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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/23388 Please be advised that this information was generated on 2017-05-06 and may be subject to change. g e n o m ic s 38, 133-140 (1996) ARTICLE NO. 0608 cDNA Cloning and Chromosomal Localization of the Genes Encoding the a- and j3-Subunits of Human Rab Geranylgeranyl Transferase: The 3' End of the a-Subunit Gene Overlaps with the Transglutaminase 1 Gene Promoter Hans van Bo k h o v e n , * ' 1,2 R o b e r t B. Ra w s o n ,+ G e r a r d F. M . M Fr a n s P. M . C r e m e r s , * a n d M ig u e l C. S e a b r a + '1 erkx,* * Department o f Human Genetics, University Hospital Nijmegen, P. 0. Box 91 Oh 6500 HB Nijmegen, The Netherlands; and f Department o f Molecular Genetics, University o f Texas Southwestern Medical Center, 5323 Harry Hines BoulevardDallas , Texas 75235-9046 Received June 10, 1996; accepted September 26, 1996 We have isolated and sequenced the complete coding sequences of the human genes for the a- and p -sub units of Rab geranylgeranyl transferase (Rab GGTase). The a- and /3-subunit genes code for proteins of 567 and 331 amino acids, respectively, showing 91 and 95% amino acid identity to their rat counterparts. We em ployed fluorescence in situ hybridization to map the ft-subunit gene to human chromosome lp31. The «-sub unit gene could be assigned to 14q ll.2, less than 2 kb upstream of the transcription initiation site of the gene for transglutaminase 1 (TGM1). The two genes are arranged in tandem in a head-to-tail orientation. The short intergenic sequence betw een the two loci contains several promoter elem ents that are involved in the induction of TGM1 gene expression in squamous cells. These results suggest that cis-acting factors for cell-type-specific transcription of one gene are located w ithin the transcribed region of a functionally unre lated gene. ©1996 Academic Press, Inc. INTRODUCTION Rab proteins constitute a large group of the Ras su perfamily of low-molecular-weight GTPases. Rab GTPases regulate vesicular transport steps in endocytic and secretory pathways (Novick and Brennwald, 1993; Zerial and Stenmark, 1993; Pfeffer et ah, 1995), by acting as molecular switches oscillating between ac tive GTP-bound and inactive GDP-bound states. To in teract with membranes, Rabs bear C20 geranylgeranyl (GG) groups attached to cysteines at or near the carSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. Y08200 and Y08201. 1To whom correspondence should be addressed. 2Telephone: 00-31-24-3614017. Fax: 00-31-24-3540488. E-mail: [email protected]. boxyl-terminal end. The attachment of the GG isoprenes is mediated by a protein prenyl transferase, Rab geranylgeranyl transferase (Rab GGTase, previously designated component B, also known as GGTase-II) (Seabra et a l, 1992a,b; Brown and Goldstein, 1993; Farnsworth et al., 1994). Rab GGTase acts on sub strates that contain a double cysteine motif at the carboxy-terminus, commonly XXCC, XCXC, or CCXX, where C is cysteine and X may be any amino acid. Rab proteins are the only known substrates for Rab GGTase. Rab GGTase is a heterodimer composed of tightly bound a- and /3-subunits, However, unlike other prenyl transferases, Rab GGTase is inactive on its own and requires an additional component, designated Rab es cort protein (REP, previously Component A of Rab GGTase). REP-1, one of two REPs present in mamma lian cells, is the gene defective in human choroideremia (CHM), an X-linked form of chorioretinal degeneration (Cremers et ah, 1990; Merry et al ., 1992; van Bokhoven et a l, 1994). Lymphoblasts of CHM patients show defi cient Rab GGTase activity that can be restored by addi tion of purified REP-1, but not Rab GGTase (Seabra et al., 1993). REP-1 and REP-2 have similar activities in the geranylgeranylation of multiple Rab proteins, suggesting that REP-2 is able to compensate for the REP-1 deficiency in most tissues of choroideremia pa tients (Cremers et a l, 1994). However, REP-1 is more efficient than REP-2 in the prenylation of at least one Rab protein, Rab27, which is present unprenylated in cells of CHM patients (Seabra et a 1995). Since Rab27 is expressed in the retinal pigment epithelium and cho roid of the eye, this protein may be the causative factor that triggers retinal degeneration in choroideremia. If so, the Rab27 gene itself is a candidate gene for retinal disease. Likewise, mutations in the a- or /3-subunits of Rab GGTase could lead to a disease phenotype includ ing retinal degeneration. To investigate this possibility, 133 0888-7543/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of I’eproduction in any form reserved. 134 VAN BOKHOVEN ET AL. Long PCRs. Long PCR was performed according to the method we isolated and mapped the human genes for the aof Barnes (1994). Briefly, reactions were run in PC2 buffer (50 mM and /3-subunits of Rab GGTase (gene symbols RABG - Tris-HCl, pH 9,1, 16 mM ammonium sulfate, 2.5 mM MgCl2l and GTA and RABGGTB ). RABGGTB was mapped to lp31 150 mg/ml BSA) supplemented with dNTPs at a final concentration by fluorescence in situ hybridization (FISH) and RAB- of 100 /¿M. Primers were diluted to 6.0 OD/ml and used at a 1:50 GGTA maps to 14qll.2. The 3' end of the a-subunit dilution (7.5 to 9 pmol) in each reaction. A 16:1 (v/v) mixture of Klentaql (AB Peptides) and cloned PFU polymerase (Stratagene) gene is located less than 2 kb from the transcription was added at 0.2 ¿¿1 per 50-^1 reaction. One hundred nanograms of initiation site of the keratinocyte transglutaminase human genomic DNA was added per reaction. Reactions were run (transglutaminase 1; TGM1 ) gene. This short in- in a final volume of 50 ¿/I in thinwall tubes using a Robocycler ther tergenic sequence between the two genes contains sev mocycler (Stratagene). Reaction parameters were 24 cycles of 99°C eral TGMl promoter elements, some of which are lo for 30 s, 67°C for 30 s, and 68°C for 300 s. Reaction products were analyzed on 1% agarose gels in l x TBE. Fragment sizes were deter cated in the 3' transcribed region of the unrelated a mined by comparison to a 1-kb DNA ladder (Bio-Rad). After electro subunit gene. phoresis, gels were stained with ethidium bromide and videographed MATERIALS AND METHODS cDNA library screening. General molecular biology procedures were performed according to Sambrook et a l (1989). Approximately 2.5 x 106 phage recombinants of a random-primed Lambda ZAPII human fetal brain cDNA library (Stratagene No, 936206) comprising 2 x 10° independent clones were plated on the bacterial host XL1Blue. Plaques were lifted onto Hybond-N (Amersham) and the filters were prehybridized at 55°C for 6 h in buffer containing 0.125 M sodium phosphate buffer (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% (w/v) SDS, 10% polyethylene glycol (PEG-6000), and 100 ¿¿g/ml soni cated and denatured herring sperm DNA. Hybridization was per formed for 16 h at 55°C in the same solution, including 3 ng/ml (5 X 108 cpm S2P/fig) of rat cv- or /?-subunit cDNA fragment (Armstrong et al,f 1993). Positive plaques were purified and cloned into plasmid Bluescript using the Stratagene excision protocol. RNA isolation and reverse transcriptase (RT)-PCR. Total RNA was isolated from HeLa cells with RNAZOL (Campro Scientific). cDNA synthesis was performed in a 10-//1 reaction volume containing 125 ng total RNA, 50 ng random hexamer primers (Pharmacia), 10 mM Tris-base, 50 mM KC1, 5 mili MgCl, 0.01% (w/v) gelatin, 1 w M dNTPs, 27 units RNasin (Pharmacia), and 0.05 units of MMLV-RT (Life Technologies) with incubations for 10 min at room temperature, 60 min at 37°C, and 6 min at 99°C. The 5' end of the /?-subunit cDNA products was PCR-amplified with sense primer 1356 (5'-GACATG~ GGCACTCCACAGAA-3f) and antisense primer 1355 (5'-CTGGAC~ AGCACTAAGAGTGTA-3'), PCRs contained 125 ng of each primer, 10 mM Tris-base, 50 mM KC1, 1.5 mM MgCl2) 0.01% (w/v) gelatin, 0.2 mM dNTPs, and 2.5 units AmpliTaq (Perkin-Elmer) in a 50-^1 reaction volume* Thirty-five cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min were carried out with an initial denaturation step of 5 min and a final extension step of 10 min. Upon electrophoresis in a 2% agarose gel, PCR products were isolated and the protruding ends were blunted with T4 DNA polymerase and phosphorylated with T4 kinase. PCR products were subcloned in plasmid bluescript that was linearized with 2?coRV and treated with calf intestinal alkaline phosphatase (Boehringer). Southern blot hybridization. Cell lines used in this study were hybrid WegRoth B3 (gift of Dr. A. Geurts van Kessel) containing a mouse genome and human chromosome 14, hybrid GM 13139 (NIGMS-Corriel repository) containing a mouse genome and human chromosome 1, and hybrid GM 10253 (NIGMS-Corriel repository) containing a hamster genome and human chromosome 3, Chromo somal DNA was isolated as described elsewhere (Aldridge et al.} 1984). Ten micrograms of DNA was digested with jScgRI^ and frag ments were resolved by electrophoresis in a 0.8% agarose gel and blotted onto Qiabrane membranes (Qiagen). 32P-labeled probes were prepared by random hexamer priming (Feinberg and Vogelstein, 1983a,b) on the appropriate DNA fragments that were isolated in low-melting-teniperature agarose (Biolabs). Prehybridization and hybridization were performed as described above, at a temperature of 65°C. Washing was performed at the same temperature in 40 mM sodium phosphate (pH 7.2) and 0.5% (w/v) SDS or 15 mM sodium phosphate (pH 7.2) and 0.5% (w/v) SDS (high stringency). (Alpha Innotech) under UV illumination. The DNA primers used in long PCRs were sense primers specific for the 3' end of the a-subunit gene RM1 (5'-CCTACCCTTGCCCTTTAACTTATTGGGAC-3') and RM3 (5'-GGTGGGCATCTTGGAGCAACTGGCTGAAC-3'); and anti sense primers specific for the 5' end of the TGMl gene (Kim et al, 1991,1992, Yamanishieia/., 1992) RM2 (5'-CCACAGACTGGATGCCGCAGGGACAGACC-3'), RM4 (5'-AGGGGCCTGGTCCTGGAACTCATCCTGC-3'), andRM13 (5'-CTCTGGCTCTGGAGATGGCGTGGTAGGGGG-3'}. JA43 and JA100 recognize sequences present in the human Rab27 gene and are described elsewhere (Anant and Seabra, in preparation). RESULTS Cloning and Sequencing of the Human Rab GGTase a and fi-Subunits - cDNAs encoding the rat a - and /3-subunits of Rab GGTase have been cloned previously (Armstrong et a l, 1993) and encode proteins of 567 and 331 amino acids, respectively. Using the rat cDNAs as probes, we screened 2,5 X 1G5 plaques of a human fetal brain cDNA library and obtained five positive clones for the human a-subunit. Sequence comparison of these hu man clones with the rat counterpart revealed that all the human cDNAs contain the complete ORF as well as part of the 5' and 3' UTRs. The 3' UTR of the human gene is approximately 850 bp shorter than that from the rat gene. The predicted amino acid sequence is very well conserved between rat and human (Fig. 1A). These two proteins are both composed of 567 amino acids and are 91% identical. The orthologous protein from Saccharomyces eerevisiae, BET4, consists of only 290 amino acids, which represents about half the size of its mammalian counterparts. The homology between the a-subunits is highest in the amino-terminus where the proteins contain five copies of a repeated sequence pre viously described (Armstrong et aL, 1993). BET4 lacks the carboxy-terminal half present in both mammalian proteins, whose function is unknown. The same cloning strategy was used to isolate five cDNAs encoding the human /3-subunit cDNA clones, which all contained the 3' portion of the open reading frame (ORF) as well as part of the 3' untranslated region (UTR), but seemed to lack the 5' end of the gene, including 300 nucleotides of the ORF. A database search revealed that human ESTs resembling the rat /3-subunit genes have been isolated, some of which con- A hgg ta rg g ta scb et4 F. F Q A F A K R I. E E E (I A E A K R L E h ggta rg g ta scb et4 E V LQ h g g ta rg g ta scb et4 ■A^Efrfn r-: s v r. EVWQ Q I. KT s ip E E L A A I, V K Sl p F E S A A I, V K Eja E El D H r. E T I ' A S r. P E P N * 4 >•WQ R | E L: E L i e A R; E L E n P E P N ■ * ♦1 iw B u E L E L C A R F L I E H S t s s P K Vilw Q T qE aE Y R S C L I, P Q l . H Y R S C L I. 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I i 1, A H c \r *:* '■.** V !., T * > .> *C.> uV C■./»' oo I i:, t * CTi J i j N V D N V D 1) V N D 1 L GK D R K V Q * Q • j G c R NT D V V V V V V V G G G G R It C SFMG D R P G S E S 14 PFU P F H vj§n G® Y s VI W V Y ! > VI W V Y S M !•) V Y 0 W WQ y s v; 17 v Y S VI V) V r, V. L. L r, L an S S 0 0 Q E L I, V ‘I" H L D li £.) E E L. V T 13 III D I, 470 470 290 Q E L "11 L C N N K E 0 E I, I , L C n ' N P- E QQP QQS 530 530 290 A V L A A I U F EA Q PL V 54 27 54 40 43 60 D D Y E Y C K'S F J D Y E V C !•; S F. D D Y R Y C MS E ii H Ej L H I, I, G r:1 i ij g e D I L‘J G E D I WG E D ii.' D T \ r .-1 !• L A V T □ T F. }• S F C A V I D T R F S F C A V I D0R F II, I. K L T, G K. L L CJ K I T, G K S I H A T HQv | HQv l HQ v | D LiI D M Ls D :Eii n T A T E /. A T E A A T I- A N C I., A S A :• '1 R F D T E i;’ N id D W -S D T s D D E!m S D D D R D L h L L I I, D I, D L D E T., T R L S I. L 1. L, !•' L F' V Q . . P V P VB • . P P V N P V F— 1 P V D E^ R Q . . P D Pn B . . QL F E C P E E V P F. E V P E I; V P0 V T A C 0 d E [ E '1' G G 0 R V Q R V 0 R V 0 K MK L pim SIv T S K F K K K S N N S Y K NT T L N o L. O l, Q L (> M 0 I, 113 86 113 99 103 119 170 143 170 156 163 176 r. K E K. A E K A P .S G P .S G PS G 5« e | G P@ G 221 200 227 213 223 233 V L) V 0 287 260 287 273 283 293 vr L d a it o d i l s G R T I. FI A A I N V A I N V A I N V N K S E V V W 1, C E R VI r.- C E 0 Vi L c: F R E !: l-l I., C E P 11, A C 0 D E E W G Ci [., A C Q D E S T G G L. A C Q D E E T G G A A A s s 352 352 231 Q ¡1 410 s El 410 V Q 270 I* F. R I. O k E T r, o 1 I. T 1, Y t) i 0 H' V I T. T L V D S 1 0 V n V A A G A :M 0 A o ESliQ c Lj61 .A sl G Q I Y C C V H A s s 9 I I. T i, y D S I H V A G Q I Y C C '1 G F A G Q I Y C C '1 <J F A (!0 I Y C G T G F G G G G G G r, * r ]■ K I'J-.'L T V' r; C :! E E K D L T v r, c H L F "l '^7 C; S F W G D DVC D V C D V C D@C d v c DVC PFH C’ • s/ ' i T-. £ F a c; S F AG 3 FA O 5 B ig C * V K K K K s C MN F D G ^ * L s' C M N F D G V L s C MN DG Kc YN F DG ■LM V rL K c Y N F D G Lip i R c Y N E3 D G /A • d f; q l d e o [. E KN * a FD90a D V I, V G ' •mc | K K 4 * d* x^ K Y G S k t Y XA S Y G S K K Y| L H D I G N R T E • *1 L D T K K R Y D Y R D S T 1' R DS A t 292 292 201 567 567 290 Sc H E CG sc H E CG cQ H E e G s cL s s c WdP K Y G Q I D T F V K E E S T F. E T 1' r 1. Y P I. r, . Y E A LT RA D ii. A L D D I L 103 L0 H lap : h o rb eta m beta h b eta sp b eta scb eta celeb eta rb eta m beta h b eta sp b eta scb eta celeb eta P1A R P L L V G S R ME R P L T V GiS R M G scb et4 rb eta m beta h b eta sp b eta scb eta celeb eta 172 172 139 L,. L Kr a R E G W- G H B 3 d R P E C W. C m m i n n d E * ♦ * V I, Q S E L E S c. K 120 120 79 S IT N K S L D K D E AC L E EACL R C E 1, S V }■: k k '1' R C E L S v r■ K S T E S R L R L P A <.) A A O AA D E R d ^e r H H a :V V N K i n r a R WL L G R A R I' lL L G R A KW I KN h g g ta rg g ta scb et4 h g g ta rg g ta scb et4 e R V N P K S Y G T W R V N p K 3 Y G 1' W K D Y P R02YEHw h g g ta rg g ta scb et4 h g g ta rg g ta scb et4 60 60 23 E R I Y S I E A U LK h g g ta rg g ta scb et4 h g g ta rg g ta scb et4 :A M E llL f Dll E? Si V :1L; {E V D D D V s 331 304 331 311 325 325 FIG. 1, (A) Deduced amino acid sequence of the human RABGGTA gene (hggta) and alignment with the orthologous proteins from rat (rggta) and »S. cerevisae (scbet4). (B) Deduced amino acid sequence of the human RABGGTB gene (hbeta) and multiple sequence alignment with the (predicted) amino acid sequences of the orthologous proteins from rat (rbeta), mouse (mbeta), S. pombe (spbeta), S, cerevisae (scbeta), and C. elegans (celebeta). The alignments were made with the PileUp method and graphically displayed by using the Prettybox program. Amino acids in black boxes are identical with the consensus sequence (not shown), while those in shaded boxes represent similar residues. The nucleotide sequences have been submitted to the EMBL database, with accession numbers Y08200 and Y08201 for RABGGTA and RABGGTB respectively. 135 , 136 VAN BOKHOVEN ET AL. tain the 5' end of the gene. This information enabled us to reconstitute the complete coding sequence of the human /?-subunit by assembling a contiguous sequence from overlapping EST sequences and the sequences from our clones. To confirm that this sequence was cor rect, we performed RT-PCR on total RNA from HeLa cells using a sense primer corresponding to nucleotide positions 1 to 20 of EST HS61279 (Accession No. R13612) and an antisense primer corresponding to nu cleotides from the most extreme 5' end of our cDNA clones. A PCR product of approximately 300 bp was subcloned and sequenced, showing that there were no discrepancies between our sequence and the sequence assembled from the EST sequences and our cDNA clones. Comparison of the human sequence with other known sequences of Rab GGTase /3-subunits revealed that it is highly conserved among species. The nucleo tide and the amino acid sequences are well conserved between human and rat (85 and 95% identity, respec tively). Furthermore, the mammalian sequences (hu man, rat, and mouse) are approximately 50% identical to the orthologous sequences from lower eukaryotes (Caenorhabditis elegans, Saecharomycespombe, and S. cerevisiae). An optimal alignment between the six se quences using the Pileup method with the Prettybox program is shown in Fig. IB. Chromosomal Localization of the Human Rab GGTase (3-Subunit Gene The gene encoding the human /3-subunit gene was hybridized to a panel of human—rodent cell hybrids to determine the chromosomal location of the gene. Hy bridization with cDNA clone p876, which contains nucle otides 300 to 1374 of the gene, followed by washing at high stringency suggested a localization on chromosome 1. Anew blot was constructed with.EcoRI-digested DNA from the hybrid cell line containing human chromosome 1 and the appropriate controls (Fig. 2, lanes 5 to 8). Hybridization under the same conditions revealed a 12.5-kb EcoRl band that maps to chromosome 1 (Fig. 2, lanes 5 to 8), Employment of probe p876 at lower stringency as well as use of the 5' end of the /3-cDNA sequence (nucleotides 1 to 300) at high stringency re vealed two signals on an EcoRI blot; a strong hybridizing band at 12.5 kb originating from chromosome 1 and a slightly fainter signal at 3 kb (Fig. 2, lanes 9 to 14). Surprisingly, the 3-kb band mapped to chromosome 3, suggesting that a /3-subunit-like sequence is located on human chromosome 3 (Fig. 2, lanes 9 to 14). To obtain a more precise localization, individual cDNA clones of the human /3-subunit gene were used for FISH on metaphase spreads from human lymphoblastoid cells. Despite the relatively small size (less than 1.5 kb) of the probes, most metaphase cells showed either single or double spots on band p31 of chromosome 1 (data not shown). The localization to human lp31 is in agreement with the recent results of Wei et al. (1995), who mapped the murine GGTase (3- a p p !s- v •*' y c'y * * 12 3 4 5 6 7 8 « 9101112 1314 FIG. 2. Assigment of the a- and ^-subunits genes to specific hu man chromosomes by hybridization to human-rodent cell hybrids. DNA from each cell line was digested with EcoRI and electrophoresed in a 1% agarose gel. DNA was transferred to Qiabrane membranes (Qiagen), which were hybridized under stringent conditions with «(lanes 1 to 4) and (lanes 5 to 14) cDNA probes. The blot in the third panel (lanes 9 to 14) was hybridized with the 5' end of the ¡3subunit gene, Lanes contain pooled DNA from human controls (lanes 1, 4, 5, 8, 9> and 12), mouse C56/BL6 DNA (lanes 2, 7, and 11), hamster A3 DNA (lane 14), hybrid WegRoth B3 containing a mouse genome and human chromosome 14 (lane 3), hybrid GM 13139 con taining a mouse genome and human chromosome 1 (lanes 6 and 10), and hybrid GM 10253 containing a hamster genome and human chromosome 3 (lane 13). Arrows indicate human signals, asterisks indicate the murine signals, and black dots indicate the hamster signals. Note that hybridization with the 5' end of the /3-subunit gene (lanes 9 to 14) reveals two human signals. The upper band (12.5 kb) originates from human chromosome 1, whereas the lower band (3.0 kb) is derived from chromosome 3. subunit gene to the distal region of mouse chromosome 3 at 2.9 cM from the Rpe65 gene. The human RPE65 gene has also been mapped to lp31 (Hamel et al., 1994X It is also in agreement with a recent report by Sanders et al. (1996), in which the B-subunit gene is placed on chromosome Ip22-p31. Colocalization of the a-Subunit and Transglutaminase 1 Genes When the human a-subunit sequence was used to search the databank with the FASTA program, signifi cant sequence homology was detected between the 3' end of the human a-subunit gene and part of a 2.9-kb fragment of the promoter of the rabbit transglutami nase 1 (TGM1; Saunders et al., 1993). The human TGM1 gene has been mapped to human chromosome 14qlL2 (Polakowska et al., 1991; Yamanishi et al, 1992, Kim et a l, 1992), indicating that the a-subunit gene is located in the same chromosomal band. Indeed, employment of the human a-cDNA clones as a probe on a Southern blot containing EcoRI-digested DNA from a panel of human-rodent cell hybrids each containing a single human chromosome confirmed the localization on chromosome 14 (Fig. 2, lanes 1 to 4). Examination of the homologous sequences raised the 137 CLONING AND MAPPING OF Rab GGTase a- AND /5-SUBUNITS A DC RM3 RM1 t= RM4 RM13 RM2 Rab GGTase u Transglutaminase I ATG B 1 JU CJ <v E 2 ‘C 2 v E -J 1 T“ JÛ 2 X CC '5 CL CL Human genomic DNA + 3 5 4 ■cf 2 cc S çc 5 CO 2 CC cc ii •’ • X CC CNJ S CC T— 2 oc - + S SE CO 5 + 6 7 8 s cc OJ 2 cc W 2 CC - + £ CC 9 CM 2 cc cn 2 CC 1 0 11 1 2 1 3 1 4 1 5 § o o CO T— £ cr TS CC o T“ 2 CC T— 2 cc o r 2 cc CO 2 tr S cc ?5 S cc -3 CO Tfà < + - + - + c5 < 1 6 17 (0 w 0) p w a o z L. i a. o z + — £* y 1 8 0 TO TJ «3 —4 ja :>'***’* > Kb ;ii M i l » : 5. 0 4. 0 3.0 • V .: ; ••' 2. 0 1.5 1.0 v ,\ , . • 0.75 0.50 0.25 H FIG. 3. (A) Model of the proposed structural relationship of the genes encoding Rab GGTase a-subunit and TGMl in the human genome. The black line represents genomic DNA and large white arrows indicate the respective transcription units. The region upstream of the TGMl gene contains one site for each of the following enzymes: Bam H l, EcoKl and flwidlll, which are shown. The possible presence of additional sites for these enzymes between the sites indicated and the 3' end of the Rab GGTase transcription unit has not been determined. Small black arrows indicate the positions of the PCR primers used relative to each other and to the stop codon (TAA) of the RABGGTA ORF and the start codon of the TGMl ORF. (B) Long PCR of human genomic DNA using the primers shown above. Human genomic DNA was amplified by long PCRs with the indicated combinations of primers. JA43 and JA100, from the RAB27 gene, were run as positive controls to ensure that we could amplify at least a 5~kb fragment under the conditions used. possibility that the RABGGTA and TGMl genes are located in a tandem head-to-tail arrangement on the human genome. If there is conservation among human, rabbit, and rat sequences, the transcriptional start site of the TGMl gene is only approximately 1400 nucleo tides downstream of the polyadenylation site of the asubunit gene. To investigate whether the human RAB GGTA and TGMl genes are in close proximity, long PCR was performed using several sense primers spe cific for the 3 ' end of the ORF of the human a-subunit gene and several antisense primers corresponding to sequences in the 5' region and ORF of the human TGMl gene (Fig. 3A). PCR products were obtained ranging in size from 2.0 to 3.5 kb, indicating that the genomic organization of the two genes, as well as the distance between them, is well conserved between rab bit and human (Fig. 3B). This result was confirmed by digestion of human genomic DNA with three distinct restriction endonucleases predicted to cleave the intergenic sequence between the a-subunit and the TGMl promoter (Fig. 3A). When the long PCR was repeated with digested genomic templates, we were able to abolish specifically the amplification of PCR products (data not shown). DISCUSSION We report here the isolation of the cDNA sequences and the chromosomal localization of the genes for the a- and /3-subunits of Rab GGTase. The predicted amino acid sequence of the 13-subunit is very well conserved, even between distantly related eukaryotes. The a-sub unit proteins of rat and human show a high degree of homology to each other, but are much less homologous to the orthologous BET4 protein from yeast. Further more, the mammalian a-subunit proteins are much larger than their yeast counterpart. This remarkable sequence divergence suggests that the mammalian asubunit proteins may have an additional function in comparison with the BET4 protein. The RABGGTB gene could be assigned to chromo some lp31 by using somatic cell hybrids and FISH analysis. This dark-staining Giemsa band on chromo some 1, which also harbors the RPE65 gene, shows synteny with mouse chromosome 3. Southern blot analysis of DNA from hum an-rodent hybrids re vealed the presence of a /3-subunit-like sequence on human chromosome 3. We believe that this is not the /6-subunit gene proper because we could not detect VAN BOKHOVEN ET AL. 138 Transglutaminase 1 ATG AP2H AP2]—I SP1 «51 6 Human Rabbit -699 ’ 2 4 1 0 " 1 9 6 1 * 1 6 3 7 ‘ - 2 6 6 -87, -61 - 2 7 \ -A 7 1 4 3 7 (A)n ------------ r w{ Rat FIG» 4. Schematic representation of the genomic organization of the intergenic region between the RABGGTA and the TGM1 genes* Transcripts from the RABGGTA and TGM1 genes are indicated by a thick horizontal line, ORF sequences are represented by shaded boxes, and polyadenylation sites are represented by the symbol (A)«. The polyadenylation site of the rabbit a-subunit gene is unknown. Dashed lines and boxes represent regions for which no sequences are available. Conserved promoter elements for keratinocyte-specific expression are indicated by circles. The relative position of these elements is presented with respect to the transcriptional start of the TGM1 gene (position 1). Conserved elements that are located within the transcribed region of the a-subunit gene are a strictly conserved KER1/AP2 box (GCCCCAGGC), a degenerate CK.-8-mer sequence (TTTGGGTT in rabbit and TTTGGGCT in rat), and a strictly conserved API sequence (TGAGTCAA). The rabbit API sites are found in the presumed 3' UTR of the ct-subunit gene. Information about the rabbit and human TGM1 promoters was extracted from Saunders et al, (1993) and Yamanishi et al. (1992), respectively, and more details can be found in these papers. it by FISH analysis and because the murine locus is syntenic with the chromosome 1 gene. It is unclear whether this /3-subunit-like sequence is derived from a pseudogene or represents a functional homologue. The presence of related sequences is not unprece dented for genes encoding protein prenyl transferases. The same observation was reported for both the aand the /3-subunits of farnesyl transferase (Andres et a l, 1993). The farnesyl transferase /3-subunit gene maps to human chromosome 8, and a related /3-sub unit sequence, which may represent a pseudogene, was detected on chromosome 9. The RABGGTA gene could be mapped to human chromosome 14 band q ll.2 , less than 2 kb away from the gene that encodes transglutaminase 1. We show here that the two genes are organized in a tandem head-to-tail orientation, where potential regulatory se quences in the promoter for the TGM1 gene lie within the Rab GGTase a-subunit transcript and possibly within the a-subunit ORF. The presence of tandem or clustered genes suggests a functional relationship and a common ancestry between them (Graham, 1995). However, in the case of RABGGTA and TGM1> it is unlikely that such a relationship exists since their ac tivity and tissue specificity are completely different. RabGGTase a-subunit is ubiquitously expressed (Arm strong et a l, 1993) while TGM1 is restricted to squa mous cells, such as epidermal keratinocytes. The region flanking the 5r end of the TGM1 gene shares several potential regulatory motifs with keratinocyte-related genes, such as KER1/AP2, API, and CK-8-mer, and SP1 binding sites which have been identified in both human and rabbit TGM1 promoter regions (Fig. 4; Ya manishi et al> 1992; Saunders et aL, 1993). Interest ingly, sequence alignments indicate that some of these promoter elements, like API, KER1/AP2, and CK-8- mer boxes; are within the transcribed region of the Rab GGTase a-subunit gene (Fig. 4). These elements are likely to be involved in the keratinocyte-specific expres sion of the TGM1 gene. Indeed, analysis of the human TGM1 promoter in bladder epithelial cells suggested that a 2.2-kb fragment upstream of the transcription start site, which includes the 3' terminal exon of the a-subunit gene, was necessary for full expression of TGM1 (Mariniello et a l 3 1995). Taken together, the present data suggest that cis-acting factors for celltype-specific transcription of one gene are located within the transcribed region of a functionally unre lated gene. However, functional testing of the con served elements for their ability to induce keratinocytespecific expression of TGM1 will be required to demon strate whether these elements act as enhancers. The tandem arrangement of two unrelated genes with over lapping ORF and regulatory elements is to our knowl edge unprecedented in mammalian genomes. The localization of RABGGTA and RABGGTB to 14qll.2 and lp31, respectively, should allow selected testing of these genes as the culprits in human diseases that are linked to the same regions. Disease loci in lp31 include nonsyndromic sensorineural deafness type II and multiple epiphyseal dysplasia (Weith et a l, 1996). The retinal degenerative disorder Stargardt disease, which is thought to be allelic with fundus flavimaculatus (Anderson e ta l, 1995; Hoyng e t a l , 1996), has been mapped to lp21} excluding the /3-subunit as a candi date gene. Furthermore, no human genetic disorders for which the a-subunit is an obvious candidate gene have been mapped to 14qll,2. Mutations in the TGM1 gene lead to autosomal recessive lamellar ichthyosis (Huber et a l 1995; Russell et a l , 1995). Due to the close distance between the RABGGTA and the TGM1 genes it is possible that microdeletions hit the two CLONING AND MAPPING OF Rab GGTase a- AND genes simultanously. Since the a-subunit protein is likely to be indispensible for many cellular processes, homozygous deletions are probably nonviable* How ever, compound heterozygous mutations of the TGMl gene, consisting of a deletion and a small mutation, could result in a contiguous gene syndrome. 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