Oligonucleotide microarray expression analysis of genes whose expression is correlated with tumorigenic and non-tumorigenic phenotype of HeLa x human fibroblast hybrid cells.

In order to understand the differences and similarities between tumorigenic and non-tumorigenic HeLaxhuman fibroblast hybrids, gene expression profiles were examined with synthetic oligonucleotide arrays containing nearly 7000 gene probe sets. We used two pairs of genetically related hybrids, each pair representing individual clones of non-tumorigenic and tumorigenic segregant hybrids, respectively. Analysis of six possible comparisons, utilizing two algorithms, identified 204 genes with differential expression. The greater number of differentially expressed genes was observed when non-tumorigenic hybrids were compared with tumorigenic segregants. Fifteen and 14 genes, respectively, were consistently found to be differentially expressed in non-tumorigenic and tumorigenic cells. Among those 29 differentially expressed genes, three (intestinal alkaline phosphatase, caveolin-1, and solute carrier family2, member3) have been reported previously to be associated with the tumorigenic phenotype, using the same hybrid pairs. In addition, among the genes previously detected by differential display, 78% of them exhibited more than 5-fold change, demonstrating a high consistency between the two methods of differential gene expression. These findings suggest that synthetic oligonucleotide arrays are a powerful and highly reproducible tool to identify those genes whose expression is associated with certain phenotypes.

The distribution of somatic H1 subtypes is non-random on active vs. inactive chromatin: distribution in human fetal fibroblasts.

Chromatin immunoprecipitation was employed to determine whether or not the previously reported depletion of histone H1 on actively transcribed sequences was selective with respect to H1 subtypes. DNA of immunofractionated chromatin was analyzed by slot-blots for repetitive sequences and PCR for single and low-copy sequences. Based on the analysis of a diverse set of sequences, we report distinct differences in subtype distributions. Actively transcribed chromatin, as well as chromatin poised for transcription, is characterized by a relative depletion of somatic H1 subtypes 2 and 4 (H1s-2 and H1s-4),whereas facultative and constitutive heterochromatin contain all four somatic subtypes. These results support a model in which subtypes are selectively depleted upon gene expression. In turn, the data also support the possibility that the somatic subtypes have different functional roles based on their selective depletion from different classes of DNA sequences.

The Treacher Collins syndrome (TCOF1) gene product, treacle, is targeted to the nucleolus by signals in its C-terminus.

The TCOF1 gene product, treacle, responsible for the craniofacial disorder Treacher Collins syndrome, has been predicted to be a member of a class of nucleolar phosphoproteins based on its primary amino acid sequence. Treacle is a low complexity protein with ten repeating units of acidic and basic residues, each of which contains a large number of putative casein kinase 2 and protein kinase C phosphorylation sites. In addition, the C-terminus of treacle contains multiple putative nuclear localization signals. The overall structure of treacle, as well as sequence similarity to several nucleolar phosphoproteins, predicts that treacle is a member of this class of proteins. Using green fluorescent protein fusion constructs with the full-length and deleted domains of the murine homolog of treacle, we demonstrate that the cellular localization of treacle is nucleolar. This localization is mediated by the last 41 residues of the C-terminus (residues 1262-1302). At least two functional nuclear localization signals have been identified in the protein, one between residues 1176 and 1270 and the second within the last 32 residues of the protein (1271-1302). The nucleolar localization signal is disrupted by two constructs that split the C-terminal region between residues 1270 and 1271. This study provides the first direct analysis of treacle and demonstrates that the protein involved in TCOF1 is a nucleolar protein.

Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

The evolutionary distribution and structural organization of the homeobox-containing repeat D4Z4 indicates a functional role for the ancestral copy in the FSHD region.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease that has been linked to deletions within a tandem array of 3.2 kb repeats adjacent to the telomere of 4q. These repeats are also present in other locations in the human genome, including the short arms of all the acrocentric chromosomes. Here, we examine two models for the role of this repeat in FSHD. First, because of the extensive similarity between the 3.2 kb repeats on 4q and those adjacent to rDNA on the acrocentric chromosomes, we investigated whether the FSHD region on 4q is involved in sub-nuclear localization, specifically to the nucleolus. The results likely exclude any involvement of nucleolar localization in the development of FSHD. Second, we investigated a model that suggests that a functional gene may be buried within the tandem array of 3.2 kb repeats. Toward this end, we evaluated the evolutionary conservation of the repeat and a double homeodomain sequence within the repeat in a variety of primate species. The genomic organization of the 3.2 kb repeat in humans, great apes and lower primates identified the FSHD-associated repeat on chromosome 4q as the likely ancestral copy. The sequence of the rhesus monkey double homeodomain reveals significant sequence identity with the human 4q sequence. These results strongly suggest a functional role for a component of the FSHD-associated repeat.

Efforts toward understanding the molecular basis of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with a frequency of 1 in 20,000. The report in 1992 of a DNA polymorphism that occurred both in familial and sporadic cases led to the pronouncement that the FSHD defect had been identified. Unfortunately, 2 years have passed without the isolation of a gene or definitive proof of the mutation. Over this time it has become clear that the region of the human genome containing the FSHD gene is a complex assemblage of mildly repetitive sequences that includes the suspected polymorphic fragment. We have employed molecular and cytogenetic techniques to initiate the structural analysis of terminal 4q35 in an effort to facilitate the isolation of the gene responsible for FSHD. As a result of these efforts and our inability to identify expressed sequences unique to 4q35 we have begun to consider alternate hypotheses for a molecular mechanism resulting in FSHD other than a simple coding sequence disruption.

Efforts toward understanding the molecular basis of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with a frequency of 1 in 20,000. The report in 1992 of a DNA polymorphism that occurred both in familial and sporadic cases led to the pronouncement that the FSHD defect had been identified. Unfortunately, 2 years have passed without the isolation of a gene or definitive proof of the mutation. Over this time it has become clear that the region of the human genome containing the FSHD gene is a complex assemblage of mildly repetitive sequences that includes the suspected polymorphic fragment. We have employed molecular and cytogenetic techniques to initiate the structural analysis of terminal 4q35 in an effort to facilitate the isolation of the gene responsible for FSHD. As a result of these efforts and our inability to identify expressed sequences unique to 4q35 we have begun to consider alternate hypotheses for a molecular mechanism resulting in FSHD other than a simple coding sequence disruption.

High resolution fluorescence in situ hybridization to linearly extended DNA visually maps a tandem repeat associated with facioscapulohumeral muscular dystrophy immediately adjacent to the telomere of 4q.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. An EcoRI fragment length polymorphism segregates with the disease in most FSHD families. Within the EcoRI fragment lies a tandem array of 3.2 kb repeats. Deletions of integral copies of this repeat have been associated with the disease. The 3.2 kbp repeat has recently been shown to cross-hybridize to several regions of heterochromatin in the human genome and DNA sequence analysis reveals strong homology to a class of heterochromatin repeats, LSau. In this report, we demonstrate that the 3.2 kbp tandem repeat lies adjacent to a subtelomeric sequence, which is within 5-14 kb of the telomeric repeat (TTAGGG)n. Direct visual fluorescence hybridization to linearly extended strands of DNA enabled the visualization of this subtelomeric sequence as a short string of signals at the end of a longer string of signals from the differentially labeled 3.2 kbp tandem repeat. Furthermore, in support of our data showing that the 3.2 kbp repeat lies in close proximity to the telomere of 4q, we demonstrated the lack of hybridization of total human DNA to this same region. Our results indicate that the tandem array of 3.2 kbp repeats, disrupted in FSHD, lies immediately adjacent to the telomere of 4q and that the gene responsible for FSHD is likely located proximal to the tandem repeat.

Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.

Achondroplasia (ACH) is the most common genetic form of dwarfism. This disorder is inherited as an autosomal dominant trait, although the majority of cases are sporadic. A gene for ACH was recently localized to 4p16.3 by linkage analyses. The ACH candidate region includes the gene encoding fibroblast growth factor receptor 3 (FGFR3), which was originally considered as a candidate for the Huntington's disease gene. DNA studies revealed point mutations in the FGFR3 gene in ACH heterozygotes and homozygotes. The mutation on 15 of the 16 ACH-affected chromosomes was the same, a G-->A transition, at nucleotide 1138 of the cDNA. The mutation on the only ACH-affected chromosome 4 without the G-->A transition at nucleotide 1138 had a G-->C transversion at this same position. Both mutations result in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.

The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. Recently, a probe was identified that detects an EcoRI fragment length polymorphism which segregates with the disease in most FSHD families. Within the EcoRI fragment lies a tandem array of 3.2 kb repeats. In several familial cases and four independent sporadic FSHD mutations, the variation in size of the EcoRI fragment was due to a decrease in copy number of the 3.2 kb repeats. To gain further insight into the relationship between the tandem array and FSHD, a single 3.2 kb repeat unit was characterized. Fluorescence in situ hybridization (FISH) demonstrates that the 3.2 kb repeat cross-hybridizes to several regions of heterochromatin in the human genome. In addition, DNA sequence analysis of the repeat reveals a region which is highly homologous to a previously identified family of heterochromatic repeats, LSau. FISH on interphase chromosomes demonstrates that the tandem array of 3.2 kb repeats lies within 215 kb of the 4q telomere. Together, these results suggest that the tandem array of 3.2 kb repeats, tightly linked to the FSHD locus, is contained in heterochromatin adjacent to the telomere. In addition, they are consistent with the hypothesis that the gene responsible for FSHD may be subjected to position effect variegation because of its proximity to telomeric heterochromatin.

YAC contigs for 4q35 in the region of the facioscapulohumeral muscular dystrophy (FSHD) gene.

We report here the construction of a genetic linkage map and an overlapping set of clones containing DNA markers linked to the causative locus for facioscapulohumeral muscular dystrophy (FSHD) on 4q35. Multipoint linkage analysis placed eight loci in the following order with odds greater than 1000:1: cen-D4S171-FXI-D4S426-D4S187-D4S130-D4S 163-D4S139-D4F35S1-qter. The most likely position of D4S809 was distal to D4F35S1. Thirty-four yeast artificial chromosomes (YACs) were isolated by PCR-based assays for STSs derived from DNA markers with known genetic and physical order. Walking from the insert ends of 2 YACs identified 7 additional YACs, bridging the gaps between three of the markers. Two new YACs were found by hybridization of a cosmid inter-Alu PCR product to dot blots of inter-Alu PCR products of YAC DNA pools. All YAC clones were positioned using the genetic and physical order of the STSs and inter-Alu PCR fingerprint data. Eleven of the YACs and two cosmids were mapped by fluorescence in situ hybridization to confirm the location of the clones and to detect chimerism. The 43 YACs were assembled into two contigs. The larger contig spans approximately 2.4 Mb and contains markers closest to the FSHD gene.

A radiation hybrid map of 15 loci on the distal long arm of chromosome 4, the region containing the gene responsible for facioscapulohumeral muscular dystrophy (FSHD).

A physical map of 4q35 was constructed through radiation hybrid analysis of 134 clones generated from the cell line HHW416, a chromosome 4-only human-hamster somatic cell hybrid. This subtelomeric region contains the as-yet-unidentified gene responsible for facioscapulohumeral muscular dystrophy. The most likely order of 15 loci within 4q35 was determined. The loci ordered on this radiation hybrid map include both genes and polymorphic loci, as well as monomorphic loci which cannot be placed on a genetic linkage map. The physical distance spanning these loci was estimated to be approximately 4.5 Mb, by using a kilobase/centiray conversion factor derived from 4p16.3 marker analysis through the same set of radiation hybrids. The comparison of this physical map to establish genetic maps suggests that this region is smaller than initially estimated and that recombination rates are increased near the telomere.

The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus.

Facioscapulohumeral muscular dystrophy (FSHD) is a relatively common autosomal dominant neuromuscular disorder. The gene for FSHD has recently been assigned to chromosome 4q35. Although abnormal mitochondrial and biochemical changes have been observed in FSHD, the molecular defect is unknown. In addition to the FSHD gene, the human muscle adenine nucleotide translocator gene (ANT1) is located on chromosome 4. Interestingly, biochemical studies recently showed a possible defect of ANT1. In order to evaluate the potential role of ANT1 in the etiology of FSHD, the human ANT1 gene was isolated by cosmid cloning and localized to 4q35, in the region containing the FSHD gene. However, in situ hybridization and physical mapping of somatic cell hybrids localized the ANT1 gene proximal to the FSHD gene. In addition, a polymorphic CA-repeat 5 kb upstream of the ANT1 gene was used as a marker in FSHD and Centre d'Etude du Polymorphisme Humain families to perform linkage analysis. These data together exclude ANT1 as the primary candidate gene for FSHD. The most likely order of the loci on chromosome 4q35 is cen-ANT1-D4S171-F11-D4S187-D4S163-D4S139-+ ++FSHD-tel.

Genetic mapping of human heart-skeletal muscle adenine nucleotide translocator and its relationship to the facioscapulohumeral muscular dystrophy locus.

The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration.

Structural organization of a src gene from Xenopus laevis.

By sequence analysis of genomic clones, the exon-intron structure of one of the two src genes from Xenopus laevis has been determined. The coding region of the gene is interrupted by 10 introns whose locations are identical to the introns in the coding regions of the src genes of human and chicken. The 5' untranslated region is contained on a separate exon with no sequence conservation relative to the corresponding region of the chicken gene. The 5' untranslated region of the Xenopus gene contains a G + C-rich stem-loop sequence and two ATG triplets. A 1.4-kb fragment containing the 5' untranslated region and sequences upstream of it acts as a promoter when introduced in the correct orientation into X. laevis cell lines. The DNA sequence of this fragment lacks the typical arrangement of TATA and CCAAT sequences but contains the ATGCAAAT octamer sequence and a (TA)39 sequence.