RESUMEN
While current sequencing efforts consider the detection of alpha satellite repeats as logical end points for map construction, detailed maps of most pericentromeric regions are lacking to confirm this hypothesis. Here we identify the different alpha satellite families present at the pericentromeric region of chromosome 12. The order, size and location of these repeats is established using radiation hybrid analysis, pulsed field gel analysis and FISH and the maps are integrated with current sequence information. For the different classes of alpha satellites present at the chromosome 12 centromere the paralogs in the human genome were mapped by FISH. Unique sequences flanking the alpha satellite repeats were identified, some of which are not represented in the current draft sequence. This mapping effort localises the different alpha satellite repeats within the pericentromeric region and anchors them in the current maps. The novel sequences identified may serve as the end point for the ongoing sequencing efforts.
Asunto(s)
Centrómero , Cromosomas Humanos Par 12 , Southern Blotting , Aberraciones Cromosómicas , Cromosomas Humanos Par 12/ultraestructura , Cósmidos/análisis , Cósmidos/química , Cósmidos/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Mapeo de Híbrido por Radiación , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Familia de Multigenes , Animales , Cósmidos/química , Cósmidos/aislamiento & purificación , Exones , Peces Venenosos , Genes , Humanos , Intrones , Complejo Mayor de Histocompatibilidad/genética , Filogenia , Complejo de la Endopetidasa Proteasomal , Proteínas/química , Proteínas/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria. It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway. For this purpose, a segment of dxr was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with the PCR segment led to the identification of a 26. 5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing. The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate=DMADP) was significantly (P
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Cianobacterias/enzimología , Cianobacterias/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Genes Bacterianos , Hemiterpenos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Compuestos Organofosforados/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Azúcar/metabolismo , Isomerasas Aldosa-Cetosa/biosíntesis , Isomerasas Aldosa-Cetosa/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cósmidos/aislamiento & purificación , Amplificación de Genes , Biblioteca de Genes , Marcadores Genéticos , Hidroliasas/biosíntesis , Hidroliasas/química , Hidroliasas/genética , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/aislamiento & purificación , Sondas de Oligonucleótidos/metabolismo , Sistemas de Lectura Abierta/genética , Operón/genética , Oxidorreductasas/biosíntesis , Oxidorreductasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transferasas/genética , Transferasas/aislamiento & purificación , Transferasas/metabolismoRESUMEN
To define control elements that regulate tissue-specific expression of the cystic fibrosis transmembrane regulator (CFTR), we have sequenced 60 kb of genomic DNA from the puffer fish Fugu rubripes (Fugu) that includes the CFTR gene. This region of the Fugu genome shows conservation of synteny with 800-kb sequence of the human genome encompassing the WNT2, CFTR, Z43555, and CBP90 genes. Additionally, the genomic structure of each gene is conserved. In a multiple sequence alignment of human, mouse, and Fugu, the putative WNT2 promoter sequence is shown to contain highly conserved elements that may be transcription factor or other regulatory binding sites. We have found two putative ankyrin repeat-containing genes that flank the CFTR gene. Overall sequence analysis suggests conservation of intron/exon boundaries between Fugu and human CFTR and revealed extensive homology between functional protein domains. However, the immediate 5' regions of human and Fugu CFTR are highly divergent with few conserved sequences apart from those resembling diminished cAMP response elements (CRE) and CAAT box elements. Interestingly, the polymorphic polyT tract located upstream of exon 9 is present in human and Fugu but absent in mouse. Similarly, an intron 1 and intron 9 element common to human and Fugu is absent in mouse. The euryhaline killifish CFTR coding sequence is highly homologous to the Fugu sequence, suggesting that upregulation of CFTR in that species in response to salinity may be regulated transcriptionally.
Asunto(s)
Cromosomas Humanos Par 7/genética , Secuencia Conservada/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Peces Venenosos/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cósmidos/genética , Cósmidos/aislamiento & purificación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/aislamiento & purificación , Humanos , Peces Killi/genética , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE: Pufferfish is a newly-established model organism in human genome research, which has been demonstrated its unique advantages in comparative genomics. Construction of a genomic library of pufferfish is the prerequisite to any studies of its genome. METHODS: Genomic DNA from Fugu rubripes, a species distribute only in Japan and China seas, was used to construct a library in a novel cosmid vector, named sCOGH2, which contains an exon-trapping cassette. RESULTS: This library is composed of 57,600 clones gridded in 60,096-well cell culture plates, one clone each well. The average size of the inserts is around 35 kb. It provides 99.2% probability to find any unique DNA fragment of pufferfish in this library. The clones of this library can survive after more than 10 times freeze-thaw, also demonstrating high stability in propagation. The clones shone highly positive signals when hybridized with the pufferfish genomic DNA. CONCLUSIONS: The library accords with qualitative demands of a cosmid library.
Asunto(s)
Cósmidos/genética , Takifugu/genética , Animales , Cósmidos/aislamiento & purificación , Biblioteca GenómicaRESUMEN
We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.
Asunto(s)
Cósmidos/genética , Repeticiones de Trinucleótidos/genética , Animales , Mapeo Cromosómico , Clonación Molecular , Cósmidos/aislamiento & purificación , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
Incremental differences in delta-aminolevulinate dehydratase (ALA-D; the second enzyme of the heme biosynthetic pathway) activity among inbred mouse strains can be attributed to variation in the number of copies of the ALA-D gene. We have cloned and characterized the Lv locus from an inbred mouse strain (DBA/2J) that has three times the normal ALA-D activity levels. The entire 12-kb ALA-D gene plus 16 kb of flanking DNA are found in 28-kb tandemly repeating units. We used the derived nucleotide sequence surrounding the internal junction of the repeats to survey wild-caught mice and demonstrate that multiple copies of the ALA-D gene occur in 7 of 24 worldwide locations of Mus domesticus mice. Data are consistent with a model that high lead (Pb) in the environment may be providing a selective advantage to mice harboring multiple copies of the ALA-D gene, since the enzyme is potently inhibited by lead.
Asunto(s)
Dosificación de Gen , Ácidos Levulínicos/metabolismo , Porfobilinógeno Sintasa/genética , Animales , Secuencia de Bases , Clonación Molecular , Cósmidos/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos DBA , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Familia de Multigenes , Porfobilinógeno Sintasa/aislamiento & purificación , Porfobilinógeno Sintasa/metabolismo , Recombinación Genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Genoma Bacteriano , Mycobacterium leprae/genética , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Metodologías Computacionales , Cósmidos/aislamiento & purificación , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Mycobacterium tuberculosis/genética , Sistemas de Lectura Abierta/genética , Operón/genética , Seudogenes , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17. The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r. We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb. The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed. We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues. We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues. The remaining genes in this region have previously been shown to be biallelically expressed.
Asunto(s)
Alelos , Cromosomas Artificiales de Levadura/genética , Cromosomas/genética , Expresión Génica/fisiología , Genes/fisiología , Impresión Genómica , Receptor IGF Tipo 2/genética , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , Cósmidos/genética , Cósmidos/aislamiento & purificación , Sondas de ADN/genética , Sondas de ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Exones/genética , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation. Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro. The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors. To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors. In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells. The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy.
Asunto(s)
Adenoviridae/genética , Cósmidos/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Cósmidos/aislamiento & purificación , Vectores Genéticos/metabolismo , Modelos Genéticos , Reacción en Cadena de la Polimerasa , beta-Galactosidasa/metabolismoRESUMEN
LCN1 gene encodes the tear lipocalin; the lipocalins are a large and growing family of proteins characterized by their ability to bind small hydrophobic molecules. We report here the location of a dinucleotide repeat microsatellite marker (D9S1826) close to LCN1 gene. Using the CEPH reference families, the position of LCN1 is located within the 9q34 genetic map between D9S23 and D9S158.
Asunto(s)
Proteínas Portadoras/genética , Mapeo Cromosómico , Cromosomas Humanos Par 9/genética , Proteínas Portadoras/aislamiento & purificación , Mapeo Cromosómico/métodos , Cósmidos/genética , Cósmidos/aislamiento & purificación , Repeticiones de Dinucleótido/genética , Humanos , Lipocalina 1 , Repeticiones de Microsatélite/genética , Polimorfismo Genético/genéticaRESUMEN
RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.
Asunto(s)
Clonación Molecular/métodos , Cósmidos/aislamiento & purificación , Biblioteca de Genes , Técnica del ADN Polimorfo Amplificado Aleatorio , Ascomicetos/genética , Ascomicetos/patogenicidad , Cósmidos/genética , ADN de Hongos/genética , Genes Fúngicos , Marcadores Genéticos , Virulencia/genéticaRESUMEN
Expression of the proto-oncogene c-myc is tightly regulated in vivo. Transcription of c-myc is assumed to be controlled by a number of positive and negative cis-acting control elements located upstream or within exon 1 and intron 1. However, these regulatory elements are not sufficient for c-myc expression after stable transfection or in transgenic mice. Transcription of c-myc in vivo thus requires additional control elements located outside the tested HindIII-EcoRI gene fragment. In order to identify these putative additional control elements, we mapped DNase I hypersensitive sites around the human c-myc gene in nine different tumor cell lines and in primary lymphocytes. Within the coding and 5' region of the gene, an almost identical pattern of DNase I hypersensitive sites was detected in the various cells. In contrast, chromatin analysis of the c-myc 3' region revealed a complex pattern of constitutive and tissue-specific DNase I hypersensitive sites. In enhancer trap experiments we identified two cis-acting control elements, both co-localizing with DNase I hypersensitive sites, that stimulated c-myc transcription after transient transfection in Raji or HeLa cells. Both regulatory elements exerted their enhancer activity in either orientation and regardless of their location within the plasmids. Both elements also conferred activation on a heterologous promoter. The association of these enhancers with DNase I hypersensitive sites, indicating their functional activity in vivo, make them potential candidates for the postulated regulatory control element(s) required for c-myc expression in vivo.
Asunto(s)
Elementos de Facilitación Genéticos , Genes myc , Animales , Células Cultivadas , Mapeo Cromosómico , Cósmidos/genética , Cósmidos/aislamiento & purificación , Desoxirribonucleasa I , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Transcripción Genética , Transfección , Translocación Genética , Células Tumorales CultivadasRESUMEN
A regional physical and transcription map involving yeast artificial chromosomes (YACs), cosmids, and cDNAs has been constructed for Xq21.3-q22 around the gene BTK (formerly atk or BPK) defective in X-linked agammaglobulinemia (XLA). With a positional cloning strategy employing direct cDNA selection, novel cDNAs were found to cluster in the region of approximately 100 kb flanking the XLA and alpha-galactosidase A loci. While these widely expressed transcripts are in the area known to contain CpG islands, a less evolutionarily conserved gene, located more than 130 kb distal of DXS178, maps to cosmid clones that could not be digested with rare-cutting restriction enzymes. The presence of transcribed sequences flanking the BTK allowed us to investigate their involvement in complex XLA phenotypes. Southern blot analysis using cDNA clones isolated from this region permitted us to exclude a contiguous deletion syndrome as an underlying defect in three patients with XLA and associated growth hormone deficiency. A single XLA patient with torsion dystonia and cosegregating X-linked deafness has been found with a deletion in the 3' part of BTK extending centromerically into the flanking expressed sequence DXS1274E. This suggests a possible involvement of the DXS1274E in this phenotype. The GenBank accession numbers for novel cDNA sequences are as follows: DXS1269E (L20773), DXS1271E (UO1923), DXS1273E (UO1925), and DXS1274E (UO1922).
Asunto(s)
Cósmidos/aislamiento & purificación , ADN Complementario/aislamiento & purificación , Proteínas Tirosina Quinasas/genética , Cromosoma X , Agammaglobulinemia Tirosina Quinasa , Agammaglobulinemia/enzimología , Agammaglobulinemia/genética , Southern Blotting , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Clonación Molecular , Femenino , Biblioteca de Genes , Marcadores Genéticos , Humanos , Cariotipificación , Datos de Secuencia Molecular , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.
Asunto(s)
Bacteriófago P1/genética , Fisura del Paladar/genética , Cósmidos/genética , Antígenos H-2/genética , Proteínas de Choque Térmico/genética , Adenosina Trifosfatasas/genética , Análisis de Varianza , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cósmidos/aislamiento & purificación , Cartilla de ADN/química , Sondas de ADN , Electroforesis en Gel de Agar , Femenino , Biblioteca de Genes , Ligamiento Genético , Genotipo , Masculino , Ratones , Datos de Secuencia Molecular , Recombinación Genética , Mapeo RestrictivoRESUMEN
A simple and rapid method for the isolation of cosmid insert DNA was developed based on triple-helix-mediated affinity capture (TAC). A modified cosmid was constructed from the SuperCos 1 cosmid vector by flanking the cloning site with two homopurine-homopyrimidine triple-helix-forming sequences. The cosmid DNA is digested with NotI restriction enzyme to release the insert DNA. The NotI-digested cosmid DNA is then combined with a biotinylated homopyrimidine oligonucleotide in an acidic buffer solution to form a triple-helix complex. The triple-helix complex is captured with streptavidin-coated magnetic beads. Insert DNA is eluted by adding a pH 9 buffered solution to the captured complex. The purified insert DNA is recovered with a yield of up to 95% and a purity of at least 95%. The isolated insert DNA was directly digested with CviJI restriction endonuclease to generate random fragments for shotgun sequencing.
Asunto(s)
Cósmidos/genética , Cósmidos/aislamiento & purificación , ADN/genética , ADN/aislamiento & purificación , Técnicas Genéticas , Secuencia de Bases , Clonación Molecular , Biblioteca de Genes , Vectores Genéticos , Genoma Humano , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The proximal portion of human chromosome 22q appears to carry genes implicated in the pathogenesis of various developmental disorders, including the cat eye syndrome (CES) and the DiGeorge syndrome (DGS). A cosmid library was prepared from a radiation hybrid selected for its content in chromosome 22 fragments. A large fraction of cosmids containing human DNA were found to derive from the juxtacentromeric region of chromosome 22, as shown by fluorescence in situ hybridization (FISH) performed using individual cosmids or cosmid pools as probes. Finer mapping was obtained for individual cosmids by hybridization to a somatic cell hybrid mapping panel which splits the long arm of the chromosome into 14 bins numbered 1 to 14 from the centromere to the telomere. Of the 10 cosmids mapped, eight belonged to group 1, the other two to group 14, in agreement with FISH data. Rare endonuclease sites and fragments conserved between species were searched in single cosmids, resulting in the selection of seven cosmid fragments which were used to screen a human fetal brain cDNA library. Three cDNAs were identified, encoded from two chromosome 22 genes which appeared to be novel, as determined from partial end sequence and comparison with the database entries. Fine localization of the 30.9 cDNA indicated that the corresponding gene was located in a segment of proximal 22q overlapping with the critical DGS region.