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Original Studies |
Department of Internal Medicine (K.K., W.E.T., R.S., M.G., C.E.F., S.B.), Division Of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University Of Medicine and Science, Los Angeles, California 90059; Karolinska Institute (S.A.), Stockholm, Sweden; and Department of Medical Genetics (P.J.C., P.V.H., G.V.C.), University of Antwerp, Antwerp B 2610, Belgium
Address all correspondence and requests for reprints to: Shalender Bhasin, M.D., Chief, Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, and Professor of Medicine, UCLA School of Medicine, 1621 E. 120th Street, Los Angeles, California 90059.
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Abstract |
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Introduction |
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The eukaryotic cilium and the sperm tail have an axonemal core that is made up of nine peripheral microtubule doublets (A and B) that surround a central pair of microtubular singlets (7). Extending from the A tubule of each doublet are the outer and inner dynein arms, which undergo ATP-dependent cycles of joining and releasing the adjacent microtubule doublet (8). The motor function of the dynein arms is achieved by an intricate assembly of approximately fifteen dynein proteins that convert the energy derived from hydrolysis of ATP into a mechanical force (9, 10). Because the absence or shortening of the dynein arms are the most common ultrastructural abnormalities in this syndrome, it is conceivable that mutations of any of these 15 proteins could impair ciliary motility. Indeed, in Chlamydomonas reinhardtii, a biflagellated green alga, mutations of the axonemal dynein genes result in the loss of flagellar motility, indicating that the dynein genes play an important role in flagellar motility (11, 12, 13, 14). Of particular interest is the gene for a 28 kDa Chlamydomonas axonemal dynein light chain protein, called p28 (14), which encodes the IDA4 locus in Chlamydomonas. Splice-site mutations of the p28 gene are associated with the absence of the inner dynein arms and impaired flagellar motility (14, 15). Because the ultrastructural abnormalities observed in the Chlamydomonas p28 mutants are similar to those seen in patients with ICS syndrome, and because loss of p28 function produces the immotile phenotype in Chlamydomonas, we hypothesized that the human homologue of the Chlamydomonas p28 gene would be a logical candidate gene for a subset of patients with this syndrome. As a first step toward determining the role of this gene in the pathogenesis of ICS, we cloned and characterized the human homologue of the Chlamydomonas p28 gene, determined its chromosome location, analyzed tissue specific distribution of gene expression, and determined its genomic structure and evolutionary conservation. To our knowledge, this is the first description of the complete nucleotide sequence of a putative human axonemal dynein gene.
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Materials and Methods |
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We retrieved Chlamydomonas reinhardtii p28 (light chain of axonemal inner dynein arms) protein sequence from the Entrez browser accession no. Z48059). By using the BLAST program at the Baylor College of Medicine Search Launcher (16), the Chlamydomonas p28 protein sequence was used as a query sequence against the Expressed Sequence Tagged (EST) database, leading to the identification of several clones including a complementary DNA (cDNA) with Gen Bank accession nos. W04922 and N70603.
PCR amplification
Oligonucleotide primers specific for various regions of the EST clones were designed using the OLIGO 4.0 program (National Biosciences, Plymouth, MN). Genomic human or rodent-human somatic cell hybrid DNAs, plasmid DNAs, and RT-PCR products were used as templates. The reactions were carried out in a total volume of 20 µl containing 10 pmol of each primer; 200 µm of each dNTP; 50 mM KCl; 10 mM Tris-HCl, pH 8.0; 1.5 mM MgCl2; and 2 U Taq DNA polymerase. Using a Crocodile Thermal-Cycler (Oncor, Gaithersburg, MD), thirty cycles of DNA amplification were performed at 94 C denaturation, variable annealing temperatures, and 72 C elongation. The amplified DNA fragments were analyzed by electrophoresis on 2% agarose gels.
PCR primers
PCR primers include the hp28.A set for a 3' end cDNA 171 bp fragment from 727–897 bp, amplified from forward (AR) primer 5'-GGCAGGTGGAGGAGAAG (727–743 bp) and reverse (AF) 5'-GGCTGCCAGAGGAACACA (896–878 bp). The 5' end hp28 cDNA probe was made as a 187-bp fragment from 113–299 bp, with primer set hp28.D, including forward (DF) primer 5'-GGAACACGGAGAAACGGA (113–130 bp) and reverse (DR) primer 5'-GTCTTCCACCCACTCCCT (299–281 bp). Primer set hp28.E amplifies a genomic 101 bp fragment from 807–907 bp, with forward (ER) primer 5'-GGCATTATTGCACCAAAGAA (807–826 bp) and reverse (EF) primer 5'-ATGATTTTATTGGCTGCCAG (907–888 bp).
Library screening
The human bacterial artificial chromosome (BAC) library from Genome Systems (St. Louis, MO) constructed in pBAC108L was screened by filter hybridization using a PCR generated cDNA probe of 171 bp (nucleotides 727 to 897), made with the hp28.A primer set.
DNA sequencing
Plasmid DNAs from the cDNA clones were prepared using Qiaquick spin columns from Qiagen, Inc. (Chatsworth, CA) and sequenced using cDNA-specific and T3/T7 vector primers with the PRISM ready reaction Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems Inc., San Diego, CA). The DNA sequencing data were analyzed with the computer software SEQED and the Wisconsin Genetic Computer Group (GCG) package (Madison, WI).
5'-end-amplification
To confirm the transcription start site of the hp28 gene, we performed rapid amplification of cDNA ends (5'-RACE) using the Marathon ready testis cDNA (Clontech, Inc., Palo Alto, CA). The PCR reaction was carried out according to the manufacturer’s instructions, except that the annealing temperature was lowered to 55 C for the 5'-RACE hp28 reverse primer 5'-GGCTGTGGGGCTGAACCTGAAGG (196–174 bp). The PCR products of 5'-RACE were separated on a 3% agarose gel, purified using a Qiagen PCR purification column, and sequenced using a reverse nested primer (5'-RACE-nest) 5'-TCCGTTTCTCCGTGTTCC (130–113 bp). These same two primers were 5'-end labeled with 32P-ATP and polynucleotide kinase for use in primer extension reactions by AMV-RT.
Southern blot analysis
Approximately 10 µg of genomic DNA was treated with restriction enzymes in a 50-µl reaction volume for 4 h at 37 C, electrophoresed on a 0.8% agarose gel, and transferred to a Sure Blot (Oncor, Gaithersburg, MD) nylon membrane by alkaline transfer (1.5 M NaCl and 0.4 N NaOH). DNA was neutralized with 0.5 M Tris-HCl and 2 x SSC and cross-linked to the membrane with a UV cross-linker (Stratagene, La Jolla, CA). Hybridization to radioactively labeled DNA probes was performed using standard protocols. The cDNA probes for the 5'-end and 3'-end of hp28 were made, respectively, by PCR ampliication with primer sets hp28.D and hp.28A.
Northern blot analysis
Human multiple tissue Northern blots, obtained from Clontech, were hybridized to hp28 cDNA probe (727–897 bp) using ExpressHyb solution (Clontech) according to the manufacturer’s instructions. Densitometry of autoradiograms was performed by scanning with a One Scanner (Apple Computers, Cupertino, CA), and OFOTO 1.1 and Scan Analysis Software (Bio Soft, Inc., Cambridge, UK).
In vitro transcription and translation
Plasmid clone containing the full length cDNA for hp28 gene was linearized with PvuI enzyme. In vitro transcription and translation were carried out using the TnT coupled kit from Promega (Madison, WI) with 35S-Met labeling of protein and the products were analyzed on a 12% polyacrylamide SDS gel. Dried gel was subjected to autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After searching the EST database with the Chlamydomonas
p28 protein sequence, we identified nine EST matches belonging to five
IMAGE consortium (Integrated Molecular Analysis of Genomes and their
Expression) cDNA clones. These five clones were derived from two
different cDNA libraries, one derived from the fetal liver and spleen
and the other from the fetal lung. The cDNA clones were obtained from
Research Genetics (Huntsville, AL) and three cDNA clones (W04922,
N76977, and H80683) were selected for sequencing based on their size
and restriction maps. Sequence analysis of these three allowed us to
compile the full length cDNA sequence of 921 bp clones (Gen Bank
accession no. AF006386) with an open reading frame of 257 amino acids
(Fig. 1A
). The 5' and 3' untranslated
regions were 56 bp and 93 bp in length, respectively.
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Sequence similarity search with the BLAST (Basic Local Alignment
Search Tool) program revealed that the amino acid sequence of the human
homologue of the Chlamydomonas p28 gene (hp28) is 66%
identical to the Chlamydomonas p28 protein and also 88% identical to
the sea urchin sperm p33 protein (19, Fig. 1B
). Regions between amino
acids 130 and 250 are highly conserved among the three proteins.
Additionally, hp28 protein contains a region of 32 amino acids near the
amino terminus of the protein that is homologous to the Src homology-3
(SH3) binding domain (Fig. 1A, underlined sequence). This
region is present in the sea urchin p33 but absent in
Chlamydomonas p28 (Fig. 1B).
Transcription start site of hp28 gene
Conventional primer extension as well as 5'RACE were performed to determine the transcription start site of the hp28 gene (data not shown). We obtained consistent results using both methodologies. The transcription start was 56 nucleotides upstream from the translation initiation codon at nucleotide position 57. Sequencing the 248 bp RACE PCR product with a nested primer gave a DNA sequence that was one nucleotide longer than the existing cDNA clone. When a plasmid containing the full length cDNA was used for in vitro transcription and translation experiments, we detected a single band of 30 kDa (data not shown). These data suggest that the open reading frame that we have is genuine because the predicted molecular weight for the hp28 protein is 28 kDa.
The hp28 gene is highly conserved during evolution.
A
PCR-generated cDNA probe spanning the region between nucleotides 727
and 897 was used to hybridize a Zoo blot containing genomic DNA from
different species digested with EcoRI restriction enzyme
(Fig. 2). We observed hybridization
signals in all species from the yeast (Sacchromyces cerevisiae) to the
human, indicating that the hp28 gene is highly conserved during
evolution.
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, A and B). A single EcoRI
band of approximately 20 kb was detected, suggesting that the hp28 gene
is a single copy gene. Additionally, because both 5' and 3'end cDNA
probes hybridized to the same 20 kb EcoRI fragment, we
conclude that the complete hp28 gene resides within this
EcoRI fragment.
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We mapped the hp28 gene to a specific region of chromosome 1 by
using one or more somatic cell hybrid panels. Initially, several cDNA
PCR primers were tested on human genomic DNA. A set of primers hp28.E
flanking the region between nucleotides 807 and 908 was able to amplify
a 101 bp product when human genomic DNA was used as template. These
primers were used on a panel of rodent-human somatic cell hybrids from
Coriell Institute (Camden, NJ) for the initial assignment of hp28 to a
particular chromosome. The expected size product was only detected in
the human control and the hybrid containing the human chromosome 1 as
the only human content, suggesting hp28 maps to human chromosome 1
(Fig. 4).
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Tissue distribution of the hp28 messenger RNA (mRNA)
When hp28.A PCR product was used as a probe on the human multiple
tissue Northern blot II (Clontech), a smaller 0.9 kb transcript and a
larger 2.5 kb transcript were detected at the highest levels in the
testis, at medium levels in the prostate, heart, liver, lung, and
pancreas, and at low levels in the ovary, skeletal muscle, and small
intestine. No hybridization signal was detected in spleen, colon
epithelium, thymus, or peripheral blood (Fig. 5, A and B). The level of expression of
the 0.9 kb transcript was 2- to 4-fold higher in the testis compared to
heart, lung, liver, and pancreas. In the testis, the 0.9 kb transcript
is expressed at a 20-fold greater abundance than the 2.5 kb transcript,
whereas the smaller transcript is only 1.2- to 1.7-fold higher in the
lung, heart, liver, and pancreas. In contrast, the amount of 2.5-kb
transcript in the brain is 4-fold higher than the 0.9 kb transcript.
The heart, skeletal muscle, and pancreas also have additional higher
molecular weight bands that are not observed in other tissues.
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A genomic bacterial artificial chromosome (BAC) clone
corresponding to most of the exons of the hp28 gene was isolated to
determine exon-intron junctional sequences. The BAC clone was subcloned
into a cosmid vector after digesting completely with EcoRI
restriction enzyme. A cosmid clone containing the approximately 20 kb
EcoRI fragment was isolated because the hp28 gene is
contained within the approximately 20 kb EcoRI fragment as
verified by PCR with primer set hp28.E. We used the Qiagen purified
cosmid DNA as a template for automated sequencing. By this procedure,
all exons were sized and sequenced at both boundaries, as summarized in
Table 1. When needed, synthetic
oligonucleotides were prepared based on intron sequences. These were
used as sequencing primers or as PCR primers to verify the results.
Thus, every exon-intron boundary was sequenced several times.
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) were identified corresponding to the 921 bp cDNA sequence.
All of the exon-intron boundary sequences conformed to the consensus
rules for such signals; all introns ended on an AG doublet, whereas all
introns started with a GT pair, followed predominantly by an A, or a
GA, a GG, or a T to a lesser degree. The first exon is 140 bp in length
as determined by 5'RACE and contains the 5'untranslated region and the
ATG start site for translation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed considerable mRNA size heterogeneity in different tissues. For example, relative to the 2.5 kb transcript, the smaller 0.9 kb transcript is expressed in greater abundance in the testis and many other tissues. However, the brain has a higher abundance of the larger 2.5 kb transcript. The molecular basis of this mRNA heterogeneity is not known. We also do not know if the alternate transcripts are translated.
The function of the hp28 gene is not known. The presence of a Src-homology-3 (SH3) binding domain in the amino terminus of hp28 protein suggests that it may bind to proteins that contain SH3 domains such as Grb2 and Shc (17, 18). The SH3 domain is a peptide domain of approximately sixty amino acids, made up of ß-sheets (17, 18). Presence of the SH3 domain is a characteristic feature of eukaryotic proteins which are involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions, such as microtubule-mediated cell division and locomotion (17). Unlike its homologs in the sea urchin and the human, the Chlamydomonas p28 protein does not have the SH3 binding domain suggesting that acquisition of SH3 binding domain is a more recent evolutionary event (19).
Chlamydomonas, a unicellular, biflagellated, green algae, is a useful model for the human ICS. Chlamydomonas flagellum and human cilium have similar ultrastructure. The immotile mutants of Chalmydomonas and the ICS patients exhibit similar ciliary ultrastructural abnormalities (14, 15). Particularly relevant are the mutations at the IDA-4 locus, that has three alleles: ida4–1, ida4–2, and ida4–3. LeDizet et al. (14, 15) demonstrated that p28, coding for a Chlamydomonas axonemal inner-dynein arm light-chain protein, is the altered gene in IDA4 mutants. Western blotting and immunostaining experiments indicate that all three IDA4 alleles give rise to Chlamydomonas that lack p28 protein in their flagella and cell bodies (15). Additionally, mutant strains regain motility by introduction of the wild type p28 protein. The p28 gene has also been linked to the IDA4 locus by restriction enzyme length polymorphism studies. The IDA4 mutations are at the intron-exon boundaries and affect the splice sites of the p28 gene (15); incorrect splicing prevents the synthesis of the p28 protein and produces the phenotype. The p28 protein has been shown to form complexes with the dynein heavy chains, actin, and caltractin/centrin molecules (14). These findings collectively suggest that the p28 gene plays an important role in ciliary motility.
More recently, the gene for a 33-kDa sea urchin sperm protein (19) that has a high degree of homology to the Chlamydomonas p28 was cloned. Antibodies against the sea urchin p33 protein inhibit the motility of the sea urchin and human sperm, providing further evidence that the dynein-like p33 gene plays an important role in sperm motility (19).
Our mapping studies using the Genebridge 4 radiation hybrid mapping panel linked the hp28 gene to a DNA marker D1S195 on chromosome 1. Another DNA marker, D1S255, that is in close proximity to the D1S195, has been linked to a locus DFNA2 for a postlingual form of progressive deafness (20). The human cochlea is a ciliated structure and impaired hearing has been described in some patients with ICS. It is, however, not clear whether the impaired hearing described in patients with the ICS is due to recurrent upper respiratory tract and middle ear infections or the result of a specific cochlear defect. We are currently screening patients with nonsyndromic deafness for mutations in the hp28 gene.
The spectrum of clinical disorders associated with defects of ciliary motility may be wider than is currently appreciated. For example, patients with Usher’s syndrome exhibit abnormal ultrastructure in their sperm and photoreceptor axonemes (21). Usher syndrome type I is sometimes associated with bronchiectasis (22, 23). Also, patients with retinitis pigmentosa have an increased incidence of abnormal nasal cilia (24).
Although the genetics and pathology of ICS have been well studied, the genes responsible for this clinical disorder have not been identified. Although both autosomal recessive and sex-linked modes of inheritance have been described, the majority of cases appear to result from autosomal recessive inheritance. The syndrome is particularly common in isolated areas with high degree of inbreeding, e.g. in remote parts of Sweden, and among Polynesians in Samoa and Maoris in New Zealand (1, 4, 5). There is heterogeneity in the clinical expression of ICS, in the pattern of inheritance, and in the ultrastructural abnormalities in different families. The severity of the disease varies considerably in the affected individuals. The variant described in Pacific Islanders differs from that described in Northern Europeans (1, 2, 3, 4, 5). The pulmonary disease appears to be less severe in the Pacific Islanders affected by this syndrome. Also, infertility appears to be uncommon among the affected Pacific Islanders. Whether the clinical phenotypes described in the Samoans, the Maoris, and the Northern Europeans are the result of distinct genetic defects or whether they represent different phenotypic expression of the same genetic defect remains to be seen. Heterogeneity of ultrastructural defects and clinical phenotype (1, 2, 3, 4, 5, 6, 25) suggests that more than one molecular defect/genetic locus may be implicated in different subsets of ICS.
With the cloning of the hp28 cDNA and the mapping of the intron-exon junctions, it is now possible to test the hypothesis that mutations of the hp28 gene are associated with ICS. We are currently screening patients with this syndrome for mutations in the hp28 gene.
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Footnotes |
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Received May 7, 1997.
Revised June 5, 1997.
Accepted June 17, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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