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R G Worton

Publications and source records attributed to R G Worton.

At least 55 records · Page 3Linked to original sources
PubMed1990

Multipoint linkage analysis and heterogeneity testing in 20 X-linked retinitis pigmentosa families.

Using multipoint linkage analysis in 20 families segregating for X-linked retinitis pigmentosa (XLRP), the lod scores on a map of eight RFLP loci were obtained. Our results indicate that under the hypothesis of homogeneity the maximal multipoint lod score supports one disease locus located slightly distal to OTC at Xp21.1. Heterogeneity testing for two XLRP loci suggested that a second XLRP locus may be located 8.5 cM proximal to DXS28 at Xp21.3. Further heterogeneity testing for three disease loci failed to detect a third XLRP locus proximal to DXS7 in any of our 20 XLRP families.

Alleles↗
PubMed1990

Age-related conversion of dystrophin-negative to -positive fiber segments of skeletal but not cardiac muscle fibers in heterozygote mdx mice.

Immunoreactive dystrophin was examined in muscle fibers of quadriceps, extraocular muscles and cardiac ventricular muscles of female heterozygote mdx mice at 10, 35 and 60 days of age, with microscopic immunoperoxidase method and by immunoblots. In quadriceps muscle fibers there was a marked gradual diminution of the dystrophin-negative fiber segments between age 10 and 60 days. We suggest that this was partly due to a spontaneous fusion of dystrophin-competent satellite cells into the dystrophin-negative fiber segments and partly to an expansion of the cytoplasmic domain of dystrophin expression related to the original myonuclei. In cardiac muscle that lacks satellite cells, there was persistence of a large number of dystrophin-negative fiber segments even at age 60 days and probably beyond. The findings of this study have implications for the detection of heterozygote female carriers of Duchenne muscular dystrophy (DMD) and for the possible therapy of DMD muscles by myoblast transfer.

Aging↗
PubMed1990

Molecular and functional analysis of the muscle-specific promoter region of the Duchenne muscular dystrophy gene.

Duchenne muscular dystrophy (DMD) gene transcripts are most abundant in normal skeletal and cardiac muscle and accumulate as normal myoblasts differentiate into multinucleated myotubes. In this report we describe our initial studies aimed at defining the cis-acting sequences and trans-acting factors involved in the myogenic regulation of DMD gene transcription. A cosmid clone containing the first exon of the DMD gene has been isolated, and sequences lying upstream of exon 1 were analyzed for homologies to other muscle-specific gene promoters and for their ability to direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs. The results indicate that the transcriptional start site for this gene lies 37 base pairs (bp) upstream of the 5' end of the published cDNA sequence and that 850 bp of upstream sequence can direct CAT gene expression in a muscle-specific manner. Sequence analysis indicates that in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. Our observation that only 149 bp of upstream sequence is required for muscle-specific expression of a chimeric CAT gene construct further implicates the CArG and myocyte-specific enhancer-binding nuclear factor 1 binding homologies as important domains in the regulation of this gene. On the other hand, the unique profile of myogenic cell line-specific induction displayed by our DMD promoter-CAT gene constructs suggests that other as yet undefined cis-acting sequences and/or trans-acting factors may also be involved.

Base Sequence↗
PubMed1990

Prenatal identification of a girl with a t(X;4)(p21;q35) translocation: molecular characterisation, paternal origin, and association with muscular dystrophy.

There are 23 females known with Duchenne or Becker muscular dystrophy (DMD or BMD) who have X;autosome translocations that disrupt the X chromosome within band p21. A female with a t(X;4)(p21;q35) translocation was identified prenatally at routine amniocentesis. At birth, she was found to have a raised CK level, consistent with a diagnosis of Duchenne muscular dystrophy. Her cells were fused with mouse RAG cells and the translocated chromosomes were separated from one another and from the normal X chromosome by segregation in the resulting somatic cell hybrids. Southern blot analysis of the hybrids indicated that the translocation occurred on the X chromosome between genomic probes GMGX11 and J-66, both of which lie within the DMD gene. Further localisation with a subfragment of the DMD cDNA clone placed the translocation breakpoint in an intron towards the middle of the gene, confirming that the de novo translocation disrupted the DMD gene. RFLP analysis of the patient, her parents, and the hybrid cell lines showed that the translocation originated in the paternal genome. This brings to six out of six the number of DMD gene translocations of paternal origin, a fact that may be an important clue in future studies of the mechanism by which X;autosome translocations arise.

Adult↗
PubMed1990

Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotypegenotype correlation.

Partial gene deletion is the major cause of mutation leading to Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Partial gene duplication has also been recognized in a few cases. We have conducted a survey for duplication in 72 unrelated nondeletion patients, analyzed by Southern blot hybridization with clones representing the entire DMD cDNA. With careful quantitative analysis of hybridization band intensity, 10 cases were found to carry a duplication of part of the gene, a frequency of 14% for nondeletion cases (10/72), or 6% for all cases (10/181). The extent of these duplications has been characterized according to the published exon-containing HindIII fragment map, and in six of the 10 duplications a novel restriction fragment that spanned the duplication junction was detected. The resulting translational reading frame of mRNA has been predicted for nine duplications. A shift of the reading frame was predicted in four of the six DMD cases and in one of the two intermediate cases, while the reading frame remained uninterrupted in both BMD cases. RFLP and quantitative Southern blot analyses revealed a grandpaternal origin of duplication in four families and grandmaternal origin in one family. In all five families, the duplication was found to originate from a single X chromosome. Unequal sister-chromatid exchange is proposed to be the mechanism for the formation of these duplications.

Adolescent↗
PubMed1989

Assignment of the gene for complete X-linked congenital stationary night blindness (CSNB1) to Xp11.3.

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by a presumptive defect of neurotransmission between the photoreceptor and bipolar cells. Carriers are not clinically detectable. A new classification for CSNB includes a complete type, which lacks rod function by electroretinography and dark adaptometry, and an incomplete type, which shows some rod function on scotopic testing. The refraction in the complete CSNB patients ranges from mild to severe myopia; the incomplete ranges from moderate hyperopia to moderate myopia. To map the gene responsible for this disease, we studied eight multigeneration families, seven with complete CSNB (CSNB1) and one with incomplete CSNB, by linkage analysis using 17 polymorphic X-chromosome markers. We found tight genetic linkage between CSNB1 and an Xp11.3 DNA polymorphic site, DXS7, in seven families with CSNB1 (LOD 7.35 at theta = 0). No recombinations to CSNB1 were found with marker loci DXS7 and DXS14. The result with DXS14 may be due to the small number of scored meioses (10). No linkage could be shown with Xq loci PGK, DXYS1, DXS52, and DXS15. Pairwise linkage analysis maps the gene for CSNB1 at Xp11.3 and suggests that the CSNB1 locus is distal to another Xp11 marker, TIMP, and proximal to the OTC locus. Five-point analysis on the eight families supported the order DXS7-CSNB1-TIMP-DXS225-DXS14. The odds in favor of this order were 9863:1. Removal of the family with incomplete CSNB (F21) revealed two most favored orders, DXS7-CSNB1-TIMP-DXS255-DXS14 and CSNB1-DXS7-TIMP-DXS255-DXS14. Heterogeneity testing using the CSNB1-M27 beta and CSNB1-TIMP linkage data (DXS7 was not informative in F21) was not significant to support evidence of genetic heterogeneity (P = 0.155 and 0.160, respectively).

Alleles↗
PubMed1989

Linkage analysis of a large Latin-American family with X-linked retinitis pigmentosa and metallic sheen in the heterozygote carrier.

An extended linkage analysis was performed on the large Latin-American kindred with X-linked retinitis pigmentosa (XLRP) and metallic sheen in the heterozygous carrier studied and reported previously by R.L. Nussbaum et al. (1985, Hum. Genet. 70:45-50) and on a smaller family with the same XLRP variant. In these kindreds the XLRP locus shows close linkage with Xp21 marker loci OTC and DXS206. The results of this linkage analysis agree with the observations made by Nussbaum et al. (1985) that an XLRP locus is distal to DXS7.

Crossing Over, Genetic↗
PubMed1989

Mapping of four translocation breakpoints within the Duchenne muscular dystrophy gene.

There are over 20 females with Duchenne or Becker muscular dystrophy (DMD or BMD) who have X-autosome translocations that break the X chromosome within band Xp21. Several of these translocations have been mapped with genomic probes to regions throughout the large (approximately 2000 kb) DMD gene. In this report, a cDNA clone from the 5' end of the gene was used to further map the breakpoints in four X-autosome translocations. A t(X;21) translocation in a patient with BMD and a t(X;1) translocation in a patient with DMD were found to break within a large 110-kb intron between exons 7 and 8. Two other DMD translocations, t(X;5) and t(X;11), were found to break between the first and the second exon of the gene within a presumably large intron (greater than 100 kb). These results demonstrate that all four translocations have disrupted the DMD gene and make it possible to clone and sequence the breakpoints. This will in turn determine whether these translocations occur by chance in these large introns or whether there are sequences that predispose to translocations.

Chromosome Mapping↗
PubMed1989

Myogenic regulation of dystrophin gene expression.

Muscle-specific transcriptional regulation of DMD gene expression has been inferred from both the histopathology of the disease and, more recently, from the use of cDNA sequences to detect DMD gene transcripts by Northern blot, RNase protection, in situ hybridization, and polymerase chain reaction (PCR) analyses. Several muscle-specific genes have been shown to be transcriptionally activated early in myogenesis and a number of cis-acting promoter elements required for muscle-specific transcriptional induction have been described. In this report we review our recent progress on studies of the mechanisms underlying myogenic regulation of dystrophin gene expression. Indirect immunofluorescence has been used to demonstrate that dystrophin is present at the muscle cell surface very early in the myogenic program. The cloning and sequencing of the dystrophin gene promoter reveals the presence of pre-defined muscle-specific cis-acting promoter elements. Functional assays provide evidence that these upstream sequences are capable of regulating DMD gene expression in a cell-and developmental stage-specific manner.

Base Sequence↗
PubMed1989

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

Animals↗
PubMed1989

Activation of the hprt gene on the inactive X chromosome in transformed diploid female Chinese hamster cells.

Treatment with 5-azacytidine, a potent inhibitor of DNA methylation, was used to induce activation of the selectable hprt gene on the inactive X chromosome in a diploid female Chinese hamster cell line. The transformed, stably diploid cell line F3B was selected in media containing the lethal purine analogue 6-thioguanine, to generate a phenotypically HPRT- mutant, F3BT1, of presumed genotype hprt-/hprt(+), where (+) represents the presumably wild-type allele on the inactive X chromosome. Treatment of F3BT1 with 5-azacytidine resulted in phenotypic reversion to HPRT+ at a frequency greater than 10(-3). Similar treatment of 6-thioguanine-resistant control lines derived from male cells, or from CHO (which has no inactive X chromosome), had no effect on the frequency of phenotypic reversion, indicating that activation of the hprt(+) allele, rather than reversion of the hprt- is responsible. This conclusion is substantiated by documentation of the low mutagenic capacity of 5-azacytidine in this system. Proof that the hprt(+) allele can be activated by 5-azacytidine treatment was obtained in somatic cell hybrids in which hprt gene products from the active and inactive X chromosomes could be distinguished by isoelectric focusing. Our results demonstrate that X-linked gene activation associated with generalized DNA demethylation occurs with high frequency in transformed diploid Chinese hamster cells.

Animals↗
PubMed1989

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles

Animals↗
PubMed1989

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.

Adolescent↗
PubMed1989

Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation.

In mdx mice, the dystrophin gene of the X chromosome is defective and, as a result, immunoreactive dystrophin is undetectable in all muscle fibers of all animals of this highly inbred strain. This study showed that implantation of suspensions of clonal cultures of normal human myoblasts into different regions of quadriceps muscles of 6-to-10-day-old mdx mice or 60-day-old mdx mice (whose muscles have been crushed 4 days before implantation) results in the appearance of scattered fiber segments containing microscopically demonstrable immunoreactive dystrophin. In the animals that received the normal myoblast implantation in the prenecrotic stage of the disease (6 to 10 days of age), the dystrophin-positive fiber segments (demonstrated at ages 35, 45, and 60 days) escaped necrosis. This was determined by the absence of the characteristic chains of central nuclei, a reliable marker of prior necrosis in mdx muscle fibers. By heavy labeling of the nuclear DNA of the transplantable human myoblasts with H3-thymidine during culturing, and by sequential performance of an immunocytochemical staining for dystrophin and autoradiography on the same sections, some dystrophin-positive fiber segments were shown to contain radiolabeled myonuclei. It was concluded that nondystrophic myoblasts fused with host muscle fibers to form mosaic muscle fibers in which the normal dystrophin gene of the implanted myoblasts was expressed. This approach may be employed for the mitigation of the deleterious consequences of a gene defect in recessively inherited human muscle diseases such as Duchenne dystrophy.

Animals↗
PubMed1988

The problem of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscular disorder. The biochemical defect remains unknown, but the gene responsible has been mapped to band Xp21. The gene has now been cloned in two laboratories solely from knowledge of its map location. L. M. Kunkel and his colleagues isolated genomic sequences (PERT 87) from within a large deletion causing DMD, whereas our group isolated genomic sequences (XJ) spanning the junction of an X-autosome translocation causing the disease. Chromosome walking by both groups has led to the isolation of over 400 kilobases of the PERT 87 and XJ region. Subclones of PERT 87 and XJ reveal restriction fragment length polymorphisms that segregate with the DMD gene in 95% of meioses, and fail to hybridize with DNA from about 8% of male patients. Selected subclones of PERT 87 and XJ contain exons that hybridize to muscle-derived complementary DNA (cDNA) clones. The cDNA clones detect a large (16 kilobase) message. Analysis of deletions, mutations and translocations suggests a DMD gene of between two million and three million base pairs. The clones obtained so far are useful for attempts to generate antibody against the gene product and for carrier identification and prenatal diagnosis.

Chromosome Mapping↗
PubMed1988

The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle.

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are allelic X-linked muscle disorders in man. The gene responsible for the disease has been cloned from knowledge of its map location at band Xp21 on the short arm of the X chromosome. The product of the DMD gene, a protein of relative molecular mass 400,000 (Mr 400K) recently named dystrophin, has been reported to co-purify with triads of mouse and rabbit skeletal muscle when assayed using polyclonal antibodies raised against fusion proteins encoded by regions of mouse DMD complementary DNA. Here we show that antibodies directed against synthetic peptides and fusion proteins derived from the N-terminal region of human DMD cDNA strongly react with an antigen present in skeletal muscle sarcolemma on cryostat sections of normal human muscle biopsies. This immunoreactivity is reduced or absent in muscle fibres from DMD patients but appears normal in muscle fibres from patients with other myopathic diseases. The same antibodies specifically react with a 400K protein in sodium dodecyl sulphate (SDS) extracts of normal human muscle subjected to Western blot analysis. We conclude that the product of the DMD gene is associated with the sarcolemma rather than with the triads and speculate that it strengthens the sarcolemma by anchoring elements of the internal cytoskeleton to the surface membrane.

Antibodies↗
PubMed1988

Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end.

The multiple copies of the human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters that map to the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. Concerted evolution of the gene family is thought to be mediated by interchromosomal recombination between rDNA repeat units, but such events would also result in conservation of the sequences distal to the rDNA on these five pairs of chromosomes. To test this possibility, a DNA fragment spanning the junction between rDNA and distal flanking sequence has been cloned and characterized. Restriction maps, sequence data, and gene mapping studies demonstrate that (i) the rRNA genes are transcribed in a telomere-to-centromere direction, (ii) the 5' end of the cluster and the adjacent non-rDNA sequences are conserved on the five pairs of chromosomes, and (iii) the 5' end of the cluster is positioned about 3.7 kb upstream from the transcription initiation site of the first repeat unit. The data support a model of concerted evolution by interchromosomal recombination.

Biological Evolution↗