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https://www.abbs.info e-mail:[email protected] ISSN 0582-9879 |
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Short Communication |
Molecular
Cloning and Characterization of Human DDX36 and Mouse Ddx36
Genes, New Members of the DEAD/H Box Superfamily
FU
Jun-Jiang, LI Lu-Yun, LU Guang-Xiu*
(
Laboratory of Human Reproductive Engineering, Central South University Xiangya
Medical College, Changsha 410078, China )
Abstract With the
strategy of homologue molecular cloning using the sequence of the maleless gene
(mle) of Drosophila, the novel homologous human and mouse genes
with longer DNA/RNA helicase box (DEAD/DEAH box), named DDX36 and Ddx36
genes, respectively, were cloned as new members of the DEAD/H box superfamily.
The predicted protein encoded by human DDX36 gene has a sequence
identity of 37 % and similarity of 58% with the MLE protein of Drosophila
and 91% and 94% with the predicted protein encoded by mouse Ddx36 gene,
respectively. Northern blotting of DDX36 shows a single strong signal of
3.8 kb in the hybridization pattern in human testis but no or very weak signal
in other tissues. The DDX36 gene is mapped to chromosome 3q25.1–3q25.2, in which
26 exons and 25 introns have been identified. DDX36 and Ddx36
genes may be involved in sex development, spermatogenesis and male
reproduction.
Key words DDX36
gene; Ddx36 gene; cDNA cloning; DEAD/H box; tissue expression pattern
Drosophila
melanogaster is one of the most intensively studied organisms in biology
and serves as a model system for the investigation of many developmental and
cellular processes common to higher eukaryotes, including human. Nearly all the
≈120-megabase euchromatic portion of the D.melanogaster
genome has been sequenced[1] so that D.melanogaster is also
an ideal model organism for the studies of structural and functional genomics.
There is evidences that up to 1 500 recessive genes contribute to male
fertility in the species[2]. Kuroda et al.[3]
found that the maleless gene (mle) with the function of the putative RNA
and DNA helicases is related to regulation of the X chromosome dosage
compensation in Drosophila and so far it has been known that four
identified regulatory genes are required for dosage compensation in Drosophila
males. The wild-type functions of these loci are necessary for male viability
and thus they are collectively designated as the male-specific lethal gene (msl).
They are mle[4–6],
msl-1[7], msl-2[7], and mle-3[8,9].
Loss of function mutations at any one of the msl loci results in male
lethality, but have no effect on the viability or fertility of females. In D.
melanogaster the mle gene, one of the loci necessary for somatic dosage
compensation, is also required for male fertility[4,7,10]. Rastelli
and Kuroda[4] suggested that mle is not involved in chromosomal
dosage compensation but may be involved in post-transcriptional gene regulation
in the germline of Drosophila although in somatic cells the mle
gene is necessary for X-chromosome dosage compensation. The protein (MLE
protein) encoded by the mle gene of Drosophila belongs to the
DEAD/H box protein family. In the DEAD/H box family, there have been more than
30 proteins from a wide range of organisms, from bacteria to human, which share
a group of conserved motifs, including the sequence Asp-Glu-Ala-Asp/His
(DEAD/H). These proteins are implicated not only in splicing but also in
diverse cellular functions, including ribosome assembly, translation
initiation, spermatogenesis and embryogenesis [OMIM 600396][11–13].
Since
the DEAD/H box proteins in Drosophila are involved in spermatogenesis,
exploring the new member of DEAD/H box family in human is helpful not only to
further study the DEAD/H box family, but also to lucubrate the mechanism of the
spermatogenesis and male infertility in human. Here we report the cloning and
characterization of human DDX36 and mouse Ddx36 genes, which
belong to the DEAD/H box superfamily, by combining the bioinformatic analysis
with the experimental techniques.
Searching
for the human homologue of Drosophila mle gene (GenBank accession
No.M74121) was performed against the human EST database of GenBank with BLAST
software in NCBI (http://www.ncbi.nlm.nih.gov/blast). Some human ESTs were
found which are homologous to the Drosophila mle gene, of them an
EST （R21093）
had a sequence identity of 56.9% in 288 nucleotides and identity of 50% and
similarity of 60.2% in 88 predicted amino acids with the mle gene,
respectively. Then the EST database searching and extending for R21093 were
performed and a longer EST（U69561）was
found. A primer pair, MLE11 (5′-TATTTTCCGAACACCCAGGAGGGGT-3′)
and MLE12 (5′-CTGTAGGCATCAGTGAATGTAAAG-GT-3′),
was designed based on the sequence of U69561. And the primer of λgt10-3
(5′-GTGGC-TTATGAGTATTTCTTCCAGGG-3′)
located on the cDNA library lgt10
vector arm was designed. The touchdown PCR amplifications were carried out
using the total cDNA of human fetal brain cDNA library (Clontech) and the human
testis Marathon-ReadyTM cDNA (Clontech) as templets, respectively.
In order to generate a good RACE product with a nonspecific background of low
level, half-nested PCR and Advantage 2 Polymerase Mix (Clontech) were adopted[14,15].
In nested PCR, a primary amplifica-tion was performed with the outer primers of
MLE12 and lgt10-3
and then aliquot of the primary PCR product was re-amplified using the inner
primers of MLE12 and lgt10-3.
For touchdown PCR amplifications, the first 5 PCR cycles were performed at 94 ℃
for 10 s, 72 ℃
for 3 min, then the second 5 PCR cycles were performed at 94 ℃
for 10 s, 70 ℃
for 3 min, finally the third 20–25
PCR cycles were performed at 94 ℃
for 10 s, 68 ℃
for 3 min, and the denature step in the first cycle was 90 s and the extension
step in the last cycle was 5 min. Finally the PCR products were kept at 4 ℃
till for PAGE electrophoresis. Then the PCR products were fragmented by 6%
polyacrylamide gel electrophoresis for analysis. The DNA fragments with strong
and clear signals and longer sizes in the gel were cut into the tubes of 1.5 ml
using a clean falchion under a UV instrument. The DNAs were eluted by ddH2O
and the eluted DNAs were re-amplified, cloned into pUCm-T vector (Sangon,
Shanghai), and sequenced with an ABI 377 XL Auto-Sequencer (ABI). The primers
were designed based on the 5′-ends
of the new sequences and nested PCR amplification were performed again for
prolonging the fragments at the 5′-ends
for several times, respectively, according to the method mentioned above. The 5′-end
outstretched primers are MLE4 (5′-CGAAGGCAGC-TTTTCTCTGAAATGCT-3′),
MLE5(5′-CTCAG-AATCTCGGTCAATATATGATC-3′),
MLE6(5′-CTCGTCGTTCATCCATGTGTACTAC-3′)
and MLE7 (5′-TCTCCGCTTCCTTGTTCTTCTGCC-3′).
And a primer pair (MLE21: 5′-AAGGCTAG-GTGGGATTGCTT-3′
and MLE22: 5′-CTGGATC-TTTAGGATTTCTAC-3′)
were designed based on the sequence of the EST U69561, in which some bases
could not be ascertained, and PCR amplification was performed using the primer
pair and the PCR product was sequenced. Nested PCR amplification, cloning and
sequencing for 5′-end
prolongation were performed for several times. Finally the sequences of the PCR
products were assembled into a novel gene with a full-length sequence of 3.6
kb, named DDX36 (DEAD/H box polypeptide 36) gene by the Human Gene
Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature/) (we previously
called it MLEL1 gene), which contained an open reading frame (ORF) of 3
024 bp, encoding 1 008 amino acids. The initiator codon (ATG) of in-frame was
located at the nucleotides 74–76
and terminator codon (TGA) at 3 098–3
100. A terminator codon in 5′
UTR sequence (“upstream
terminator”)
was located at the nucleotides 59–61.
Two trailing signals and poly A in the 3′
UTR were found [Fig.1 (A)]. Three PAC clones (AC072034, AC018452, AC041012)
from the work draft sequence and the newest Homo sapiens chromosome 3 working
draft sequence segment (ref|NT-005678.3|Hs3-5835) were obtained by BLAST
searching against the GenBank database using the sequence of DDX36 gene
and the exon-intron structure was confirmed. The exons differ in size, the
largest is 686 bp
and the smallest 39 bp
(see Table 1).
Fig.1 cDNA and deduced amino acid sequences
of human DDX36 gene (A) and mouse Ddx36 gene (B)
(A) The sequence of DDX36 cDNA and
the encoded protein. (B) The sequence of Ddx36 cDNA and the encoded
protein. Glycine-rich domain and DNA/RNA helicase domain are underlined. The
stop codon is marked by “asterisk”.
The polyadenylation signals (AATAAA) in 3′UTR
are underlined with wave. The molecular weights of DDX36 and Ddx36
proteins are 114 776.19
and 113 882.42,
respectively, and their deduced iso-electric points are 7.57 and 8.63,
respectively, being alkaline proteins. The sequences of DDX36 and Ddx36
were registered in GenBank, EMBL and DDBJ Databases under accession No.AF217190
and AF448804, respectively.
In
order to obtain the full-length cDNA (Ddx36) of mouse homologue of the
novel human DDX36 gene, the same techniques were applied using the
sequence of DDX36 gene. Some mouse ESTs (accession No.AI390533, BE650099
and mouse UniGene Cluster: Mm.334158) were obtained by homologous searching
against the mouse EST database in GenBank. The 5′–
and 3′-RACE (nested-PCR) techniques were
performed to determine the upstream and downstream sequences of the novel mouse
gene. As a result, the full-length cDNA of mouse Ddx36 gene, covering 3
534 bp with an ORF of 3 003 bp and encoding 1 001 amino acids, was obtained.
The initiator codon (ATG) in-frame was located at the nucleotides 50–52
and terminator codon (TGA) at 3 053–3
055. A terminator codon in 5′
UTR sequence was located at the nucleotides 35–37.
Two trailing signals and poly A in the 3′
UTR were also found [Fig.1 (B)].
BLASTp
searching indicated the predicted protein encoded by human DDX36 gene
had a sequence identity of 37 % and similarity of 58% with the MLE protein of Drosophila
and 91% and 94% with the predicted protein encoded by mouse Ddx36 gene,
respectively. Searching against the conserved domain database with reverse
position specific BLAST (http://www.ncbi.nlm.nih.gov/blast) and with
profilescan tool in Prosite database[16] revealed some significant
motifs in DDX36 and Ddx36 genes: the DNA/RNA helicase
domain(DEAD/H box) at the amino acid residues 259–614
and 252–607,
and the glycine-rich regions at 10–63
and 13–44,
respectively, so that the genes may belong to DEAD/H-like helicase superfamily,
like the mle gene in Drosophila. Multiple sequence alignment
against prosite database with CLUSTAL W Program[17] was performed
using the human DDX36 protein, mouse Ddx36 protein, Drosophila
maleless protein, Sciara ocellaris maleless protein, human RNA
helicase A and bovine RNA helicase A. The results showed that they all contain
DEAD/H box and are sequence highly conservative, and hence indicated that they
belong to DEAD/H box superfamily [Fig.2(A)]. Fig.2(A) and (B) show a very high
homology between DDX36 and Ddx36.
Fig.2 The comparison of predicted amino acid sequences of the DDX36
and Ddx36 with other DEAD box family members by Clustal W program
(A) Multiple sequence alignment results.
The protein encoded by human DDX36 gene has a sequence identity of 37 %
and similarity of 58% with the MLE protein of Drosophila and 91% and 94%
with the protein encoded by mouse Ddx36 gene, respectively. (B)
Phylogenetic tree result. DROMLE, Drosophila maleless protein (M74121);
SOCY18119–Sciara
ocellaris maleless protein (Y18119); DDX9, human
RNA helicase A (AL13848); BTNDNAHII, Bovine RNA helicase A (X8289); DDX36,
human DEAD/H box polypeptide 36 (AF217190); Ddx36, mouse DEAD/H box
polypeptide 36 (AF448804).
Prediction
of transmembrane region and protein orientation with TMpred program found
strong transmembrane helices at the amino acid residues 700–725
and 698–717
in DDX36 and Ddx36 proteins, respectively, and a strongly
preferred model with N-terminus outside. Prediction of cleavage site of signal
peptide indicated that they belong to the non-secrete protein without signal
peptide sequence at the N-terminus. The PKC, CK2, cAMP and TYR phospho-sites
may be involved in signal conduction. These results hint that DDX36 and Ddx36
proteins are membrane proteins with biological functions.
Northern
blot analysis of DDX36 expression in multiple human tissues (MTN I and
MTN II, Clontech) shows a very strong signal in testis and no signal or very
weak signal in other tissues (Fig.3) and indicates a single transcript of ≈3.8
kb in testis. Considering the information that the mle gene is not
involved in chromosomal dosage compensation but may be involved in
post-transcriptional gene regulation in the germline of Drosophila, it
was suggested that the DDX36 and Ddx36 genes might have a latent
function in sex development, spermatogenesis and post-transcriptional
regulation in human testis.
Fig.3 Expression pattern of the DDX36
gene in multiple human tissues
2 mg
of poly A+ RNA per lane was run on a denaturing formaldehyde 1.0%
agarose gel, transferred to a nylon membrane by Northern blotting.
Hybridization was performed with [32P]-labeled probe by PCR labeling
at the DDX36 nuecleotides 2 368–2
532 using the primer pair of MLE22 (5′-CTGGATCTTTAGGATTTCTAC-3′)
and MLE13（5′-TGGGAAGAGGCTAGGCGA-3′）.The
membrane was washed in a solution containing 2×SSC
and 0.05% SDS at the room temperature twice, each for 5 min, washed in 0.1×SSC
and 0.1% SDS at 42 ℃
twice, each for 5 min. Finally the hybridized membrane was wrapped and exposed
at -70 ℃
for 48 h. Lane 1–16
contain, in order, RNA from pancreas, thymus, prostate, testis, ovary, small
intestine, colon, peripheral blood leukocyte, heart, brain, placenta, lung,
liver, skeletal muscle, kidney and spleen. Arrow shows the 3.8 kb hybridized
signal.
In
order to map the DDX36 gene, PCR amplification was performed using the
primer pair (EX 13-1: 5′-GCTTGCCTCAGTTTGAAATAC-3′
and EX 13-2: 5′-GCTTTTCTGCAACTCTTTAT-CT-3′)
of exon 14 and using human/mouse somatic cell hybrid DNAs（PCRable
DNA PSC Hybrid Panel, BIOS product）as
templets, respectively. The results indicated that DDX36 is located on
the chromosome 3. PCR amplification was performed with GB4 panel and the
results were checked by agarose electro-phoresis and then statistically
analyzed. The RH results are as follows:
120020211022000011110000-1001100100100101000210001111001002000111000-00200100100120000100010100.
These data shows that the DDX36 gene is mapped between the
microsatellite markers D3S1280 and D3S1275. Referring to GDB database
(http://gdb.org/hugo) for comprehensive Map and FISH map of PAC, finally we
assigned DDX36 to chromosome 3q25.1–3q25.2.
In
summary, the DDX36 and Ddx36 genes are highly homologous to each
other at both the nucleotide and amino acid levels, which belong to the DEAD/H
box superfamily, like mle in Drosophila. The DDX36 and Ddx36
genes may be involved in sex development, spermatogenesis[18] and
male reproduction[19].
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Received:
February 28, 2002
Accepted: April 22, 2002
This
work was supported by a grant from the Special Funds for Major State Basic
Research Program of China (973 Program) (No.G1999055901)
*Corresponding
author: Tel,86-731-4497661;Fax,86-731-4497661;e-mail, [email protected]