Physical analysis of murine albino deletions that disrupt liver-specific gene regulation or mesoderm development

Complementation analyses of radiation-induced deletion mutations involving the albino (c) locus in Chromosome (Chr) 7 of the mouse have identified several loci, in addition toc, that have important roles in development. The mesoderm-deficient (msd) and hepatocyte-specific developmental regulation-1 (hsdr-1) loci, which are proximal and tightly linked toc, are important in the formation of mesoderm and in the regulation of liver- and kidney-specific induction of various enzymes and proteins, respectively. Cloning deletion-breakpoint-fusion fragments caused by lethal albino deletions that genetically define the extents of themsd andhsdr-1 loci is one way of generating molecular probes for studying the gene(s) involved in these phenotypes. The distal breakpoints of five such deletions were positioned on a long-range (PFGE) map of 1.7 Mb of wild-type DNA surrounding thec, D7Was12, andEmv-23 loci. In addition, the distal breakpoints of two viable albino deletions, which remove part of the tyrosinase gene and extend distally, were localized in the vicinity of the lethal deletion breakpoints. Therefore, the viable deletions can be exploited to generate additional DNA probes that should facilitate the isolation of breakpoint clones from chromosomes carrying lethal deletions defininghsdr-1 andmsd.     

Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations within the Pid-Hbb Region of Mouse Chromosome 7

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).     

Molecular Analysis of Radiation-Induced Albino (C)-Locus Mutations That Cause Death at Preimplantation Stages of Development

Deletion mutations at the albino (c) locus have been useful for continuing the development of fine-structure physical and functional maps of the Fes-Hbb region of mouse chromosome 7. This report describes the molecular analysis of a number of radiation-induced c deletions that, when homozygous, cause death of the embryo during preimplantation stages. The distal extent of these deletions defines a locus, pid, (preimplantation development) genetically associated with this phenotype. The proximal breakpoints of eight of these deletions were mapped with respect to the Tyr (tyrosinase; albino) gene as well as to anonymous loci within the Fah-Tyr region that are defined by the Pmv-31 viral integration site and by chromosome-microdissection clones. Rearrangements corresponding to the proximal breakpoints of two of these deletions were detected by Southern blot analysis, and a size-altered restriction fragment carrying the breakpoint of one of them was cloned. A probe derived from this deletion fusion fragment defines a locus, D7Rn6, which maps within (or distal to) the pid region, and which discriminates among the distal extents of deletions eliciting the pid phenotype. Extension of physical maps from D7Rn6 should provide access both to the pid region and to loci mapping distal to pid that are defined by N-ethyl-N-nitrosourea-induced lethal mutations.     

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.     

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.     

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.     

Construction and characterization of deletions with defined end points in Drosophila using P elements in trans

We used P-element transposase-mediated "male recombination" between two P elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide crustacean cardioactive peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one-quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 bp of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end points. Importantly, it can use preexisting P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

Molecular analysis of the Adh region of the genome of Drosophila melanogaster

A small region of the genome of Drosophila melanogaster has been cloned in a series of overlapping phage. A length of 165 X 10(3) base-pairs of contiguous DNA that spans polytene chromosome region 35A4 to 35B1 and includes the structural gene for alcohol dehydrogenase (Adh) as well as at least two other genes, outspread (osp) and no-ocelli (noc), has been characterized by mapping chromosome aberrations to the DNA. The relationship between osp and Adh is surprising: of nine osp alleles associated with chromosome breakpoints, five map distal (i.e. 5') to Adh and four map proximal (i.e. 3') to this gene. None affects the expression of Adh. As defined by these and other breakpoints, the osp gene spans at least 52 X 10(3) base-pairs and overlaps the Adh gene. The noc gene, as defined by the mapping of nearly 30 breakpoints, is at least 50 X 10(3) base-pairs in size. Alleles of noc and noc- deletions show either of two kinds of interaction with the recessive lethality of l(2)br29ScoR+1, a lethal that maps immediately distal to noc. One class of noc allele is viable when heterozygous with ScoR+1, while the other class is lethal or semi-lethal. Both classes, however, are homozygous or hemizygous viable. The locations of these two classes of noc allele on the DNA fall into two clusters, with those that are viable with ScoR+1 located proximal to those that are not. The physical boundary between these classes lies at a site just distal to that of the breakpoint of the inversion associated with ScoR+1 itself.     

The albino-deletion complex in the mouse defines genes necessary for development of embryonic and extraembryonic ectoderm

A detailed embryological analysis has been undertaken on embryos carrying the c4FR60Hd-, c5FR60Hg- or c2YPSj-albino deletions of mouse chromosome 7. Embryos homozygous for the c4FR60Hd deletion are abnormal at day 7.5 of gestation. The extraembryonic ectoderm does not develop, and primitive-streak formation and mesoderm production do not occur. In contrast, extensive development of the extraembryonic ectoderm, as well as mesoderm production, are observed in the c5FR60Hg- and c2YPSj-homozygous embryos. The mesoderm does not, however, organize into somites and the neural axis does not form. The embryos are grossly abnormal by day 8.5 of development. There are two other albino deletions (c6H and c11DSD) that are known to affect the embryo around the time of gastrulation (Niswander et al. 1988), and the lethal phenotype observed for the c4FR60Hd-homozygous embryos is similar to that described for c6H-homozygous embryos, whereas the c5FR60Hg- and c2YPSj-homozygous embryos display a phenotype that is similar to c11DSD-homozygous embryos. A detailed complementation analysis using these five deletions revealed that the c5FR60Hg, c2YPSj and c11DSD deletions could partially complement the phenotype produced by the c4FR60Hd and c6H deletions in any combination. Extensive development of the extraembryonic structures and production of mesoderm occurs in the compound heterozygotes. These results suggest that the distal breakpoints of the c5FR60Hg, c2YPSj and c11DSD deletions lie more proximal than the distal breakpoints of the c4FR60Hd and c6H deletions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine Physical Mapping of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

The diploid-like chromosome pairing in polyploid wheat is controlled by the Ph1 (pairing homoeologous) gene that is located on chromosome arm 5BL. By using a combination of cytogenetic and molecular techniques, we report the physical location of the Ph1 gene to a submicroscopic chromosome region (Ph1 gene region) that is flanked by the breakpoints of two deletions (5BL-1 and ph1c) and is marked by a DNA probe (XksuS1). The Ph1 gene region is present distal to the breakpoint of deletion 5BL-1 but proximal to the C-band 5BL2.1. Two other DNA probes (Xpsr128 and Xksu75) flank the region-Xpsr128 being proximal and Xksu75 being distal. The estimated size of the region is less than 3 Mb. The chromosome region around the Ph1 gene is high in recombination as the genetic distance of the region between 5BL-1 breakpoint and C-band 5BL2.1 (not resolved by the microscope) is at least 9.3 cM.     

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.     

Physical mapping studies on the human X chromosome in the region Xq27-Xqter

We have characterized three terminal deletions of the long arm of the X chromosome. Southern analysis using Xq27/q28 probes suggests that two of the deletions have breakpoints near the fragile site at Xq27.3. Flow karyotype analysis provides an estimate of 12 X 10(6) bp for the size of the deleted region. We have not detected the deletion breakpoints by pulsed-field gel electrophoresis (PFGE) using the closet DNA probes, proximal to the fragile site. The physical distance between the breakpoints and the probes may therefore be several hundred kilobases. The use of the deletion patients has allowed a preliminary physical map of Xq27/28 to be constructed. Our data suggest that the closest probes to the fragile site on the proximal side are 4D-8 (DXS98), cX55.7 (DXS105), and cX33.2 (DXS152). PFGE studies provide evidence for the physical linkage of 4D-8, cX55.7, and cX33.2. We have also found evidence for the physical linkage of F8C, G6PD, and 767 (DXS115), distal to the fragile site.     

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.     

Molecular and cytogenetic analysis of an interstitial 20p deletion associated with syndromic intrahepatic ductular hypoplasia (Alagille syndrome)

Summary High-resolution chromosome analysis of a 19-year-old female proband with syndromic intrahepatic ductular hypoplasia (Alagille syndrome, AWS) revealed an interstitial deletion of chromosome 20p with breakpoints provisionally located in or close to p11.22 and p12.2. Southern blots from digests of DNA of the proband and her chromosomally normal parents were hybridized with the human DNA probes pR12.21, HuPrPcDNA2, and pDS6-SgI, which have been mapped to the region 20 (p12-pter), and rehybridized with the F IX probe for calibration. Comparing the hybridization signals of the normally sized DNA fragments of the familiy, we found no evidence for loss of any of the three tested distal chromosome 20p loci in our proband. Furthermore, in situ hybridization with HuPrPcDNA2 revealed a specific accumulation of grains at or around the faint distal G band suspected to represent all or most of band p12.3 of the proband's deleted 20p and at p12 of the normal chromosome 20. Thus the AWS of our proband is associated with an interstitial deletion that preserved the three tested distal loci on 20p. Since nine further reported cases of 20p deletion are clinically similar, we propose AWS as a further contiguous gene syndrome and assign it to an approximately 8-Mb-large chromosome 20p segment (provisionally, p11.23–p12.1).     

The albino deletion complex and early postimplantation survival in the mouse

The albino deletion complex in the mouse represents 37 overlapping chromosomal deficiencies that have been arranged into at least twelve complementation groups. Many of the deletions cover regions of chromosome 7 that contain genes necessary for early embryonic development. The work reported here concentrates on two of these deletions (c6H, c11DSD), both of which were known to be lethal around the time of gastrulation when homozygous. A detailed embryological analysis has revealed distinct differences in the lethal phenotype associated with the c6H and c11DSD deletions. c6H homozygous embryos are grossly abnormal at day 7.5 of gestation, whereas c11DSD homozygous embryos appear abnormal at day 8.5 of gestation. There is no development of the extraembryonic ectoderm in c6H homozygotes, whereas extensive development of this tissue type occurs in c11DSD homozygotes. The visceral endoderm is abnormally shaped and the parietal endoderm appears to be overproduced in c6H homozygotes; these structures are not affected in c11DSD homozygotes. The embryonic ectoderm is runted in both types of embryo and it is not possible to obtain homozygous embryo-derived stem-cell lines for either deletion. Mesoderm formation occurs in the c11DSD but not in the c6H homozygotes. The c11DSD deletion chromosome complements the c6H chromosome in that the lethal phenotype of the compound heterozygote is similar to that of the c11DSD homozygote. These results suggest that a gene(s) necessary for normal development of the extraembryonic ectoderm is present in the c11DSD but deficient in the c6H deletion chromosome.     

242 breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread.

Using whole cosmids as probes, we have mapped 242 DMD/BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.     

A deletion map of the WAGR region on chromosome 11.

The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.