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Cloning, Mapping, and Tissue Distribution of a Human Homologue of the Mouse Jerky Gene Product
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 389–395 (1997)
RC976935
Cloning, Mapping, and Tissue Distribution of a Human Homologue of the Mouse Jerky Gene Product1 Zhizhen Zeng, Hla Kyaw, Karen R. Gakenheimer, Meena Augustus, Ping Fan, Xiaochun Zhang, Kui Su, Kenneth C. Carter, and Yi Li2 Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, Maryland 20850
Received June 5, 1997
Inactivation of the jerky gene by insertion of a transgene into the mouse genome results in epileptic seizures in transgenic mice. This finding indicates that the jerky gene plays an important role in inducing epilepsy syndromes in mice. We report here our efforts in cloning, chromosomal mapping, and analysis of tissue distribution of a novel human gene, the HHMJG, a homologue to the mouse jerky gene product. We have successfully identified a full length cDNA clone encoding a novel human protein homologous to the mouse jerky gene product. The finding was based on the result of an analysis of EST (expressed sequence tag) sequences of a clone from a human tonsil cDNA library. A 4.0 kb mRNA species of the HHMJG is abundantly expressed in the majority of human tissues examined, including brain and skeletal muscle. However, in the testes, two mRNA species of the HHMJG, approximately 2.0 and 4.0 kb, are abundantly expressed. Sequence analysis of the HHMJG cDNA indicates that it encodes a putative protein of 51 kD, which shares significant sequence homology to not only the mouse jerky gene product but also some nuclear regulatory proteins, such as centromere binding protein-B. The predicted nuclear localization of the HHMJG product suggests that this protein may function as a nuclear regulatory protein. The result of human chromosomal mapping shows that the HHMJG is located on human chromosome 11q21. Our identification of the HHMJG cDNA provides a potential gene candidate to further investigate the biological significance and clinical implications of the HHMJG in human epilepsy. q 1997 Academic Press
in the absence of acute precipitating factors and affects approximately 4% of individuals at some time in their lives. According to its etiology, epilepsy can be defined as idiopathic and symptomatic. Symptomatic epilepsies are usually caused by other brain disorders. However, a certain proportion of idiopathic epilepsies are considered to be genetic consequences (1). Although familial occurrence of epilepsy has been known for a long time, little progress in mapping epilepsy genes has been reported until recently because of complexity in the genetic contribution to epilepsy. The first epilepsy locus was identified in a common benign idiopathic generalized epilepsy syndrome, juvenile myoclonic epilepsy (2). Since then, the chromosomal loci for several epilepsy genes have been determined (3–11). Furthermore, evidence of genetic heterogeneity in epilepsy suggests complexity of its etiology (12), and indicates that the same phenotype of the epilepsy syndrome may derive from different genetic mechanisms or vice versa. Therefore, it is critical to explore the genetic events related to epilepsy for a better understanding of their roles in both physiological and pathological conditions. We report our effort at cloning, mapping, and characterizing a novel human gene, named the human homologue of the mouse jerky gene (HHMJG). Sequencing analysis of a cDNA clone of the HHMJG shows that it shares good homology to the mouse jerky gene product reported by Toth and colleagues (13), in which they found that inactivation of the jerky gene caused epileptic seizures in transgenic mice. MATERIALS AND METHODS
Epilepsy is one of the most common neurological disorders characterized by recurrent unprovoked seizures 1 The nucleotide sequence of the gene reported in this article has been submitted to GenBank under Accession No. AF004715. 2 To whom correspondence should be addressed at Department of Protein Therapeutics, Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, MD 20850. Fax: (301) 340-7159.
cDNA cloning. Expressed sequence tag (EST) analysis (14– 16) of cDNA clones derived from a human tonsil cDNA library oligo(dT)primed and constructed in the pCMVSport 2.0 (GIBCO BRL) identified a 400-bp clone demonstrating significant homology to the jerky gene product of mouse. This cDNA clone encoding a complete HHMJG was sequenced to completion by a ABI sequencer (14) (GenBank accession number L76380). It contains a gene of 2889-bp encoding a human protein homologous to the mouse jerky gene prod-
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FIG. 1. The cDNA sequence and deduced amino acid sequence of HHMJG. (A) The nucleotide sequence is indicated by the number on the top of each row. Amino acids are represented by the one-letter code and indicated below their respective codons. Two potential in-frame ATG initiation codons (#207 and 303) are underlined. The termination codon is indicated by an asterisk. (B) Result of alignment of the deduced amino acid sequence (HHMJG) to the mouse jerky gene product (MJERKY).
uct. cDNA library construction was carried out essentially as described (17). Isolation of the HHMJG genomic clones. Genomic clones of the HHMJG were isolated by plaque hybridization of the human Lambda DASH II genomic library (Stratagene) with a full length cDNA clone of the HHMJG as a probe. Standard techniques were used for genomic library screening (18). The Lambda DASH II human genomic library (2 1 106 phage) was plated at a density of 5 1 104 plaque forming units/150 mm plate for primary screening. The plaques were transferred onto nylon transfer membrane (Amersham) and prehy-
bridized at 427C for a minimum of 2 hours in 50% formamide buffer (50% formamide, 51 SSC, 21 Denhardts’, 1% SDS, 20 mM NaH2PO4 , and 250 mg/ml denatured salmon sperm DNA). Hybridization was performed in fresh 50% formamide buffer containing [32P] a-dCTPlabeled probe (Prime-It II Random Primer Labeling Kit, Stratagene) at 427C for 18 hours. Following hybridization, the membranes were rinsed twice with 21 SSC containing 0.1% SDS at room temperature for 10 minutes, and then washed twice at 427C for 10 minutes in 0.21 SSC containing 0.1% SDS at high stringency. The membranes were exposed to Kodak autoradiography film overnight at 0 807C with an intensifying screen. To proceed to a secondary screening,
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FIG. 1—Continued
putative positive clones were picked, and put into 500 ml of SM buffer with 5.8 gm NaCl/L, 2.0 gm MgSO4/L, 50 ml 1M Tris (pH 7.5)/L, and 5 ml 2% gelatin/L and 10 ml of chloroform. Secondary tertiary screening was completed using the same method as primary screening. Positive lambda phages in SM buffer were titered, and then plated in duplicate. Once the bacteriophage suspension was recovered and pooled in a sterile conical tube, 5% of chloroform was added. Tubes were shaken gently, incubated for 15 minutes at room temperature and centrifuged for 15 minutes at 2000 1 g. Supernatants were transferred to new sterile tubes to which 5% polyethylene glycol (M.W. 8,000) and 5% NaCl were added. Tubes were gently shaken, incubated at 377C for 30 minutes and centrifuged for 20 minutes at 10,000 1 g. Supernatants were discarded. The genomic DNA pellets were resuspended in TE buffer, extracted twice with phenol:chloroform [1:1 (v/v)], and precipitated by ethanol. Pellets were resuspended in sterile water. Four genomic clones, each more than twelve kb in length, were obtained and confirmed by sequencing with EST primers of the HHMJG using an ABI sequencer (14). Northern blot analysis. Human Multiple Tissue Northern (MTN) Blots (Cat. No. 7760-1 & 7750-1) and Human Brain Tissue Northern Blot (Cat. No. 7755-1) were purchased from Clontech for analysis of expression of the HHMJG mRNA. Hybridization was carried out essentially as described (18) using [32P]a-dCTP-labeled probe (PrimeIt II Random Primer Labeling Kit, Stratagene), which represents the full length cDNA of the HHMJG.
Chromosome mapping. Both a 2.9 kb cDNA and a 30 kb genomic DNA of the HHMJG were nick-translated using Digoxygenin- 11 dUTP (Boehringer Mannheim) and fluorescence in situ hybridization (FISH) was done as described by Johnson and colleagues (19). Chromosomes were counterstained with DAPI. Color digital images containing both DAPI and gene signal detected using anti-Digoxygenin conjugated Rhodamine, were recorded using a triple-band pass filter set (Chroma Technology, Inc., Brattleburo, VT) in combination with a charged coupled-device camera (Photometrics, Inc., Tucson, AZ) and variable excitation wave length filters (20). Images were analyzed using the ISEE software package (Inovision Corp. Durham, N.C.) and the cytogenetic map location was assigned (21).
RESULTS Cloning of HHMJG. The clone of 2889-bp nucleotides contains a open reading frame of 1329 bp, encoding a polypeptide of 442 amino acids. The deduced amino acid sequence of the HHMJG shows 55% similarity and 35% identity to the mouse jerky gene product (Fig. 1). There are four potential in-frame ATG initiation codons (#171, 183, 207, and 303). Among them two (#207 and 303) are in a favorable context for initiating
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FIG. 2. Tissue expression of the HHMJG mRNA. Multiple Tissue Northern (MTN) blots containing poly-A RNA’s from various human tissues were obtained from Clontech, and examined with 32P-dCTP, random priming labeled probe using the full length cDNA clone of the HHMJG as a template. ExpressHyb hybridization solution (Clontech) was applied to the blots according to Clontech’s protocol, PT1190-1. The results showed that one species of the HHMJG mRNA, about 4.0 kb, was obviously expressed in abundance in most of the human tissues examined. The exceptions are the lung, liver, and peripheral leukocyte tissues, in which expression of HHMJG mRNA is less abundant. In the testes, there are two species of the HHMJG mRNA, approximately 2.0 kb and 4.0 kb, between which the 2.0 kb mRNA species is predominant. The 4.0 kb mRNA species of the HHMJG is expressed at very significant level in all eight brain tissues from different parts of the human brain.
translation; the presence of a purine at position 03 conforms to the Kozak translation initiation consensus sequence (22). The results using the BLAST algorithm of the sequence database with the HHMJG cDNA nucleotide sequence revealed that the HHMJG shares very significant homology with both nucleotide and amino acid sequences to the jerky gene product of mouse, and to several other genes which encode nuclear regulatory proteins, such as putative transposase (23) and centromere binding protein B (24). Seven genomic clones of the HHMJG were confirmed by partial sequencing (data not shown). Tissue expression of HHMJG mRNA. Northern blot analysis of multiple human tissue Northern blots using the full-length cDNA of the HHMJG as a hybridization probe reveals that a 4.0 kb mRNA of the HHMJG is abundantly expressed in the heart, brain, placenta, skeletal muscle, pancreas, thymus, prostate, ovary, and small intestine, but in the testes, two mRNA species of the HHMJG, approximately 2.0 kb and 4.0 kb, are abundantly expressed. Between them, the 2.0 kb mRNA species is predominant. However, expression of the HHMJG mRNA is less abundant in the lung, liver and peripheral leukocytes (Fig. 2). Using the human
brain tissue Northern blot, the 4.0 kb mRNA species of the HHMJG is expressed virtually in the same abundance in all eight brain tissues included (Fig. 2). Chromosomal localization of the HHMJG. The chromosomal location of the HHMJG was established using single-copy gene fluorescence in situ hybridization to normal human male metaphase chromosome spreads (25). For the genomic probe, approximately 16 spreads were analyzed by eye, most of which had a doublet signal characteristic of genuine hybridization on both homologues of chromosome 11 (Fig. 3A-B). Doublet signal was not detected on any other chromosome. Detailed analysis of 10 individual chromosomes, using fluorescence banding combined with high-resolution image analysis, indicated that the HHMJG is positioned largely within band 11q21. Since this band is flanked very closely by q14.3 and 22.1, some of the signal appeared to overlap into these bands (Fig. 3C). DISCUSSION There are many studies which suggest a genetic contribution to human epilepsy and evidence exists for linkage of susceptibility loci to specific chromo-
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somal regions (2 – 12). Studies on the mouse jerky gene have demonstrated that inactivation of the gene by insertion of a transgene into the mouse genome results in epileptic seizures in the transgenic mice (13). Based on the similarity between the human and the mouse genomes, it is obvious that identification of the human homologue of the mouse jerky gene will be beneficial to the discovery of potential candidate genes related to human epilepsy and to study their role in the etiology. We have successfully identified a full length cDNA clone encoding a novel human protein homologous to the mouse jerky gene product. The finding of the HHMJG full length cDNA was based on the results of the analysis of ‘‘EST’’ (expressed sequence tag) sequences of a clone from a human tonsil cDNA library. Although the sequence homology between the mouse jerky gene product and the HHMJG product may not necessarily correspond to the similarity of functions of the proteins encoded by these genes, it provides researchers with a novel gene candidate for further investigation of its role in human epilepsy. In recent years there is an increasing interest in finding potential genes associated with human epilepsy and in studying their biological significance and the clinical implications. We believe that our enunciation of the HHMJG will benefit the investigation of human epilepsy. Based on the results of sequence analysis, the HHMJG cDNA encodes a putative protein of 51 kD, which is possibly located in the nucleus of the cell as predicted by PSORT software (26). The probable nuclear localization of the HHMJG product corresponds well with the results derived from the BLAST algorithm of the HHMJG sequences in the data base. The results indicate that in addition to the similarity to the jerky gene of mouse, the HHMJG also shares good sequence homology with genes known as nuclear regulatory proteins, such as centromere binding protein-B. As we know, CENP-B, a highly conserved centromere-associated protein, binds to alpha-satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENPB box (27). CENP-B participates in and is necessary for mitotic chromosome movement (28). Because of the fundamental effects of the nuclear regulatory proteins on cells, (such as regulation of transcription on gene expression, cell proliferation, and their physiological functions) it is possible that the HHMJG product may have similar effects on the target cells. The tissue distribution of the HHMJG from our study also indicates that the HHMJG mRNAs are expressed in the majority of tissues examined, therefore, protein encoded by the HHMJG may have an influence on cells in these tissues. Interestingly, the HHMJG mRNA shows two species of different sizes, about 2.0 and 4.0 kb, respectively, in the tes-
tis. It is not clear what the biological significance of the alternatively spliced form of the HHMJG mRNA is in the testis. We have successfully applied the FISH mapping technology to identify location of the HHMJG on human chromosomes. The result of chromosomal mapping reveals that the HHMJG is located to human chromosome 11 at band q21. However, since signals for possible homologous sequences to the HHMJG appeared consistently on 11q21 and sporadically on 6p21.3-22.1 to which we considered as a weak secondary signal (data not shown), it could well be that this suggests a locus heterogeneity of sorts. Both linkage and association studies provide strong evidence that a gene locus on chromosome 6 is involved in the expression of juvenile myoclonic epilepsy (JME) (11, 29, 30). It has also been demonstrated that the inactivation of the jerky gene results in whole body jerks, generalized clonic seizures, and epileptic brain activity in affected mice (13). Considering the facts which include the sequence homology between the HHMJG and the mouse jerky gene, the results of chromosomal mapping of the HHMJG, and the similarity of the HHMJG product to several nuclear regulatory proteins, we believe that HHMJG may be a potential candidate gene associated with human epilepsy. If it can be established in kindreds of epileptic individuals that the HHMJG is either inactivated or mutated, some clinico-pathological correlations could be made. It is also interesting to note that genes related to cerebellar and spino cerebellar ataxia have been mapped to 11q (31) and 6p. However, since this is the first report of a human homologue to the mouse jerky gene to our knowledge, and there is no report thus far to indicate that a potential candidate gene associated to human epilepsy is located at chromosome 11q21, we do not at present know the correlation between the chromosomal localization of the HHMJG and its biological significance in human epilepsy. Therefore, further investigation is necessary to address these questions. There is no question about the importance of genetic mechanisms in epilepsy. However, in most forms of epilepsy, patients do not segregate within families following a simple memdelian pattern of inheritance. The complexity of the disease suggests that besides genetic factors, others, such as environmental factors and modifying genes may also be important for disease expression in susceptible individuals. It is reasonable to believe that both genetic and non-genetic influences on susceptibility are required. Therefore, the identification of the HHMJG provides a novel candidate gene associated to human epilepsy. Further investigation of the role of the HHMJG will give us a better understanding of the biological significance and potential clinical implications of this gene in the human epilepsy syndrome.
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FIG. 3. Fluorescence in situ hybridization mapping of the HHMJG. The genomic DNA probe of the HHMJG was hybridized to normal human male metaphase chromosomes. (A) A chromosome spread from a single cell showing hybridization to the q arm of both chromosomes # 11 (arrows). (B) Examples of 2 chromosomes #11 with hybridisation signal. The left panel of each set shows a DAPI stained chromosome (blue) with gene signal (red). The right panels shows the same chromosome displayed as an inverse black and white image. The G-band like DAPI pattern was enhanced using an edge sharpening algorith. (C) ideogram of chromosome #11 showing the position of the HHMJG.
ACKNOWLEDGMENTS We thank the following individuals for their support of the work: Drs. William Haseltine, Craig Rosen, Michael Antonaccio, Pat Dillon, Gianni Garotta, and Reiner Gentz, and individuals in the laboratories of gene discovery and bioinformatics at Human Genome Sciences, Inc.. In particular, we thank Dr. Steve Ruben, Department of Protein Therapeutics, Human Genome Sciences Inc., for his support and helpful discussion.
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Cloning, Mapping, and Tissue Distribution of a Human Homologue of the Mouse Jerky Gene Product1 Zhizhen Zeng, Hla Kyaw, Karen R. Gakenheimer, Meena Augustus, Ping Fan, Xiaochun Zhang, Kui Su, Kenneth C. Carter, and Yi Li2 Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, Maryland 20850
Received June 5, 1997
Inactivation of the jerky gene by insertion of a transgene into the mouse genome results in epileptic seizures in transgenic mice. This finding indicates that the jerky gene plays an important role in inducing epilepsy syndromes in mice. We report here our efforts in cloning, chromosomal mapping, and analysis of tissue distribution of a novel human gene, the HHMJG, a homologue to the mouse jerky gene product. We have successfully identified a full length cDNA clone encoding a novel human protein homologous to the mouse jerky gene product. The finding was based on the result of an analysis of EST (expressed sequence tag) sequences of a clone from a human tonsil cDNA library. A 4.0 kb mRNA species of the HHMJG is abundantly expressed in the majority of human tissues examined, including brain and skeletal muscle. However, in the testes, two mRNA species of the HHMJG, approximately 2.0 and 4.0 kb, are abundantly expressed. Sequence analysis of the HHMJG cDNA indicates that it encodes a putative protein of 51 kD, which shares significant sequence homology to not only the mouse jerky gene product but also some nuclear regulatory proteins, such as centromere binding protein-B. The predicted nuclear localization of the HHMJG product suggests that this protein may function as a nuclear regulatory protein. The result of human chromosomal mapping shows that the HHMJG is located on human chromosome 11q21. Our identification of the HHMJG cDNA provides a potential gene candidate to further investigate the biological significance and clinical implications of the HHMJG in human epilepsy. q 1997 Academic Press
in the absence of acute precipitating factors and affects approximately 4% of individuals at some time in their lives. According to its etiology, epilepsy can be defined as idiopathic and symptomatic. Symptomatic epilepsies are usually caused by other brain disorders. However, a certain proportion of idiopathic epilepsies are considered to be genetic consequences (1). Although familial occurrence of epilepsy has been known for a long time, little progress in mapping epilepsy genes has been reported until recently because of complexity in the genetic contribution to epilepsy. The first epilepsy locus was identified in a common benign idiopathic generalized epilepsy syndrome, juvenile myoclonic epilepsy (2). Since then, the chromosomal loci for several epilepsy genes have been determined (3–11). Furthermore, evidence of genetic heterogeneity in epilepsy suggests complexity of its etiology (12), and indicates that the same phenotype of the epilepsy syndrome may derive from different genetic mechanisms or vice versa. Therefore, it is critical to explore the genetic events related to epilepsy for a better understanding of their roles in both physiological and pathological conditions. We report our effort at cloning, mapping, and characterizing a novel human gene, named the human homologue of the mouse jerky gene (HHMJG). Sequencing analysis of a cDNA clone of the HHMJG shows that it shares good homology to the mouse jerky gene product reported by Toth and colleagues (13), in which they found that inactivation of the jerky gene caused epileptic seizures in transgenic mice. MATERIALS AND METHODS
Epilepsy is one of the most common neurological disorders characterized by recurrent unprovoked seizures 1 The nucleotide sequence of the gene reported in this article has been submitted to GenBank under Accession No. AF004715. 2 To whom correspondence should be addressed at Department of Protein Therapeutics, Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, MD 20850. Fax: (301) 340-7159.
cDNA cloning. Expressed sequence tag (EST) analysis (14– 16) of cDNA clones derived from a human tonsil cDNA library oligo(dT)primed and constructed in the pCMVSport 2.0 (GIBCO BRL) identified a 400-bp clone demonstrating significant homology to the jerky gene product of mouse. This cDNA clone encoding a complete HHMJG was sequenced to completion by a ABI sequencer (14) (GenBank accession number L76380). It contains a gene of 2889-bp encoding a human protein homologous to the mouse jerky gene prod-
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FIG. 1. The cDNA sequence and deduced amino acid sequence of HHMJG. (A) The nucleotide sequence is indicated by the number on the top of each row. Amino acids are represented by the one-letter code and indicated below their respective codons. Two potential in-frame ATG initiation codons (#207 and 303) are underlined. The termination codon is indicated by an asterisk. (B) Result of alignment of the deduced amino acid sequence (HHMJG) to the mouse jerky gene product (MJERKY).
uct. cDNA library construction was carried out essentially as described (17). Isolation of the HHMJG genomic clones. Genomic clones of the HHMJG were isolated by plaque hybridization of the human Lambda DASH II genomic library (Stratagene) with a full length cDNA clone of the HHMJG as a probe. Standard techniques were used for genomic library screening (18). The Lambda DASH II human genomic library (2 1 106 phage) was plated at a density of 5 1 104 plaque forming units/150 mm plate for primary screening. The plaques were transferred onto nylon transfer membrane (Amersham) and prehy-
bridized at 427C for a minimum of 2 hours in 50% formamide buffer (50% formamide, 51 SSC, 21 Denhardts’, 1% SDS, 20 mM NaH2PO4 , and 250 mg/ml denatured salmon sperm DNA). Hybridization was performed in fresh 50% formamide buffer containing [32P] a-dCTPlabeled probe (Prime-It II Random Primer Labeling Kit, Stratagene) at 427C for 18 hours. Following hybridization, the membranes were rinsed twice with 21 SSC containing 0.1% SDS at room temperature for 10 minutes, and then washed twice at 427C for 10 minutes in 0.21 SSC containing 0.1% SDS at high stringency. The membranes were exposed to Kodak autoradiography film overnight at 0 807C with an intensifying screen. To proceed to a secondary screening,
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FIG. 1—Continued
putative positive clones were picked, and put into 500 ml of SM buffer with 5.8 gm NaCl/L, 2.0 gm MgSO4/L, 50 ml 1M Tris (pH 7.5)/L, and 5 ml 2% gelatin/L and 10 ml of chloroform. Secondary tertiary screening was completed using the same method as primary screening. Positive lambda phages in SM buffer were titered, and then plated in duplicate. Once the bacteriophage suspension was recovered and pooled in a sterile conical tube, 5% of chloroform was added. Tubes were shaken gently, incubated for 15 minutes at room temperature and centrifuged for 15 minutes at 2000 1 g. Supernatants were transferred to new sterile tubes to which 5% polyethylene glycol (M.W. 8,000) and 5% NaCl were added. Tubes were gently shaken, incubated at 377C for 30 minutes and centrifuged for 20 minutes at 10,000 1 g. Supernatants were discarded. The genomic DNA pellets were resuspended in TE buffer, extracted twice with phenol:chloroform [1:1 (v/v)], and precipitated by ethanol. Pellets were resuspended in sterile water. Four genomic clones, each more than twelve kb in length, were obtained and confirmed by sequencing with EST primers of the HHMJG using an ABI sequencer (14). Northern blot analysis. Human Multiple Tissue Northern (MTN) Blots (Cat. No. 7760-1 & 7750-1) and Human Brain Tissue Northern Blot (Cat. No. 7755-1) were purchased from Clontech for analysis of expression of the HHMJG mRNA. Hybridization was carried out essentially as described (18) using [32P]a-dCTP-labeled probe (PrimeIt II Random Primer Labeling Kit, Stratagene), which represents the full length cDNA of the HHMJG.
Chromosome mapping. Both a 2.9 kb cDNA and a 30 kb genomic DNA of the HHMJG were nick-translated using Digoxygenin- 11 dUTP (Boehringer Mannheim) and fluorescence in situ hybridization (FISH) was done as described by Johnson and colleagues (19). Chromosomes were counterstained with DAPI. Color digital images containing both DAPI and gene signal detected using anti-Digoxygenin conjugated Rhodamine, were recorded using a triple-band pass filter set (Chroma Technology, Inc., Brattleburo, VT) in combination with a charged coupled-device camera (Photometrics, Inc., Tucson, AZ) and variable excitation wave length filters (20). Images were analyzed using the ISEE software package (Inovision Corp. Durham, N.C.) and the cytogenetic map location was assigned (21).
RESULTS Cloning of HHMJG. The clone of 2889-bp nucleotides contains a open reading frame of 1329 bp, encoding a polypeptide of 442 amino acids. The deduced amino acid sequence of the HHMJG shows 55% similarity and 35% identity to the mouse jerky gene product (Fig. 1). There are four potential in-frame ATG initiation codons (#171, 183, 207, and 303). Among them two (#207 and 303) are in a favorable context for initiating
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FIG. 2. Tissue expression of the HHMJG mRNA. Multiple Tissue Northern (MTN) blots containing poly-A RNA’s from various human tissues were obtained from Clontech, and examined with 32P-dCTP, random priming labeled probe using the full length cDNA clone of the HHMJG as a template. ExpressHyb hybridization solution (Clontech) was applied to the blots according to Clontech’s protocol, PT1190-1. The results showed that one species of the HHMJG mRNA, about 4.0 kb, was obviously expressed in abundance in most of the human tissues examined. The exceptions are the lung, liver, and peripheral leukocyte tissues, in which expression of HHMJG mRNA is less abundant. In the testes, there are two species of the HHMJG mRNA, approximately 2.0 kb and 4.0 kb, between which the 2.0 kb mRNA species is predominant. The 4.0 kb mRNA species of the HHMJG is expressed at very significant level in all eight brain tissues from different parts of the human brain.
translation; the presence of a purine at position 03 conforms to the Kozak translation initiation consensus sequence (22). The results using the BLAST algorithm of the sequence database with the HHMJG cDNA nucleotide sequence revealed that the HHMJG shares very significant homology with both nucleotide and amino acid sequences to the jerky gene product of mouse, and to several other genes which encode nuclear regulatory proteins, such as putative transposase (23) and centromere binding protein B (24). Seven genomic clones of the HHMJG were confirmed by partial sequencing (data not shown). Tissue expression of HHMJG mRNA. Northern blot analysis of multiple human tissue Northern blots using the full-length cDNA of the HHMJG as a hybridization probe reveals that a 4.0 kb mRNA of the HHMJG is abundantly expressed in the heart, brain, placenta, skeletal muscle, pancreas, thymus, prostate, ovary, and small intestine, but in the testes, two mRNA species of the HHMJG, approximately 2.0 kb and 4.0 kb, are abundantly expressed. Between them, the 2.0 kb mRNA species is predominant. However, expression of the HHMJG mRNA is less abundant in the lung, liver and peripheral leukocytes (Fig. 2). Using the human
brain tissue Northern blot, the 4.0 kb mRNA species of the HHMJG is expressed virtually in the same abundance in all eight brain tissues included (Fig. 2). Chromosomal localization of the HHMJG. The chromosomal location of the HHMJG was established using single-copy gene fluorescence in situ hybridization to normal human male metaphase chromosome spreads (25). For the genomic probe, approximately 16 spreads were analyzed by eye, most of which had a doublet signal characteristic of genuine hybridization on both homologues of chromosome 11 (Fig. 3A-B). Doublet signal was not detected on any other chromosome. Detailed analysis of 10 individual chromosomes, using fluorescence banding combined with high-resolution image analysis, indicated that the HHMJG is positioned largely within band 11q21. Since this band is flanked very closely by q14.3 and 22.1, some of the signal appeared to overlap into these bands (Fig. 3C). DISCUSSION There are many studies which suggest a genetic contribution to human epilepsy and evidence exists for linkage of susceptibility loci to specific chromo-
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somal regions (2 – 12). Studies on the mouse jerky gene have demonstrated that inactivation of the gene by insertion of a transgene into the mouse genome results in epileptic seizures in the transgenic mice (13). Based on the similarity between the human and the mouse genomes, it is obvious that identification of the human homologue of the mouse jerky gene will be beneficial to the discovery of potential candidate genes related to human epilepsy and to study their role in the etiology. We have successfully identified a full length cDNA clone encoding a novel human protein homologous to the mouse jerky gene product. The finding of the HHMJG full length cDNA was based on the results of the analysis of ‘‘EST’’ (expressed sequence tag) sequences of a clone from a human tonsil cDNA library. Although the sequence homology between the mouse jerky gene product and the HHMJG product may not necessarily correspond to the similarity of functions of the proteins encoded by these genes, it provides researchers with a novel gene candidate for further investigation of its role in human epilepsy. In recent years there is an increasing interest in finding potential genes associated with human epilepsy and in studying their biological significance and the clinical implications. We believe that our enunciation of the HHMJG will benefit the investigation of human epilepsy. Based on the results of sequence analysis, the HHMJG cDNA encodes a putative protein of 51 kD, which is possibly located in the nucleus of the cell as predicted by PSORT software (26). The probable nuclear localization of the HHMJG product corresponds well with the results derived from the BLAST algorithm of the HHMJG sequences in the data base. The results indicate that in addition to the similarity to the jerky gene of mouse, the HHMJG also shares good sequence homology with genes known as nuclear regulatory proteins, such as centromere binding protein-B. As we know, CENP-B, a highly conserved centromere-associated protein, binds to alpha-satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENPB box (27). CENP-B participates in and is necessary for mitotic chromosome movement (28). Because of the fundamental effects of the nuclear regulatory proteins on cells, (such as regulation of transcription on gene expression, cell proliferation, and their physiological functions) it is possible that the HHMJG product may have similar effects on the target cells. The tissue distribution of the HHMJG from our study also indicates that the HHMJG mRNAs are expressed in the majority of tissues examined, therefore, protein encoded by the HHMJG may have an influence on cells in these tissues. Interestingly, the HHMJG mRNA shows two species of different sizes, about 2.0 and 4.0 kb, respectively, in the tes-
tis. It is not clear what the biological significance of the alternatively spliced form of the HHMJG mRNA is in the testis. We have successfully applied the FISH mapping technology to identify location of the HHMJG on human chromosomes. The result of chromosomal mapping reveals that the HHMJG is located to human chromosome 11 at band q21. However, since signals for possible homologous sequences to the HHMJG appeared consistently on 11q21 and sporadically on 6p21.3-22.1 to which we considered as a weak secondary signal (data not shown), it could well be that this suggests a locus heterogeneity of sorts. Both linkage and association studies provide strong evidence that a gene locus on chromosome 6 is involved in the expression of juvenile myoclonic epilepsy (JME) (11, 29, 30). It has also been demonstrated that the inactivation of the jerky gene results in whole body jerks, generalized clonic seizures, and epileptic brain activity in affected mice (13). Considering the facts which include the sequence homology between the HHMJG and the mouse jerky gene, the results of chromosomal mapping of the HHMJG, and the similarity of the HHMJG product to several nuclear regulatory proteins, we believe that HHMJG may be a potential candidate gene associated with human epilepsy. If it can be established in kindreds of epileptic individuals that the HHMJG is either inactivated or mutated, some clinico-pathological correlations could be made. It is also interesting to note that genes related to cerebellar and spino cerebellar ataxia have been mapped to 11q (31) and 6p. However, since this is the first report of a human homologue to the mouse jerky gene to our knowledge, and there is no report thus far to indicate that a potential candidate gene associated to human epilepsy is located at chromosome 11q21, we do not at present know the correlation between the chromosomal localization of the HHMJG and its biological significance in human epilepsy. Therefore, further investigation is necessary to address these questions. There is no question about the importance of genetic mechanisms in epilepsy. However, in most forms of epilepsy, patients do not segregate within families following a simple memdelian pattern of inheritance. The complexity of the disease suggests that besides genetic factors, others, such as environmental factors and modifying genes may also be important for disease expression in susceptible individuals. It is reasonable to believe that both genetic and non-genetic influences on susceptibility are required. Therefore, the identification of the HHMJG provides a novel candidate gene associated to human epilepsy. Further investigation of the role of the HHMJG will give us a better understanding of the biological significance and potential clinical implications of this gene in the human epilepsy syndrome.
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FIG. 3. Fluorescence in situ hybridization mapping of the HHMJG. The genomic DNA probe of the HHMJG was hybridized to normal human male metaphase chromosomes. (A) A chromosome spread from a single cell showing hybridization to the q arm of both chromosomes # 11 (arrows). (B) Examples of 2 chromosomes #11 with hybridisation signal. The left panel of each set shows a DAPI stained chromosome (blue) with gene signal (red). The right panels shows the same chromosome displayed as an inverse black and white image. The G-band like DAPI pattern was enhanced using an edge sharpening algorith. (C) ideogram of chromosome #11 showing the position of the HHMJG.
ACKNOWLEDGMENTS We thank the following individuals for their support of the work: Drs. William Haseltine, Craig Rosen, Michael Antonaccio, Pat Dillon, Gianni Garotta, and Reiner Gentz, and individuals in the laboratories of gene discovery and bioinformatics at Human Genome Sciences, Inc.. In particular, we thank Dr. Steve Ruben, Department of Protein Therapeutics, Human Genome Sciences Inc., for his support and helpful discussion.
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