Loss of heterozygosity at the dilute-short ear (Myo5a-Bmp5) region of the mouse: mitotic recombination or double non-disjunction?

The occurrence of homozygous-viable dilute-short ear (Myo5a-Bmp5) double mutants in mouse specific locus mutation experiments has generally been assumed to be the result of double non-disjunction such that the mutant inherits two copies of chromosome 9 carrying the recessive alleles from the test-stock. A homozygous viable Myo5a-Bmp5 double mutant was recovered recently in our laboratory. We were able to genetically analyse both the Myo5a-Bmp5 region and proximal and distal markers in the original mutant as well as in offspring of the original mutant. Our results indicate the mutational event to be due to mitotic recombination and not double non-disjunction.     

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.     

Synthesis and secretion of kidney beta-galactosidase in mutant le/le mice.

We have found that the pigmentation mutant light ear in the mouse has a striking effect on lysosomes in the kidney. Male mice homozygous for the le mutant allele have a 4-fold elevated concentration of kidney beta-galactosidase (EC 3.2.1.23). The abnormal elevation of kidney beta-galactosidase is the net result of two processes. First, beta-galactosidase is elevated due to the defective urinary secretion of lysosomal enzymes which is a specific effect of the le mutation. Secondly, this effect is most evident in males and testosterone-treated females because of the induction of beta-galactosidase synthesis by testosterone, which occurs in +/+ as well as in mutant mice. The pale-ear mutation (ep) which mimics the pigmentation phenotype of le also has a similar effect on kidney lysosomes.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.     

Spontaneous and induced chromosomal damage and mutations in Bloom Syndrome mice

Bloom Syndrome (BS) is characterized by both cancer and genomic instability, including chromosomal aberrations, sister chromosome exchanges, and mutations. Since BS heterozygotes are much more frequent than homozygotes, the issue of the sensitivity of heterozygotes to cancer is an important one. This and many other questions concerning the effects of BLM (the gene responsible for the BS) are more easily studied in mice than in humans. To gain insight into genomic instability associated with loss of function of BLM, which codes for a DNA helicase, we compared frequencies of micronuclei, somatic mutations, and loss of heterozygosity (LOH) in Blmtm3Brd homozygous, heterozygous, and wild-type mice carrying a cII transgenic reporter gene. It should be noted that the Blmtm3Brd is inserted into the endogenous locus with a partial duplication of the gene, so some function of the locus may be retained. The cII reporter gene was introduced from the Big Blue mouse by crossing them with Blmtm3Brd mice. All measurements were made on F2 mice from this cross. The reticulocytes of Blmtm3Brd homozygous mice had more micronuclei than heterozygous or wild-type mice (4.5, 2.7, and 2.5 per thousand, respectively; P < 0.01) but heterozygotes did not differ significantly from wild-type. Unlike spontaneous chromosome damage, spontaneous mutant frequencies did not differ significantly among homozygous, heterozygous, and wild-type mice (3.2 x 10(-5), 3.1 x 10(-5), and 3.1 x 10(-5), respectively; P > 0.05). Mutation measurements were also made on mice that had been treated with ethyl-nitrosourea (ENU) because Bloom Syndrome cells are sensitive to ethylating agents. The ENU-induced mutation frequency in Blmtm3Brd homozygous, heterozygous, and wild mice were 54 x 10(-5), 35 x 10(-5), and 25 x 10(-5) mutants/plaques, respectively. ENU induced more mutations in Blmtm3Brd homozygous mice than in wild-type mice (P < 0.01), but not significantly more in heterozygous mice (P = 0.06). Spontaneous LOH did not differ significantly among the genotypes, but ENU treatment induced much more LOH in Blmtm3Brd homozygous mice, as measured by means of the Dlb-1 test of Vomiero-Highton and Heddle. Hence, these Blmtm3Brd mice resemble Bloom Syndrome except that they have normal frequencies of spontaneous mutation. The fact that these mice have elevated rates of both cancer and chromosomal aberrations (as shown by more micronuclei and LOH) but normal rates of spontaneous mutation, shows the greater importance of chromosomal events than mutations in the origin of their cancers.     

Construction and characterization of Chinese hamster cell EmtA EmtB double mutants.

Starting with hybrid cell lines between a Chinese hamster cell EmtA mutant and a Chinese hamster cell EmtB mutant, we have constructed cell lines that are homozygous for mutant alleles at both the emtA locus and the emtB locus, by using a two-step segregation protocol. The EmtA EmtB double mutants are approximately 10-fold more resistant to emetine inhibition than either of the parental mutants. Having both the EmtA mutation and the EmtB mutation expressed in the same cell also results in a level of resistance to cryptopleurine that is significantly higher than a simple additive effect of the two mutations alone. Analysis of ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis demonstrated that a parental hybrid and a first-step segregant, which has lost the wild-type emtA allele, synthesize both a normal and an altered form of ribosomal protein S14, whereas an EmtA EmtB double mutant synthesizes only the altered form of this ribosomal protein. This result confirms that the emtB locus is the structural gene for ribosomal protein S14. Our results also suggest that the products of the emtA and emtB loci interact directly, indicating that the emtA locus, like the emtB locus, encodes a component of the ribosome.     

Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice

A group of mouse aristaless-related genes has been implicated in functions in the development of the craniofacial skeleton. We have generated an Alx3 mutant allele in which the lacZ coding sequence is inserted in-frame in the Alx3 gene and the sequences encoding the conserved protein domains are deleted. Mice homozygous for this null allele are indistinguishable from wild-type mice. Compound mutants of Alx3 and Alx4, however, show severe craniofacial abnormalities that are absent in Alx4 single mutants. Alx3/Alx4 double mutant newborn mice have cleft nasal regions. Most facial bones and many other neural crest derived skull elements are malformed, truncated or even absent. The craniofacial defects in Alx3/Alx4 double mutant embryos become anatomically manifest around embryonic day 10.5, when the nasal processes appear to be abnormally positioned. This most probably leads to a failure of the medial nasal processes to fuse in the facial midline and subsequently to the split face phenotype. We detected a significant increase in apoptosis localised in the outgrowing frontonasal process in embryonic day 10.0 double mutant embryos, which we propose to be the underlying cause of the subsequent malformations.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

Derivative Alleles of the Arabidopsis Gibberellin-Insensitive (gai) Mutation Confer a Wild-Type Phenotype

The gai mutation of Arabidopsis confers a dwarf phenotype resembling that of mutants defective in gibberellin (GA) biosynthesis. However, gai mutant plants differ from GA biosynthesis mutants because they fail to respond to exogenous GAs and accumulate endogenous GA species to higher (rather than lower) levels than found in wild-type controls. The gai mutation, therefore, identifies a gene that modulates the response of plant cells to GA. We have mapped gai with respect to visible and restriction fragment length polymorphism (RFLP) markers from chromosome 1. To observe the phenotype exhibited by individuals potentially lacking wild-type (GAI) function, we have also isolated novel irradiation-induced derivative alleles of gai. When homozygous, these alleles confer a revertant phenotype that is indistinguishable from the wild type. gai is a semidominant mutation that exerts its effects either because it is a gain-of-function mutation or because it is a loss-of-function or reduced-function mutation. The genetic and physiological properties of the derivative alleles are considered with reference to these alternative modes of dominance of gai. Because these alleles are potential deletion or rearrangement mutations, together with the closely linked RFLP markers identified in the linkage mapping experiments, they provide useful resources for the isolation of the gai locus via a map-based cloning approach.     

Radiation and haematopoiesis in Harwell steel mice

Haematological information on steel (Sl) mice is limited largely to Sl/Sld mice of Bar Harbor stock (WC.B6 F1). Therefore, two Harwell alleles, SlgbH and Slcon, were investigated. In the steady state both heterozygotes were modestly anaemic, homozygous Slcon and compound Slcon/SlgbH more so. On perturbation by X-irradiation Slcon/SlgbH showed a decrease in median lethal dose (MLD)--6.5 Gy, Slcon/+ and Slcon/Slcon slightly less change (7.5 Gy) compared with +/+, 8 Gy. In recovery from sublethal doses single heterozygotes, double heterozygotes with Wv, and compounds showed no delay in restoration of the count of red blood corpuscles (RBC) such as that seen in typical W mice (e.g. Wv/+, W/Wv). Effects on Slcon/Slcon and Slcon/SlgbH differ from those reported for Sl/Sld in that they show normal growth of spleen colonies when used as lethally irradiated recipients of bone marrow, they support growth of implanted bone marrow to form radiation chimaeras. When Harwell steel mice are donors of bone marrow to lethally irradiated +/+ mice the chimaeras ultimately are not anaemic; when lethally irradiated Harwell steel mice are recipients of +/+ marrow they remain macrocytically anaemic. One deduces that, for normal development and production of normal RBC in the steady state, the erythron requires intrinsic factors determined by wild type alleles at the W locus and extrinsic factors determined by wild type alleles at the Sl locus. Mutant alleles at either locus may determine macrocytosis. Two mutant alleles at either locus are still more deleterious, often lethal. Whereas mutant W alleles may also influence the pluripotent haematopoietic stem cell (HSC) leading to reduced MLD on X-irradiation, a similarly reduced MLD for Sl mutants may represent an increased need for and consumption of products of the haematopoietic stem cells rather than truly increased radiosensitivity, since the Do for spleen colony-forming units is the same for Slcon/SlgbH as +/+ mice.     

Ctr1 and its role in body copper homeostasis

Members of the Cu transporter (Ctr) family have been reported to be part of the copper uptake machinery in several organisms. Recently it has been suggested that human Ctr1 (hCtr1) may act as a copper transporter in several tissues including the intestine. hCtr1 is a 190 amino acid protein and is predicted to have three transmembrane-spanning domains and exist in the plasma membrane as a homo-trimer. Ctr1-transfected cell lines exhibit saturable, pH-dependent Cu(I) uptake indicating a role in copper transport. Recent studies with Ctr1 knockout mice have highlighted an essential function in mammalian embryonic development since homozygous mutants die in utero. Heterozygotes are indistinguishable from wild-type littermates but have a severely reduced brain copper content, suggesting that Ctr1 is a key component of the copper uptake pathway in the brain. However, its role in other tissues remains elusive.     

Lysosomal Dysfunctions Associated with Mutations at Mouse Pigment Genes

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.     

Combinatorial control of meristem identity in maize inflorescences

The architecture of maize inflorescences, the male tassel and the female ear, is defined by a series of reiterative branching events. The inflorescence meristem initiates spikelet pair meristems. These in turn initiate spikelet meristems which finally produce the floret meristems. After initiating one meristem, the spikelet pair and spikelet meristem convert into spikelet and floret meristems, respectively. The phenotype of reversed germ orientation1 (rgo1) mutants is the production of an increased number of floret meristems by each spikelet meristem. The visible phenotypes include increased numbers of flowers in tassel and ear spikelets, disrupted rowing in the ear, fused kernels, and kernels with embryos facing the base of the ear, the opposite orientation observed in wild-type ears. rgo1 behaves as single recessive mutant. indeterminate spikelet1 (ids1) is an unlinked recessive mutant that has a similar phenotype to rgo1. Plants heterozygous for both rgo1 and ids1 exhibit nonallelic noncomplementation; these mutants fail to complement each other. Plants homozygous for both mutations have more severe phenotypes than either of the single mutants; the progression of meristem identities is retarded and sometimes even reversed. In addition, in rgo1; ids1 double mutants extra branching is observed in spikelet pair meristems, a meristem that is not affected by mutants of either gene individually. These data suggest a model for control of meristem identity and determinacy in which the progress through meristem identities is mediated by a dosage-sensitive pathway. This pathway is combinatorially controlled by at least two genes that have overlapping functions.     

A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.     

The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGF beta superfamily.

The mouse short ear gene is required for normal growth and patterning of skeletal structures, and for repair of bone fractures in adults. We have carried out an extensive chromosome walk in the chromosome region that surrounds this locus. Here we show that the short ear region contains the gene for a TGF beta-related protein called bone morphogenetic protein 5 (Bmp-5). This gene is deleted or rearranged in several independent mutations at the short ear locus. Mice homozygous for large deletions of the Bmp-5 coding region are viable and fertile. Mutations at the short ear locus provide an important new tool for defining the normal functions of BMPs in mammals. The specific skeletal defects seen in short-eared animals, which occur against a background of otherwise normal skeletal structures, suggest that particular aspects of skeletal morphology may be determined by individual members of a family of signaling factors that can induce the formation of cartilage and bone in vivo.