Mutations in the humanAIREgene (hAIRE) result in the development of an autoimmune disease named A... more Mutations in the humanAIREgene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmunepolyendocrinopathycandidiasisectodermaldystrophy; OMIM 240300). Previously, we have cloned hAIREand shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization ofAire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of theAIREgene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT–PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.
We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, wh... more We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6. The protein sequence of 270 amino acids was deduced from the nucleotide sequence of an open reading frame containing the homeobox. The open reading frame is interrupted at the genomic level by a 960 bp intron and is organized in two exons. The Hox 1.3 protein was found to contain extensive sequence homology with the murine homeodomain protein Hox 2.1, which is encoded on chromosome 11. There are two homologous regions in the first exon, i.e. a hexapeptide conserved in many homeobox-containing genes and the N-terminal domain, which was found to be homologous only to Hox 2.1. Furthermore, in exon 2 the homologies of the homeodomain regions are extended up to the carboxy terminus of Hox 1.3 and Hox 2.1. Durin...
We have used an approach based on the observation of CpGrich regions near the 5' end of many gene... more We have used an approach based on the observation of CpGrich regions near the 5' end of many genes to screen a panel of cosmids derived from the t-complex and tested candidate sequences for evidence of transcription in a number of different mouse tissues. One gene so identified is expressed specifically in testicular germ cells and maps to a subregion of the t-complex also containing loci involved in transmission ratio distortion and male sterility. The transcript is first detected during the pachytene stage of the first meiotic division, but is expressed in highest levels in the later haploid spermatogenic stages. Sequence analysis verified the existence of a CpG-rich element on the 5' end of the gene and predicts a unique protein species with no significant homologies to those previously determined.
X-linked dominant hypophosphatemic rickets (HYP) is the most common form of hereditary rickets. R... more X-linked dominant hypophosphatemic rickets (HYP) is the most common form of hereditary rickets. Recently we have cloned thePEX gene and shown it to be mutated and deleted in HYP individuals. We have now completely sequenced a 243-kb genomic region containing PEX and have identified all intron–exon boundary sequences. We show that PEX, homologous to members of a neutral endopeptidase family, has an exon organization that is very similar to neprilysin. We have performed an extensive mutation analysis examining all 22 PEX coding exons in 29 familial and 14 sporadic cases of hypophosphatemia. Sequence changes include missense, frameshift, nonsense, and splice site mutations and intragenic deletions. A mutation was found in 25 (86%) of the 29 familial cases and 8 (57%) of the 14 sporadic cases. Our data provide the first evidence that most of the familial and also a large number of the sporadic cases of hypophosphatemia are caused by loss-of-function mutations in PEX.[The sequence data d...
Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has bee... more Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.
Proceedings of the National Academy of Sciences, 1990
We have isolated two genes, Hox-2.8 and Hox-2.9, from the mouse homeobox cluster Hox-2, located o... more We have isolated two genes, Hox-2.8 and Hox-2.9, from the mouse homeobox cluster Hox-2, located on chromosome 11. A 120-kilobase yeast artificial chromosome (YAC) containing a large region of the murine Hox-2 cluster, including 45 kilobases of sequence upstream of the most 5' gene, was cloned. The DNA sequence of the YAC is unrearranged relative to the genomic map. We have subcloned from the YAC insert a homeobox gene, Hox-2.8, whose homeodolmain is highly related to that of the Drosophila homeotic gene proboscopedia (pb). The expression pattern of Hox-2.8 during embryogenesis extends the trend established by genes from Hox-2.5 to -2.7 of successively anterior domains of expression in the neural tube. We have also subcloned and sequenced from a cosmid the labial (lab)-related Hox-2.9, the most 3' member of the cluster to date. These data lend further support to the idea of a common evolutionary origin of the mouse Hox and Drosophila HOM clusters. The YAC will enable us to co...
Background: Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines ... more Background: Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell's gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Methodology/Principal Findings: Gene expression measurements by real-time quantitative PCR revealed a remarkable coexpression of TDRD1 and ERG (r 2 = 0.77) but not ETV1 (r 2 ,0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (r = 20.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERGnegative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. Conclusions/Significance: We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.
Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the ... more Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to $500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation.
Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial ... more Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial list of personnel. A full list of contributors at each centre is available as Supplementary Information.
transfection, cells were collected and processed for CAT or luciferase activity using standard te... more transfection, cells were collected and processed for CAT or luciferase activity using standard techniques 14. GST pull downs and immunoprecipitations GST±Rb (wild type and mutants 15) and other GST fusion proteins were expressed and puri®ed from Escherichia coli XA90 (ref. 16). GST fusion proteins that were immobilized on glutathione-sepharose (Pharmacia), or H3-derived peptides 3 bound to Sulfolink beads (Pierce), were incubated with extract in IPH buffer 16. Complexes were washed four times in IPH buffer before processing for methylase assays or western blotting. Antibodies against HA (12CA5, Roche), Gal4±DBD (DNA-binding domain; sc-510, Santa Cruz), SUV39H1 (M. Cleary), Rb (G3-245; XZ55, PharMingen) or HP1 (ref. 3) were used. For immunoprecipitations antibodies were incubated with HeLa nuclear extract (Cell Culture Center) or U2OS nuclear extract in IPH buffer at 4 8C (ref. 17). After 2 h a 50:50 mixture of protein A/G-sepharose beads (Pharmacia) was added. To avoid the possibility that DNA mediates the interaction between SUV39H1 and Rb, the immunoprecipitations were probed for the presence of histones with negative results. Histone methylase assays and protein sequencing Precipitations from pull downs or immunoprecipitations were incubated with 20 mg histones (Sigma) and 1 ml [3H-Me]-S-adenosyl methionine (NEN, 80 Ci mmol-1) in PBS at 30 8C for 1 h. Assays were analysed by SDS±PAGE followed by western blotting and autoradiography or were spotted onto P-81 cationic exchange paper (Whatman), washed in carbonate buffer and quanti®ed by scintillation counting 3. For amino-terminal sequencing, radiolabelled H3 was blotted to polyvinylidene¯uoride and sequenced by Edman degradation (Protein Sequencing Facility, University of Cambridge). We counted fractions for the presence of tritium. Supplementary information (with changes to the original Supplementary Information) is available on Nature's WorldWide Web site (http://www.nature.com).
As part of an integrated mapping and sequencing analysis of genomes, we have developed an approac... more As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (>0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database. The nucleotide sequence data reported in this paper have been submitted to the EMBL database and have been assigned the accession numbers X65374-X65393 and X65268-X65275.
Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been ... more Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that, takes advantage of methylation differences between genomic DNA and vector. Thirdly. two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.
An X;8 translocation was identified in a 27-year-old female patient manifesting multiple exostose... more An X;8 translocation was identified in a 27-year-old female patient manifesting multiple exostoses and autism accompanied by mental retardation and epilepsy. Through molecular analysis using yeast artificial chromosomes (YACs) and cosmid clones, the translocation breakpoint was isolated and confirmed to be reciprocal within a 5′-GGCA-3′ sequence found on both X and 8 chromosomes without gain or loss of a single nucleotide. The translocation breakpoint on the X chromosome occurred in the first intron of the gastrin-releasing peptide receptor (GRPR) gene and that on chromosome 8 occurred ∼30 kb distal to the 3′ end of the Syndecan-2 gene (SDC2), also known as human heparan sulfate proteoglycan or fibroglycan. The GRPR gene was shown to escape X-inactivation. A dosage effect of the GRPR and a position effect of the SDC2 gene may, however, contribute the phenotype observed in this patient since the orientation of these genes with respect to the translocation was incompatible with the formation of a fusion gene. Investigation of mutations in these two genes in unrelated patients with either autism or multiple exostoses as well as linkage and association studies is needed to validate them as candidate genes.
Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosph... more Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosphatemic rickets and have been shown to be deleted for the 5′ and 3′ end of the mouse homolog of PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome; formerly called PEX), respectively. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. The human SMS (spermine synthase) gene maps ∼39 kb upstream of PHEX and is transcribed in the same direction. To elucidate the complex phenotype of Gy mice, we characterized the genomic region upstream of Phex. By establishing the genomic structure of mouse Sms, a 160-190 kb deletion was shown in Gy mice, which includes both Phex and Sms. There are several pseudogenes of SMS/Sms in man and mouse. Northern analysis revealed three different Sms transcripts which are absent in Gy mice. Measurement of polyamine levels revealed a marked decrease in spermine in liver and pancreas of affected male Gy mice. Analysis of brain tissue revealed no gross or histological abnormalities. Gy provides a mouse model for a defect in the polyamine pathway, which is known to play a key role in cell proliferation.
Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurode... more Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3′-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.
GenBank accessions nos U73910-U73915, and Y09419 X-linked hypophosphatemic rickets in humans is c... more GenBank accessions nos U73910-U73915, and Y09419 X-linked hypophosphatemic rickets in humans is caused by mutations in the PEX gene which codes for a protein homologous to neutral endopeptidases. Hyp and Gy mice both have X-linked hypophosphatemic rickets, although genetic data and the different phenotypic spectra observed have previously suggested that two different genes are mutated. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. We now report the cloning of the mouse homolog of PEX which is highly conserved between man and mouse. The 3′ end of this gene is deleted in Hyp mice. In Gy mice, the first three exons and the promotor region are deleted. Thus, Hyp and Gy are allelic mutations and both provide mouse models for X-linked hypophosphatemia.
Mutations in the humanAIREgene (hAIRE) result in the development of an autoimmune disease named A... more Mutations in the humanAIREgene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmunepolyendocrinopathycandidiasisectodermaldystrophy; OMIM 240300). Previously, we have cloned hAIREand shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization ofAire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of theAIREgene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT–PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.
We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, wh... more We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6. The protein sequence of 270 amino acids was deduced from the nucleotide sequence of an open reading frame containing the homeobox. The open reading frame is interrupted at the genomic level by a 960 bp intron and is organized in two exons. The Hox 1.3 protein was found to contain extensive sequence homology with the murine homeodomain protein Hox 2.1, which is encoded on chromosome 11. There are two homologous regions in the first exon, i.e. a hexapeptide conserved in many homeobox-containing genes and the N-terminal domain, which was found to be homologous only to Hox 2.1. Furthermore, in exon 2 the homologies of the homeodomain regions are extended up to the carboxy terminus of Hox 1.3 and Hox 2.1. Durin...
We have used an approach based on the observation of CpGrich regions near the 5' end of many gene... more We have used an approach based on the observation of CpGrich regions near the 5' end of many genes to screen a panel of cosmids derived from the t-complex and tested candidate sequences for evidence of transcription in a number of different mouse tissues. One gene so identified is expressed specifically in testicular germ cells and maps to a subregion of the t-complex also containing loci involved in transmission ratio distortion and male sterility. The transcript is first detected during the pachytene stage of the first meiotic division, but is expressed in highest levels in the later haploid spermatogenic stages. Sequence analysis verified the existence of a CpG-rich element on the 5' end of the gene and predicts a unique protein species with no significant homologies to those previously determined.
X-linked dominant hypophosphatemic rickets (HYP) is the most common form of hereditary rickets. R... more X-linked dominant hypophosphatemic rickets (HYP) is the most common form of hereditary rickets. Recently we have cloned thePEX gene and shown it to be mutated and deleted in HYP individuals. We have now completely sequenced a 243-kb genomic region containing PEX and have identified all intron–exon boundary sequences. We show that PEX, homologous to members of a neutral endopeptidase family, has an exon organization that is very similar to neprilysin. We have performed an extensive mutation analysis examining all 22 PEX coding exons in 29 familial and 14 sporadic cases of hypophosphatemia. Sequence changes include missense, frameshift, nonsense, and splice site mutations and intragenic deletions. A mutation was found in 25 (86%) of the 29 familial cases and 8 (57%) of the 14 sporadic cases. Our data provide the first evidence that most of the familial and also a large number of the sporadic cases of hypophosphatemia are caused by loss-of-function mutations in PEX.[The sequence data d...
Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has bee... more Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.
Proceedings of the National Academy of Sciences, 1990
We have isolated two genes, Hox-2.8 and Hox-2.9, from the mouse homeobox cluster Hox-2, located o... more We have isolated two genes, Hox-2.8 and Hox-2.9, from the mouse homeobox cluster Hox-2, located on chromosome 11. A 120-kilobase yeast artificial chromosome (YAC) containing a large region of the murine Hox-2 cluster, including 45 kilobases of sequence upstream of the most 5' gene, was cloned. The DNA sequence of the YAC is unrearranged relative to the genomic map. We have subcloned from the YAC insert a homeobox gene, Hox-2.8, whose homeodolmain is highly related to that of the Drosophila homeotic gene proboscopedia (pb). The expression pattern of Hox-2.8 during embryogenesis extends the trend established by genes from Hox-2.5 to -2.7 of successively anterior domains of expression in the neural tube. We have also subcloned and sequenced from a cosmid the labial (lab)-related Hox-2.9, the most 3' member of the cluster to date. These data lend further support to the idea of a common evolutionary origin of the mouse Hox and Drosophila HOM clusters. The YAC will enable us to co...
Background: Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines ... more Background: Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell's gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Methodology/Principal Findings: Gene expression measurements by real-time quantitative PCR revealed a remarkable coexpression of TDRD1 and ERG (r 2 = 0.77) but not ETV1 (r 2 ,0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (r = 20.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERGnegative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. Conclusions/Significance: We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.
Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the ... more Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to $500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation.
Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial ... more Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial list of personnel. A full list of contributors at each centre is available as Supplementary Information.
transfection, cells were collected and processed for CAT or luciferase activity using standard te... more transfection, cells were collected and processed for CAT or luciferase activity using standard techniques 14. GST pull downs and immunoprecipitations GST±Rb (wild type and mutants 15) and other GST fusion proteins were expressed and puri®ed from Escherichia coli XA90 (ref. 16). GST fusion proteins that were immobilized on glutathione-sepharose (Pharmacia), or H3-derived peptides 3 bound to Sulfolink beads (Pierce), were incubated with extract in IPH buffer 16. Complexes were washed four times in IPH buffer before processing for methylase assays or western blotting. Antibodies against HA (12CA5, Roche), Gal4±DBD (DNA-binding domain; sc-510, Santa Cruz), SUV39H1 (M. Cleary), Rb (G3-245; XZ55, PharMingen) or HP1 (ref. 3) were used. For immunoprecipitations antibodies were incubated with HeLa nuclear extract (Cell Culture Center) or U2OS nuclear extract in IPH buffer at 4 8C (ref. 17). After 2 h a 50:50 mixture of protein A/G-sepharose beads (Pharmacia) was added. To avoid the possibility that DNA mediates the interaction between SUV39H1 and Rb, the immunoprecipitations were probed for the presence of histones with negative results. Histone methylase assays and protein sequencing Precipitations from pull downs or immunoprecipitations were incubated with 20 mg histones (Sigma) and 1 ml [3H-Me]-S-adenosyl methionine (NEN, 80 Ci mmol-1) in PBS at 30 8C for 1 h. Assays were analysed by SDS±PAGE followed by western blotting and autoradiography or were spotted onto P-81 cationic exchange paper (Whatman), washed in carbonate buffer and quanti®ed by scintillation counting 3. For amino-terminal sequencing, radiolabelled H3 was blotted to polyvinylidene¯uoride and sequenced by Edman degradation (Protein Sequencing Facility, University of Cambridge). We counted fractions for the presence of tritium. Supplementary information (with changes to the original Supplementary Information) is available on Nature's WorldWide Web site (http://www.nature.com).
As part of an integrated mapping and sequencing analysis of genomes, we have developed an approac... more As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (>0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database. The nucleotide sequence data reported in this paper have been submitted to the EMBL database and have been assigned the accession numbers X65374-X65393 and X65268-X65275.
Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been ... more Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that, takes advantage of methylation differences between genomic DNA and vector. Thirdly. two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.
An X;8 translocation was identified in a 27-year-old female patient manifesting multiple exostose... more An X;8 translocation was identified in a 27-year-old female patient manifesting multiple exostoses and autism accompanied by mental retardation and epilepsy. Through molecular analysis using yeast artificial chromosomes (YACs) and cosmid clones, the translocation breakpoint was isolated and confirmed to be reciprocal within a 5′-GGCA-3′ sequence found on both X and 8 chromosomes without gain or loss of a single nucleotide. The translocation breakpoint on the X chromosome occurred in the first intron of the gastrin-releasing peptide receptor (GRPR) gene and that on chromosome 8 occurred ∼30 kb distal to the 3′ end of the Syndecan-2 gene (SDC2), also known as human heparan sulfate proteoglycan or fibroglycan. The GRPR gene was shown to escape X-inactivation. A dosage effect of the GRPR and a position effect of the SDC2 gene may, however, contribute the phenotype observed in this patient since the orientation of these genes with respect to the translocation was incompatible with the formation of a fusion gene. Investigation of mutations in these two genes in unrelated patients with either autism or multiple exostoses as well as linkage and association studies is needed to validate them as candidate genes.
Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosph... more Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosphatemic rickets and have been shown to be deleted for the 5′ and 3′ end of the mouse homolog of PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome; formerly called PEX), respectively. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. The human SMS (spermine synthase) gene maps ∼39 kb upstream of PHEX and is transcribed in the same direction. To elucidate the complex phenotype of Gy mice, we characterized the genomic region upstream of Phex. By establishing the genomic structure of mouse Sms, a 160-190 kb deletion was shown in Gy mice, which includes both Phex and Sms. There are several pseudogenes of SMS/Sms in man and mouse. Northern analysis revealed three different Sms transcripts which are absent in Gy mice. Measurement of polyamine levels revealed a marked decrease in spermine in liver and pancreas of affected male Gy mice. Analysis of brain tissue revealed no gross or histological abnormalities. Gy provides a mouse model for a defect in the polyamine pathway, which is known to play a key role in cell proliferation.
Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurode... more Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3′-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.
GenBank accessions nos U73910-U73915, and Y09419 X-linked hypophosphatemic rickets in humans is c... more GenBank accessions nos U73910-U73915, and Y09419 X-linked hypophosphatemic rickets in humans is caused by mutations in the PEX gene which codes for a protein homologous to neutral endopeptidases. Hyp and Gy mice both have X-linked hypophosphatemic rickets, although genetic data and the different phenotypic spectra observed have previously suggested that two different genes are mutated. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. We now report the cloning of the mouse homolog of PEX which is highly conserved between man and mouse. The 3′ end of this gene is deleted in Hyp mice. In Gy mice, the first three exons and the promotor region are deleted. Thus, Hyp and Gy are allelic mutations and both provide mouse models for X-linked hypophosphatemia.
Uploads
Papers by Hans Lehrach