Cloning
and Expression Analysis of a Novel Gene, UBAP1, Possibly Involved in
Ubiquitin Pathway
QIAN
Jun, ZHANG Xiao-Hui, YANG Jian-Bo, WANG Jie-Ru
, ZHANG Bi-Cheng,
TANG Ke, LI Gui-Yuan*
( Cancer Research Institute, Hunan
Medical University, Changsha 410078, China )
Abstract The
9p21-22 region shows loss of heterozygosity in up to 60% of human
nasopharyngeal carcinomas (NPC), indicating the presence of a tumor suppressor
gene in this region. We have identified a novel minimal common deletion region
at 9p21-22. Twenty-two epithelial-derived expressed sequence tags(ESTs) in this
critical region were systematically screened by differential RT-PCR to
investigate the expression patterns in NPC cell line HNE1 and primary cultures
of normal nasopharyngeal epithelial cells. One of these ESTs was found
down-expressed in HNE1, whose differential expression was confirmed by Northern
blot. Subsequently the corresponding gene sequence for this EST was established
by cDNA cloning and RACE procedures (GenBank Accession No.AF222043).
Furthermore, a mouse homologue of this gene was identified (GenBank Accession
No.AF275549). This gene is 2.7 kb long and contains two UBA domains. It is a
new member of UBA domain protein family, encoding a putative protein of 502
amino acids with a theoretical molecular mass of 55 kD, so we have named this
gene UBAP1 for ubiquitin associated protein 1 (HUGO Gene Nomenclature
Committee-approved symbol). Northern blot and RT-PCR analysis demonstrated a
ubiquitous pattern of gene expression in human and mouse tissues. Direct
sequencing analysis of the coding region of hUBAP1 following RT-PCR
failed to reveal any mutations in a preliminary screening of NPC cell line HNE1
and primary nasopharyngeal carcinomas samples. However, more detailed analysis
is to be performed to reveal if fine mutations of this gene are present in NPC.
Key
words nasopharyngeal neoplasms; gene
expression; gene cloning; chromosome 9p; UBA domain
The
short arm of chromosome 9 has been shown to be involved in nonrandom genomic
alterations in a variety of human cancers[1―7].
Observations of structural chromosome rearrangements and loss of heterozygosity(LOH)
studies involving chromosome 9p have implied the presence of tumor suppressor
genes along its length[1―7].
In particular, LOH for the 9p21-22 region has been observed up to 61% of
nasopharyngeal carcinoma(NPC)[7]. The known tumor suppressor genes,
such as p53, pRB2 and p16/MTS1 located in 9p21-22
are not likely to play an important role in NPC pathogenesis[8,9].
Therefore, the real tumor suppressor gene of NPC is unclear[10]. To
isolate putative tumor suppressor gene(s), we earlier performed detailed
deletion mapping of 9p21-22 using 11 high-density microsatellite polymorphic
DNA probes in 25 paired blood and NPC samples. A novel minimal common region of
deletion at 9p21-22 was identified subsequently, and LOH for this refined
region was found up to 68.0%(17/25)[11].
To
date, the human expressed sequence tags(ESTs) sequencing project has generated
large numbers of partial gene sequences and some proportion of these have so
far been assigned to specific chromosomes (http: //www.ncbi.nlm.nih.gov/dbEST/dbEST-summary.html).
In present study, to identify novel genes in 9p21-22, we systematically
screened 22 ESTs in above-mentioned refined region to investigate their
expression patterns in NPC cell line HNE1, primary cultured nasopharyngeal
epithelial cells and biopsies using differential RT-PCR. One of these ESTs
(GenBank entry: w56112) was found down-expressed in NPC. Subsequently the
corresponding full-length cDNA was isolated and characterized (GenBank
Accession Number: AF222043) by positional candidate approach[12] and
RACE procedures. Furthermore, a mouse homologue of this gene(GenBank Accession
Number: AF275549) was identified by EST-based analysis. Here we describe the
cloning and characterization of this gene, designated UBAP1 for
ubiquitin associated protein 1 (HUGO Gene Nomenclature Committee-approved
symbol), which is a novel member of UBA domain protein family.
1 Materials and Methods
1.1 Materials
1.1.1 Specimens Primary NPC biopsies were collected from Xiangya Hospital,
Hunan Medical University, Changsha, Hunan. All tumors were undifferentiated NPC
according to the World Health Organization(WHO) classification. Twenty tumor
samples were checked by microscopy that they contain more than 70% malignant
cells. No patient had been treated with chemo- or radiotherapy before sampling.
The NPC cell line HNE1 was grown as monolayer in RPMI 1640 medium supplemented
with 10% fetal bovine serum.
1.2 Methods
1.2.1 5′ RACE and clone sequencing RACE was carried out with the
Marathon-Ready brain cDNA RACE kit and Advantage PCR kit (Clontech, Palo Alto,
CA) as described by the manufacturer. The gene-specific primer1(GSP1) ,
5′-AGAAGC TCATTTTGCTATCG-3′, was 150―170
bp of cDNA clone 326360 from the 5′ end. The GSP1 was used in combination with
the adapter primer AP1 to amplify the 5′ end of the gene. The PCR amplification
was performed for 35 cycles of 94℃
for 30 s and 68 ℃
for 4 min in a reaction volume of 50 ml, according to the manufacturer’s
protocol. These PCR products were reamplified using a nested GSP2
(5′-CACTCTTAGAATTCACTTTA-3′) and adaptor primer AP2. Conditions were identical
to first-round amplification. The final RACE products were subcloned using the
pGEM-T Easy system (Promega) and sequenced. All sequencing was performed on an
ABI377 DNA sequencer (Perkin-Elmer, Norwalk, CT, USA).
1.2.2 Expression analysis For Northern analysis, a human
multiple-tissue Northern blot (Clontech, PaloAlto, CA) was hybridized with a
radiolabeled 430 bp RACE product or with the PCR product corresponding to the sequence
of coding region of hUBAP1 according to the manufacturer’s
specifications. Probes were labeled with [a-32P]dCTP using the
Random prime kit (Bochringer Mannbeim). The filter was washed to a final
stringency of 1×SSC,
1 g/L SDS at 65 ℃
and exposed to film (Eastman Kodak Company) for 4 days at –80 ℃.
For RT-PCR, RNA from human and mouse tissues was isolated using Trizol reagent,
1―2 mg of total RNA or 500 ng of mRNA was
reverse transcribed using cDNA synthesis kit (Promega). All PCRs were performed
using Taq polymerase and the buffer (Promega) supplied by with 0.2
mmol/L dNTPs and 0.2 μmol/L
primers. PCR products were analyzed on 1.5% agarose gels.
1.2.3 Mutational analysis RT-PCR and direct sequencing were
performed to screen tumors for mutational analysis in the hUBAP1 gene.
The cDNA obtained from NPC cell line HNE1 and ten primary nasopharyngeal
carcinoma samples were taken as templates for each PCR. The primer set was
designed to amplify the coding region of hUBAP1 cDNA from nucleotides 173
to 1 681. PCR products were purified and cloned for direct sequencing as
described above.
1.2.4 Database searches and sequence
analysis Database searches were
carried out using BLAST and Swiss-Prot programs. Motif searches were performed
with PROSITE and SMART programs. Comparison of proteins was carried out using
Clustal W 1.7. All bioinformatic tools including the open reading frame(ORF)
identification in the paper are at the ExPASy molecular biology WWW server of
the Swiss Institute of Bioinformatics(www.expasy.ch).
2 Results
2.1 Identification and cloning of the
full-length cDNA of UBAP1 from human and mouse
In
the latest release of the human genome map, large amount of these ESTs has been
previously assigned map locations. We searched the Homo sapiens UniGene
database (http: //www.ncbi.nih.gov/UniGene/index) to select 22 ESTs mapped to
the overlapping region most commonly undergoing LOH in NPC[11]. The
expression of these ESTs in normal nasopharyngeal epithelial cells and NPC cell
line HNE1 were investigated by using differential RT-PCR (data not shown). We
found one EST (GenBank entry: W56112) was expressed in high level in primary
culture of normal nasopharyngeal epithelial cells, whereas very low in NPC cell
line HNE1. The result of Northern hybridization about this EST was consistent
with that of RT-PCR (Fig.1). Because this EST is a potential candidate for the
NPC gene in 9p21-22, we characterized it more fully.
Fig.1 Down-regulated expression of hUBAP1
in NPC cell line HNE1 comparison with that in normal nasopharyngeal epithelial
cell
1,
NPC cell line HNE1; 2, normal nasopharyngeal epithelial cell. GAPDH
served as controls for RNA quality.
To
obtain the full-length cDNA, we took the cDNA clone 326360 corresponding the
human EST W56112 obtained from IMAGE Consortium. The sequencing of this clone
indicated the insert length was about 2.4 kb and there was a putative open
reading frame (ORF) from 143 nt to 1349 nt, and a poly(A) tail was identified
at the 3′ end. But an inframe stop codon was not detected at 5′ end. When cDNA
326360 was used to probe a multiple tissue Northern blot, an approximately 2.7
kb transcript was observed in all tissues studied (Fig.2). Because this
transcript was about 300 bp larger than the cDNA clone insert, we used the
Marathon-Ready brain cDNA RACE approach to find the 5′ end as described in
methods. When the 430 bp fragment of RACE was used to probe the same Northern blot
, a 2.7 kb transcript was identified. When this fragment was combined into the
5′ sequence of the cDNA clone 326360, a 2 701 bp gene sequence was generated
(GenBank Accession No.AF222043). Conceptual translation of this sequence
revealed a 1 508 bp open reading frame spanning 173―1681
nt.
Fig.2 Multiple tissue Northen blots
analysis of hUBAP1 gene expression
1,
heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney;
8, pancreas. Approximate size of the band is 2.7 kb.
Similarity
search with full length of cDNA AF222043 against the UniGene databases detected
one orthologous mouse UniGene clusters, Mm.29877. The cluster contains 76 ESTs
from various mouse tissues. When these ESTs were assembled into a virtual cDNA
of 2672 bp, this showed an average 89% match to our human sequence. Searching
of the mouse EST division of GenBank with this virtual mouse sequence using the
Blastn program retrieves two ESTs (AI530128 and AI255233), overlapping the 5′
and 3′ end of the mouse sequence respectively, corresponding to the same cDNA
clone IMAGE 1889594. Sequencing analysis of this EST clone suggests the 2 678
bp of insert segment represent a full-length mouse homology cDNA of human UBAP1.
The predicted ORF is from 203 nt to 1711 nt, which was obtained from the RT-PCR
product of nine mouse tissues (Fig.3). The data clearly indicated that the
corresponding mouse gene was a homolog of human UBAP1. The nucleotide
sequence data of mouse cDNA will appear in the GenBank database with the
accession number of AF275549.
Fig.3 Expression analysis of mUBAP1
gene in mouse tissues using RT-PCR
A housekeeping gene, GAPDH, was
amplified to normalize the relative levels of the cDNA. 1, heart; 2, liver; 3,
brain; 4, kidney; 5, spleen; 6, skeletal muscle; 7, stomach; 8, lung; 9,
testis. M: PCR marker.
2.2 Sequencing analysis of
the hUBAP1 cDNA
Analysis
of the human UBAP1 sequence revealed the existence of an ORF of 1508 bp
encoding a putative protein of 502 amino acids (Fig.4). The predicted protein
has a theoretical molecular mass of 55 kD and a calculated isoelectric point of
5.2. The initiation codon ATG is surrounded by a partial translation initiation
context[13]. There is a potential polyadenylation signal (AATAAA) at
nucleotide position 2649. The deduced amino acid sequence of mouse UBAP1 shares
90% identity with that of human UBAP1. When UBAP1 was analyzed to search for
functional domains using the bioinformatics tools available at
http://www.expasy.ch, the result revealed the presence of two tandem UBA
domains at carboxy-terminal (Fig.4). In addition, the PROSITE database
identified two possible N-glycosylation sites, five PKC phosphory-lation sites,
seven casein kinase II phosphorylation sites and three N-myristoylation sites.
Homology
searches using the Swiss-Prot program revealed no homology between UBAP1 and
any known proteins. But multiple sequence alignment with UBA domains of UBAP1
with that of other members of UBA domain protein family such as UBP5 and RAD23
etc. indicated high homology within the important domains(Fig.4).
Fig.4 Sequencing analysis of
the hUBAP1
(A) Predicted amino acid sequence of
hUBAP1. The tandem UBA domains are in bold type and underlined. (B) UBA domains
of hUBAP1 cDNA aligned with other UBA domain family members,
UBP5_Human(accession number p45974); CBL_Mouse(accession number p22682);
R23A_Human(accession number P54725); RA23_Yeast(accession number: P32628);
UBC4_Drome(accession number P52486). Conserve residues are shaded.
2.3 Tissue expression profile
of human and mouse UBAP1 gene
Human
adult multiple tissue Northern blot was performed with hUBAP1 as probe
(Fig.2). A single signal of 2.7 kb was detected in all tissues studied.
Expression was strongest in heart, sketletal muscle and liver(Fig.2). In
addition, we examined the tissue distribution of mouse transcript in various
tissues by reverse transcrption-coupled polymerase chain reaction (RT-PCR).
Primers used for RT-PCR correspond to the coding region of the mouse cDNA
(5′-CAGGTTCTAAATGGCTTCT-3′ and 5′-ATGTGCTATGGAAAGTGC-3′). The PCR product was
generated in all tissues examined indicating that the transcript is ubiquitouly
expressed in a wide variety of mouse tissues (Fig.3). Therefore, hUBAP1
gene and its mouse homologue described in the present study seems to be
involved in a basic housekeeping function of cells.
2.4 Mutational analysis
Northern
blot analysis demonstrate that hUBAP1 is expressed in normal
nasopharyngeal epithelial cells (Fig.1). In a preliminary inves-tigation on
possible mutation of hUBAP1 in NPC, we undertook a limited screened
mutation of coding sequence of the hUBAP1 gene in NPC cell line HNE1 and
ten primary nasopharyngeal carcinomas using RT-PCR and direct sequencing
analysis. Because we do not have true information about exonic structure of hUBAP1,
we analyzed RNA transcripts from these samples. The coding region of hUBAP1(173―1
681 nt) was amplified using RT-PCR. Then the RT-PCR products were consturcted
into TA-Vector system to direct sequencing analysis. We were unable to detect
any differences suggesting mutations or polymor-phisms in these limited
samples.
3 Discussion
This
paper reports the cloning of a novel gene UBAP1, which is highly
conserved between human and mouse. UBAP1 encodes a putative protein of
502 amino acids with a theoretical molecular mass of 55 kD and a calculated
isoelectric point of 5.2. The Nothern blot and RT-PCR analysis showed the genes
were expressed ubiquitously in human and mouse tissues, although heart,
sketletal muscle and liver seemed to express this gene more abundantly than
other tissues in human. Because the predicted amino acid sequence of UBAP1
has no significant homology to known proteins, we were unable to speculate on
the function of this protein. However, some bioinfomatic tools employed in the
PROSITE database identified important domains including two tandem UBA domians
and a coiled-coil domain.
UBA
domain is a novel sequence motif which occurs in a large protein family. It has
been suggested that UBA domains are involved in conferring target specificity
to multiple enzymes of the ubiquitination system[14―16].
The total size of the UBA domain is about 55 residues, the conserved core
region spans about 45 residues. So far, there are about 125 proteins found to
have UBA domain which is highly conserved in Saccharomyces cerevisiae
and Droso-phila melanogaster[15―16].
These members are largely involved in the ubiquitin-dependent pathway which has
been shown to be required for growth control, cell cycle regulation, receptor
function, development and the stress response[17,18]. More and more
studies have described the ubiquitin-dependent pathway has a decisive role in
understanding in pathological states including abnormal cellular proliferation
and tumor growth[19]. The shared role of this family raises
considerable interest in identifying novel members of this family as candidate
cancer genes. For example, the hyperplastic discs (hyd) gene and lats
gene have been identified as tumor suppressors in Drosophila melanogaster[20].
Furthermore, one recent study has showed that human LATS1, the mammalian
homologues of Drosophila lats gene, could suppress tumor growth and
rescue all developmental defects, including embryonic lethality in flies[21].
Therefore, as a novel member of UBA domain family and taking its ubiquious
expression pattern into account, the protein of UBAP1 may act as a important
role in ubiquitn pathway and cell progression. UBAP1 does not appear to be a
member of a smaller subfamily, since no further closely related members of the
UBA domain family were found on any searches. We designated the novel gene as UBAP1
for ubiquitin associated protein 1, that has been approved by HUGO Gene
Nomenclature Committee.
Human
chromosome band 9p21-22 is thought to harbor tumor suppressor genes. Gene
deletion or loss of heterozygosity in this region was found in several types of
human cancers including NPC[1―7].
Together with the known involvement of the UBA domain family in cell
progression and cancer, the chromosomal location of UBAP1 suggests a
possible improtant role for UBAP1 in NPC. One unequivocal way of implicating a
candidate gene in tumorigenesis is to identify mutations consistently in a
specific tumor type. Because LOH is the observation for 9p21 in NPC, the
prediction is that these would be inactivating mutations, although this is not
always the case. Our preliminary analysis of the coding region of hUBAP1
following RT-PCR failed to reveal any mutations. Clearly, because of its map
location, a more extensive analysis is to be done to establish whether subtle
mutations are present in NPC. In summary, the chromosomal position and
expression profile of such a gene might contribute toward ongoing positional
candidate approaches for disease genes linked to this genomic locus.
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Received: November 1, 2000 Accepted: December 18, 2000
This work is supported by a grant form
State 863 High Technology R&D Project of China (No. 102-10-01-05;
Z19-01-01-03), the Special Funds for Major State Basic Research of China,
Fundamental Investigation on Human Carcinogenesis (No. G1998051008) and Chinese
Medicine Board of New York, Inc.(No. 96655)
*Corresponding author: Tel,
86-731-4805383; Fax, 86-731-4805383; e-mail, [email protected]