Biochimica et Biophysics Acta, 1220 (1994) 219-222 0 1994 Elsevier Science B.V. All rights reserved 0167-4889/94/$07.00 219 BBAMCR 10315 Short Sequence-Paper Isolation and sequence of a full length cDNA encoding a novel rat inositol 1,4,5trisphosphate 3-kinase Stephen Thomas a, Brigitte Brake b, J. Paul Luzio ‘, Keith Stanley d and George Banting a~* a Department of Biochemistry, University of Bristol, Bristol, BS8 1 TD (UK), b Institut fiir Arzeneimittel, Berlin (Germany), c Department of Clinical Biochemistry University of Cambridge, Cambridge (UK) and d Heart Research Institute, Sydney (Australia) (Received 10 August 1993) Key words: IP, 3kinase; cDNA, Nucleotide sequence; PCR, (Rat) Immunoscreening a rat liver cDNA expression library has led to the isolation of a full-length cDNA clone encoding a novel isoform of rat inositol 1,4,5trisphosphate 3-kinase (IP, 3-kinase). Sequence comparison shows it (i) to be 93% identical to human hippocampus IP, 3-kinase B over 468 residues at the protein level, and (ii) to encode a protein 204 amino acids larger than the published sequence of its human homologue. Immunoscreening of a rat liver cDNA expression library using a strategy designed to identify clones encoding organelle specific integral membrane proteins led to the isolation and identification of seven immuno- logically non cross-reactive groups of clones [ll. The library was constructed in the plasmid expression vec- tor pUEX [2] which allows (i) the regulated expression of in frame cDNA inserts as the carboxy-terminal portion of a P-galactosidase fusion protein, and (ii) direct double strand DNA sequencing. The sequences of full-length clones from two of these groups have been presented elsewhere and shown to encode the rat polymeric immunoglobulin receptor and the trans Golgi network (TGN) integral membrane protein TGN38 [1,3]. Two of the remaining groups of clones encode rat microsomal glutathione-S-transferase and the Hl sub- unit of the rat asialoglycoprotein receptor (Ref. 1; Luzio, Banting and Stanley, unpublished observations). We now show that clones in a fifth group encode a novel isoform of rat inositol 1,4,5_trisphosphate 3- kinase (IP, 3-kinase). This group originally comprised only two clones, designated GORF34 and GORF40. Double stranded plasmid DNA sequence analysis of these clones, using dideoxy chain termination sequencing with ‘Sequenase’ * Corresponding author. Fax: +44 272 288274. The nucleotide sequence data presented here have been submitted to the EMBL/Genbank database under the accession number X74227 RNIP3. (United States Biochemical) indicated that the entire sequence of GORF40 lies within the 1.2 kb of GORF34, and that neither contains sequences corresponding to the extreme 5’ end of the coding sequence. In order to obtain these sequences, a 401 bp BamHI fragment was excised from the 5’ end of the GORF34 insert (Fig. l), gel purified [4], labelled with 32P (Amersham, UK) by random priming [5] and used as probe in a DNA screen of 300,000 independent clones from the original rat liver cDNA library [1,6]. Five positive clones were isolated, and the longest (GORF34.1) chosen for fur- ther analysis. The entire sequence of the 3.05 kb insert was obtained in both directions by double strand DNA sequence analysis, and aligned with that of GORF34. The sequence of GORF34 lies within that of GORF34.1 (Fig. 1). The DNA sequence of GORF34 is identical to that of GORF34.1 except for base 1795 of the GORF34.1 sequence which is a cytosine in GORF34 and a thymine in GORF34.1 (Fig. 2). 667 1077 1832 GORF34 m region of GORFE.4 used as probe to ikohte GORF34.1 = coding regbi~ of GORF34 1 m noncoding region of GCflF34.1 Fig. 1. Cartoon indicating (i) the position of the GORF34 sequence within that of GORF34.1, and (ii) the position of the BamHI fragment from GORF34 used in the isolation of GORF34.1. Num- bers correspond to nucleotide positions in GORF34.1 SSDI 0167-4889(93)E0188-6
Transcript
Biochimica et Biophysics Acta, 1220 (1994) 219-222 0 1994 Elsevier Science B.V. All rights reserved 0167-4889/94/$07.00
219
BBAMCR 10315 Short Sequence-Paper
Isolation and sequence of a full length cDNA encoding a novel rat inositol 1,4,5trisphosphate 3-kinase
Stephen Thomas a, Brigitte Brake b, J. Paul Luzio ‘, Keith Stanley d and George Banting a~*
a Department of Biochemistry, University of Bristol, Bristol, BS8 1 TD (UK), b Institut fiir Arzeneimittel, Berlin (Germany), c Department of Clinical Biochemistry University of Cambridge, Cambridge (UK) and d Heart Research Institute, Sydney (Australia)
Immunoscreening a rat liver cDNA expression library has led to the isolation of a full-length cDNA clone encoding a novel isoform of rat inositol 1,4,5trisphosphate 3-kinase (IP, 3-kinase). Sequence comparison shows it (i) to be 93% identical to human hippocampus IP, 3-kinase B over 468 residues at the protein level, and (ii) to encode a protein 204 amino acids larger than the published sequence of its human homologue.
Immunoscreening of a rat liver cDNA expression library using a strategy designed to identify clones encoding organelle specific integral membrane proteins led to the isolation and identification of seven immuno- logically non cross-reactive groups of clones [ll. The library was constructed in the plasmid expression vec- tor pUEX [2] which allows (i) the regulated expression of in frame cDNA inserts as the carboxy-terminal portion of a P-galactosidase fusion protein, and (ii) direct double strand DNA sequencing. The sequences of full-length clones from two of these groups have been presented elsewhere and shown to encode the rat polymeric immunoglobulin receptor and the trans Golgi network (TGN) integral membrane protein TGN38 [1,3]. Two of the remaining groups of clones encode rat microsomal glutathione-S-transferase and the Hl sub- unit of the rat asialoglycoprotein receptor (Ref. 1; Luzio, Banting and Stanley, unpublished observations). We now show that clones in a fifth group encode a novel isoform of rat inositol 1,4,5_trisphosphate 3- kinase (IP, 3-kinase).
This group originally comprised only two clones, designated GORF34 and GORF40. Double stranded plasmid DNA sequence analysis of these clones, using dideoxy chain termination sequencing with ‘Sequenase’
* Corresponding author. Fax: +44 272 288274. The nucleotide sequence data presented here have been submitted to the EMBL/Genbank database under the accession number X74227 RNIP3.
(United States Biochemical) indicated that the entire sequence of GORF40 lies within the 1.2 kb of GORF34, and that neither contains sequences corresponding to the extreme 5’ end of the coding sequence. In order to obtain these sequences, a 401 bp BamHI fragment was excised from the 5’ end of the GORF34 insert (Fig. l), gel purified [4], labelled with 32P (Amersham, UK) by random priming [5] and used as probe in a DNA screen of 300,000 independent clones from the original rat liver cDNA library [1,6]. Five positive clones were isolated, and the longest (GORF34.1) chosen for fur- ther analysis. The entire sequence of the 3.05 kb insert was obtained in both directions by double strand DNA sequence analysis, and aligned with that of GORF34. The sequence of GORF34 lies within that of GORF34.1 (Fig. 1). The DNA sequence of GORF34 is identical to that of GORF34.1 except for base 1795 of the GORF34.1 sequence which is a cytosine in GORF34 and a thymine in GORF34.1 (Fig. 2).
667 1077 1832
GORF34
m region of GORFE.4 used as probe to ikohte GORF34.1
= coding regbi~ of GORF34 1
m noncoding region of GCflF34.1
Fig. 1. Cartoon indicating (i) the position of the GORF34 sequence within that of GORF34.1, and (ii) the position of the BamHI fragment from GORF34 used in the isolation of GORF34.1. Num-
bers correspond to nucleotide positions in GORF34.1
Since GORF34 had been isolated during an im- munoscreening strategy it was possible to identify the open reading frame encoding the protein product of this cDNA clone (i.e., it is that reading frame which is in frame with the @-gaIactosidase coding sequences in the pUEX vector). Conceptual translation of this read- ing frame in GORF34.1 indicated the presence of a ‘stop’ codon (TAG) within the open reading frame previously identified in GORF34. The thymine in this codon is the base which was observed to be a cytosine in GORF34; the CAG codon present in GORF34 encodes glutamine. Thus conceptual translation of the
2. DNA (upper line) and predicted protein (lower line) se- quences of GORF34.1. Numbers indicate nucleotide or amino acid positions in the sequences. The in frame ‘stop’ codon upstream of the initiator methionine is indicated by a solid bar. The methionine residue corresponding to that designated as the initial methionine residue in human IP, 3-kinase [Ill is indicated by a hatched box. The position of sequence ambiguity between GORF34 and GORF34.1 is indicated by an open box, and the positions corresponding to the oligonucleotide primers used in PCR analysis are indicated by arrows
above the DNA sequence.
GORF34.1 DNA sequence in the same reading frame as that used for GORF34 suggests that this clone encodes a truncated protein. A PCR experiment was performed in order to ascertain which of the two nucleotide sequences is present in rat liver mRNA. mRNA was isolated from rat liver by standard tech- niques [7], and 1 mg used as template in a reverse transcriptase (RT) PCR reaction using Tuq polymerase (Boehringer), previously published procedures [8] and oligonucleotide primers flanking the suspect base (Fig. 2). The RTPCR was designed to amplify a 165 bp
D NA fragment which would contain only one recogni- tion site for the restriction enzyme PvulI (CAGCTG) if it contained the 'stop' codon (TAGCTG)(giving frag- ments of 120 bp and 45 bp upon PvuII digestion), but two if it contained the glutamine codon (CAGCTG) (giving fragments of 90 bp, 45 bp and 30 bp upon PvuII digestion). Undigested and PvuII digested RT- PCR products were electrophoresed on a 4% agarose gel alongside similarly treated PCR products derived from GORF34.1 plasmid DNAs using the same oligo- nucleotide primers. Ethidium bromide stained DNA fragments were visualised on a UV transilluminator. The undigested RTPCR product (Fig. 3, lane 4) was of the same size (165 bp) as that obtained from GORF34.1 (Fig. 3, lane 2). PvuII digestion of the GORF34.1 PCR product yielded the predicted fragments of 120 bp and 45 bp (Fig. 3, lane 3), whilst PvuII digestion of the RTPCR product produced fragments of 90 bp, 45 bp and 30 bp (Fig. 3, lane 5). Thus, unlike the GORF34.1 PCR product, the RTPCR product was doubly di- gested by PvuII. This implies that the codon in ques- tion reads CAG rather than TAG, a fact which was confirmed by cloning and sequencing the RTPCR frag- ment.
The predicted protein sequence of GORF34.1 was used to search the Swissprot Database (Release 25) using the University of Wisconsin Genetics Computer
1 2 3 4 5 Fig. 3. Analysis of PCR products by electrophoresis on a 4% agarose gel. GORF34.1 plasmid DNA and first strand eDNA synthesised from rat liver RNA were used as templates for a PCR reaction with the oligonucleotide primers indicated in Fig. 2. Boehringer V DNA size standards were loaded in lane 1. Undigested PCR products were loaded in lanes 2 (GORF34.1) and 4 (rat liver RNA), PvulI digested PCR products were loaded in lanes 3 (GORF34.1) and 5 (rat liver RNA). Ethidium bromide stained DNA fragments were identified by
UV illumination.
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205 14I~PI~ ~ B S I ~ V ~ L ~ - ~ I W l ~ D T 254
z55 Gzax~a, ri~s..~eousvaoxD .... .r.,~..?s~','p~Tgav, i T 298 I,I .111 I I1*11 I1-, . .11- . IIIIIII IIIIII a4 a v ~ s o ~ s ~ u a ~ ~ n . . . x v A v z z 1̀30
599 K C B E V I G S S ~ ' l r ' F I I I D K I ~ I D FGK'~LPEGQTL011DVPWQEGIq~ 648
( ( l l ( l ( ( l i ( l l l l l l l l l l l i l l l l l l l l l l l l l l l l l i i l l l l l l l l 431 KCI~EVIGSSLLF IHDKKE~ IDFGKTTPLPEGQTLQHDVPWQEGNR 480
Fig. 4. Alignment of the homologous regions of the GORF34.1 (upper line) and human hippocampus IP 3 3-kinase B (lower line) amino acid sequences. Numbers refer to the positions of the amino acids within the respective sequences. Amino acid identities are indicated. The alignment was performed using the default parame-
ters within the 'Bestfit' program of the UWGCG package [9].
Group (UWGCG) FASTA program [9] and shown to have significant homology to two other proteins• It is 68% identical to rat brain IP a 3-kinase [10] and 93% identical (between residues 205 and 673 of the GORF34.1 amino acid sequence) to human hippocam- pus IP 3 3-kinase B [11]. An alignment of the relevant regions of the GORF34.1 and human hippocampus IP 3 3-kinase B amino acid sequences is presented in Fig. 4.
The published sequence of human hippocampus IP 3 3-kinase B is acknowledged to be incomplete, since it lacks an in frame 'stop' codon upstream of the ATG codon encoding first methionine residue [11]. It is also notable that this codon does not lie within a sequence corresponding to the consensus often found to flank initiating ATGs (G/ACCATGG)[12] . The sequence of GORF34.1 extends 1100 bp upstream of this ATG codon. Several in frame ATGs exist within this region, the most 5' of which (i) resides within a sequence which more closely matches the Kozak consensus se- quence and (ii) is 342 bp downstream of an in frame 'stop' codon (Fig. 2). We suggest this to be the initiator codon for the synthesis of full-length rat IP 3 3-kinase B, which is predicted to have a molecular mass of 74 kDa.
We thank Dr Barbara Reaves, Dr. Milena Girotti, Jenny Walker and Andrew Wilde for critical comments on the manuscript, and the DKFZ and MRC for finan- cial support.
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