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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-
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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
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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
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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. Therefore,
it is worthwhile to screen the RABGGTA and TGMl
genes carefully for heterozygous deletions in syn­
dromes that include lamellar ichthyosis (Rayner e t a l ,
1978, and cases described therein).
ACKNO WLED GMENTS
We thank J. Kissing for help in the RT-PCR experiments, B. Jans­
sen and Dr. A. Geurts van Kessel for kindly providing human-rodent
cell hybrids, and Dr. Helen Hobbs for helpful comments. This work
was supported in part by the National Institutes of Health, the Perot
Family Foundation, and the Foundation Fighting Blindness.
F.P.M.C. is the recipient of a fellowship of the Royal Netherlands
Academy of Arts and Sciences and M.C.S. is a Pew Scholar in the
Biomedical Sciences.
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