(Received for publication, March 7, 1995; and in revised form, May 26, 1995)
From the
Nuclear respiratory factor 1 (NRF-1) is a transcription factor
that acts on nuclear genes encoding respiratory subunits and components
of the mitochondrial transcription and replication machinery. Here we
describe the isolation and characterization of the human gene encoding
NRF-1. The human genomic sequences detected with NRF-1 cDNA probes at
high stringency are all contained within seven overlapping recombinant
The electron transport chain and oxidative phosphorylation
system rely upon the functional interplay of gene products expressed
from both nuclear and mitochondrial genetic systems. Because of the
limited coding capacity of the mitochondrial genome, nuclear genes must
provide the majority of the respiratory subunits and all of the gene
products necessary for mtDNA A transcriptional analysis of nuclear
cytochrome c(6, 7) and cytochrome oxidase
subunit (8, 9) genes was undertaken to identify
regulatory factors that might serve such an integrative function. These
studies led to the identification, purification, and molecular cloning
of nuclear respiratory factors NRF-1 (10, 11) and
NRF-2(9, 12) . Functional recognition sites for one or
both of these nuclear transcription factors reside in the promoters of
nuclear genes that encode many of the respiratory subunits, mtDNA
transcription and replication factors, and the rate-limiting heme
biosynthetic enzyme (10, 11, 13, 14) . These findings
led to the hypothesis that NRFs may facilitate nuclear-mitochondrial
interactions through their activation of these nuclear target
genes(11, 13) . Recently, a fatal human genetic
defect resulting in the depletion of mtDNA has been
described(15, 16) . Affected individuals have severely
reduced levels of mtDNA in affected tissues, leading to a diminished
respiratory phenotype. The majority of known mitochondrial diseases are
associated with mutations in the mitochondrial genome and can be
genetically transmitted through maternal inheritance(2) . By
contrast, mtDNA depletion appears to be inherited as a nuclear gene
defect(15) , thus implicating a component of the mtDNA
replication machinery itself or a regulatory gene required for the
maintenance of mtDNA. It is intriguing in this context that mtTFA
levels have been shown to vary with mtDNA in patients displaying the
extremes of depletion and pathological proliferation of mtDNA (17, 18) and that NRF-1 has been found to be an
important activator of the mtTFA promoter(13) . Here, we
describe the structure, expression, and chromosomal assignment of the
human gene encoding NRF-1. This information should be useful in
elucidating the potential involvement of the NRF-1 gene in human
mitochondrial diseases resulting from nuclear gene defects.
Figure 3:
Analysis of the 5`-terminus of the NRF-1
cDNA by RACE-PCR and genomic organization of the 5`-terminal exons of
the NRF-1 gene. A, nucleotide sequence of the 5`-untranslated
region of the NRF-1 cDNA cloned by RACE-PCR (GenBank
Figure 4:
Mapping of the NRF-1 transcription
initiation site. A uniformly labeled, single-stranded antisense probe
was generated from an M13 template containing a 1.1-kb NRF-1 genomic
fragment extending upstream from the priming site at the 3`-terminus of
5`UTR-1 (see Fig. 3A). Yeast tRNA (lanes1 and 2) and total HeLa RNA (lane3) (20
µg each) were hybridized to the probe, and the hybrids were
digested with 0 (lane1) or 500 (lanes2 and 3) units of S1 nuclease for 60 min. The products were
analyzed on a 6% urea-acrylamide gel adjacent to a sequencing ladder (lanes 4-7) generated by extension of the same primer.
Note that the sequence indicated alongside the sequencing ladder is the
complement of the gene sequence described for Fig. 3A.
The transcription initiation sites (arrows) and the
5`-terminal nucleotide of the RACE-PCR product (filledcircle) are indicated.
COS-1, HeLa, and L6 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% (v/v)
bovine calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. COS-1 and HeLa cells were allowed to grow to 80%
confluence prior to transfections, while L6 cells were grown to
Figure 1:
Genomic
organization of the human NRF-1 gene. The diagram depicts the
intron/exon structure of the human NRF-1 gene with coding regions (closedboxes), 5`- and 3`-untranslated regions (openboxes), and introns (solidline). The coding exons (CE-1 through CE-9) and introns
(I-3 through I-10) are numbered consecutively. The coordinates at the
5`- and 3`-ends of each exon indicate their position within the cDNA
(mRNA) relative to the NRF-1 transcription initiation site (see Fig. 3and Fig. 4). Approximate intron sizes are as
follows: I-3, 15 kb; I-4, 5 kb; I-5, 15 kb; I-6, 5 kb; I-7, 1.2 kb;
I-8, 0.8 kb; I-9, 1.3 kb; and I-10, 11 kb. The phage isolates (
Figure 7:
PCR analysis of genomic DNA from
human-hamster somatic cell hybrids. Total genomic DNA from human,
hamster, or the indicated hybrid cell line was amplified with primers
NRF-1 6-2F and NRF-1 6-2R as described under ``Experimental
Procedures.'' An aliquot (20 µl) of each PCR was analyzed on a
1% agarose gel containing ethidium bromide and visualized under UV
light. A description of the human chromosome content of each of the
hybrid cell lines is listed in Table 2. Size markers are
indicated at the left.
Purified genomic DNA from Chinese hamster ovary cells and a panel of
human-hamster hybrids (see Table 2) from BIOS Laboratories (New
Haven, CT) were subjected to PCR amplification using primers NRF-1 6-2F
and NRF-1 6-2R. Human (Sigma) and Chinese hamster ovary genomic DNAs
(100 ng each) along with 250 ng of DNA from each of the hybrid lines
were amplified in 100-µl reactions containing 40 pmol of each
primer, 2.5 units of Taq polymerase, 200 µM each
dNTP, 10 mM Tris, 50 mM KCl, 1.5 mM MgCl
Genomic hybridization analysis performed under normal stringency
conditions using a NRF-1 cDNA probe revealed eight prominent PstI restriction enzyme fragments in human genomic DNA (Fig. 2, lane1). To determine whether the six
phage isolates can account for these genomic fragments, DNA from each
phage was digested with PstI and run simultaneously. The
results demonstrate a one-to-one correspondence between the NRF-1 PstI fragments in genomic DNA and those present in the phage
isolates (compare lane1 with lanes
2-6). Moreover, the relative intensities of the
hybridization signals were very similar between genomic and phage DNAs.
Although sequences more distantly related to NRF-1 may be present in
the human genome, these results demonstrate that those most closely
related to the NRF-1 cDNA are likely present in a single contiguous
locus.
Figure 2:
Hybridization analysis of NRF-1
restriction fragments in genomic DNA and in recombinant
At its 5`-end, CE-1 has only 6 bp of 5`-untranslated region
matching that of the original NRF-1 cDNA. The point of divergence with
this cDNA coincides with the consensus for an intron acceptor splice
junction, indicating that additional upstream exon(s) contain the
remainder of the 5`-untranslated region. RACE-PCR was performed on HeLa
cell poly(A)
If the sites revealed by S1 nuclease mapping are authentic
initiation sites, the 5`-flanking DNA adjacent to these sites should
contain promoter activity. To test this possibility, a restriction
fragment containing
Figure 6:
Comparison of NRF-1 and cytochrome mRNA
levels in rat tissues. A, total RNA (20 µg) prepared by
the urea/LiCl method from the tissues indicated above lanes
1-7 was assayed for cytochrome c and NRF-1 mRNAs by
RNase protection analysis using specific antisense riboprobes as
described under ``Experimental Procedures.'' Lane8 contained 20 µg of yeast tRNA treated identically
to the tissue RNA samples. Lanes9 and 10 contained the undigested cytochrome c and NRF-1
riboprobes, respectively. The positions of the RNase-protected NRF-1
(157 bp) and cytochrome c (Cyt c; 108 bp) products
obtained with the riboprobes are indicated (arrows). B, shown is the extended exposure (3 days) of the same
autoradiogram in A to facilitate better visualization of the
NRF-1-protected product.
To verify that the transfected promoter
utilizes the same initiation sites as the endogenous human gene,
promoter-dependent 5`-end formation was assayed by RNase protection in
transfected COS cells and L6 myoblasts. The riboprobe used in this
assay is specific for the detection of primate NRF-1 genes (Fig. 5). No transcript of the size expressed from the
endogenous NRF-1 gene in HeLa cells (Fig. 5, lanes1 and 6) was detected in mock-transfected L6
myoblasts (lane7) under conditions where it is
readily detected in mock-transfected COS cells (lane2). Transfection of the fusion gene into the rat L6 cells
yielded a transcript of identical size to that expressed in the primate
HeLa or COS cells (lane8). Likewise, the level of
NRF-1 transcript was elevated by transfection of the fusion gene into
COS cells (lane3). These results establish that 1)
both the endogenous and transfected NRF-1 genes initiate transcription
at the identical positions; 2) the cloned fragment contains at least a
portion of the NRF-1 promoter utilized in vivo; and 3) 5`UTR-1
is the 5`-terminal exon of the human NRF-1 gene.
Figure 5:
Transcription initiation sites of
endogenous and transfected NRF-1 genes by ribonuclease protection
mapping. Total RNA was prepared from untransfected HeLa cells (2
µg) (lanes1 and 6) or from COS and L6
cells (10 µg) transfected with either pGEM-4 blue carrier plasmid
alone (lanes2 and 7) or the same carrier in
combination with pNRF-1CAT (lanes3 and 8).
RNA samples were hybridized to an antisense T7 riboprobe spanning the
NRF-1 transcription start site, and the hybrids were digested with
RNase A and RNase T1 as described under ``Experimental
Procedures.'' Yeast tRNA (10 µg) was treated identically as a
negative control (lanes4 and 9). Lanes5 and 10 contained undigested probe. The
position of the protected product is indicated (arrow).
The NRF-1 gene may ultimately prove to be a target for
mutations that affect mitochondrial function. To determine its
chromosomal location, a panel of human-hamster hybrid DNAs were
subjected to PCR amplification using NRF-1 gene-specific primers
complementary to segments of introns I-6 and I-8 as indicated in Fig. 1. Electrophoresis of the PCR products on a 1% agarose gel
followed by ethidium bromide staining revealed the expected size
fragment of 1.5 kb in the lane containing amplification products from
human genomic DNA (Fig. 7). The size of the amplified product
corresponded to the sum of the sizes of exons CE-5 (158 bp) and CE-6
(197 bp) and the intervening intron I-7 of 1.2 kb (see Fig. 1).
No amplification product was observed when hamster DNA was used as the
PCR template (Fig. 7), indicating that the primers are
human-specific. Analysis of the amplification products obtained using a
panel of DNA samples extracted from human-hamster cell hybrids as
templates for PCR showed the 1.5-kb fragment only in cell lines 756 and
1006 (Fig. 7). Examination of the human chromosomal composition
of the various hybrid lines (Table 2) revealed that the only
human chromosome present in lines 756 and 1006 and not in the other
hybrids is chromosome 7. This result was verified using a second set of
human-specific primers derived from the 5`-end of the gene (data not
shown). To confirm the assignment of the NRF-1 locus to chromosome 7
and to further map its position within this chromosome, fluorescence in situ hybridization analysis of human metaphase chromosomes
was performed. Hybridizations were carried out using
biotin-dUTP-labeled
Figure 8:
Fluorescent in situ hybridization
analysis of human metaphase chromosomes with a NRF-1 genomic probe. A-D, in situ hybridization of biotin-labeled
The results presented here are consistent with a single-copy
gene encoding NRF-1 in humans. Its genomic organization consists of 11
exons spread over 65 kb, and the polypeptide specified by the coding
exons is identical to that predicted from the NRF-1 cDNA(11) .
The mRNA 5`-terminus cloned by RACE-PCR contains the two 5`-terminal
exons, and genomic clones containing these exons are overlapping and
contiguous with the remainder of the gene. An active promoter residing
in a genomic fragment containing the 5`-terminal exon initiates
transcription at the same sites utilized by the endogenous gene and
therefore represents a functional promoter in vivo. These
results establish that the gene described here is responsible for the
expression of human NRF-1. There is no evidence that the NRF-1 gene
is a member of a transcription factor family of related genes. Despite
a striking degree of sequence conservation within the NRF-1 DNA-binding
domains between humans and lower eukaryotes(11) , no related
human family members were detected by genomic hybridization analysis.
All of the recombinant phage clones obtained represented overlapping
segments of a single genomic locus. The restriction fragments from
these genomic clones that hybridized to the NRF-1 cDNA probe accounted
for all of those detected in total genomic DNA. Finally, only a single
NRF-1 chromosomal locus mapping to 7q31 was detected by fluorescence in situ hybridization analysis using a probe encompassing a
highly conserved region of the DNA-binding domain (Fig. 1, CE-2 and CE-3). The presence of NRF-1 recognition
sites in many nuclear genes whose products function in the mitochondria
suggests a role for NRF-1 in nuclear-mitochondrial
interactions(10, 11, 13) . In at least two of
these genes, mtTFA and 5-aminolevulinate synthase, the NRF-1 sites have
been found to be important determinants of promoter
function(13, 14) . Transcriptional activation of
D-loop promoters by mtTFA facilitates the synthesis of mitochondrial
RNAs as well as heavy strand replication
primers(1, 4) . Thus, NRF-1 may help coordinate the
synthesis of respiratory subunits from both genomes and contribute to
the maintenance of mtDNA through its effects on mtTFA expression. Recently, mtTFA levels were found to be reduced in patients with
mtDNA depletion(17, 18) . Elevated mtTFA levels have
also been found in the ragged-red muscle fibers of patients with mtDNA
deletions that lead to the abnormal proliferation of
mtDNA(17) . Thus, mtTFA seems to vary with mtDNA in these
individuals. One possibility is that the reduction in mtTFA is not a
primary cause of mtDNA depletion, but rather a consequence of reduced
mtDNA levels. The fact that a patient with reduced mtDNA and mtTFA also
had low mtTFA RNA levels argues that the primary defect may be in the
synthesis or degradation of mtTFA RNA (18) . Cell lines that
are transiently depleted of mtDNA with dideoxycytidine (32) had
reduced mtTFA but increased mtTFA RNA, suggesting that expression is
up-regulated at a pre-translational level in response to the loss of
mtDNA(18) . One intriguing possibility is that mutation in a
regulatory gene encoding NRF-1 or some other factor may lead to reduced
expression of mtTFA and consequently the loss of mtDNA. Similarly,
reduction of mtDNA by dideoxycytidine treatment or impaired respiratory
function from mtDNA mutation may lead to the activation of mtTFA
transcription by NRF-1. However, the fact that mtTFA RNA levels are
unchanged in established cell lines lacking mtDNA as a result of
propagation in ethidium bromide (17, 33) is
inconsistent with such a feedback mechanism and with the results
obtained with dideoxycytidine(18) . It will be of considerable
interest to determine whether mutations in the NRF-1 gene can lead to
aberrant mtDNA copy number control and to impaired oxidative
metabolism. The isolation, characterization, and chromosomal assignment
of the human NRF-1 gene should aid in evaluating its potential role in
mtDNA depletion and possibly other nuclear gene defects resulting in
mitochondrial disease.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
clones. The NRF-1 gene encompassed by these recombinants spans
65 kilobases (kb) and has 11 exons and 10 introns that range in
size from 0.8 to 15 kb. A rapid amplification of cDNA ends-polymerase
chain reaction product containing the 5`-terminus of the NRF-1 cDNA has
two exons from the 5`-untranslated region and terminates at a major
transcription initiation site identified by S1 nuclease mapping. A
genomic fragment containing a portion of the 5`-terminal exon and an
additional 1 kb upstream had a functional promoter that was active in
transfected COS cells, HeLa cells, and L6 myoblasts. The transcription
initiation site utilized by the transfected promoter corresponded to
that used by the endogenous gene in vivo. NRF-1 mRNA was
expressed at very low levels in rat tissues compared with cytochrome c and, unlike cytochrome c, was most abundantly
expressed in lung and testis. The NRF-1 gene was localized to human
chromosome 7 by analysis of DNA from a panel of human-hamster cell
hybrids with human-specific NRF-1 polymerase chain reaction primers.
This assignment was further refined to 7q31 by cohybridization of
NRF-1- and chromosome 7-specific probes to human metaphase chromosomes.
These analyses should be useful in evaluating the potential role of
NRF-1 in mitochondrial diseases resulting from defects in the nuclear
control of mitochondrial function.
(
)transcription and
replication(1, 2) . Several of the latter have been
characterized from vertebrates in recent years. These include a
mitochondrial RNA-processing endonuclease that has been implicated in
mtDNA heavy strand replication(1) , a mitochondrial termination
factor that may contribute to the proper rRNA and mRNA stoichiometries
within the mitochondria(3) , and a mitochondrial transcription
factor (mtTFA) that is required for efficient heavy and light strand
transcription(1, 4) . The latter may also participate
in the replication and maintenance of
mtDNA(1, 4, 5) . Regulatory mechanisms that
coordinate the expression of these gene products with the expression of
respiratory subunits may, in part, serve to integrate nuclear and
mitochondrial genetic systems.
Isolation and Sequencing of Genomic NRF-1
Clones
A human placenta genomic library constructed from Sau3AI partial digestion products cloned into EMBL-3 SP6/T7
(CLONTECH) was screened with random primer-labeled radioactive probes (19) derived from the NRF-1 cDNA (11) as described
previously(20) . Phage DNA was prepared from purified positive
isolates by an established procedure(21) . Hybridizing
fragments were first cloned into pGEM7zf(+) or pGEM5zf(+)
(Promega) and then subcloned into M13 vectors (New England Biolabs
Inc.) for DNA sequencing on both strands by the dideoxy chain
termination method(22) .Genomic Blotting
Human genomic DNA (10 µg) was
digested with PstI; electrophoresed on a 1% agarose gel along
with PstI-digested DNA from 9-1,
47-1,
39-1,
6-2, and
12-1; and transferred to nitrocellulose.
The filter was probed with a random primer-labeled (19) NRF-1
cDNA probe containing the entire coding region (11) as
described previously(23) . Hybridization was carried out as
described(23) , except that the filter was washed consecutively
in 2
SSC, 0.5% SDS and in 2
SSC, 0.1% SDS at room
temperature and in 0.1
SSC, 0.5% SDS for 30 min at 65 °C.
RACE-PCR
The following primers were used for the
amplification of the 5`-end of NRF-1 by the method of RACE-PCR: primer
1 (cDNA synthesis), 5`-CCACTGCATGTGCTTCTATGGTAG-3`; and primer 2
(nested primer for PCR amplification), 5`-CACTCCGTGTTCCTCCATG-3`.
RACE-PCR was carried out with reagents provided in the 5`-Amplifinder
RACE kit obtained from CLONTECH according to the manufacturer's
instructions. Briefly, cDNA was synthesized from 2 µg of HeLa
poly(A) RNA using a primer within the NRF-1 coding
region extending from positions 263 to 286 in an antisense direction
(primer 1). Amplification was performed with a ``nested''
primer (primer 2) complementary to the NRF-1 coding region (positions
225-243 in an antisense direction) and an anchor primer provided
with the kit. PCR mixtures were heated at 82 °C for 1 min prior to
addition of primers. After addition of primers, amplification was
carried at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2
min for 35 cycles with a final extension time of 7 min at 72 °C.
The PCR product was extracted once with chloroform/isoamyl alcohol
(24:1) and precipitated with ethanol. A portion of the PCR product was
cloned into pGEM-T (Promega), and the insert was sequenced on both
strands by the dideoxy chain termination method(22) .
S1 Nuclease Mapping
Uniformly labeled,
single-stranded probes were generated by the method of Burke (24) using an M13 template containing sequences upstream of
position 91 and the antisense primer shown in Fig. 3A.
Hybridization to RNA and S1 nuclease digestion were carried out as
described previously(25) .
accession number U27701) as described under ``Experimental
Procedures.'' Solidlines above the sequence
mark the positions of two 5`-untranslated region exons (5`UTR-1 and
5`UTR-2) and the first coding exon (CE-1). Numbers refer to
nucleotide positions relative to the transcription initiation sites (arrows) as determined by S1 nuclease analysis (see Fig. 4). The filledcircle at position +6
indicates the first nucleotide of the RACE-PCR product. Translated
frame is in boldfaceletters. B, diagram of
the intron/exon organization of the 5`-terminus of the NRF-1 gene
including the coding region (closedbox),
5`-untranslated sequences (openboxes), introns (solidlines), and the 5`-flanking region (dashedline). The coordinates at the 5`- and 3`-ends of each
exon indicate their position within the cDNA (mRNA) relative to the
NRF-1 transcription initiation site (A; see Fig. 4).
Regions encompassed by the overlapping genomic
clones
2-1
and
9-1 are indicated at the top (solidboldfacelines). Approximate intron sizes are as follows: I-1, 2.5
kb; and I-2, 5 kb.
Promoter Plasmids and CAT Assays
A 2-kb HindIII fragment containing 5`UTR-1 and upstream sequences was
excised from 2-1 and cut at the NaeI site at position
+38. The resulting 1.1-kb fragment was cloned into the HindIII and SmaI sites in the polylinker of
pGEM7zf(+). An NsiI-Asp718I fragment was excised
from this construction and cloned into the corresponding sites in the
polylinker of RC4CAT/+17(9) , thus fusing the first 38 bp
of 5`UTR-1 to position +17 of the RC4CAT first exon to generate
NRF-1CAT.
50% confluence in 100-mm dishes. Transfections were performed in
100-mm plates by the CaPO
precipitation method as
previously described (8) using 20 µg of DNA consisting of 5
µg of test plasmid, 1 µg of pRSVLacZ(26) , and 14
µg of pGEM-4 blue carrier plasmid. The CaPO
precipitate
was prepared by the dropwise addition of 60 µg of DNA (in the
proportions described above) and 180 µl of 2 M CaCl
to a 15-ml polypropylene tube containing 1.5 ml of 208 mM HEPES, 136 mM NaCl, 5 mM KCl, 11.2 mM glucose, and 1.4 mM Na
HPO
(pH
7.1) and water to give a final volume of 3 ml. Addition of DNA was
accompanied by the bubbling of air into the mixture in order to
facilitate the formation of a uniform precipitate. The precipitate was
incubated at room temperature for 30 min with vortexing every 10 min.
CaPO
/DNA precipitate (1 ml) was added to each of three
100-mm plates of cells. Twenty-four hours following transfection, cells
were washed twice with 5 ml of phosphate-buffered saline and then
refed. After an additional 24 h, cells from triplicate plates were
harvested into 5 ml of phosphate-buffered saline. CAT assays were
performed as described previously(27) , and cell extracts were
assayed for
-galactosidase activity (28) to normalize for
transfection efficiency.
RNA Preparation and Ribonuclease Protection
Assays
Total tissue RNA was prepared from adult Sprague-Dawley
rats (Harlan Bioproducts for Science, Inc.) by LiCl precipitation as
described(29) . Equal amounts of RNA were analyzed for NRF-1
and cytochrome c message levels by RNase protection
assays(30) . The cytochrome c transcript was assayed
with a SP6-generated riboprobe derived from a cloned 167-bp fragment
spanning the splice junction between the rat cytochrome c first intron and second exon. The NRF-1 antisense riboprobe was
generated from a cloned 305-bp fragment containing CE-8 of the rat
NRF-1 gene and some flanking intron sequences. Hybridization of probe
and RNA was carried out as described(6) , except for a
reduction in the RNase digestion time from 1 h to 30 min.PCR Amplification of Somatic Cell Hybrids
The
following oligonucleotide primers were used: NRF-1 6-2F,
5`-GATGACCACACTGTTCTCTTCC-3`; and NRF-1 6-2R,
5`-CTGGTAGCCCTCAGGTTTACTC-3`. NRF-1 6-2F is a sense primer specific for
intron sequences immediately preceding the 5`-splice junction of CE-5,
while NRF-1 6-2R is an antisense primer specific for the region
downstream of the 3`-splice junction of CE-6 (see Fig. 1).
)
containing genomic sequences and the extent of their overlap are
indicated at the bottom (boldsolidlines).
The arrows flanking CE-5 and CE-6 indicate the approximate
positions of the PCR primers discussed for Fig. 7. The dashedline upstream of CE-1 represents
5`-untranslated region and intron sequences contained within
9-1
as described for Fig. 3B. The sequences of CE-1 through
CE-9 and their respective 5`- and 3`-splice junctions have been
deposited in GenBank
under accession numbers
U18375-U18383. Exons are not drawn to
scale.
, and 0.001% (w/v) gelatin. After an initial
denaturation at 95 °C for 5 min, Taq polymerase was added
at 72 °C. Amplification was carried out at 95 °C for 1 min, 65
°C for 1 min, and 72 °C for 1 min for 35 cycles, followed by a
15-min extension at 72 °C. A 20-µl aliquot of each PCR was
electrophoresed on a 1% agarose gel, and the products were visualized
by ethidium bromide staining.
Fluorescence in Situ Hybridization
Chromosomal
localization of the NRF-1 locus by fluorescence in situ hybridization was performed at BIOS Laboratories. Cesium
chloride-banded DNA from 47-1 (see Fig. 1) was
nick-translated with biotin-dUTP, combined with sheared human DNA, and
hybridized to normal metaphase chromosomes derived from
phytohemagglutinin-stimulated peripheral blood lymphocytes.
Cohybridizations with a centromere-specific probe were performed to
confirm the identity of the labeled chromosome. Hybridization signals
were detected by incubation of the post-hybridization slides in
fluorescein-conjugated avidin. Upon detection of the signal, the slides
were counterstained with propium iodide and analyzed.
Isolation and Structural Organization of the Human
NRF-1 Gene
Screening of a human placenta genomic library with a
probe derived from the human NRF-1 cDNA clone (11) led to the
initial isolation of six overlapping phages spanning 57 kb of
genomic DNA (Fig. 1). Regions of overlap were determined by
restriction enzyme mapping and hybridization analysis of phage DNA
(data not shown). The DNA sequences were determined for subcloned
segments from each isolate that hybridized to the NRF-1 cDNA and
included coding regions, untranslated regions, and intron/exon
junctions. These sequences have been deposited in GenBank
under accession numbers U18375-U18383. The human gene
consists of nine protein coding exons (CE-1 through CE-9) interrupted
by introns ranging in size from 0.8 to 15 kb (Fig. 1).
clones.
Human genomic DNA (lane1) and DNA isolated from the
indicated recombinant
clones (lanes 2-6) were
digested with PstI and subjected to hybridization analysis as
described under ``Experimental Procedures.'' Numbers
1-9 to the right of each hybridizing fragment refer to CE-1
through CE-9 depicted in Fig. 1. Size markers at left are from HindIII-digested
DNA.
RNA for the purpose of obtaining a
complete 5`-untranslated region that would facilitate the
identification and isolation of the 5`-end of the NRF-1 gene.
Hybridization of the RACE-PCR product (Fig. 3A) to
9-1 revealed a single fragment containing a 5`-untranslated
region exon designated 5`UTR-2 (Fig. 3, A and B). This exon was separated from CE-1 by an intron of
5
kb (Fig. 3B, I-2). The remainder of the
RACE-PCR product was not present on
9-1. However, rescreening
the library with a synthetic oligomer containing a sequence within the
RACE-PCR product just upstream from 5`UTR-2 yielded
2-1, which
overlapped the 5`-end of
9-1 (Fig. 3B). An
additional exon, designated 5`UTR-1, was present on a single
restriction fragment from
2-1. This exon was colinear with the
remainder of the RACE-PCR product, and no acceptor splice junction
consensus was present in the genomic sequence coinciding with the
5`-RACE-PCR end. Therefore, 5`UTR-1 and 5`UTR-2 are contiguous with the
NRF-1 gene and likely represent the 5`-terminal exons.
Identification of the NRF-1 Gene Promoter and
Transcription Initiation Sites
S1 nuclease mapping was performed
using total HeLa cell RNA to determine whether the 5`-ends of 5`UTR-1
and the RACE-PCR product coincide with a transcription initiation site.
As shown in Fig. 4, S1 nuclease cleavage revealed a
heterogeneous cluster of initiation sites within 8 bp. These initiation
sites overlapped the 5`-end of the RACE-PCR product, and one coincided
precisely with the 5`-terminal nucleotide (Fig. 3A). 1.1 kb of the 5`-untranslated region and 38 bp
of 5`UTR-1 was cloned into a rat cytochrome c/CAT expression
vector. In this construct, 5`UTR-1 is fused at nucleotide +38 to
position +17 of the cytochrome c gene 5`-untranslated
region(9) . Thus, any NRF-1 promoter activity within this
fragment would be expected to initiate transcription through the NRF-1
start sites revealed by S1 nuclease mapping. As shown in Table 1,
significant levels of CAT activity above the promoterless control
resulted from transfection of the fusion gene into COS cells, HeLa
cells, and L6 myoblasts. The NRF-1 promoter activity is
18-25-fold higher than the negative control RC4CAT/+17, but
6-11-fold lower than the cytochrome c promoter activity
under identical conditions of transfection. The relative differences in
these transfected promoters are consistent with the low steady-state
level of NRF-1 mRNA compared with that of cytochrome c mRNA
(see Fig. 6).
Tissue Distribution of NRF-1 mRNA Expression and
Assignment of the NRF-1 Gene to Human Chromosome 7q31
In light
of the proposed role for NRF-1 in nuclear-mitochondrial interactions,
it was of interest to determine the tissue distribution of NRF-1 mRNA
expression. As expected for a regulatory factor, the NRF-1 transcript
is expressed at very low abundance in all cells and tissues tested.
Therefore, to assay its relative distribution in rat tissues, a portion
of the rat NRF-1 gene that is homologous to human CE-8 was cloned and
sequenced for use in a sensitive riboprobe assay. The sequence of this
fragment has been deposited in GenBank under accession
number U27700. Cytochrome c transcripts, whose levels of
expression are well established in rat tissues(31) , were
measured simultaneously as an internal control. It is evident from Fig. 6A that NRF-1 transcripts are barely detectable
under conditions where cytochrome c transcripts are easily
visualized. The NRF-1 transcripts are observed upon extended exposure
of the autoradiogram (Fig. 6B). The highest levels of
NRF-1 mRNA expression are in lung and testis, followed by intermediate
levels in kidney, heart, and brain, with the lowest levels in muscle
and liver. This contrasts with cytochrome c mRNA, which is
expressed abundantly in heart and kidney, at an intermediate level in
muscle and brain, and at the lowest levels in testis, lung, and liver.
Aside from both having their lowest expression in liver, there is
little correlation between the expression of cytochrome c and
NRF-1 mRNAs.
47-1 DNA and resulted in the specific
labeling of the long arm (q) of a group C chromosome, the size and
morphology of which were consistent with chromosome 7 (data not shown).
To further establish the identity of the labeled chromosome, a
chromosome 7 centromere-specific probe was cohybridized to metaphase
chromosomes along with labeled
47-1 DNA from the NRF-1 gene (see Fig. 1). In each of the four separate determinations shown in Fig. 8, hybridizing bands were detectable at the centromere and
at a position distal to the centromere in the chromosome 7 pair.
Measurements performed on 10 specifically labeled chromosomes 7
indicated the NRF-1 locus to be present at a position that corresponded
to 59% of the distance from the centromere to the telomere of 7q. This
position corresponds to band 7q31.
47-1 DNA to four separate sets of human metaphase chromosomes
derived from phytohemagglutinin-stimulated peripheral blood
lymphocytes. Signals were detected by incubation of the
post-hybridization slides in fluorescein-conjugated avidin. The
specific labeling of the chromosomes at 7q31 is indicated by the arrows. The chromosome 7 homologues are identified by
centromere-specific staining.
We thank Dr. J. K. Reddy for the human placenta
genomic library and Dr. C. A. Bradfield for the human-hamster somatic
cell hybrid DNA panel used in the PCR experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.