Zoo Blot Research Articles

073_16735-H5 Reverse Electrical Remodeling Induced by Sinus Rhythm and Calcium Channel Blockers May Play a Role for Better Long-Term Follow-Up Results in Patients With Long Standing Persistent Atrial Fibrillation and Treated With Cryo-Balloon Ablation

To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient9s leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On zoo blots, this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.

Gene Mapping: Basics, Techniques and Significance

Watson says, Like the system of interstate highways spanning our country, the map of the human genome will be completed stretch by stretch. It may be possible to use genetic information to diagnose the disease accurately and to predict a patient's likely response to a particular medicine or treatment. For whole genome mapping development and application of mapping, sequencing and computational tools are very essential and also linkage, physical and sequence maps are required to put the information together. For most genome mapping projects involve markers consisting of a unique site in the genome and should be independent of any particular experimental resource. For mapping purpose the DNA and RNA identification is essential. These genes are identified by hybridizing DNA clones against Northern blot, cDNA libraries, Zoo blot, Western blot and Southern blot of genomic DNA digested with rare cutter restriction endonuclease. The various experimental studies of gene mapping have extended our understanding of the genetics. This has allowed the investigators to detect a particular gene, which is responsible for the disease. Recent studies have shown the various effective and scientific gene mapping techniques and gene identification methods, which are helpful to diagnose a particular disease. It is easy for the doctor to give right medicine to the right patient to cure the disease when he can identify the defective gene responsible for disease. This article reviews the details of identification techniques of genes, gene mapping with broad applications. KEY WORDS: Gene mapping; Restriction mapping; Fluorescent in situ hybridization (FISH); Sequenced tagged site (STS) mapping; Somatic cell hybridization.

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared approximately 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an approximately 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.

Variable number of tandem repeats in the growth hormone gene of Sparus aurata: association with growth and effect on gene transcription

The GH gene of Sparus aurata(saGH) contains variable number of tandem repeats (VNTR). The hyper‐variable minisatellites in the first and third introns segregate in a Mendelian manner and exhibit numerous alleles. Analysis by PCR and sequencing of the two introns in several wild Sparidae species revealed comparable minisatellites with some variations. ‘Zoo blot’ with the first intron unit as a probe showed this sequence to be characteristic of several families from the Perciformes order. Unexpectedly, a similar minisatellite was found in the first intron of the GH gene in flounder, which belongs to a different order. Transfection of constructs containing a reporter gene and first intron of different length to four cell lines resulted in an inhibitory effect of the longer intron relative to the short intron. A (CA)n microsatellite (saGHpCA) is found in the GH promoter. A similar repeat at the same location is present in GH promoters of several other fish species. High variability (11 alleles) of the saGHpCA was found in a hatchery population. Full‐sib family genotyping showed a Mendelian inheritance of these alleles. A significant association was found between allele distribution and body mass in large and average size fishes from a hatchery population. The intron minisatellites may serve as markers for hybrid population and parental assignment. Its presence in families and orders of the higher teleosts may help solving classification uncertainties. Their conservation and inhibitory effect suggest a biological role. The saGHpCA is correlated with growth and may be a good candidate for predicting growth performance.

Identification of human cells in brain xenografts and in neural co-cultures of rat by in situ hybridisation with Alu probe

Transplantation of human cells into animal models of neurodegenerative disorders is an important scientific application to analyse the survival and developmental capacity of grafted human cells under in vivo conditions. It is critical, therefore, to have a reliable method to distinguish between human and animal cells. In the present study, we describe a combined in situ hybridisation and immunocytochemistry method for the identification of human cells in cultured rat brain cells and xenografts. The specific Alu probe we utilised, which corresponds to the consensus sequence of human Alu repeats was evaluated by southern blot hybridisation of zoo blot and by in situ hybridisation of primary and neoplastic cells from man, rat, mouse, and hamster. This method allows a definite identification of human cells in neural xenografts and, in combination with additional in situ techniques, a further detection of grafted cells.

Peas-Mea1-Ppp2r5d overlapping gene complex: a transposon mediated-gene formation in mammals.

Human and mouse MEA1/Mea1 is flanked by two overlapping genes, a novel PEAS/Peas in a head-to-head orientation and PPP2R5D/Ppp2r5d in a tail-to-tail orientation making a Peas-Mea1-Ppp2r5d overlapping gene complex (PMP-complex). Genomic zoo blot analyses and database searching revealed that Mea1 exists only in mammals, while Peas and Ppp2r5d are conserved in eukaryotes. Mea1 and Peas are transcribed from a testis-expressed bidirectional promoter. Mea1-Ppp2r5d overlapping segment (MPOS) contains polyadenylation signals for both genes and shows marked conservation throughout mammals. Furthermore, the MPOS occupies 3'-region of transcripts of both genes is expected to form a clover-like intramolecular structure. Mouse genomic library Screening and database searches identified two MPOS-derived sequences in Odf2 gene and RP23-86H7 cosmid clone, respectively, in which MPOS might be a core segment for the retropositions. Thus, a key role of MPOS, a short transposable element containing polyadenylation signals on both strands, in the formation of the Mea1 during mammalian evolution is suggested.

Gene expression for a novel protein RGPR-p117 in various species: the stimulation by intracellular signaling factors.

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors.

Regional and Developmental Expression of Epm2a Gene and Its Evolutionary Conservation

Lafora's disease, an autosomal recessive progressive myoclonus epilepsy, is caused by mutations in the EPM2A gene encoding a dual-specificity phosphatase (DSP) named laforin. Here, we analyzed the developmental and regional expression of murine Epm2a and discussed its evolutionary conservation. A phylogenetic analysis indicated that laforin is evolutionarily distant from other DSPs. Southern zoo blot analysis suggested that conservation of Epm2a gene is limited to mammals. Laforin orthologs (human, mouse, and rat) display more than 94% similarity. All missense mutations known in Lafora disease patients affect conserved residues, suggesting that they may be essential for laforin's function. Epm2a is expressed widely in various organs but not homogeneously in brain. The levels of Epm2a transcripts in mice brains increase postnatally, attaining its highest level in adults. The most intense signal was detected in the cerebellum, hippocampus, cerebral cortex, and the olfactory bulb. Our results suggest that Epm2a is functionally conserved in mammals and is involved in growth and maturation of neural networks.

Cloning and Characterization of a Golgin-Related Gene from the Large-Scale Polymorphism Linked to the PML Gene

Megabase-scale mapping of the PML gene locus revealed the presence of a large-scale insertion–deletion polymorphism located 25 kb downstream of the PML gene. The polymorphism is organized as a head-to-tail tandem 25-kb repeat containing one to five units. Characterization of the first repeat unit downstream of PML revealed the presence of a gene with strong homology to a family of Golgin-related proteins. The gene, designated GLP (for Golgin linked to PML), is strongly expressed as a 6-kb transcript in normal human testis. In situ hybridization of normal human testis demonstrated that the expression of GLP was restricted to late meiotic germ cells. There was weak expression in late pachytene spermatocytes and strong expression in spermatids. GLP is 50% homologous to other Golgin-related proteins including the vesicle docking protein GM130. Southern blot hybridization of genomic DNA with a GLP probe demonstrated numerous homologous bands outside the PML locus. Three of these loci have been mapped by fluorescence in situ hybridization to chromosome loci 9q34.1, 15q11–q13, and 15q22–q24. Hybridization of a GLP cDNA probe to a zoo blot demonstrated multiple signals in nonhuman primates but not in other species and suggested the duplication of an ancestral locus around 20 million years ago.

Biochemical modifications and transcriptional alterations attendant to sterol feeding in Phytophthora parasitica.

Phytophthora species are eukaryotic sterol auxotrophs that possess the ability to grow, albeit poorly, in the complete absence of sterols. Growth of Phytophthora is often improved substantially when an exogenous source of sterol is provided. Additionally, sterols may be required for sexual and asexual sporulation in Phytophthora. Our research has been focused on identifying and characterizing the immediate physiological effects following sterol addition to cultures of P. parasitica. Through gas chromatographic analysis of extracts from P. parasitica cultures that were fed various sterols, we have obtained evidence for sterol C5 desaturase and delta7 reductase activities in this organism. Zoo blots were probed with DNA sequences encoding these enzymes, from Saccharomyces cerevisiae and Arabidopsis thaliana. Hybridization of a S. cerevisiae ERG3 probe to P. parasitica DNA was observed, implicating sequence similarity between the sterol C5 desaturase encoding genes. Differential display experiments, using RNA from P. parasitica, have demonstrated a pattern of altered gene expression between cultures grown in the presence and absence of sitosterol. Characterization of sterol-related metabolic effects and sterol functions in Phytophthora should lead to improved measures for control of this important group of plant pathogens.

Male-specific DNA markers from African catfish (Clarias gariepinus).

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species.

Psx homeobox gene is X-linked and specifically expressed in trophoblast cells of mouse placenta.

We previously isolated a cDNA clone for a homeobox-containing gene with its expression restricted to the extraembryonic tissues. In this study, Psx gene expression was further examined using in situ hybridization to determine the cellular distribution of Psx transcripts during embryo development. Psx expression was first detected at embryonic day 8.5 only in trophoblast giant cells and chorionic ectoderm. At E 9.5 and E 13.5, the expression was restricted to the giant cells and the labyrinthine trophoblast layer. In addition, the gene expression was detected in differentiated Rcho-1 trophoblast cells in vitro, which is typical of trophoblast giant cells in vivo, but not in proliferating Rcho-1 cells and HRP-1 cells. Interestingly, rat Psx homologue mRNA is about 200 bp shorter than mouse Psx, suggesting that there is a high degree of sequence divergence between the mouse and rat Psx homologues. The sequence divergence, perhaps as a result of rapid evolution, is further supported by the zoo blot analysis because the Psx gene was detectable only in mouse and rat but not in other vertebrate species tested. Psx is localized to the murine X chromosome. Taken together, our results suggest that Psx gene plays a unique role in the function of differentiated trophoblast cells and also serves as a useful model for studying trophoblast cell lineages and the rapid evolution of homeobox genes.

Cloning and Characterization of ZNF236, a Glucose-Regulated Kruppel-like Zinc-Finger Gene Mapping to Human Chromosome 18q22–q23

We report the cDNA cloning and characterization of ZNF236, a novel Kruppel-like zinc-finger gene initially identified by its glucose-regulated expression in human mesangial cells using mRNA differential display. Using the differential display fragment as a probe, we screened a human fetal kidney cDNA library and isolated several clones representing two differently spliced mRNA transcripts, designated ZNF236a and -b. Both transcripts were identical apart from the presence of an additional exon in ZNF236a that truncates the open reading frame. RT-PCR analysis confirmed the expression of both transcripts to be upregulated in human mesangial cells in response to elevated levels of d-glucose. ZNF236a and -b cDNAs encode polypeptides of 174 and 204 kDa, containing 25 and 30 C2H2 zinc-finger motifs, respectively. Northern blot analysis showed that ZNF236 is ubiquitously expressed in all human tissues tested. Expression levels were highest in skeletal muscle and brain, intermediate in heart, pancreas, and placenta, and lowest in kidney, liver, and lung. Southern zoo blot analysis indicated that ZNF236 is conserved in the genomes of all mammalian species tested, but not in yeast. The mapping of ZNF236 to human chromosome 18q22–q23, close to the IDDM6 locus, coupled with the glucose-regulated expression of the gene in human mesangial cells, suggests that ZNF236 may be a candidate gene for diabetic nephropathy.

Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development

We have characterized a retroviral promoter-trap insertion into a novel mammalian septin gene, Sep3. Its predicted amino acid sequence shares significant homology to that of Saccharomyces cerevisiae CDC3, CDC10, CDC11, CDC12, the Drosophila genes Pnut, Sep1, Sep2, and the mammalian genes BH5, CDC10, Nedd5, Diff6, and Sep2, which are implicated in cytokinesis and cell polarity. Sep3 encodes a protein of 465 amino acids, and contains an evolutionary conserved ATP/GTP-binding motif, two coiled-coil domains, and a highly hydrophobic domain at the C terminus. Alkaline phosphatase reporter gene expression in transgenic embryos was first detected at E8.5 in the neural fold, and high levels of expression continued throughout embryogenesis in the neural tube and brain. In addition, a low level of transient expression was detected in the somites, gut, and branchial arches of mouse embryos. Overall, reporter gene expression recapitulated Sep3 mRNA expression during mouse embryogenesis. In adults, Sep3 transcripts were only detected in the brain and testis. Zoo blot analysis revealed that Sep3-related sequences exist in several vertebrate species including zebrafish, frog, chicken, mouse and human. Consistent with the retroviral insertion into the 3′ UTR of the Sep3 gene, no obvious phenotypes associated with the promoter trap were detected in transgenic embryos or adult mice. In summary, we report the first isolation of a novel full-length Sep3 cDNA and extensive characterization of its expression during mouse embryogenesis and in adult tissues.

Orthologues of the Caenorhabditis elegans Longevity Gene clk-1 in Mouse and Human

The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al., 1997, Science 275: 980–983). The primary structure of CLK-1 showed a significant homology to Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes. In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene. Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively. Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C. elegans CLK-1, and 37% identity to S. cerevisiae Coq7p/Cat5p. Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes. Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle. Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene. Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3–p13.11.

The Sox10Dom Mouse: Modeling the Genetic Variation of Waardenburg-Shah (WS4) Syndrome

Hirschsprung disease (HSCR) is a multigenic neurocristopathy clinically recognized by aganglionosis of the distal gastrointestinal tract. Patients presenting with aganglionosis in association with hypopigmentation are classified as Waardenburg syndrome type 4 (Waardenburg-Shah, WS4). Variability in the disease phenotype of WS4 patients with equivalent mutations suggests the influence of genetic modifier loci in this disorder. Sox10(Dom)/+ mice exhibit variability of aganglionosis and hypopigmentation influenced by genetic background similar to that observed in WS4 patients. We have constructed Sox10(Dom)/+ congenic lines to segregate loci that modify the neural crest defects in these mice. Consistent with previous studies, increased lethality of Sox10(Dom)/+ animals resulted from a C57BL/6J locus(i). However, we also observed an increase in hypopigmentation in conjunction with a C3HeB/FeJLe-a/a locus(i). Linkage analysis localized a hypopigmentation modifier of the Dom phenotype to mouse chromosome 10 in close proximity to a previously reported modifier of hypopigmentation for the endothelin receptor B mouse model of WS4. To evaluate further the role of SOX10 in development and disease, we have performed comparative genomic analyses. An essential role for this gene in neural crest development is supported by zoo blot hybridizations that reveal extensive conservation throughout vertebrate evolution and by similar Northern blot expression profiles between mouse and man. Comparative sequence analysis of the mouse and human SOX10 gene have defined the exon-intron boundaries of SOX10 and facilitated mutation analysis leading to the identification of two new SOX10 mutations in individuals with WS4. Structural analysis of the HMG DNA-binding domain was performed to evaluate the effect of human mutations in this region.

Human ULK1, a Novel Serine/Threonine Kinase Related to UNC-51 Kinase ofCaenorhabditis elegans:cDNA Cloning, Expression, and Chromosomal Assignment

Theunc-51gene, isolated from mutants ofCaenorhabditis elegansexhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura,et al.,1994,Genes Dev.8: 2389–2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pIof 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p ofSaccharomyces cerevisiae.Phylogenetic analysis of ULK1, UNC-51, and Apglp suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that theULK1gene spans 30–40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system ofC. elegans.Both FISH and RH mapping confirmed the regional localization ofULK1to human chromosome 12q24.3.

Molecular Cloning and Expression Analysis of Five Novel Genes in Chromosome 1p36

The human chromosome 1p36 region displays frequent nonrandom chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumor suppressor genes. To identify these putative tumor suppressors we employed exon trapping, cDNA selection, and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated C1orf1 and xylan 1,4-β-xylosidase 1 (XBX1). Two further genes represented new members of known gene families: PTPRZ2 was a tyrosine phosphatase and FRAP2 represented a FKBP12–rapamycin-associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1, and to enolase 1 (ENO1). It colocalized with α enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organization of its 5′ untranslated sequences. Second, MBP-1 contained two single-base insertions not present in either ENO1 or ENO1L1 sequences, which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain-specific transcripts of 7.9 and 6.5 kb for PTPRZ2. In contrast, C1orf1, FRAP2, ENO1L1, and XBX1 appeared to be expressed ubiquitously in the tissues tested, with transcript sizes of 4.5, 8.7, 1.75, and 4.5 kb, respectively. Using fluorescencein situhybridization, we mapped the five novel genes relative to chromosome 1p36 breakpoints present in three established tumor cell lines and one nontumor cell line. The karyotypic abnormalities in these cell lines were exploited as chromosomal landmarks; we could thus show that the telomere to centromere gene order was PTPRZ2–(MBP-1/ENO1/ENO1L1)–(C1orf1/XBX1)–FRAP2. The localization of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumor suppressors.

Y;16 translocation breakpoint associated with a partial Turner phenotype identifies a foamy virus insertion

We have previously identified YACs containing the Y breakpoint in a male patient with Turner-like hydrops in the newborn period and a Y;16 translocation (Erickson et al., 1995). We have now subcloned these YACs and built a lambda contig across the Y breakpoint. A subclone near the Y;16 breakpoint of our patient shows great evolutionary conservation by hybridization to a zoo blot. We have sequenced the region responsible for the hybridization and found a foamy (spuma) virus sequence. These viruses are known to be xenotropic and a very similar cross-species hybridization (using the same commercial zoo blot) was recently reported with a human foamy virus clone (Cordonnier et al., 1995). Thus, we have identified a foamy virus insertion near the Y heterochromatin boundary.

The Charcot–Marie–Tooth Binary Repeat Contains a Gene Transcribed from the Opposite Strand of a Partially Duplicated Region of theCOX10Gene

Misalignment between the two elements of the CMT1A-REP binary repeat on chromosome 17p11.2–p12 causes two inherited peripheral neuropathies, Charcot–Marie–Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies. This binary repeat contains repetitive DNA elements that include LINES, SINES, medium reiteration frequency repeats, and a transposon-like element. TheCOX10gene has been mapped 10 kb centromeric to the distal CMT1A-REP element, and a portion of this gene is present in both the proximal and the distal CMT1A-REP elements. We report the isolation and characterization of a novel cDNA (C170RF1), which maps centromeric to and partially within the proximal CMT1A-REP element. Part of C170RF1 is transcribed from the opposite strand of theCOX10partial gene duplication present in the proximal CMT1A-REP element. This finding shows that C170RF1 andCOX10are being transcribed from opposite strands of identical DNA sequences that are separated by 1.5 Mb in the genome. RT-PCR analysis showed the proximal transcript was expressed in skeletal muscle. Sequence analysis identified an open reading frame encoding a 199-amino-acid protein. Zoo blot analysis showed that the transcript is conserved in nonhuman primates. The presence of a binary repeat contributes to the instability of this region of chromosome 17, yet two CMT1A-REP elements are present in the chimpanzee and all human populations. The presence of expressed sequences in both elements of the CMT1A-REP binary repeat could explain the maintenance of this repeat in humans.