Search PubMed⌕ Search

Biomedical subjects

A Schedl

Publications and source records attributed to A Schedl.

34 records · Page 2Linked to original sources
PubMed1997

The reticulocalbin gene maps to the WAGR region in human and to the Small eye Harwell deletion in mouse.

We describe the localization of the gene encoding reticulocalbin, a Ca2+-binding protein of the endoplasmic reticulum, on human chromosome 11p13 midway between the WT1 and the PAX6 genes and show that it is hemizygously deleted in WAGR individuals. The mouse reticulocalbin gene is also shown to map to the region of conserved synteny on mouse chromosome 2 and to be deleted in the Small eye Harwell (SeyH) mutation. Loss of the reticulocalbin gene could contribute to the early lethality of SeyH and SeyDey homozygotes.

Abnormalities, Multiple↗
PubMed1996

Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities.

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

Animals↗
PubMed1995

A high-resolution integrated physical, cytogenetic, and genetic map of human chromosome 11: distal p13 to proximal p15.1.

We describe a detailed physical map of human chromosome 11, extending from the distal part of p13 through the entirety of p14 to proximal p15.1. The primary level of mapping is based on chromosome breakpoints that divide the region into 20 intervals. At higher resolution YACs cover approximately 12 Mb of the region, and in many places overlapping cosmids are ordered in contiguous arrays. The map incorporates 18 known genes, including precise localization of the GTF2H1 gene encoding the 62-kDa subunit of TFIIH. We have also localized four expressed sequences of unknown function. The physical map incorporates genetic markers that allow relationships between physical and genetic distance to be examined, and similarly includes markers from a radiation hybrid map of 11. The cytogenetic location of cosmids has been examined on high-resolution banded chromosomes by fluorescence in situ hybridization, and FLpter values have been determined. The map therefore fully integrates physical, genic, genetic, and cytogenetic information and should provide a robust framework for the rapid and accurate assignment of new markers at a high level of resolution in this region of 11p.

Base Sequence↗
PubMed1994

Germ line transmission of yeast artificial chromosomes in transgenic mice.

Several groups have recently reported the successful generation of transgenic mice harbouring yeast artificial chromosomes (YACs). Different methodological approaches have been shown to produce similar results, namely, the faithful expression of the transgenes carried on YAC DNA. In this paper, we compare the reported techniques for obtaining transgenic mice carrying YACs using a 250-kb YAC bearing the mouse tyrosinase gene. These methods include: microinjection of gel-purified YAC DNA into pronuclei of fertilized mouse oocytes, yeast spheroblast fusion with embryonic stem (ES) cells and lipofection of YAC DNA into ES cells. Taken together, these reports show that the delivery of large genomic regions covering a gene of interest (such as those cloned in YAC vectors) is feasible, and will ensure appropriate temporal and spatial expression of the transgene at a level comparable to that of the endogenous counterpart.

Animals↗
PubMed1994

Wilms' tumour--a case of disrupted development.

Wilms' tumour is a paediatric kidney malignancy that arises through aberrant differentiation of nephric stem cells. We are studying the role of one Wilms' tumour predisposition gene, WT1. This is a tumour suppressor gene whose function is required for normal development of the genitourinary system. WT1 encodes a putative transcriptional repressor of the zinc finger family. Here we discuss how one of the normal functions of WT1 may be to suppress myogenesis during kidney development. Furthermore, we describe how we are proposing to use YAC (yeast artificial chromosome) transgenesis to analyse WT1 regulation and function in mice. We also discuss the evolution of the WT1 gene amongst different vertebrate classes and how this may provide insights into genitourinary evolution.

Animals↗
PubMed1993

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

Animals↗
PubMed1993

A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice.

Expression of transgenes in mice often fails to follow the normal temporal and spatial pattern and to reach the same level as the endogenous copies. Only in exceptional cases has position-independent and copy number-dependent expression been reproduced. The size constraint of standard constructs may prevent the inclusion of important remote regulatory elements. Yeast artificial chromosomes (YACs) provide a means of cloning large DNA fragments and the transfer of YAC DNA into somatic cells has been reported. We have previously produced transgenic mice carrying a 35 kilobase YAC construct. Here we report the transfer of a 250 kilobase YAC covering the mouse tyrosinase gene into mice by pronuclear injection of gel-purified YAC DNA. The YAC was inserted into the mouse genome without major rearrangements and expression of the YAC-borne tyrosinase gene resulted in complete rescue of the albino phenotype of the recipient mice. Expression from the transgene reached levels comparable to that of the endogenous gene and showed copy number dependence and position independence.

Animals↗
PubMed1992

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.

Animals↗
PubMed1992

Physical mapping of the albino-deletion complex in the mouse to localize alf/hsdr-1, a locus required for neonatal survival.

The albino-deletion complex in the mouse defines a genetically well-characterized region of chromosome 7 in which a number of loci essential for normal development and viability reside. One locus, designated alf or hsdr-1, is necessary for neonatal survival. Its absence results in hypoglycemia associated with biochemical and ultrastructural abnormalities in hepatocytes and proximal tubule cells of the kidney. We constructed a long-range physical map of the region defined by the proximal segment of the albino-deletion complex as a step toward localizing alf/hsdr-1. Sixteen markers, including 11 whose isolation is described here and in the accompanying paper (A. Schedl et al., 1992, Genomics 14, 288-297), were ordered on a panel of albino-deletion DNAs and their distribution was examined by pulsed-field gel electrophoresis. The resulting approximately 4300-kb physical map covers the entire region absent from the prototypic alf/hsdr-1 deletion c14CoS, estimated as approximately 3600 kb. Since the deletion c11DSD complements and overlaps most of c14CoS, alf/hsdr-1 was mapped at the proximal extreme of c14CoS, approximately 3000 kb from the albino locus. The density of CpG islands was found to be very heterogeneous across the region mapped.

Albinism↗
PubMed1992

Chromosome jumping from flanking markers defines the minimal region for alf/hsdr-1 within the albino-deletion complex.

The locus alf/hsdr-1, defined by the albino-deletion complex on mouse chromosome 7, is essential for neonatal survival. Animals homozygous for a subset of the deletions die shortly after birth due to impaired gene expression in liver parenchymal cells and kidney proximal tubular cells. Here, we describe a detailed analysis of the region containing alf/hsdr-1 by means of chromosome jumping from flanking markers. Three chromosome jumping libraries based on the restriction enzymes XmaI and SalI were constructed. Isolation of eight jumping clones distributed over 450 kb allowed more than 240 kb to be cloned in genomic lambda and cosmid libraries. Five of the probes map within the minimal genetic interval for alf/hsdr-1, which is defined by the proximal borders of the deletions c10R75M and c11DSD. The breakpoints of these deletions were precisely mapped, which allowed alf/hsdr-1 to be localized to a 310-kb interval.

Albinism↗
PubMed1992

Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice.

Mice homozygous for albino deletions encompassing the locus alf/hsdr-1 die shortly after birth. Lethality is thought to be the consequence of hypoglycemia, which results from the failure to activate hormone-dependent genes in liver and kidney encoding enzymes important for gluconeogenesis. Within the region in which alf/hsdr-1 has been defined by physical mapping, we identified the gene encoding fumarylacetoacetate hydrolase (FAH), an enzyme of tyrosine metabolism. Lack of FAH activity should lead to accumulation of toxic tyrosine metabolites. In man, genetically determined FAH deficiency is the primary defect in tyrosinemia type I, a fatal liver disease of infants. Northern blot and in situ hybridization analysis of mouse tissues showed that the cell types that normally express FAH correspond to those that exhibit a phenotype in alf/hsdr-1 deletion mice. Moreover, we could mimic aspects of the alf/hsdr-1 deletion phenotype in vitro by treating primary hepatocyte cultures with an intermediate of tyrosine metabolism. These findings strongly suggest that alf/hsdr-1 encodes FAH and that absence of FAH is responsible for neonatal lethality in albino deletion mice. Mechanisms by which this metabolic defect might bring about alterations in gene expression characteristic of the alf/hsdr-1 deletion phenotype are discussed.

Amino Acid Sequence↗
PubMed1992

Multiple effects on liver-specific gene expression in albino lethal mice caused by deficiency of an enzyme in tyrosine metabolism.

alf/hsdr-1 is a locus in the mouse defined by albino deletions to be essential for neonatal viability. Homozygous deletion of alf/hsdr-1 leads to a pleiotropic phenotype in liver and kidney, including impaired perinatal activation of hormone-dependent genes, and the induction of detoxifying enzymes and early-response genes. To elucidate the molecular basis of this complex phenotype, we have identified the gene mapping at alf/hsdr-1 by positional cloning, using overlapping albino locus deletions to define the location of alf/hsdr-1. The gene encodes fumarylacetoacetate hydrolase, FAH, an enzyme of tyrosine metabolism. Genetically determined FAH deficiency in man leads to a severe liver failure in infants. In mice, we find that the normal sites of expression of FAH correlate tightly with cell-types which display abnormalities in albino lethal mice. The identification of the Fah gene as a candidate for alf/hsdr-1 offers a novel explanation for the complex phenotype, one into which all aspects can be accommodated. The phenotype can now be understood as a sequence of responses to toxic electrophilic metabolites.

Animals↗
PubMed1991

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse.

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.

Albinism↗
PubMed1991

The albino-deletion complex of the mouse: molecular mapping of deletion breakpoints that define regions necessary for development of the embryonic and extraembryonic ectoderm.

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.

Albinism↗