Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. II. Complementation Analyses of Lethal Brown Deletions

Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.     

Molecular and functional mapping of the piebald deletion complex on mouse chromosome 14

The piebald deletion complex is a set of overlapping chromosomal deficiencies surrounding the endothelin receptor B locus collected during the Oak Ridge specific-locus-test mutagenesis screen. These chromosomal deletions represent an important resource for genetic studies to dissect the functional content of a genomic region, and several developmental defects have been associated with mice homozygous for distinct piebald deletion alleles. We have used molecular markers to order the breakpoints for 20 deletion alleles that span a 15.7-18-cM region of distal mouse chromosome 14. Large deletions covering as much as 11 cM have been identified that will be useful for regionally directed mutagenesis screens to reveal recessive mutations that disrupt development. Deletions identified as having breakpoints positioned within previously described critical regions have been used in complementation studies to further define the functional intervals associated with the developmental defects. This has focused our efforts to isolate genes required for newborn respiration and survival, skeletal patterning and morphogenesis, and central nervous system development.     

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.     

A genome-wide resource of cell cycle and cell shape genes of fission yeast

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.     

Y chromosome and male infertility: what is a normal Y chromosome?

The human Y chromosome contains a number of genes and gene families that are essential for germ cell development and maintenance. Many of these genes are located in highly repetitive elements that are subject to rearrangements. Deletion of azoospermia factor (AZF) regions AZFa, AZFb, and AZFc are found in approximately 10-15% of men with severe forms of spermatogenic failure. Several partial AZFc deletions have been described. One of these, which removes around half of all the genes within the AZFc region, appears to be present as an inconsequential polymorphism in populations of northern Eurasia. A second deletion, termed gr/gr, also results in the absence of several AZFc genes and it may be a genetic risk factor for spermatogenic failure. However, the link between these partial deletions and fertility is unclear. The gr/gr deletion is not a single deletion but a combination of deletions that vary in size and complexity and result in the absence of different genes. There are also regional or ethnic differences in the frequency of gr/gr deletions. In some Y-chromosome lineages, these deletions appear to be fixed and may have little influence on spermatogenesis. Most of these data (gene content and Y chromosome structure) have been deduced from the reference Y chromosome sequence deposited in NCBI. However, recently there have been attempts to define these types of structural rearrangements in the general population. These have highlighted the considerable degree of structural diversity that exist. Trying to correlate these changes with the phenotypic variability is a major challenge and it is likely that there will not be a single reference (or normal) Y chromosome sequence but many.     

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).     

Physical Mapping of Genetic Markers on the Short Arm of Chromosome 5

The deletion of the short arm of chromosome 5 is associated with the cri-du-chat syndrome. In addition, loss of this portion of a chromosome is a common cytogenetic marker in a number of malignancies. However, to date, no genes associated with these disorders have been identified. Physical maps are the first step in isolating causative genes, and genes involved in autosomal recessive disorders are now routinely mapped through the identification of linked markers. Extensive genetic maps based upon polymorphic short tandem repeats (STRs) have provided researchers with a large number of markers to which such disorders can be genetically mapped. However, the physical locations of many of these STRs have not been determined. Toward the goal of integrating the human genetic maps with the physical maps, a 5p somatic cell hybrid deletion mapping panel that was derived from patients with 5p deletions or translocations was used to physically map 47 STRs that have been used to construct genetic maps of 5p. These data will be useful in the localization of disease genes that map to 5p and may be involved in the etiology of the cri-du-chat syndrome.     

Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7

Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.     

Identification of protein complexes required for efficient sister chromatid cohesion

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.     

Ionizing radiation and genetic risks. X. The potential "disease phenotypes" of radiation-induced genetic damage in humans: perspectives from human molecular biology and radiation genetics.

Estimates of genetic risks of radiation exposure of humans are traditionally expressed as expected increases in the frequencies of genetic diseases (single-gene, chromosomal and multifactorial) over and above those of naturally-occurring ones in the population. An important assumption in expressing risks in this manner is that gonadal radiation exposures can cause an increase in the frequency of mutations and that this would result in an increase in the frequency of genetic diseases under study. However, despite compelling evidence for radiation-induced mutations in experimental systems, no increases in the frequencies of genetic diseases of concern or other adverse effects (i.e., those which are not formally classified as genetic diseases), have been found in human studies involving parents who have sustained radiation exposures. The known differences between spontaneous mutations that underlie naturally-occurring single-gene diseases and radiation-induced mutations studied in experimental systems now permit us to address and resolve these issues to some extent. The fact that spontaneous mutations (among which are point mutations and DNA deletions generally restricted to the gene) originate through a number of different mechanisms and that the latter are intimately related to the DNA organization of the genes, are now well-documented. Further, spontaneous mutations include those that cause diseases through loss of function as well as gain of function of genes. In contrast, most radiation-induced mutations studied in experimental systems (although identified through the phenotypes of the marker genes) are predominantly multigene deletions which cause loss of function; the recoverability of an induced deletion in a livebirth seems dependent on whether the gene and the genomic region in which it is located can tolerate heterozygosity for the deletion and yet be compatible with viability. In retrospect, the successful mutation test systems (such as the mouse specific locus test) used in radiation studies have involved genes which are non-essential for survival and are also located in genomic regions, likewise non-essential for survival. In contrast, most of the human genes at which induced mutations have been looked for, do not seem to have these attributes. The inference therefore is that the failure to find induced germline mutations in humans is not due to the resistance of human genes to induced mutations but due to the structural and functional constraints associated with their recoverability in livebirths. Since the risk of inducible genetic diseases in humans is estimated using rates of "recovered" mutations in mice, there is a need to introduce appropriate correction factors to bridge the gap between these rates and the rates at which mutations causing diseases are potentially recoverable in humans. Since the whole genome is the "target" for radiation-induced genetic damage, the failure to find increases in the frequencies of specific single-gene diseases of societal concern does not imply that there are no genetic risks of radiation exposures: the problem lies in delineating the phenotypes of recoverable genetic damage that are recognizable in livebirths. Data from studies of naturally-occurring microdeletion syndromes in humans and those from mouse radiation studies are instructive in this regard. They (i) support the view that growth retardation, mental retardation and multisystem developmental abnormalities are likely to be among the quantitatively more important adverse effects of radiation-induced genetic damage than mutations in a few selected genes and (ii) underscore the need to expand the focus in risk estimation from known genetic diseases (as has been the case thus far) to include these induced adverse developmental effects although most of these are not formally classified as "genetic diseases". (ABSTRACT TRUNCATED)     

The Prevalence of Chromosomal Deletions Relating to Developmental Delay and/or Intellectual Disability in Human Euploid Blastocysts

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34–149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.Key words: aging, genomic, screen, lifespan, yeast, C. elegans, pH, chronological, replicative     

Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue

Large-scale deletions of mitochondrial DNA (mtDNA) have been associated with aging and disease in post-mitotic tissues. These post-mitotic tissues, including skeletal muscle, heart and brain, are heavily dependent on intact functional mitochondria. The cochlear tissues are known to contain an abundance of mitochondria. This observation stimulated a search for mtDNA deletions in the cochlea and its elements using a sensitive nested PCR methodology and long range PCR to explain the functional deficits observed in age-related hearing loss. The presence of the so-called “common” deletion (CD) was detected in cochlear tissue from two individuals with age-related hearing loss, 73 and 78 years of age. Three additional deletions, that to our knowledge have not been previously reported, were also identified in these two individuals, including a 5354 bp deletion flanked with a 3 bp repeat, a 9682 bp deletion flanked by a 10 bp repeat and a 5142 bp deletion without a flanking repeat. The 9682 and 5142 bp deletions were also detected in an individual 39 years of age with normal hearing, however, these two deletions were not detected in a normal hearing individual 9 years of age. In contrast, the 5354 bp deletion was detected in all four of the individuals studied. To localize the deletions within the cochlea, the cochlear elements were removed by laser capture microdissection (LCM) and the mtDNA from these tissues was studied. The 5142 and 5354 bp deletions were detected in the organ of corti, spiral ligament, and ganglion cells, but not in the stria vascularis. These findings correlate with the reduction in the number of spiral ganglion cells and outer hair cells, and the normal stria vascularis volume observed in this individual. All four of these deletions involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. These observations suggest that multiple mtDNA deletions may contribute to a deficit in mitochondrial function in the cochlea and result in hearing loss if a level of physiological significance is reached.     

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 to c, 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 to c, 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 the msd and hsdr-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 approximately 1.7 Mb of wild-type DNA surrounding the c, D7Was12, and Emv-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 defining hsdr-1 and msd.     

Isolation and characterization of mouse hepatocyte lines carrying a lethal albino deletion.

Mice homozygous for chromosomal deletions at or around the albino locus on chromosome 7 express reduced levels of a group of liver genes, including tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), and generally die perinatally. Sequences within the deleted region are thought to encode a regulatory factor(s) that affects expression of these genes in trans. To facilitate study of the putative factors, we immortalized hepatocytes derived from newborn cch wild-type and c14CoS deletion homozygous mice as well as cch/c14CoS heterozygous mice using a SV40 temperature-sensitive A255 mutant virus. Three c14CoS deletion homozygous hepatocyte lines were characterized and compared with the homozygous wild-type and heterozygous lines. The SV40 tsA255 mutant-transformed hepatocyte lines were temperature-sensitive for maintenance of transformation and expressed many liver-specific genes. In agreement with in vivo studies, hepatocyte lines derived from mice homozygous for the deletion expressed reduced mRNA levels of a number of liver genes including TAT, PEPCK, X1, X2, and X7 in comparison with heterozygous and wild-type cell lines. Similar mRNA levels of transferrin and albumin, genes whose expression is unaffected by the mutation in vivo, were observed in all cell lines. The expression of two genes, X5 and metallothionein, reported to be reduced in newborn mutant mice, did not differ appreciably among cell lines. TAT and PEPCK have been shown to respond poorly to glucocorticoids and cAMP in newborn mutant mice. Interestingly, all affected liver genes tested were responsive to glucocorticoids and dibutyryl cAMP in deletion homozygous cell lines as well as in wild-type and heterozygote-derived cell lines. This may suggest that effects of the deletion on expression of liver-specific genes do not cause loss of responsiveness to glucocorticoids and cAMP. These immortalized hepatocyte lines, which express most, if not all, liver-specific genes, should provide a useful means for further investigation of the effects of the albino lethal deletion.     

Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster

Loss of function mutations in genes of the achaete-scute complex (ASC) or in the gene vnd of D. melanogaster result in neural hypoplasia. Two types of defects contribute to the development of the neural hypoplasic phenotype: a lower than normal proportion of neuroblasts delaminate from the neuroectoderm, and there is abundant cell death in the neural primordium during later stages. In addition, we found that increasing the copy number of ASC wild-type alleles leads to effects opposite to those caused by their deletion. All of these results indicate that the function of these genes is required for the commitment of neuroectodermal cells as neuroblasts and that the loss of these genetic functions causes the cells either to take on an epidermal fate or to die.     

Molecular analysis of aniridia patients for deletions involving the Wilms' tumor gene

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.