A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins.

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.     

SMA-3 smad has specific and critical functions in DBL-1/SMA-6 TGFbeta-related signaling

A TGFbeta signal transduction cascade controls body size and male tail morphogenesis in the nematode Caenorhabditis elegans. We have analyzed the function of the sma-3 Smad gene, one of three Smad genes that function in this pathway. Null mutations in sma-3 are at least as severe as null mutations in the ligand and type I receptor genes, dbl-1 and sma-6, indicating that the other Smads do not function in the absence of SMA-3. Furthermore, null mutations in sma-3 do not cause defects in egg laying or in regulation of the developmentally arrested dauer larva stage, indicating no overlapping function with another C. elegans TGFbeta signaling pathway. The sma-3 gene is widely expressed at all developmental stages in hermaphrodites and males. The molecular lesions associated with eight sma-3 alleles of varying severity have been determined. The missense mutations cluster in two previously identified regions important for Smad function.     

Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii

Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.     

Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation

Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogues, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. We have analysed the genetic interactions between Cdx1 and Cdx2 in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. We conclude that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminding of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice.     

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.     

Three New Classes of Mutations in the Caenorhabditis Elegans Muscle Gene Sup-9

We are studying five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. A distinctive ``rubber-ban'''' muscle-defective phenotype was previously shown to result from rare altered-function mutations in either of two of these genes, unc-93 and sup-10. Null mutations in sup-9, sup-10, sup-18 or unc-93 act as essentially recessive suppressors of these rubber-band mutations. In this work, we identify three new classes of sup-9 alleles: altered-function rubber-band, partial loss-of-function and dominant-suppressor. The existence of rubber-band mutations in sup-9, sup-10 and unc-93 and the suppression of these mutations by null mutations in any of these three genes suggest that these proteins are required at the same step in muscle contraction. Moreover, allele-specific interactions shown by the partial loss-of-function mutations indicate that the products of these interacting genes may physically contact each other in a multiple subunit protein complex. Finally, the phenotypes of double rubber-band mutant combinations suggest that the rubber-band mutations affect a neurogenic rather than a myogenic input in excitation-contraction coupling in muscle.     

The promiscuous MLL gene links chromosomal translocations to cellular differentiation and tumour tropism

MLL is a promiscuous gene involved in a diversity of chromosomal fusions in haematological malignancies, usually resulting from chromosomal translocations. MLL-associated chromosomal rearrangements usually occur in tumours of specific haematological lineages, suggesting a crucial role for the MLL fusion partner in determining disease phenotype (or tumour tropism). The MLL gene is homologous to Drosophila trithorax, and is likewise involved in embryo pattern formation. Common themes linking several of the MLL partners include a possible involvement in embryo patterning via Hox gene regulation and chromatin remodelling. These findings reinforce the link between developmental regulation and chromosomal translocations, and indicate the role of chromosomal translocation in activating genes capable of determining tumour phenotype in leukaemias and sarcomas.     

Folate modulates Hox gene-controlled skeletal phenotypes

Hox genes are well-known regulators of pattern formation and cell differentiation in the developing vertebrate skeleton. Although skeletal variations are not uncommon in humans few mutations in human HOX genes have been described. If such mutations are compatible with life, there may be physiological modifiers for the manifestation of Hox gene-controlled phenotypes, masking underlying mutations. Here we present evidence that the essential nutrient folate modulates genetically induced skeletal defects in Hoxd4 transgenic mice. We also show that chondrocytes require folate for growth and differentiation and that they express folate transport genes, providing evidence for a direct effect of folate on skeletal cells. To our knowledge, this is the first report of nutritional influence on Hox gene-controlled phenotypes, and implicates gene-environment interactions as important modifiers of Hox gene function. Taken together, our results demonstrate a beneficial effect of folate on skeletal development that may also be relevant to disorders and variations of the human skeleton.     

Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins.

Microtubules in yeast are essential components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. The relative importance in these processes of the two divergent alpha-tubulin genes of the budding yeast Saccharomyces cerevisiae, TUB1 and TUB3, was examined through the construction of null mutations and by increasing their copy number on chromosomes and on plasmids. Experiments with null alleles of TUB3 showed that TUB3 was not essential for mitosis, meiosis, or mating. Null alleles of TUB3, however, did cause several phenotypes, including hypersensitivity to the antimicrotubule drug benomyl and poor spore viability. On the other hand, the TUB1 gene was essential for growth of normal haploid cells. Even in diploids heterozygous for a TUB1 null allele, several dominant phenotypes were evident, including slow growth and poor sporulation. This functional difference between the two genes is apparently due to different levels of expression, because extra copies of either gene could suppress the defects caused by a null mutation in the other. We conclude that in spite of the 10% divergence between the products of the two genes, there is no essential qualitative functional difference between them.     

Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants

Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.