Characterization of the Murine Hyaluronidase Gene
Region Reveals Complex Organization and Cotranscription of
Hyal1 with Downstream Genes, Fus2 and
Hyal3*
Tamara L.
Shuttleworth
§,
Michael D.
Wilson¶
,
Brandy A.
Wicklow,
John A.
Wilkins**, and
Barbara L.
Triggs-Raine
From the Departments of Biochemistry and Medical
Genetics, and ** Internal Medicine and Immunology, University
of Manitoba, Winnipeg, Manitoba R3E 0W3, and the ¶ Department of
Biology, Centre for Environmental Health, University of Victoria,
Victoria, British Columbia V8W 3N5, Canada
Received for publication, September 17, 2001, and in revised form, February 21, 2002
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ABSTRACT |
Hyaluronidases are required for the
breakdown of hyaluronan (HA), an abundant component of the
extracellular matrix of vertebrate tissues. Multiple hyaluronidase
genes have been identified, but the only clue to the function of their
products has come from the identification of hyaluronidase 1 deficiency
in a single patient with a mild clinical phenotype. As a first step in
the generation of mice with hyaluronidase deficiency, we have used
experimental and bioinformatic approaches to examine the organization
of the mouse chromosome 9 region containing, in order,
Hyal2, Hyal1, and Hyal3. This
region was found to be complex, with Fus2 partially embedded in Hyal3, and Ifrd2 immediately
downstream from Hyal3. The Hyal genes were all
found to have four exons, and exons 2-4 exhibited the highest sequence
conservation. Northern blot analysis demonstrated that the tissue
expression profile for Hyal1 was similar in mice and
humans, but a greater number of transcripts was detected in mouse
tissues. Hyal3 was expressed more broadly in mice compared
with humans and again exhibited additional transcripts. Reverse
transcription-PCR demonstrated that some of the larger Hyal1 transcripts, seen on the Northern blot, were the
result of cotranscription of Hyal1 with downstream genes,
Fus2 or Hyal3. In vitro
transcription/translation of one of the high abundance bicistronic
transcripts produced Hyal 1, suggesting that Hyal 1 could be produced
from all of the bicistronic transcripts. Characterization of the region
including mouse Hyal1 and Hyal3 revealed
complex organization and transcription that must be considered in the development and interpretation of mouse models involving genes in this region.
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INTRODUCTION |
Hyaluronidases are endoglycosidases essential for the breakdown of
hyaluronan (HA),1 a high
molecular mass glycosaminoglycan abundant in the extracellular matrix
of vertebrate tissues (for review, see Ref. 1). High levels of HA
confer important structural and functional properties to tissues,
including viscosity to synovial fluid (2) and fluidity to the matrix
surrounding proliferating and migrating cells involved in processes
such as embryogenesis and wound healing (3, 4). HA degradation is an
important component of these processes, and the recent identification
of multiple related hyaluronidase genes (5, 6) suggests that this
requirement is fulfilled through duplication and divergence.
Several mammalian hyaluronidases have now been recognized. The most
extensively studied is PH-20, a neutrally active hyaluronidase that is
specific to sperm and degrades the HA-rich cumulus enveloping the egg,
allowing fertilization to occur (7). At least two acid-active
hyaluronidases, HYAL 1 and HYAL 2, have been recognized (8, 9), whereas
the identification of two other enzymes, HYAL 3 and HYAL 4, as
hyaluronidases is based on homology to previously characterized
hyaluronidases. They are believed to be lysosomal enzymes, but with
differing substrate specificities (9) and expression profiles (5, 6).
The existence of more than one functional lysosomal hyaluronidase is
suggested by the mild phenotype and cell-specific HA accumulation
observed in a patient with a deficiency of HYAL 1 (5, 10).
The existence of multiple hyaluronidases provides vast potential for
both constitutive and regulated turnover of HA. At this time, little is
known about the role of these individual hyaluronidases. It is
interesting that three of the hyaluronidase-encoding genes, HYAL1, HYAL2, and HYAL3, were
originally identified as candidates in a search for a tumor suppressor
for small cell lung carcinoma, on human chromosome 3p21 (11).
HYAL2 and HYAL1 remain in the minimum interval
that has been defined for this disease (12), making it important to
evaluate their potential role in lung carcinoma. Given that only a
single hyaluronidase-deficient patient has been identified to date, the
most direct way to examine the roles of these enzymes in health and
disease is through the generation of mice deficient in each enzyme.
As a first step in the generation of mouse models with targeted
disruptions of the Hyal1 or Hyal3 genes, we have
isolated the mouse Hyal1, 2, and 3 genes and characterized the organization and expression profiles of
Hyal1 and Hyal3. The mouse Hyal2 gene was characterized previously (13). During our analysis, unusual complexity in both the structure and expression of the hyaluronidases was detected. Hyal3 was found to overlap with another gene
in the minimal interval for lung carcinoma, Fus2; the second
exon of Fus2 was found imbedded in the first intron of
Hyal3. This unusual organization was accompanied by complex
transcription profiles that were accounted for by cotranscription of
Hyal1 with the downstream genes, Fus2 or
Hyal3. We used in vitro transcription/translation to show that one of the bicistronic mRNA species could be
translated. Although cotranscription is considered to be rare in
mammals, the abundance of the bicistronic transcripts encoding Hyal 1 and their ability to be translated indicate that they are biologically relevant. These factors must be considered in gene targeting strategies aimed at the generation of mouse models for hyaluronidase deficiency or
lung carcinoma susceptibility, as the disruption of one gene in this
region could impact on the expression of others.
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EXPERIMENTAL PROCEDURES |
Primer Extension--
To determine the transcription start site
of Hyal1, a synthetic oligonucleotide
(5'-ACTTTCTGAGCATGCTGTGTAAGCCCAAGCATGTC-3') was purified and 5'-end
labeled with [
-32P]dATP and T4 polynucleotide kinase
(14). Approximately 1 µg of poly(A)+ RNA from mouse liver
(see below) and 1 × 106 cpm of
-32P-labeled oligonucleotide were annealed at 42 or
48 °C after a 10-min denaturation at 85 °C (14). The annealed
primer was extended with ExpandTM Reverse Transcriptase
(Roche Molecular Biochemicals) at 37 °C for 2 h. The products
were analyzed on a 6% denaturing polyacrylamide gel and sized using a
concurrently run sequencing ladder.
Isolation of mRNA and Reverse Transcription
(RT)-PCR--
Poly(A)+ mRNA for primer extension
analysis (see above), RT-PCR, and 3'-rapid amplification of cDNA
ends (RACE) (see below) was isolated from the livers and testes of CD1
adult mice using a FastTrack 2.0 Kit (Invitrogen), following the
manufacturer's instructions; first strand cDNA was synthesized
with ExpandTM reverse transcriptase using
oligo(dT)17 as a primer. To amplify Hyal3 from
mouse testis cDNA by PCR, we used Taq polymerase
(Invitrogen) with forward (WPG207: 5'-TGGGAGGAGTGGTGTCCACTC-3') and
reverse (WPG245: 5'-CCACCAGGTAGTCATGGAGATGCCA-3') primers to generate a
636-bp fragment beginning at the equivalent of nucleotide 655 of the
human HYAL3 cDNA; the primers were based on the
published human HYAL3 cDNA sequence, GenBank accession
no. AF036035. The PCR program consisted of 35 cycles: 94 °C for
80 s, 50 °C for 90 s, and 72 °C for 90 s.
Transcripts containing both Hyal1 and Fus2 were
PCR amplified from mouse liver cDNA using DyNAzyme EXT (Finnzymes)
and forward (WPG199: 5'-CTGGAGCACAGCCTGGGGGAG-3') and reverse (WPG288:
5'-AGTGGTGAGGTGTAGCCGTC-3') primers; the PCR program consisted of an
initial 3-min denaturation at 94 °C followed by 35 cycles of
94 °C for 45 s, 55 °C for 45 s, 72 °C for 5 min, and
a final extension at 72 °C for 8 min. Forward (WPG199) and reverse
(WPG306: 5'-GAGGCTGCCAGGTAGACTTGTCG-3') primers were used to amplify
transcripts containing both Hyal1 and Hyal3 from liver cDNA with DyNAzyme EXT, as above, except with annealing at
50 °C. A fragment required to generate the 5'-end of the
Hyal1/Fus2 in vitro transcription/translation
construct was amplified from liver cDNA using DyNAzyme EXT, as
above, and with forward (WPG333: 5'-CAGCTTCTCTAAGCTGGTGCAACT-3') and
reverse (WPG183: 5'-CTTGTGTGAGGTAAGTGAATG-3') primers and an extension
of 2 min 30 s. RT-PCR fragments were cloned using the Original TA
Cloning Kit (Invitrogen) following the manufacturer's instructions and
sequenced as described below.
3'-RACE of Murine Hyal3--
For the identification of the
3'-end of the Hyal3 mRNA, first strand cDNA was
synthesized from liver poly(A)+ mRNA using an
oligo(dT)17 adapter primer
(5'-GACTCGAGTCGACATCGA(T)17-3') and ExpandTM
reverse transcriptase. The RNA template was subsequently degraded with
RNase H. A 2-µl aliquot of first strand cDNA was PCR amplified using a forward primer (WPG345: 5'-CTGAGCCCAACATAGAGTCAGGA-3') within
exon 4 of mouse Hyal3 and a reverse adapter primer
(5'-GACTCGAGTCGACATCGA-3'). The PCR program consisted of an initial
3-min denaturation at 94 °C followed by 35 cycles of 94 °C for
45 s, 55 °C for 45 s, 72 °C for 90 s, and a final
extension at 72 °C for 8 min. RT-PCR products were cloned using the
Original TA Cloning Kit and sequenced as described below.
P1 Artificial Chromosome (PAC) Library Screening and
Characterization--
To identify a mouse genomic DNA fragment
containing the Hyal gene family, a mouse RPCI-21 C129/Sv PAC
library (15) was screened at the MRC Genome Facility (Hospital for Sick
Children, Toronto, ON) by hybridization using the 2,200-bp
EcoRI/XhoI insert of the human HYAL1
expressed sequence tag (EST) clone, GenBank AA223264, and the 1,506-bp
PacI/EcoRI insert of the human HYAL2
EST clone, GenBank AA001817, as probes. PAC DNA from hybridizing clones was prepared using a modified alkaline lysis procedure (16), restriction enzyme digested, and separated by pulse field gel electrophoresis on a CHEF system (Bio-Rad Laboratories) at 200 V for
19 h (switch interval conditions: initial time, 3 s; final time, 8 s). The DNA was transferred to nylon membranes (14) and
probed with the 1,183-bp NotI/EcoRI fragment of
mouse Hyal1 EST, GenBank AA716890, the 380-bp
NotI/EcoRI fragment of mouse Hyal2
EST, GenBank AA014559, as well as the mouse Hyal3 cDNA fragment obtained by RT-PCR with WPG207/WPG245 as described above. Hybridizations were performed under stringent conditions and following the procedures of Sambrook et al. (14). One
Hyal1-hybridizing fragment (8.5-kb SalI fragment)
was subcloned into the SalI site of pBS- to generate pHLS1.
DNA Sequencing, PCR Amplification, and Cloning--
Automated
sequencing reactions were performed on double stranded DNA using the
Sanger dideoxy sequencing method and separated with an ABI 377 gene
analyzer at the Centre for Applied Genomics (Toronto). A fragment
required to generate the 3'-end of the Hyal1/Fus2 in
vitro transcription/translation construct, described below, was
amplified from PAC genomic DNA (452D10) with DyNAzyme EXT and
forward (WPG287: 5'-CAGCTGAGCTTACCCTGGA-3') and reverse (WPG305: 5'-CACCACCATCATTAGGCCTAGGTGCATGATCATG-3') primers; the PCR program consisted of an initial 3-min denaturation at 94 °C followed by 35 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for
6 min, and a final extension at 72 °C for 8 min. To determine the
sequence of the gap between the unassembled sequence contigs containing Hyal2 and Hyal1 (GenBank AC025353), PCR
amplification of PAC DNA was performed as above but using forward
(WPG352: 5'-CTGGATATCAGTCCAGCTGGCCTC-3') and reverse (WPG348:
5'-ATGAGTCACAGGTGTGAGGAAGAC-3') primers, an annealing temperature of
65 °C, and a 5-min extension at 72 °C. All other PCR
amplifications of PAC DNA were performed using Taq
polymerase and a PCR program consisting of 35 cycles: 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. PCR fragments were
cloned using the Original TA Cloning Kit as described above.
Bioinformatic Analyses--
Comparative sequence alignments were
generated using PipMaker (bio.cse.psu.edu) (17). DNA and protein
sequences were compared with available public data bases using the
various BLAST programs available through the network server at the
National Center for Biotechnology Information website
(www.ncbi.nlm.nih.gov). DNAStar software was used for DNA and protein
alignment. Multiple alignments of protein sequences were done with
ClustalW (18). Repeat elements were characterized using RepeatMasker
(ftp.genome.washington.edu/cgi-bin/RepeatMasker).2
Compiled, overlapping sets of mouse and human ESTs were downloaded as
UniGene clusters (19), and the individual ESTs were assembled using
DNAStar software. Additional overlapping EST sequences were then
obtained by submitting the consensus sequence of the assembled
cDNAs to BLAST. This allowed the extension of previously published
cDNA sequences and helped to avoid chimeric reads and genomic
contaminants found in the EST data base. Differences, including single
nucleotide polymorphisms, alternative splicing events, and predictions
not supported by EST data, were investigated further by performing
comparisons with mouse and human genomic data.
The human genomic sequence from the HYAL2/SEMA3B locus at
3p21.3 was assembled from two separate GenBank sequences (U73167 and
U90094). Mouse genomic sequence data (GenBank AC025353) came from a
high throughput phase 1 draft of mouse clone RP23-93E19 and is found on
three unordered contiguous stretches of DNA (contigs). Within GenBank
AC025353, the sequence gap between the contigs containing
Hyal2 and Hyal1 was determined to be 275 bp by
PCR with primers WPG352 and WPG348, as described earlier. The second sequence gap within the Hyal3 gene was filled using the
sequence that we generated from a full-length EST clone (GenBank
AY048681).
Northern Blot Analysis--
Adult mouse poly(A)+ RNA
Northern blots (Swiss Webster) and embryo multiple tissue Northern
(MTNTM) blots (Swiss Webster), each containing ~2 µg of
poly(A)+ RNA per lane, were purchased from Origene and
CLONTECH, respectively. Hybridizations were
performed under stringent conditions in UltraHyb (Origene) for the
adult blots or Express Hyb (CLONTECH) for the embryo blot, according to the manufacturers' instructions. The embryo
and the two adult blots were probed with a 353-bp
BsaHI/XhoI fragment within exon 2 of mouse
Hyal1. Subsequently, one of the adult blots was probed with
a 211-bp HincII/BsrGI fragment located 2.5 kb
downstream from Hyal1, within Fus2; the other
adult blot was probed with a 636-bp Hyal3 RT-PCR fragment
(WPG207/WPG245). The Northern blots were probed with human 
-actin
cDNA as a control. Probes were labeled with
[
-32P]dATP by random priming (14). The hybridized
blots were exposed to BioMax MS film (Kodak) between two intensifying
screens at 
80 °C. Stripping the blots consisted of heating the
membrane in a 0.5% SDS solution, at 90-100 °C, for 10 min; the
subsequent absence of a signal was confirmed by autoradiography.
In Vitro Transcription/Translation--
A cotranscript with the
potential to encode Hyal 1 and Fus 2 was in vitro translated
using the TNT SP6 Coupled Reticulocyte Lysate System (Promega). DNA
encoding luciferase, provided with the kit, was used as a positive
control; negative controls contained pSP64(poly(A)) vector DNA
(Promega) or no DNA. A construct containing a cDNA sequence for a
Hyal1/Fus2 cotranscript, under the control of an SP6
promoter, was generated in three steps (Fig.
1A). First, the internal
region of the transcript was obtained by restriction enzyme digestion
of a clone (generated by RT-PCR with WPG199 and WPG288 as described
earlier) with SpeI and EcoRV to generate a 1.6-kb
fragment. The 5'-overhangs of the fragment were filled in with Klenow
(New England Biolabs) and subcloned into the HincII site of
pSP64(poly(A)) to generate pSP64(a). The 3'-end of the transcript was
obtained by digestion of a clone (generated by PCR with WPG287 and
WPG305 as described above) with SmaI and EarI to
obtain a 0.8-kb fragment; the 5'-overhangs were filled in with Klenow
and inserted into pSP64(a) digested with SmaI, to generate pSP64(b). To
introduce the 5'-end of the transcript, a RT-PCR product, generated
with WPG333 and WPG183 as described earlier, was treated with Vent DNA
polymerase (New England Biolabs) to remove the 3'-overhangs.
Subsequently, the product was digested with NcoI to produce
a 1.3-kb WPG333/NcoI fragment that was inserted into
pSP64(b) digested with HindIII (filled in with Klenow) and NcoI, to generate pSP64(Hyal1/Fus2). A construct containing
only Hyal1 cDNA was also generated (Fig. 1B).
To obtain the Hyal1 cDNA, pSP64(Hyal1/Fus2) was
digested with EcoRI, and the ends were filled in with
Klenow. The product was subsequently digested with BsrGI and
inserted into the BsrGI/HincII site of
pSP64(poly(A)) to generate pSP64(Hyal1). To generate pSP64(Fus2) (Fig.
1C), a construct containing Fus2 cDNA,
pSP64(Hyal1/Fus2), was digested with EcoRI and treated with
Klenow to fill the ends. The Fus2 cDNA was then inserted into pSP64(poly(A)) after a digestion of the vector with
BamHI and EcoRI and treatment with Klenow.
In vitro transcription/translation was performed using 1 µg of pSP64(Hyal1/Fus2), pSP64(Hyal1), pSP64(Fus2), or control
plasmid DNA in a 50-µl total reaction containing 25 µCi of
L-[4,5-3H]leucine (Amersham Biosciences) and
1 µl of TNT SP6 polymerase; the reaction was incubated at 30 °C
for 90 min. 5-µl aliquots of the products were separated by 12%
SDS-PAGE and transferred to a nitrocellulose membrane (20). The
membrane was exposed to BioMax MS film with a TranScreen-LE
intensifying screen (Kodak) and subsequently analyzed by Western blot
(see "Western Blot Analysis").
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Fig. 1.
Schematic diagram depicting the
construction of the bicistronic Hyal1/Fus2
cDNA template and the constructs containing only
Hyal1 or Fus2 cDNA, used in the
in vitro transcription/translation studies.
A, multiple cloning steps were performed to construct the
Hyal1/Fus2 cDNA template, pSP64 (Hyal1/Fus2).
B and C, cloning steps performed in the
generation of the constructs containing only Hyal1 cDNA
(pSP64(Hyal1)) or Fus2 cDNA (pSP64(Fus2)), respectively.
Abbreviations: B, BamHI; E,
EarI; GI, BsrGI; H,
HincII; Hd, HindIII; Nc,
NcoI; RI, EcoRI; RV,
EcoRV; S, SpeI; Sm,
SmaI. Brackets surround sites that were lost
during the cloning steps. Oligonucleotide primers used for PCR
amplification of the fragments for cloning are shown as
arrows.
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Production of HYAL 1 Monoclonal Antibody--
To generate an
antibody against human HYAL 1, the 1,100-bp NcoI (filled in
with Klenow) fragment of the HYAL1 EST clone (GenBank AA223264) was subcloned into the Klenow-filled HindIII site of pET-28a(+) (Novagen), and the construct was transformed into BL21
(DE3) cells. The expression of the HYAL 1 (amino acids
1-366)-His6 tag fusion was induced for 3 h with 1 mM isopropyl--D-thiogalactoside, and
insoluble inclusion bodies were purified following the manufacturer's (Novagen) instructions. The fusion protein was purified further by 10%
SDS-PAGE. The protein bands were visualized by soaking the gel in 4 M sodium acetate for 1 h; the HYAL 1-His6
tag fusion was excised from the gel, reequilibrated with
phosphate-buffered saline, and homogenized with Titer-Max Gold
(CedarLane Laboratories Inc.). Monoclonal antibodies against
HYAL 1 were produced from BALB/c mice as described previously (21), in
the Canadian Genetic Diseases Network Immunoprobes Facility
(Winnipeg, MB). Clones that produced antibodies toward HYAL 1 were identified by enzyme-linked immunosorbent assay screening of the
culture medium from individual clones for a reaction against amino
acids 1-289 of HYAL 1 fused to maltose-binding protein. Positive
clones were tested for their ability to recognize HYAL 1 but not HYAL 2 or HYAL 3 fusion proteins on a Western blot (data not shown). Clone
1D10 was found to work well for Western blot analysis and was specific
for HYAL 1. Further characterization of the specificity of this
antibody will be presented in a separate manuscript.
Western Blot Analysis--
The nitrocellulose membrane was
blocked for 1 h with 5% skim milk powder in Tris-buffered saline
(10 mM Tris, 150 mM NaCl, pH 7.5) and 0.1%
Tween 20 (TBST). The membrane was incubated with 0.5 µg/ml anti-HYAL
1 in TBST for 1 h and subsequently washed with TBST. This was
followed by a 1-h incubation in horseradish peroxidase-conjugated sheep
anti-mouse (1:15,000) in TBST and several washes with TBST, before
visualization of the bound secondary antibody with the ECL Western blot
detection system (Amersham Biosciences).
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RESULTS |
The development of strategies for the generation of mouse models
deficient in Hyal1, Hyal2, or Hyal3
necessitated a careful evaluation of the organization and transcription
profiles of their genes. Initial characterization of Hyal1
and Hyal3 gene organization and structure was performed (see
"Experimental Analysis of Hyaluronidase Gene Structure and
Organization"). As genomic sequence in the hyaluronidase region of
mouse chromosome 9F became available, bioinformatic approaches were
used to confirm and extend our knowledge of this region (see
"Bioinformatic Comparisons and Analysis of the
Hya12/Sema3b Region"). The complex mRNA expression
profiles of mouse Hyal1 and Hyal3 were
characterized and their potential biological significance assessed (see
"Expression Profile of the Hyal1 to Hyal3
Region"). The murine Hyal2 gene structure and expression had already been examined in a previous study (13) and therefore was
not included in the experimental analysis.
Experimental Analysis of Hyaluronidase Gene Structure and
Organization
Characterization of Murine Hyal1 and Hyal3 cDNA
Sequences--
To determine the organization of the genes in the
mouse chromosome 9 region and the intron/exon structures of murine
Hyal1 and Hyal3, the full-length cDNA
sequences were required. A 2,125-bp cDNA sequence containing the
entire 1,386-bp open reading frame of murine Hyal1 had been
reported previously (22), but it was not known whether this was the
full-length cDNA. To determine this, the putative transcription
start site of Hyal1 was obtained by primer extension as
described under "Experimental Procedures." A reproducible
105-nucleotide extension product, as seen in Fig. 2, corresponded to an initiation site 73 nucleotides upstream from the translation initiation codon and 30 nucleotides upstream from the published cDNA sequence (GenBank
AF011567), extending the Hyal1 cDNA to a length of 2,155 bp. Although the 105-bp extension product is clearly the major species,
other less prominent bands were detected and may be additional
extension products. Because all of the ESTs corresponding to the 3'-end
of Hyal1 terminated at position 2155, we concluded that a
poly(A) cleavage site was present at this position, although no
consensus poly(A) signal was identified upstream.
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Fig. 2.
Primer extension of Hyal1. The
105-nucleotide extension product ( ) was seen at both 42 and 48 °C
annealing temperatures after an 8-day exposure. The nucleotide position
of the predicted transcription initiation site of Hyal1 is
shown at the bottom of the Fig. (). Other less prominent
extension products (*) were either inconsistent or failed to produce a
sharp band. The transcription initiation site of HexB was
determined as a control (H). S, sequencing ladder
run concurrently for sizing.
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No sequences representing murine Hyal3 cDNA were
initially available within the mouse EST data base; therefore, a
fragment of the Hyal3 cDNA was isolated by RT-PCR
amplification of cDNA from mouse testis using primers
(WPG207/WPG245) that were based on the human HYAL3 sequence.
A BLAST search with the sequence of this RT-PCR fragment subsequently
identified a single EST corresponding to Hyal3 (GenBank
BE376250) which had become available in the data base. Complete
sequencing of this EST clone and comparison with human HYAL3
(GenBank AF036035) suggested an ATG translation initiation codon at
position 48 (numbering corresponding to GenBank AY048681) and a TGA
stop codon at position 1,284, giving an open reading frame of 1,236 bp.
Primer extension to determine the Hyal3 transcription
initiation site was not performed because of the low level of
Hyal3 transcripts.
Sequencing of the Hyal3 EST clone, GenBank BE376250,
revealed a string of A nucleotides at the 3'-end which was initially believed to represent the poly(A) tail. However, this string was also
found in the corresponding genomic DNA sequence (GenBank AC025353),
suggesting that it may be an artifact resulting from priming upstream
from the true poly(A) tail. To determine the true poly(A) cleavage site
of Hyal3, we used 3'-RACE, and two products of ~0.53 and
0.3 kb were cloned and sequenced (data not shown). The 0.53-kb fragment
identified a poly(A) cleavage site at position 1817-1819 (numbers
corresponding to GenBank AY048681); the site could not be
determined specifically because of the presence of A nucleotides at
positions 1818 and 1819 in the cDNA as well as in the genomic
sequence. A poly(A) signal (CATAAA) was located upstream from this
cleavage site. An alternative poly(A) cleavage site, between
nucleotides 1582 and 1604, was indicated by the 0.3-kb product and
could not be specified because of the string of A nucleotides within
the cDNA and genomic sequence. This site corresponded to the one
predicted by the sequence of GenBank BE376250. The authenticity of this
poly(A) site was confirmed by the presence of a lengthy poly(A) tail,
at the 3'-end of the RACE product, which was much longer than the
sequence of A nucleotides in the genomic DNA and by the absence of
amplification in the negative control (i.e. no reverse
transcriptase). A weak poly(A) signal (TATACA) was identified upstream
from this cleavage site. The full-length Hyal3 cDNA
(GenBank AY048681) is determined to be 1.8 kb, and the upstream
alternative poly(A) site generates a 1.6-kb cDNA.
Isolation and Localization of Mouse Hyal1, Hyal2, and
Hyal3--
As a first step in characterizing the structure and
organization of the Hyal1 and Hyal3 genes, mouse
genomic C129/Sv clones containing the Hyal1 and
Hyal2 genes were identified by screening a mouse PAC library
using human HYAL1 and HYAL2 cDNAs as
hybridization probes. Three PAC clones, 452D10, 52408, and 562N23, were
identified which hybridized to both probes. These clones exhibited
identical hybridization patterns when the PAC DNA was digested with
EcoRI, and multiple bands were found to be shared by all
three clones when several restriction enzymes were used (data not
shown). Taken together, these results suggested that the genomic region
containing Hyal1 and Hyal2 was present in all of
the clones. The PAC clone, 452D10, was chosen for further
characterization because it had the smallest insert.
A restriction enzyme map of 452D10 was generated using a combination of
single and double restriction enzyme digests. Summation of the fragment
sizes generated by individual restriction enzymes gave a genomic DNA
insert size of ~145 kb (Fig. 3). To
localize Hyal1, Hyal2, and Hyal3 genes
within this PAC clone, restriction enzyme fragments from mouse
Hyal1 and Hyal2 ESTs and a Hyal3
cDNA fragment (WPG207/WPG245) were used as hybridization probes
against restriction enzyme digests of 452D10 (data not shown). As
indicated in Fig. 3, the genes were found to span ~23 kb, and the
order of the genes was Hyal2, Hyal1, and
Hyal3. Hyal1 and Hyal2 have been
mapped previously to mouse chromosome 9F1-F2 (21, 13), suggesting that
the PAC clone, 452D10, including the Hyal3 gene, can also be
localized to chromosome 9F1-F2.
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Fig. 3.
Restriction enzyme map and localization of
Hyal1, 2, and 3 on
mouse genomic C129/Sv PAC clone, 452D10. Hyal1,
2, and 3 are designated 1,
2, and 3, respectively. N,
NotI; P, PacI; S,
SalI; Pm, PmeI.
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Hyal1 and Hyal3 Gene Structures--
The Hyal1 and
Hyal3 gene structures were determined by restriction enzyme
mapping, PCR amplification, and DNA sequencing of the Hyal1
genomic subclone, pHLS1, and the PAC clone, 452D10, respectively.
Genomic DNA containing human HYAL3 (GenBank U73167) was
aligned with the available sequence of the HYAL3 cDNA
(GenBank AF036035) to first determine the gene structure of human
HYAL3 (data not shown). Based on the available sequence of
the mouse Hyal1 cDNA (GenBank AF011567), the human and
mouse Hyal3 cDNAs (GenBank AF036035 and AY048681) and
guided by the human HYAL1 (10) and HYAL3 gene
structures, PCR primers were specified to amplify the predicted
Hyal1 and Hyal3 exons and introns, from pHLS1 and
the PAC clone, 452D10, respectively (Fig.
4, A and B). Sequencing of the PCR fragments with the primers shown in Fig. 4,
A and B, determined that both the
Hyal1 and Hyal3 genes consisted of four exons,
and conventional splice signals were found at each of the exon/intron
junctions (Tables I and
II). The mouse Hyal1 and
Hyal3 genes spanned ~3.2 and 5.7 kb, respectively.
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Fig. 4.
Murine Hyal1 and
Hyal3 gene structures. Strategies used to
determine exon and intron sizes and boundaries of Hyal1
(A) and Hyal3 (B) are shown. Primers
a-d and e-h were used to PCR amplify and
sequence the Hyal1 and Hyal3 exons from pHLS1 and
452D10 PCR fragments, respectively. Nucleotide numbering of
Hyal1 and Hyal3 corresponds to the cDNA
sequences in GenBank accession numbers AF011567 and AY048681,
respectively.
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Table I
Nucleotide sequences of the exon/intron boundaries of the mouse Hyal1
gene
Exon sequences are in uppercase; intron sequences are in lowercase.
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Table II
Nucleotide sequences of the exon/intron boundaries of the mouse Hya13
gene
Exon sequences are in uppercase; intron sequences are in lowercase.
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Localization of Murine Fus2 within the Hyal Family--
To
localize the Hyal3 gene specifically with reference to
Hyal1, manual and automated sequencing were used to analyze
2,496 bp of noncontiguous sequence downstream from Hyal1
(GenBank AF290431 and AF296733). During the analysis of this sequence,
the first exon of mouse Fus2 (GenBank AF172275) was found
starting 719 bp downstream from the 3'-end of Hyal1. The
first exon of Hyal3, 1,212 bp downstream from the 3'-end of
Hyal1, was found within the first intron of Fus2.
The second exon of Fus2, 2,345 bp downstream from the 3'-end
of Hyal1, was within the first intron of Hyal3. The Fus2 sequence was reported previously downstream from
Hyal2 (13); this may be consistent with our findings because
the distance between Hyal2 and Fus2 was not
stated, and we have identified Hyal2 upstream from
Hyal1.
Bioinformatic Comparisons and Analysis of the
Hyal2/Sema3b Region
Genomic Sequence Comparisons and Analysis--
Further analysis of
the organization and structure of the hyaluronidase region was
performed by directly comparing 40 kb of mouse genomic sequence with
57.3 kb of a human sequence contig. This revealed the conserved linkage
of six genes (Hyal2 to Sema3b). The data are
shown as a traditional dot-plot (Fig.
5A) and percent identity plot
(PIP; Fig. 5B), generated by submitting sequences and
annotations to the PipMaker web site (see "Experimental
Procedures").
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Fig. 5.
Comparative analysis of the murine
Hyal2/Sema3b region and the
orthologous region in human. A, dot-plot display of
mouse (y axis) and human (x axis) sequence
identity. The coding exons 2, 3, and 4 of Hyal2 are
highlighted (as in B) in dark pink to
illustrate their similarity to the corresponding exons in the
HYAL1 and HYAL3 genes. B, PIP of the
Hyal2/Sema3b genomic region. Nucleotide positions
for the mouse sequence are shown on the x axis, and the
percent sequence identity with the corresponding human sequence
(50-100%) is shown on the y axis as horizontal
lines and dots. The gene orientation is shown using
arrows, and exons are drawn as black (translated)
and light gray (untranslated) boxes.
Arrowed boxes of different shades depict the long
interspersed nuclear elements LINE1 (light gray) and LINE2
(black). Dark gray boxes show long terminal
repeat elements (LTRs), and dark gray triangles
designate other interspersed repeats such as DNA transposons.
Light gray triangles show SINEs (short interspersed nuclear
elements) other than MIRs (mammalian interspersed repeats), which are
in turn drawn as black triangles. Tall open boxes
depict simple repeats, short open boxes depict regions with
CpG/GpC contents greater than or equal to 60%, and short dark
gray boxes depict regions with CpG/GpC contents greater than or
equal to 75%. Dark pink and dark yellow on the
upper and lower halves of the PIP represent the locations of exons on
the + and strands, respectively, whereas the light
pink and light yellow areas represent untranslated
regions (UTRs). The red stripe indicates an
unfilled 275-bp gap in the mouse sequence. Genes are labeled based on
cDNAs, ESTs, and comparative genomic information. RepeatMasker (see
Footnote 2) was used to locate repeat elements.
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The dot-plot display (Fig. 5A) was generated using
parameters that allowed one block of DNA to align to more than one
region on the second sequence. This not only reveals the similarity
between the orthologous Hyal2 and HYAL2 genes,
but also the similarity between the coding regions of exons 2-4 of
Hyal2 and the corresponding exons from the paralogous human
genes HYAL1 and HYAL3 (see highlighted region in Fig. 5A). Similarity between exons 2-4 of
Hyal1 and Hyal3 and the human paralogs is also
evident, although this was not highlighted in Fig. 5A.
In Fig. 5B, the mouse sequence is drawn along the
x axis with exons and repetitive elements being labeled. The
percent identity between mouse and human genomic DNA is indicated as a
series of horizontal lines ranging from 50 to 100%
similarity (y axis). The parameter of "single coverage"
was used so as to allow only one alignment at each sequence position to
be displayed on the PIP. Because of gaps in the alignment, the
horizontal lines on the x axis are discontinuous
and are most visible in regions corresponding to exons. Outside of the
exon regions, the horizontal lines appear as a spattering of
dots that correspond to smaller gap-free DNA alignments.
Dark pink (translated) and light pink
(untranslated) regions in Fig. 5B represent alignments of
exons found on the top DNA strand, including Hyal2,
Hyal1, Fus2, Hyal3, and
Ifrd2. Dark (translated) and light
(untranslated) yellow coloring identifies alignments for
Sema3b found on the bottom strand. The red stripe marks the 275-bp gap between Hyal2 and Hyal1
whose size was defined by PCR amplification; this fragment was highly
repetitive and could not be fully sequenced. The blue shaded
box represents an assembly of 14 ESTs that are unspliced and
appear to be noncoding. Through the above comparisons and analyses, the
sizes of Hyal1 and Hyal3, as well as the order of
the mouse genes, determined experimentally, were confirmed.
The clustering of the Hyal1, Fus2,
Hyal3, and Ifrd2 genes, together with evidence
that Hyal1, Fus2, and Hyal3 are
sometimes coordinately regulated, led us to search for a putative
promoter for this region. A 498-bp sequence (GenBank AF290430),
upstream from the published cDNA sequence of Hyal1
(GenBank AF011567), was examined by visual scanning and using TFSEARCH
(23). No classical TATA box was identified, although a potential CAAT
box was detected, with reference to GenBank AF290430, at position 411 and two GC boxes at positions 397 and 402. A number of small alignments
upstream from Hyal1 were evident in the PIP (Fig.
5B) which may be significant, but only two of these
conserved regions are predicted to bind known transcription factors
(Sp1 sites at positions 368 and 386) (GenBank AF290430). Functional
studies are needed to identify DNA segments in this region which may be involved in the coordinated regulation of Hyal1,
Fus2, and Hyal3.
Analysis of Expressed Sequences--
The analysis of ESTs using
BLAST was used to confirm the intron/exon junctions as well as the
organization of the Fus2 and Hyal3 genes. These
comparisons also suggested a slightly different organization in humans,
with FUS2 and HYAL3 sometimes sharing the same
first exon. There is no evidence for these findings in mouse, possibly
because of the limited number of Hyal3 ESTs. It was also
noted that the 5'-coding region of IFRD2 was within the 3'-untranslated region of HYAL3, but no evidence of this has
been seen in the mouse.
Alignment of Protein Sequences--
To demonstrate the homology
between the predicted protein sequence of mouse Hyal 3 and the
previously identified hyaluronidases, the amino acid sequences of human
hyaluronidases, HYAL 1, 2, and 3 and mouse Hyal 1, 2, and 3 were
aligned using ClustalW (Fig. 6).
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Fig. 6.
Multiple alignment of human and mouse Hyal 1, Hyal 2, and Hyal 3 predicted protein sequences. Sequences of human
hyaluronidases, HYAL 1 (GenBank AAD04190), HYAL 2 (GenBank AAC62823),
and HYAL 3 (GenBank AAD04257), and mouse Hyal 1 (GenBank AAC15949),
Hyal 2 (GenBank AAK28481), and Hyal 3 were aligned using ClustalW.
Asterisks represent identical residues in all of the
proteins; colons and periods indicate strong and
weak conservation, respectively.
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Expression Profile of the Hyal1 to Hyal3 Region
Tissue Expression Profiles of Hyal1 and Hyal3--
To determine
whether the tissue expression profiles of the mouse Hyal1
and Hyal3 genes were similar to their human counterparts, we
used Northern blot analysis. The steady-state expression of Hyal1 and Hyal3 mRNA in adult mouse tissues
was examined by hybridization using mouse cDNA fragments as probes
under stringent conditions. The highest Hyal1 expression
levels in adult tissues were in liver, followed by kidney and skin
(Fig. 7A), consistent with
similar studies by Csoka et al. (22) and revealing an
expression profile similar to the human gene (5). For Hyal3,
the expression profile in the mouse tissues (Fig. 7D) was
broader than in human tissues (5), with an expression profile similar
to Hyal1, except also present in brain, stomach, and testis.
Hyal1 mRNA was detected in mouse embryos of all
developmental stages, with the strongest and the greatest number of
bands at the 7 day stage (Fig. 7E).
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Fig. 7.
Tissue expression profiles of adult
Hyal1, Fus2, and Hyal3, and embryo
Hyal1 mRNA. A and C,
adult mouse Northern blot probed with Hyal1 and
Fus2, respectively; 17-h exposures for both.
Arrows denote bands shared between Hyal1 and
Fus2. B and D, adult Northern blot
(different from A and C) probed with
Hyal1 (17.5-h exposure) and Hyal3 (22-h
exposure), respectively. * denotes a band shared between
Hyal1 and Hyal3. E, mouse embryo
Northern blot probed with Hyal1. F, adult
Northern blots probed with actin. Developmental stages are shown above
the embryo blot. Molecular weight markers in kb are shown on the
right.
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For both Hyal1 and Hyal3, the number of
transcripts in the mouse tissues was greater than in the human tissues,
and the higher expression of Hyal1 compared with
Hyal3 made it easier to characterize the Hyal1
transcripts. Major Hyal1 transcripts of 6.2, 4.5, 3.3, and
1.3 kb in size, as well as a weak signal at 2.1 kb, were detected in
both embryo and adult mouse tissues (Fig. 7, A and
E). A 2.1-kb Hyal1 mRNA corresponds to the
size of the published Hyal1 cDNA sequence, GenBank
AF011567, and a mouse Hyal1 EST, GenBank AA716890, may be
represented by the 1.3-kb transcript (data not shown). No ESTs
corresponding to the larger Hyal1 transcripts were
identified. The consistent 1.8-kb Hyal3 transcript observed on the Northern blot (Fig. 7D) putatively corresponds to the
Hyal3 full-length cDNA (GenBank AY048681). Additional
Hyal3 transcripts (3.8, 3.3, and 2.4 kb) were also detected
in tissues with higher expression levels (Fig. 7D).
Alternative Polyadenylation of Hyal1--
The high abundance of
the large Hyal1 transcripts (6.2, 4.5, and 3.3 kb) suggested
that they might have biological relevance. We were unable to explain
the larger sized transcripts by alternate splicing within the gene or
alternative transcription initiation (data not shown). To determine
whether alternative polyadenylation could explain the larger
transcripts observed on the Hyal1 Northern blot, we used
several fragments downstream from Hyal1 to probe the adult
Northern blot. One probe, a genomic HincII/BsrGI
fragment located 3.0 kb downstream from Hyal1 and containing
sequence from the coding region of mouse Fus2, detected five
transcripts (Fig. 7C). The 1.5-kb transcript that is highly
expressed in testis is likely to correspond to the Fus2
published cDNA, GenBank AF172275. Three larger bands on the
Fus2 Northern blot (6.2, 4.5, and 2.4 kb) (Fig.
7C) appeared to be of the same size and tissue distribution as three from the Hyal1 Northern (Fig. 7A),
suggesting that some of the larger transcripts resulted from
cotranscription of Hyal1 and Fus2. Similar
comparisons of Hyal1 and Hyal3 Northern blots revealed a transcript of 3.3 kb in both blots (Fig. 7, B and
D), implying that the two genes could be within a single
transcript as well and use the poly(A) signal of Hyal3.
Cotranscription of Hyal1/Fus2 and Hyal1/Hyal3--
To determine
whether Hyal1 and downstream genes, Fus2 or
Hyal3, are sometimes cotranscribed, RT-PCR was performed on
mouse liver cDNA. The cDNA was amplified using a forward primer
(WPG199) within exon 3 of Hyal1 and a reverse primer
(WPG288) within exon 2 of Fus2 (Fig.
8A). A number of PCR
amplification products were observed (data not shown). These products
were cloned and sequenced, and the sequences of the RT-PCR products
have all been submitted to GenBank. The products demonstrated that
Hyal1 and Fus2 were both found within seven
differently spliced transcripts (Fig. 8B,
i-vii). Using a forward Hyal1 primer (WPG199)
and a reverse primer (WPG283) within exon 3 of Hyal3 (Fig.
8C), a single RT-PCR product was amplified from mouse liver
cDNA (data not shown); sequencing revealed that Hyal1
and Hyal3 were found within one transcript as well (Fig.
8D).
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Fig. 8.
Cotranscription of
Hyal1/Fus2 and
Hyal1/Hyal3. RT-PCR products of
mouse liver cDNA were generated using a forward primer in
Hyal1 ( ) and reverse primers in Fus2 and
Hyal3 (). Hyal1 and Fus2
(A) and Hyal1 and Hyal3 (C)
genomic arrangements are shown. B, i-vii, RT-PCR
products consisting of Hyal1 and Fus2 within the
same transcripts. Sequences were deposited as GenBank AF417494,
AF417493, AF417495, AF417492, AF417498, AF417497, and AF417496.
D, RT-PCR product containing Hyal1 and
Hyal3 within a single transcript. The abundance of the
RT-PCR products for Hyal1/Fus2, determined
qualitatively (with 1 being the most abundant and
4, the least) and the predicted putative protein products
for both are stated within the tables. Hyal1 exons are
depicted by unshaded boxes, Fus2 exons are shown
by light gray boxes, and dark gray boxes
represent Hyal3 exons. Black boxes depict the
sequence between Hyal1 and Fus2 which is present
in some of the bicistronic transcripts and may contain an internal
ribosome entry site. Start (ATG) and stop (TGA) codons are indicated.
Hyal 1 followed by the caret or prime or
asterisk correspond to putative Hyal 1 proteins with
different C termini. Fus 2** and Fus 2'' represent putative
Fus 2 proteins with different N termini.
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At least seven bicistronic mRNA species containing Hyal1
and Fus2 were identified in mouse liver using RT-PCR. The
level of these transcripts was not quantitated, but the intensity of
the ethidium bromide staining of these species allowed a qualitative assessment of their abundance (Fig. 8). These various mRNA species have the potential to encode several novel protein products (Fig. 8).
In Vitro Transcription/Translation of Hyal1 and Fus2--
The
bicistronic transcripts containing both Hyal1 and
Fus2 are expressed at relatively high levels and thus may
contribute significantly to biological function if they are translated.
To determine whether the bicistronic transcripts containing both Hyal1 and Fus2 could be translated, we generated
a construct expressing the most abundant mRNA species
(Fig. 8Bi), as well as constructs containing only
Hyal1 or Fus2 cDNA, and tested their ability
to be translated in vitro. pSP64(Hyal1/Fus2) DNA (expressing
the abundant Hyal1/Fus2 transcript),
pSP64(Hyal1), pSP64(Fus2), positive control DNA expressing luciferase
and negative controls with the pSP64(poly(A)) vector or no added DNA,
were introduced into the TNT Coupled Reticulocyte Lysate System, and
the DNA templates were transcribed and translated. Aliquots of the
resulting samples were separated on a SDS-polyacrylamide gel,
transferred to a nitrocellulose membrane (20), and analyzed by both
autoradiography and Western blot. Autoradiography detected three
identical bands in the lanes containing in vitro
transcribed/translated pSP64(Hyal1/Fus2) and pSP64(Hyal1) (Fig.
9A). Bands of different
molecular mass were also detected in the lane containing
in vitro transcribed/translated pSP64(Fus2) (Fig.
9A), suggesting that Hyal 1, but not Fus 2, could be
expressed from the bicistronic mRNA. The predicted molecular mass
of Fus 2 expressed from pSP64(Fus2) is 34.6 kDa, correlating to the top
band detected in the lane containing in vitro
transcribed/translated pSP64(Fus2) (Fig. 9A). The smaller
bands observed in the positive control and experimental samples are
assumed to result from leaky scanning, resulting in translation
initiation at downstream AUG codons. Products smaller than the
full-length protein are commonly observed with these systems
(Reticulocyte Lysate System technical notes). No bands were detected in
the negative control lanes.
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Fig. 9.
In vitro transcription/translation
and Western blot analysis of Hyal 1. A bicistronic mRNA
species encoding both Hyal1 and Fus2 was
translated in vitro using [3H]leucine, SP6
polymerase, and the TNT-coupled reticulocyte lysate system. Constructs
containing Hyal1 or Fus2 cDNA were also
transcribed and translated. Luciferase cDNA was used as a positive
control, and negative controls consisted of pSP64(poly(A)) vector alone
or no added DNA. The samples were separated on a 12%
SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and
analyzed by autoradiography and Western blot. A,
autoradiograph of 3H-labeled cotranscript products;
2.5-h exposure. B, Western blot detection of in
vitro translated products using anti-HYAL 1 (-HYAL
1). In vitro transcribed/translated products from
pSP64(Hyal1/Fus2) (lane 1), pSP64(Hyal1) (lane
2), pSP64(Fus2) (lane 3), expressed luciferase protein
(lane 4), pSP64(poly(A)) negative control (lane
5), and no DNA (lane 6) are shown. Molecular mass
markers in kDa are shown on the left.
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Western Blot Analysis--
To determine which protein bands
corresponded to Hyal 1, Western blot analysis of the in
vitro translated samples was performed, using anti-HYAL 1. In the
lanes containing the in vitro translated proteins from
pSP64(Hyal1/Fus2) and pSP64(Hyal1), the top three bands were detected
with anti-HYAL 1 (Fig. 9B).
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DISCUSSION |
We have analyzed the organization of the Hyal1,
Hyal2, and Hyal3 genes on mouse chromosome 9F1-F2
using both experimental and bioinformatic approaches. The region
encompassing these genes was found to be complex and gene dense (one
gene/6 kb). Six mouse genes were identified on 40 kb of mouse
chromosome 9, and their order was determined to be Hyal2,
Hyal1, Fus2, Hyal3, Ifrd2,
and Sema3b (Figs. 3 and 5). This is identical to the order
of the orthologous human genes located on chromosome 3p21.3, but the interval in humans was 17 kb larger, in part because of the insertion of 10 kb of repeat elements into the intergenic region between HYAL2 and HYAL1 (Fig. 5A). Our
analysis revealed the second exon of Fus2, a gene encoding
an N-acetyltransferase, within the first intron of
Hyal3. Further, Ifrd2, a gene encoding an
interferon-related developmental regulator, was found immediately 3' to
Hyal3 (Fig. 5B). Although the general gene
organization was conserved in humans and mice, the analysis of ESTs
from this region suggested some differences. Human HYAL3 and
FUS2 sometimes share the same first exon, but no ESTs were
identified to support this usage in the mouse. Further, in humans, the
3'-untranslated region of HYAL3 overlaps with the putative
5'-coding region of IFRD2 (GenBank Y12395), again a finding
not observed in the mouse. Northern blot analysis revealed further
complexity in the transcription of this region, which is discussed later.
The order of a second group of three related HYAL genes on
human chromosome 7q31 has been determined to be SPAM1,
HYAL4, and HYALP1 (6). Mouse Spam1 has
been localized to chromosome 6 (24), suggesting that there is a second
group of hyaluronidase genes, as in humans, on mouse chromosome 6. An
analysis has not been done to determine whether genes related to
Fus2, Ifrd2, and Sema3b are also
duplicated with the hyaluronidase genes in these regions. If the six
linked genes (Hyal2, Hyal1, Fus2,
Hyal3, Ifrd2, and Sema3b) are
completely duplicated, it would be interesting to determine whether
there is increased transcriptional complexity in any parts of this
region. A full characterization of this region may also provide insight
into the evolutionary history of these two groupings of hyaluronidase genes.
The mouse Hyal1, Hyal2, and Hyal3
genes were found, using bioinformatic approaches, to consist of four
exons and three introns (Fig.
5B). This general
structure was confirmed for Hyal1 and Hyal3 using
experimental approaches and published information for Hyal2
(13). This structure is also conserved in human HYAL2 and
SPAM1 (5, 25), mouse Spam1 (26), and human
HYAL3 (data not shown). The human HYAL1 structure
is similar, except that the first exon junction in the 5'-untranslated
region is not always recognized, leading to the retention of the first
intron a significant proportion of the time (5, 27). There is no
evidence in the EST data base that this intron is retained in the mouse
Hyal1 gene, although RT-PCR does detect a low level of
alternative splicing in this region (data not shown). Surprisingly, in
contrast to mouse Hyal2 and Hyal3 and human
HYAL2, HYAL3, and SPAM1 where the
translation start codon (ATG) is found near the 5'-end of exon 2, the
addition of a new upstream sequence in the mouse gene places the ATG
start codon within exon 1. The strongest sequence conservation among
the members of the Hyal gene family is found in exons 2-4
(Fig. 5A), which, except for Hyal1, contains the entirety of the coding sequence.
Northern blot analysis of mouse Hyal1 demonstrated a tissue
expression profile similar to that of the human gene, but more transcripts were detected. In humans, only two major HYAL1
transcripts (1.9 and 2.6 kb) have been identified (5) compared with
four major transcripts in mice (6.2, 4.5, 3.3, and 1.3 kb) (Fig. 7, A and B), indicating that transcriptional
regulation differs between mice and humans. During a search for the
basis of the larger Hyal1 transcripts, shared bands between
Hyal1 and Fus2 (Fig. 7, A and C), as well as between Hyal1 and Hyal3
(Fig. 7, B and D), were recognized.
RT-PCR confirmed the cotranscription of Hyal1 and Fus2 as well as Hyal1 and Hyal3 (Fig.
8). In humans, no evidence for
significant levels of cotranscription of HYAL1 with other genes is indicated from Northern blot analysis, suggesting that this
region has evolved since the divergence of humans and mice.
Hyal3 expression was found to be lower than that of
Hyal1 (Fig. 7, B and D), as had been
observed previously for the human gene (5). However, the mouse
Hyal3 tissue expression profile differed from that of human
HYAL3 in that in addition to expression in the same tissues
as the human gene, Hyal3 had also acquired expression in the
same tissues as Hyal1. Interestingly, a similar phenomenon
was observed for Fus2, suggesting some type of coordinate regulation for this region in mouse.
The steady-state levels of bicistronic mRNA species containing both
Hyal1 and Fus2 are relatively high (Fig. 7),
suggesting that they may have biological relevance. Several of the
bicistronic species, characterized by RT-PCR (Fig. 8), encode unique
protein products. "Wild-type" Hyal 1 protein is encoded by the
cDNAs depicted in Fig. 8B, v-vii, but
because of alternative splicing to form different bicistronic mRNA
species, Hyal 1 proteins with different C termini would be generated by
species i-iv of Fig. 8. Differing forms of Fus 2 are
encoded by the bicistronic transcripts (Fig. 8B,
ii, vi, and vii), but our in
vitro transcription/translation studies suggest that they are not
produced. The bicistronic transcript comprised of both Hyal1
and Hyal3 encodes a Hyal 1::Hyal 3 fusion protein
(Fig. 8D). It is interesting that the most abundant
bicistronic transcript, detected by RT-PCR (Fig. 8), encodes Hyal 1 that has the last eight amino acids removed and replaced by four novel amino acids. Given that the C termini of other hyaluronidases (PH-20
and HYAL 2) were found to be important for its localization (28, 29),
this could have implications for the targeting and localization of Hyal 1.
In vitro transcription/translation studies of the most
abundant bicistronic transcript, identified by RT-PCR (Fig. 8), showed that only Hyal 1 could be expressed from this transcript. A protein product, translated in the in vitro system from pSP64(Fus2),
corresponded to the predicted size of Fus 2 but was not detected in the
lane containing protein products expressed from the bicistronic
transcript (Fig. 9A). Based on the analysis of this
transcript, it is likely that Hyal 1, but not Fus 2 protein products,
can be generated from all of the cotranscripts.
Reports of polycistronic transcription in mammals are rare (30-33),
but more will probably arise as complete analysis of mRNA species
becomes possible with the availability of the human genome sequence.
Polycistronic transcripts may result from the close proximity of
unrelated genes. In prokaryotes, the proteins encoded on operons are
functionally related, but whether this is the case in eukaryotes is yet
to be determined. We currently have no evidence that Hyal 1 and Fus 2 have a related biological function.
In contrast to Hyal2, the Hyal1, Fus2,
Hyal3, and Ifrd2 genes on mouse chromosome 9 form
a cluster that exhibits complex organization and transcription. The
cotranscription of Hyal1 with downstream genes,
Fus2 and Hyal3, indicates that the same promoter
can be used to express multiple genes in this region. It is interesting that the tissue distribution of Fus2 and Hyal3 in
mouse differs from that in humans and now appears similar to the
profile of Hyal1. It is possible that this entire cluster of
genes has evolved some coordinate regulation in mouse or lost this
regulation in humans.
The characterization of the region encompassing Hyal2,
Hyal1, and Hyal3 revealed unexpected complexity
in structure and expression which has significant implications for the
targeting of all of the genes in this region. We found the genes in
this region to be densely packed, in both humans and mice, and in some
cases, overlapping. In mice, the dense organization was accompanied by significant levels of cotranscription as well as coordinated tissue expression of some of the genes. Experimental studies showed that Hyal
1 is likely to be translated from the bicistronic transcripts encoding
Hyal 1 and either Fus 2 or Hyal 3. The high abundance of the
cotranscripts, together with their ability to be translated, suggests
that they contribute significantly to the production of Hyal 1. The
complexity in this region of mouse chromosome 9 may make it difficult
to target genes without impacting on neighboring genes. Strategies for
targeting genes in this region and analyzing the mouse knockouts of
genes in this region should take this information into account.
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ACKNOWLEDGEMENTS |
We thank Dr. Steven Scherer and
Jack Huizenga for screening the mouse PAC library, Tim Salo
and Patty Sauder for technical assistance, and Dr. Steven Pind and Dr.
Klaus Wrogemann for critical review of the manuscript.
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FOOTNOTES |
*
This work was supported in part by Grant MT-15463 from the
Medical Research Council of Canada. The HYAL 1 antibody was generated in, and subsidized by, the Canadian Genetic Diseases Network
Immunoprobes Facility.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF417492, AF417493, AF417494, AF417495,
AF417496, AF417497, AF417498, and AY048681.
§
Supported by a Manitoba Health Research Council studentship.
Supported by the Michael Smith Foundation for Health Research.
To whom correspondence should be addressed: Dept. of
Biochemistry and Medical Genetics, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.:
204-789-3218; Fax: 204-789-3900; E-mail: traine@ms.umanitoba.ca.
Published, JBC Papers in Press, April 2, 2002, DOI 10.1074/jbc.M108991200
2
A. F. A. Smit and P. Green, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
HA, hyaluronan;
EST, expressed sequence tag;
PAC, P1 artificial chromosome;
PIP, percent
identity plot;
RACE, rapid amplification of cDNA ends;
RT, reverse
transcription.
| |
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ABSTRACT
INTRODUCTION
