Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrP(C)

Creutzfeldt-Jakob disease (CJD) in Libyan Jews, linked to the E200K mutation in PRNP (E200KCJD), is the most prevalent of the inherited prion diseases. As other prion diseases, E200KCJD is characterized by the brain accumulation of PrP(Sc), a pathologic conformational isoform of a normal glycoprotein denominated PrP(C). To investigate whether the E200K mutation is enough to de novo confer PrP(Sc) properties to mutant PrP, as suggested by experiments in Chinese hamster ovary cells, we examined the biochemical behavior of E200KPrP in brains and fibroblasts from sporadic as well as homozygous and heterozygous E200KCJD patients, asymptomatic transgenic mice carrying the E200K mutation, as well as in normal and scrapie-infected mouse neuroblastoma cells expressing E200KPrP. E200KPrP was examined for protease sensitivity, solubility in detergents, releasibility by phosphoinositol phospholypase-C and localization in cholesterol enriched membrane microdomains (rafts). In all tissues except in brains of CJD patients and ScN2a cells, E200KPrP displayed properties similar to those of PrP(C). Our results indicate that the E200K mutation does not automatically convey the properties of PrP(Sc) to new PrP molecules. A conversion process occurs mainly in the prion disease affected brain, suggesting the presence of a tissue-specific or age-dependent factor, in accord with the late onset nature of inherited CJD.     

Congenital malformations in a common marmoset (Callithrix jacchus) similar to human 13-trisomy syndrome

This paper describes the finding of a number of malformations in a live-born common marmoset. The malformations included microphthalmia, ocular hypertelorism, cleft lip and palate and polydactyly. The findings are consistent with and strongly suggest chromosome trisomy similar to that which has been seen in man.     

Mapping the mouse craniofacial mutation first arch (Far) to chromosome 2

First arch (Far) is a semidominant mutation that causes severe craniofacial defects in mice. Here we report the results of linkage studies with the chromosome 2 markers nonagouti, pallid, and Ulnaless. Far is loosely linked to nonagouti (24-37 cM), more closely linked to pallid (13-28 cM), and closely linked to Ulnaless (2.3 +/- 1.5 cM). The embryological defect in Far mutants is confined to one segmentally-derived region of the head, the anterior first branchial arch. It may therefore be significant that, in mapping near Ulnaless, Far also maps in the vicinity of the Hox-4 gene cluster.     

ENU-induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.     

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.     

Two-hit model for sporadic congenital anomalies in mice with the disorganization mutation.

Congenital anomalies have complex etiologies involving both genetic and nongenetic components. Many are sporadic, without obvious evidence for heritability. An important model for these anomalies is a mutation in laboratory mice that is called "disorganization" (Ds), which functions as a variable autosomal dominant and leads to a wide variety of congenital anomalies involving many developmental processes and systems. Variable expressivity, asymmetrical manifestations, and low penetrance suggest that somatic events determine the location and nature of these anomalies. A statistical analysis suggests that occurrence of anomalies in mice with the Ds mutation follows a Poisson distribution. These results suggest that congenital anomalies in mice with the Ds mutation occur independently of each other. We propose that Ds causes a heritable predisposition to congenital anomalies and that Ds and appropriate somatic events combine to compromise normal development. We also propose that some sporadic, nonheritable congenital anomalies involve somatic mutations at Ds-like loci. Ds may therefore serve not only as a model for developmental anomalies in cell fate and pattern formation but also for complex developmental traits showing variable expressivity, low penetrance, and sporadic occurrence in mice and humans.     

Effects of the lethal yellow (Ay) mutation in mouse aggregation chimeras

The Ay allele is a recessive lethal mutation at the mouse agouti locus, which results in embryonic death around the time of implantation. In the heterozygous state, Ay produces several dominant pleiotropic effects, including an increase in weight gain and body length, a susceptibility to hepatic, pulmonary and mammary tumors, and a suppression of the agouti phenotype, which results in a yellow coat color. To investigate the cellular action of Ay with regard to its effects upon embryonic viability and adult-onset obesity, we generated a series of aggregation chimeras using embryos that differ in their agouti locus genotype. Embryos derived from Ay/a x Ay/a matings were aggregated with those derived from A/A x A/A matings, and genotypic identification of the resultant chimeras was accomplished using a molecular probe at the Emv-15 locus that distinguishes among the three different alleles, Ay, A, and a. Among 50 chimeras, 25 analyzed as liveborns and 25 as 9.5 day embryos, 29 were a/a in equilibrium A/A and 21 were Ay/a in equilibrium A/A. The absence of Ay/Ay in equilibrium A/A chimeras demonstrates that Ay/Ay cells cannot be rescued in a chimeric environment, and the relative deficiency of Ay/a in equilibrium A/A chimeras suggests that, under certain conditions, Ay heterozygosity may partially affect cell viability or proliferation. In the 25 liveborn chimeras, Ay/a in equilibrium A/A animals became obese as adults and a/a in equilibrium A/A animals did not. There was no correlation between genotypic proportions and rate of weight gain, which shows that, with regard to its effects on weight gain, Ay heterozygosity is cell non-autonomous.     

A cell line derived from sphingomyelinosis mouse shows alterations in intracellular cholesterol metabolism similar to those in type C Niemann-Pick disease.

Cell lines derived from the sphingomyelinosis (gene symbol, spm) mouse were established from homozygous (spm/spm) and heterozygous (spm/+) embryos according to a rigid 3T3 transfer schedule. The SPM-3T3 cells derived from a homozygous embryo showed extensive accumulation of intracellular cholesterol, attenuated esterification of exogenously added cholesterol and increased de novo cholesterol synthesis, when compared to SPMH-3T3 cells derived from a heterozygous embryo. The phenotypic abnormalities were very similar to those observed in fibroblasts from patients with Niemann-Pick disease type C (NP-C), in which a defect in the intracellular transport of unesterified cholesterol is suggested. The genetic defect in SPM-3T3 cells should be closely related to that in NP-C. The SPM-3T3 cell line is useful for biochemical and genetic studies on the regulation of intracellular cholesterol metabolism.     

The inner mitochondrial membrane contains ion-conducting channels similar to those found in bacteria

Patch-clamp experiments were performed on rat liver mitochondria inner membranes. Application of voltage gradients of either polarity revealed the presence of several different conductances, ranging up to 1.3 nS in symmetrical 150 mM KCl. Evidence is presented that at least those higher than 0.3 nS are substates of the highest conductance channel. Increasing matrix-side-positive (unphysiological) transmembrane voltage gradients favored the switch of the 1.3 nS channel to operation in lower conductance states. The size of these conductances, the presence of substates and the channel behavior are strongly reminiscent on one hand of the observations on the membrane of protoplasts from the gram-positive bacterium Streptococcus faecalis, [Zoratti, M. and Petronilli, V. (1988) FEBS Lett. 240, 105-109], and on the other of some properties of previously described channels of mitochondrial origin.     

Vertebrate golgi complexes have beads in a similar position to those found in arthropods

Insects and other arthropods have bead-like structures in Golgi complexes from all cell types. They are arranged in rings at the base of transition vesicles located near the smooth surface of the rough endoplasmic reticulum making the forming face of the Golgi complex and are only seen easily after staining in bismuth salts. Procedures used to demonstrate the beads in arthropod Golgi complexes do not selectively stain any structures where they would be expected to occur in several mouse and tadpole tissues. However, a faint pattern similar to the arthropod GC beads can be made out in the large GCs concerned in the formation of acrosomes during mouse spermatogenesis. Uranyl staining shows particles of about the same size and spacing as the beads of arthropod GCs. We conclude that vertebrate GCs may have beads that differ from arthropods in their staining properties.     

The intramembranous domains of lipophilin in phosphatidylcholine vesicles are similar to those in the myelin membrane

A membrane-permeable photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (125I-TID) has been used to label lipophilin in normal human myelin and after incorporation of purified lipophilin into phosphatidylcholine (PC) vesicles. The labelled protein was isolated and the specific activities for lipophilin from myelin and from PC vesicles was found to be 1.2 X 10(11) and 1.5 X 10(11) cpm/mol, respectively. The chromatographic profiles of tryptic peptides were similar in both cases and the specific activities of the C-terminal intramembranous fragments (residues 205-268) the same. We concluded that the organization of lipophilin in PC vesicles was similar to its organization in myelin and that the PC-vesicle system represents a good system in which to study the orientation and interaction of lipophilin with lipids.     

Evidence for allelism of the recessive insertional mutation add and the dominant mouse mutation extra-toes (Xt)

A recessive mutant caused by insertional mutagenesis in transgenic mice has been detected in which the anterior part of the forelimb is disorganized. The morphology of the thumb is always altered and sometimes the adjacent finger has an extra phalanx. This phenotype suggests that a body plan gene is affected. We have named the mutation add (anterior digit-pattern deformity). Using the cloned DNA from the flanking region of the integrated transgene, add has been mapped close to the centromere of chromosome 13. This position links add to a genetically mapped locus called extra-toes (Xt). The phenotype of the double-mutant add/Xt as well as the molecular analysis suggest that add and Xt are allelic.     

Human immunodeficiency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA alkylating agent, nitrogen mustard.

The product of the human immunodeficiency virus type 1 (HIV-1) vpr gene induces cell cycle arrest in the G2 phase of the cell cycle and is characterized by an accumulation of the hyperphosphorylated form of cdc2 kinase. This phenotype is similar to the effect of DNA-damaging agents, which can also cause cells to arrest at G2. We previously reported that Vpr mimicked some of the effects of a DNA alkylating agent known as nitrogen mustard (HN2). Here we extend these earlier observations by further comparing the activation state of cdc2 kinase, the kinetics of G2 arrest, and the ability to reverse the arrest with chemical compounds known as methylxanthines. Infection of cells synchronized in the G1 phase of the cell cycle with a pseudotyped HIV-1 resulted in arrest at G2 within 12 h postinfection, before the first mitosis. Similar to that induced by HN2, Vpr-induced arrest led to a decrease in cdc2 kinase activity. Vpr-mediated G2 arrest was alleviated by methylxanthines at concentrations similar to those needed to reverse the G2 arrest induced by HN2, and cells proceeded apparently normally through at least one complete cell cycle. These results are consistent with the hypothesis that Vpr induces G2 arrest through pathways that are similar to those utilized by DNA-damaging agents.