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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(5): 655-661                                     CN 31-1300/Q

 

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]