Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene.

Oculocutaneous albinism (OCA) is the most common autosomal recessive disorder among southern African Blacks. There are three forms that account for almost all OCA types in this region. Tyrosinase-positive OCA (OCA2), which is the most common, affects approximately 1/3,900 newborns and has a carrier frequency of approximately 1/33. It is caused by mutations in the P gene on chromosome 15. Brown OCA (BOCA) and rufous OCA (ROCA) account for the majority of the remaining phenotypes. The prevalence of BOCA is unknown, but for ROCA it is approximately 1/8,500. Linkage analysis performed on nine ROCA families showed that ROCA was linked to an intragenic marker at the TYRP1 locus (maximum LOD score = 3.80 at straight theta=.00). Mutation analysis of 19 unrelated ROCA individuals revealed a nonsense mutation at codon 166 (S166X) in 17 (45%) of 38 ROCA chromosomes, and a second mutation (368delA) was found in an additional 19 (50%) of 38 chromosomes; mutations were not identified in the remaining 2 ROCA chromosomes. In one family, two siblings with a phenotypically unclassified form of albinism were found to be compound heterozygotes for mutations (S166X/368delA) at the TYRP1 locus and were heterozygous for a common 2.7-kb deletion in the P gene. These findings have highlighted the influence of genetic background on phenotype, in which the genotype at one locus can be influenced by the genotype at a second locus, leading to a modified phenotype. ROCA, which in southern African Blacks is caused by mutations in the TYRP1 gene, therefore should be referred to as "OCA3," since this is the third locus that has been shown to cause an OCA phenotype in humans.     

Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs

Down-regulation of the Drosophila ribosomal protein S21 gene (rpS21) causes a dominant weak Minute phenotype and recessively produces massive hyperplasia of the hematopoietic organs and moderate overgrowth of the imaginal discs during larval development. Here, we show that the S21 protein (RpS21) is bound to native 40S ribosomal subunits in a salt-labile association and is absent from polysomes, indicating that it acts as a translation initiation factor rather than as a core ribosomal protein. RpS21 can interact strongly with P40, a ribosomal peripheral protein encoded by the stubarista (sta) gene. Genetic studies reveal that P40 underexpression drastically enhances imaginal disc overgrowth in rpS21-deficient larvae, whereas viable combinations between rpS21 and sta affect the morphology of bristles, antennae, and aristae. These data demonstrate a strong interaction between components of the translation machinery and showed that their underexpression impairs the control of cell proliferation in both hematopoietic organs and imaginal discs.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

The open-eyelid mutation, lidgap-Gates, is an eight-exon deletion in the mouse Map3k1 gene

The BALB/cGa mouse strain and its descendants, now called the SELH/Bc strain, have produced two waves of high frequency of spontaneous heritable mutations. One of these, the recessive lidgap-Gates (lg(Ga)) mutation, causes the same open-eyelids-at-birth phenotype as the gene knockout mutations of Map3k1 and co-maps to distal Chr 13. The lg(Ga) mutation is demonstrated to be a 27.5-kb deletion of exons 2-9 in the Map3k1 gene, the first spontaneous mutant allele described at this locus. The lg(Ga) mutation is consistent with a pattern suggesting that the waves of mutation in BALB/cGa and its descendants tend to be large deletions or ETn insertions, whose elevated rate of occurrence is due to an unknown mechanism.     

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 X 10(-5) in post-spermatogonial stages and 3.7 X 10(-5) in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 X 10(-6) in the post-spermatogonial stages and 1.1 X 10(-6) in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 X 10(-5) and for dominant cataract mutations 0.9 X 10(-6). In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 X 10(-5). In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 518 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.     

Genetics of an Unstable White Mutant in Drosophila Simulans: Reversion, Suppression and Somatic Instability

A spontaneous white mutation, white-milky (wmky) of Drosophila simulans is moderately unstable and is associated with a 16-kb long DNA insertion into the white gene. wmky, which is an unstable mutation found in D. simulans, has been genetically analyzed. Among nine spontaneous, partial reversions toward wild type, five were white locus mutations. They are phenotypically different from each other and three show eye color sexual dimorphism indicating a failure of the dosage compensation mechanism. Two w locus mutations whose eye color appeared identical between males and females were also isolated. Of the other back-mutants, three were associated with a recessive suppressor of wmky and one was a semidominant suppressor. These suppressor loci are located on the third chromosome at map positions about 90 and 120, respectively. The suppressor mutations demonstrate specific effects on w locus mutations derived from wmky which lack in the gene dosage compensation. Somatic instability was detected at the frequency of 5.6 X 10(-4) in wmky flies heterozygous for the recessive suppressor and the frequency was increased 10-fold when the suppressor mutation was placed in a different genetic background.     

Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.     

Chromosomal superkiller mutants of Saccharomyces cerevisiae.

Yeast strains carrying a 1.5 X 10(6)-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e., increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated ski1 through ski4 (for superkiller), ski3 and ski4 are located on chromosome XIV, and ski1 is on chromosome VII. A ski1 mutation results in a decreased rate of cell growth. The kex1 and kex2 mutations are epistatic to each ski mutation.     

Elimination of Introns at the Drosophila Suppressor-of-Forked Locus by P-Element-Mediated Gene Conversion Shows That an RNA Lacking a Stop Codon Is Dispensable

The suppressor of forked [su(f)] locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci. It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3' end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor. Three su(f) mRNAs are produced by alternative polyadenylation. The 2.6- and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon. Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns. This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function. Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.     

The Genetic Analysis of Achiasmate Segregation in Drosophila Melanogaster. III. the Wild-Type Product of the Axs Gene Is Required for the Meiotic Segregation of Achiasmate Homologs

The regular segregation of achiasmate chromosomes in Drosophila melanogaster females is ensured by two distinct segregational systems. The segregation of achiasmate homologs is assured by the maintenance of heterochromatic pairing; while the segregation of heterologous chromosomes is ensured by a separate mechanism that may not require physical association. Axs(D) (Aberrant X segregation) is a dominant mutation that specifically impairs the segregation of achiasmate homologs; heterologous achiasmate segregations are not affected. As a result, achiasmate homologs frequently participate in heterologous segregations at meiosis I. We report the isolation of two intragenic revertants of the Axs(D) mutation (Axs(r2) and Axs(r3)) that exhibit a recessive meiotic phenotype identical to that observed in Axs(D)/Axs(D) females. A third revertant (Axs(r1)) exhibits no meiotic phenotype as a homozygote, but a meiotic defect is observed in Axs(r1)/Axs(r2) females. Therefore mutations at the Axs(D) locus define a gene necessary and specific for homologous achiasmate segregation during meiosis. We also characterize the interactions of mutations at the Axs locus with two other meiotic mutations (ald and ncd). Finally, we propose a model in which Axs(+) is required for the normal separation of paired achiasmate homologs. In the absence of Axs(+) function, the homologs are often unable to separate from each other and behave as a single segregational unit that is free to segregate from heterologous chromosomes.     

Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.     

Detection of new genes participating in the trans-regulation of locus yellow of MDG-4 in Drosophila melanogaster

The aim of the present work was to obtain mutations in the genes involved in regulation of the yellow locus and mdg4. For this purpose, we searched for mutations changing phenotypic expression of the y(2) mutation induced by mdg4 insertion into the regulatory region of the yellow locus. Mutations have been obtained in the earlier described system of prolonged genome instability, sometimes combined with P-M hybrid dysgenesis. The mutation mod(mdg4) in a novel gene, modifier of mdg4, was detected which either enhanced or suppressed a phenotypic expression of several mutations induced by mdg4 insertion. We suggest that mod(mdg4) controls the expression of mdg4. In addition, the mutations in five loci located on the X chromosome have been found which enhanced the mutation phenotype of several y alleles induced by insertions of different mobile elements in the regulatory region of the latter. Possibly, the protein products of these genes designated as enhancers of yellow-1, 2, 3, 4 and 5 are directly or indirectly involved in control of the yellow locus expression.     

Chediak-Higashi syndrome mutation and genetic testing in Japanese black cattle (Wagyu)

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disorder that affects several species including mice, humans, and cattle. Evidence based on clinical characteristics and somatic cell genetics suggests that mutations in a common gene cause CHS in the three species. The CHS locus on human chromosome 1 and mouse chromosome 13 encodes a lysosomal trafficking regulator formerly known as LYST, now known as CHS1, and is defective in CHS patients and beige mice, respectively. We have mapped the CHS locus to the proximal region of bovine chromosome 28 by linkage analysis using microsatellite markers previously mapped to this chromosome. Furthermore, we have identified a missense A:T-->G:C mutation that results in replacement of a histidine with an arginine residue at codon 2015 of the CHS1 gene. This mutation is the most likely cause of CHS in Wagyu cattle. In addition, we describe quick, inexpensive, PCR based tests that will permit elimination of the CHS mutation from Wagyu breeding herds.     

Molecular and Genetic Organization of the Suppressor of Sable and Minute(1)1b Region in Drosophila Melanogaster

Recessive mutations at the suppressor of sable [su(s)] locus in Drosophila melanogaster result in suppression of second site mutations caused by insertions of the mobile element 412. In order to determine whether su(s) mutations might have other phenotypes, a saturation mapping of the su(s) region was carried out. The screen yielded 76 mutations that comprise ten genetic complementation groups ordered distal to proximal as follows: l(1)1Bh, l(1)1Bi, M(1)1B, su(s), l(1)1Bk, l(1)1Ca, mul, tw, l(1)lDa and brc. Twenty-three of the mutations are su(s) alleles, and all are suppressors of the 412-insertion-caused v1 allele. Although the screen could have detected su(s) mutations causing sex-specific dominant lethality or sterility as well as all types of recessive lethality or sterility, the only other phenotype observed was male sterility that is enhanced by cold temperature. This type of sterility is exhibited only by alleles induced by base-substitution-causing mutagens. Genetic functions of the poly(A+) messages transcribed from the su(s) microregion were identified by the reintroduction of cloned sequences into embryos by P element transformation. su(s) function has been attributed to a 5-kb message. The segment of DNA encoding only this 5-kb message rescues both the suppression and cold-sensitive male sterility phenotypes of su(s). Minute (1) 1B has been provisionally identified as encoding a 3.5-kb message; lethal (1)1Bi encodes a 1-kb message; and lethal (1)1Bk encodes a 4-kb message. The possible functions of su(s) and M(1)1B are discussed.     

Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly, deterioration of motor functions with ataxia, and spasticity, eventuating in mental decline. The brain appears swollen on magnetic resonance imaging, with diffuse white-matter abnormalities and the invariable presence of subcortical cysts. MLC was recently localized on chromosome 22q(tel). We have narrowed down the critical region by linkage analysis of 11 informative families with MLC to a region of approximately 250 kb, containing four known genes. One family with two patients who were siblings did not display linkage between the MLC phenotype and any of the analyzed microsatellite markers on chromosome 22q(tel), suggesting genetic heterogeneity and the existence of at least a second MLC locus. The maximum two-point LOD score for the 11 families was 6.6 at recombination fraction .02. Twelve different mutations in seven informative and six uninformative families were found in one of the candidate genes, KIAA0027, which we renamed "MLC1." The gene encodes a putative membrane protein with eight predicted transmembrane domains. The patients of one family were compound heterozygotes for mutations that both introduced stop codons. The mutations further included frameshifts, splice-acceptor mutations, a putative splice-donor mutation, and amino acid substitutions of residues in predicted transmembrane domains. These data provide strong evidence that mutations of MLC1 cause the disease.