30344–30352. w8x M. Zeiner, U. Gehring, Biotechniques 17 Ž1994. 1050–1052. w9x F.U. Hartl, N. Pfanner, D.W. Nicholson, W. Neupert, Biochim. Biophys. Acta 988 Ž1989. 1–45. w10x A. Eden, G. Simchen, N. Benvenisty, J. Biol. Chem. 271 Ž1996. 20242–20245. w11x S. Sugio, G.A. Petsko, J.M. Manning, K. Soda, D. Ringe, Biochemistry 34 Ž1995. 9661–9669. w12x S.L. Naylor, T.B. Shows, Somat. Cell Genet. 6 Ž1980. 641–652. w13x J.H. Priest, J.M. Rust, P.M. Fernhoff, Am. J. Med. Genet. 42 Ž1992. 820–824.