Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development

The Candida albicans genes, CST20 and HST7, were cloned by their ability to suppress the mating defects of Saccharomyces cerevisiae mutants in the ste20 and ste7 genes, which code for elements of the mating mitogen-activated protein (MAP) kinase pathway. These Candida genes are both structural and functional homologs of the cognate Saccharomyces genes. The pattern of suppression in Saccharomyces is related to their presumptive position in the MAP kinase cascade. Null alleles of these genes were constructed in Candida. The Candida homozygous null mutants are defective in hyphal formation on some media, but are still induced to form hyphae by serum, showing that serum induction of hyphae is independent of the MAP kinase cascade. The Candida heterozygotes CST20/cst20 and HST7/hst7 are also defective in hyphal formation. This lack of dominance of the wild-type allele suggests that gene dosage is important in Candida.

Mutation rates among RNA viruses

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μg ≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.

Emergence of FY*Anull in a Plasmodium vivax-endemic region of Papua New Guinea

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is α+-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that α+-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that α+-thalassemia may facilitate so-called “benign” Plasmodium vivax infection to act later in life as a “natural vaccine” against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of α+-thalassemia ≥0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a−b−]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*Anull) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*Anull (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47–8.91). Emergence of FY*Anull in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise α+-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.

The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.

Molecular characterization of four induced alleles at the Ednrb locus

The piebald locus on mouse chromosome 14 encodes the endothelin-B receptor (EDNRB), a G protein-coupled, seven-transmembrane domain protein, which is required for neural crest-derived melanocyte and enteric neuron development. A spontaneous null allele of Ednrb results in homozygous mice that are predominantly white and die as juveniles from megacolon. To identify the important domains for EDNRB function, four recessive juvenile lethal alleles created by either radiation or chemical mutagens (Ednrb27Pub, Ednrb17FrS, Ednrb1Chlc, and Ednrb3Chlo) were examined at the molecular level. Ednrb27Pub mice harbor a mutation at a critical proline residue in the fifth transmembrane domain of the EDNRB protein. A gross genomic alteration within the Ednrb gene in Ednrb3Chlo results in the production of aberrantly sized transcripts and no authentic Ednrb mRNA. Ednrb17FrS mice exhibited a decreased level of Ednrb mRNA, supporting previous observations that the degree of spotting in piebald mice is dependent on the amount of EDNRB expressed. Finally, no molecular defect was detected in Ednrb1Chlc mice, which produce normal levels of Ednrb mRNA in adult brain, suggesting that the mutation affects important regulatory elements that mediate the expression of the gene during development.

Btk dosage determines sensitivity to B cell antigen receptor cross-linking

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl–Ficoll response, anti-IgM-induced proliferation, B1 cell development, and serum IgM and IgG3 levels remained significantly impaired in these animals. B cells from Btk −/− transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl–Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.

Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to β2-microglobulin (β2M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with β2M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/β2M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.

Identification of morc (microrchidia), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse

The microrchidia, or morc, autosomal recessive mutation results in the arrest of spermatogenesis early in prophase I of meiosis. The morc mutation arose spontaneously during the development of a mouse strain transgenic for a tyrosinase cDNA construct. Morc −/− males are infertile and have grossly reduced testicular mass, whereas −/− females are normal, indicating that the Morc gene acts specifically during male gametogenesis. Immunofluorescence to synaptonemal complex antigens demonstrated that −/− male germ cells enter meiosis but fail to progress beyond zygotene or leptotene stage. An apoptosis assay revealed massive numbers of cells undergoing apoptosis in testes of −/− mice. No other abnormal phenotype was observed in mutant animals, with the exception of eye pigmentation caused by transgene expression in the retina. Spermatogenesis is normal in +/− males, despite significant transgene expression in germ cells. Genomic analysis of −/− animals indicates the presence of a deletion adjacent to the transgene. Identification of the gene inactivated by the transgene insertion may define a novel biochemical pathway involved in mammalian germ cell development and meiosis.

Altered regulation of platelet-derived growth factor receptor-α gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-α gene (PDGFRα). Mice heterozygous for the PDGFRα-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRα and Pax1. Using the human PDGFRα promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRα gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln → His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRα-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRα expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein–DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRα gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRα expression may be causally related to NTDs.

The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice

Neuropeptide Y (NPY) and the endogenous melanocortin receptor antagonist, agouti gene-related protein (AGRP), coexist in the arcuate nucleus, and both exert orexigenic effects. The present study aimed primarily at determining the brain distribution of AGRP. AGRP mRNA-expressing cells were limited to the arcuate nucleus, representing a major subpopulation (95%) of the NPY neurons, which also was confirmed with immunohistochemistry. AGRP-immunoreactive (-ir) terminals all contained NPY and were observed in many brain regions extending from the rostral telencephalon to the pons, including the parabrachial nucleus. NPY-positive, AGRP-negative terminals were observed in many areas. AGRP-ir terminals were reduced dramatically in all brain regions of mice treated neonatally with monosodium glutamate as well as of mice homozygous for the anorexia mutation. Terminals immunoreactive for the melanocortin peptide α-melanocyte-stimulating hormone formed a population separate from, but parallel to, the AGRP-ir terminals. Our results show that arcuate NPY neurons, identified by the presence of AGRP, project more extensively in the brain than previously known and indicate that the feeding regulatory actions of NPY may extend beyond the hypothalamus.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in Cpefat mice associated with a carboxypeptidase E mutation

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia–diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.