Normal Gene Product Research Articles

The abnormal isoform of the prion protein accumulates in late‐endosome‐like organelles in scrapie‐infected mouse brain

The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

Platelet phenotyping in carriers for Glanzmann's thrombasthenia: a simple screening test for assessment of the molecular defect

Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by the abnormality of aggregation due to a platelet glycoprotein (GP) IIb-IIIa deficiency or a dysfunctional complex. Molecular abnormalities have been localized on the gene coding for GP IIb or IIIa. The aim of our work was an attempt to obtain indirectly information on the putative localization of the molecular defect in patients with GT type I or II by the determination of the HPA-1 (GP IIIa) and HPA-3 (GP IIb) alloantigenic systems' expression in GT carriers. If GT results from a defective GP IIb gene, a GT carrier would appear homozygous for HPA-3 by serology, because the normal gene product will be expressed while the abnormal GP IIb gene product will not be present. Conversely, if the abnormality is in the GP IIIa gene, such an individual would appear homozygous for HPA-1. Therefore, the heterozygous status for HPA would result from the normal expression of the two genes for the considered alloantigenic system. Among the four families studied with informative members, our presumptions were strengthened by the preliminary genetic results in one family showing a mutation in the GP IIb gene. Thus, serology could be a simple screening test for the possible defective gene responsible for GT allowing molecular investigation focusing only on GP IIb or IIIa gene.

Widespread expression of Huntington's disease gene (IT15) protein product

Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Localization of the Tp53 gene on Syrian hamster chromosome 9 by fluorescence in situ hybridization.

Mutations of the Tp53 tumor suppressor gene have been detected in 50% of all human cancers. The normal Tp53 gene product acts as a negative regulator of cell division or is associated with abnormal growth in certain differentiated cell types. The Tp53 gene was mapped by FISH to Syrian hamster chromosome 9 bands qb2.2-->2.3 using a genomic DNA probe with a high degree of sequence identity to the human Tp53 gene.

Molecular Genetics of Alzheimer Disease: Identification of Genes and Gene Mutations

Alzheimer disease (AD), the major form of senile dementia, is rapidly becoming a major health problem in developed countries since as populations live longer the number of elderly people continues to grow. Simultaneously, the number of AD cases is increasing since AD is mainly a late-onset disorder and since no effective therapies are available. The primary causes of AD have not yet been elucidated. However, several researchers in different disciplines are actively engaged in AD research with one ultimate goal, i.e. trying to understand the disease pathology. Knowledge of the biochemical basis of the disease may eventually lead to a therapy which either prevents AD or allows AD patients to be treated. One such discipline is molecular genetics. The aim of molecular genetic studies is to identify genes that are either responsible for or contribute to the expression of AD. Once such a gene is identified, studies of its normal and/or abnormal gene product may help us understand the primary disease mechanisms and lead to more effective therapy. Currently, 3 genetic loci have been identified that predispose to AD: AD1 on chromosome 21, AD2 on chromosome 19, and AD3 on chromosome 14. This review aims at summarizing the progresses that have made in identifying genes and gene mutations that predispose to AD.

Co-infusion of normal bone marrow partially corrects the gld T-cell defect. Evidence for an intrinsic and extrinsic role for Fas ligand.

Ipr and gld mice develop systemic autoimmune diseases with nearly indistinguishable manifestations, including the accumulation of massive numbers of CD4-CD8- T lymphocytes. In vivo chimera experiments have shown that the Ipr mutation is functionally expressed in both T and B cells. When lethally irradiated Ipr mice were given a combination of normal and Ipr bone marrow, only Ipr-derived B cells produced autoantibodies and only Ipr-derived T cells hyperproliferated. In contrast, analogous experiments with gld mice showed that the co-infusion of normal bone marrow greatly reduced autoantibody production. These results indicated that the gld B cell defect was extrinsic to those cells producing autoantibodies, in agreement with the recent molecular data showing that the normal gene products of the Ipr and gld loci form an interacting receptor-ligand pair. In the present study, we have extended our functional studies with gld mice using T cell-marked congenic donors. Lymphadenopathy was reduced three- to fourfold in gld mice given a combination of congenic normal and gld bone marrow compared with mice given gld bone marrow alone, and the absolute number of CD4-CD8- T cells was reduced by a factor of 7. Surprisingly, the residual CD4-CD8- T cells present in the mixed chimeras were derived entirely from the gld donor marrow. This suggests that the gld mutation results in both an extrinsic and intrinsic defect in T cells.

Two forms of the mb-1 gene transcript in cattle.

We have isolated two forms of bovine mb-1 cDNA clones from a cDNA library of a bovine leukemia virus-infected B-lymphoblastoid cell line. The nucleotide sequence analysis of these cDNA clones indicated that the shorter form of cDNA had a 119-bp deletion that precisely corresponded to the exon III domain of the human and murine mb-1 genes. This deletion would result in a frame shift, producing a premature translation termination leading to the lack of the entire transmembrane and cytoplasmic domains of the normal mb-1 molecule. The existence of two species of mRNA transcripts corresponding to the isolated cDNA clones were confirmed in all of the bovine leukemia cell lines examined by PCR with the use of the primers flanking the deletion site. Furthermore, the shorter mb-1 transcript was induced in normal PBMC by stimulation with mitogens or culture supernatants of bovine leukemia virus-infected cell lines. These findings indicate the presence of the truncated mb-1 gene product which may have some function different from that of the normal mb-1 gene product.

Somatic cell genetic analysis of two classes of CHO cell mutants expressing opposite phenotypes in sterol-dependent regulation of cholesterol metabolism.

Two different classes of hamster cell mutants (25RA cells and M1 cells) express opposite phenotypes in sterol dependent regulation. In 25RA cells, sterols added in growth medium fail to cause down-regulation of sterol synthesis rate and low density lipoprotein (LDL) receptor activity, while in M1 cells, removal of lipids from growth medium fail to cause up-regulation of sterol synthesis rate and LDL receptor activity. Cell hybridization analysis showed that the 25RA phenotype is semidominant, while the M1 phenotype is recessive. Using 25RA as the parental cells, we isolated eight independent mutant cells (DM cells) and showed that all of them belong to the same genetic complementation group as the M1 mutant, indicating that the normal (unmutated) M1 gene product(s) is required to express the 25RA phenotype. We next performed gene transfer experiments using hamster cell genomic DNAs containing the functional human M1 gene as the donor, and the double mutant cell DM7 as the recipient. The resultant transfectant cells express the 25RA cell phenotype (instead of the wild-type cell phenotype). This result, along with the results obtained from cell hybridization analysis, shows that the 25RA and M1 cell phenotypes are caused by mutations at two different genes.

P53 Expression in Epithelial Ovarian Neoplasms: Relationship to Clinical and Pathological Parameters, Ki-67 Expression and Flow Cytometry

Epithelial ovarian tumors of varying malignancy as well as normal ovaries were examined for their expression of p53 with the monoclonal antibody PAb1801. Immunohistochemical detection of p53 protein is possible when the gene has been mutated, but not when the normal gene product alone is present. Our results indicate that this tumor suppressor gene may be involved in tumorigenesis, as its expression was detected in both borderline and malignant tumors while normal ovaries and benign ovarian tumors were unstained with the p53 antibody. The presence of p53 was also related to dissemination of disease, residual tumor bulk, and poor differentiation as well as the presence of the proliferation variable Ki-67, another negative prognostic variable. No significant relation could be detected to S-phase fraction or DNA ploidy. Furthermore, the presence of p53 in malignant epithelial ovarian tumors was related to significantly decreased patient survival, with only 36% alive compared to 70% in the p53-negative group (P = 0.002). In the subgroup of patients with residual tumor burden after surgery, those with p53-positive tumors had a significantly (P = 0.05) decreased survival compared to those with p53-negative neoplasms, which further supports an independent role in ovarian cancer malignancy.

High incidence of uterine inversion in mast cell-deficient osteopetrotic mutant mice of mi/mi genotype.

Mutations at either W or mi (microphthalmia) loci in the mouse can lead to a deficiency in melanocytes and mast cells. In addition, W mutants can be anemic and sterile, whereas mi mice are osteopetrotic because of a monocyte/macrophage/osteoclast defect. Since c-kit receptor tyrosine kinase is the gene product of the W locus and mi mutation has been suggested to affect the transduction of signals from the c-kit and c-fms receptors, we here examined the effect of mi mutation on fertility. Testes and ovaries from mi/mi mice were histologically normal, and the pattern of c-kit protein expression was not different from that of +/+ mice. Homozygous mutant crosses (mi/mi x mi/mi) were fertile, but inversion of the uterus occurred in 86% of the deliveries. In some cases, the placenta was found still attached to the inverted uterus after delivery. Decidual cells were present and expressed c-kit protein normally in the placenta of mi/mi mice. The inversion was also observed in mi/mi females mated to +/+ males. No uterine inversion was noted when +/mi females were crossed with mi/mi or +/mi males, suggesting that the genotype of the mother but not of the father or fetus is important for the pathogenesis. The numbers and body weights of mi/mi newborns were less than those of +/mi littermates. Mast cells were absent, but c-kit-positive cells were present, in the uterine muscle layers of pregnant mi/mi mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Obesity, diabetes, and neoplasia in yellow A vy /‐ mice: ectopic expression of the agouti gene

The viable yellow A(vy) mutation results in a mottled yellow mouse that is obese, slightly larger than its nonyellow sibs, and more susceptible to tumor formation in those tissues sensitized by the strain genome. The mutation exhibits variable expressivity resulting in a continuum of coat color phenotypes, from clear yellow to pseudoagouti. The mouse agouti protein is a paracrine signaling molecule that induces hair follicle melanocytes to switch from the synthesis of black pigment to yellow pigment. Molecular cloning studies indicate that the obesity and growth effects of the A(vy) mutation result from ectopic expression of the normal agouti gene product. This review seeks to summarize the current state of knowledge regarding the obesity, stimulation of somatic growth, and enhancement of tumor formation caused by the A(vy) mutation, and to interpret these pleiotropic effects in terms of the normal function of the agouti protein.

Molecular Feminization of Mouse Seminal Vesicle by Prenatal Exposure to Diethylstilbestrol: Altered Expression of Messenger RNA

Exposure to estrogens during critical stages of development has been reported to cause irreversible changes in estrogen target tissues such as the reproductive tract. In fact, recent studies using mice describe prenatal estrogen exposure resulting in the expression of the major estrogen-inducible uterine secretory protein, lactoferrin (LF), by the seminal vesicles of the male offspring. Thus, we have studied the role of estrogens in abnormal and normal gene expression in the developing male reproductive tract using LF and seminal vesicle secretory protein IV (SVS IV), an androgen-regulated murine seminal vesicle secretory protein, as markers. Lactoferrin and SVS IV protein and mRNA expression were studied in histological samples by using the techniques of in situ hybridization (ISH) and immunohistochemistry (IHC). Seminal vesicle secretory protein IV was expressed in all (100%) epithelial cells of the control seminal vesicle, but this protein was decreased by castration. However, LF expression was undetectable by ISH or IHC in control seminal vesicle epithelium. Lactoferrin was inducible in 2% of the seminal vesicle epithelial cells from adult castrated mice treated with estradiol 17ß (E2; 20µg./kg./day for 3 days), indicating that a small percentage of the seminal vesicle cells could be induced to secrete LF after modification of the endocrine environment. Prenatal DES treatment (100µg./kg. maternal body weight on days 9 through 16 of gestation) resulted in the male offspring exhibiting constitutive expression of LF in 5% of the seminal vesicle epithelial cells, while expression of the androgen-regulated protein SVS IV was slightly decreased. The maximal contrast between LF and SVS IV expression was observed in prenatally DES-treated mice that were subsequently castrated as adults and further treated with E2; LF was detected in 40% of the epithelial cells in these mice. Double immunostaining techniques revealed that epithelial cells which were making LF had ceased production of SVS IV. Since a large percentage of the epithelial cells in the intact prenatal DES exposed male was capable of expressing the normal gene product, SVS IV, it was concluded that DES treatment during prenatal development appears to imprint or induce estrogenic sensitivity in the adult seminal vesicle, causing increased production of LF. The results suggest that this altered protein response may be an example of atypical gene expression in male reproductive tract tissues following hormonal manipulation early in development.

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.

Cloning and expression of the gene for the melanoma-associated ME20 antigen.

Human melanoma cells, but not tumor cells of other histological origin, express a unique membrane-associated glycoprotein, designated ME20-M, and secrete a soluble glycoprotein, designated ME20-S, defined by monoclonal antibody ME20. Here we report the isolation and characterization of a cDNA clone that when transfected into COS cells directs the expression of ME20-M and ME20-S. This cDNA contains an open reading frame which encodes a 661-amino-acid-long precursor that contains a 23-amino-acid signal peptide and a 26-amino-acid transmembrane domain, separated by a hydrophilic region containing 5 potential Asn-linked and 14 predicted Pro-associated, Thr-linked glycosylation sites. The transmembrane domain is followed by a carboxy-terminal 45-amino-acid putative intracellular domain rich in Ser residues. Analysis of ME20-M by amino acid sequencing identified the proteolytic processing site. Signal peptide cleavage occurs at the Thr-24-Lys-25 peptide bond of the precursor and results in the 637-amino-acid ME20-M with a calculated molecular weight of 67,782. ME20-M is derived from a single 3.3- to 3.4-kb mRNA transcript that is expressed at varying levels in melanoma cell lines, correlating with immunofluorescence determination of protein expression. The amino acid sequence of the ME20 antigen deduced from the cDNA differs from the human neonatal melanocyte-specific Pmel 17 gene product by a single amino acid substitution and deletion of 7 amino acid residues, and it is 80% homologous with the bovine retinal pigment RPE1 cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of the Tunicate locus on maize floral development

AbstractThe co‐dominant Tunicate (Tu) mutation in maize causes nonreproductive structures in both the male and female inflorescences to be enlarged. This mutation also affects sex determination, permitting the development of pistils in the normally staminate tassel. In order to characterize the role of the normal tu gene product, we have analysed genetic interaction between Tu and other mutations that perturb specific stages of floral development. Synergistic interactions observed suggested that the tu product functions in at least three stages of floral development–determination of spikelet primordia, differentiation of non‐reproductive organs and pistil abortion in the tassel. © 1994 Wiley‐Liss, Inc.

Defective Glia in the Drosophila Brain Degeneration Mutant drop-dead

To understand better the cellular basis of late-onset neuronal degeneration, we have examined the brain of the drop-dead mutant of Drosophila. This mutant carries an X-chromosomal recessive mutation that causes severe behavioral defects and brain degeneration, manifested a few days after emergence of the adult. Analysis of genetically mosaic flies has indicated that the focus of the drop-dead mutant phenotype is in the brain and that the gene product is non-cell autonomous. We examined the adult drop-dead mutant brain prior to onset of symptoms and found that many glial cells have stunted processes, whereas neuronal morphology is essentially normal. Adult mutant glial cells resemble immature glia found at an earlier stage of normal brain development. These observations suggest that defective glia in the drop-dead brain may disrupt adult nervous system function, contributing to progressive brain degeneration and death. The normal drop-dead gene product may prevent brain degeneration by providing a necessary glial function.

The role of arginine in the conserved polar loop of the c-subunit of the Escherichia coli H +-ATPase

The Arg-41 of the c-subunit of the F oF 1-ATPase of Escherichia coli has been changed by site-directed mutagensis to Glu, Leu or Lys. None of the mutants can carry out oxidative phosphorylation. No detectable F 1-ATPase activity is found on the membranes and only small amounts in the cytoplasm. Two-dimensional gel electrophoresis shows that in all three mutant strains the assembly of the F oF 1-ATPase has been affected. When plasmids carrying the mutant genes, together with other normal unc genes, were inserted into strains each carrying a mutation in one of the unc genes other than uncE their capacity for oxidative phosphorylation was reduced or eliminated, the effect being most pronounced with the uncG and uncC mutants and least pronounced with the plasmid giving the Arg → Lys substitution. The c-subunit is a multimer in the ATP synthase complex and it appears that a mixture of normal and mutant gene products allows assembly of a functional complex.

Amino-terminal parathyroid hormone-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells.

PTH-related protein (PTHrP), originally identified through its causative role in human humoral hypercalcemia of malignancy, is now known to be a normal gene product expressed in a wide variety of neuroendocrine, epithelial, and mesoderm-derived tissues. PTHrP gene expression has recently been demonstrated in fetal and adult, benign and malignant, as well as human and rodent pancreatic islets. As in other tissues, the role of PTHrP expression in the normal islet is only beginning to be explored. In the current report, PTHrP expression in the normal rat pancreatic islet was confirmed using an affinity-purified antiserum directed against the N-terminal, biologically active region of the molecule. The effects of PTHrP on the islet were then explored using rat insulinoma (RIN m5F) cells. Synthetic PTHrP-(1-36) bound specifically, but with low affinity (Kd, approximately 10(-7) M) to RIN cell membranes. PTHrP-(1-36) failed to stimulate cAMP production in RIN cells, although RIN cells displayed a normal adenylate cyclase response to glucagon-like peptide-1-(7-36). In contrast, PTHrP-(1-36) induced a rapid dose-dependent rise in intracellular calcium in RIN cells in doses as low as 10(-12)-10(-10) M. These findings 1) confirm that PTHrP is expressed by islet cells, 2) demonstrate that the effects of PTHrP on the pancreatic islet are mediated, as in keratinocytes and lymphocytes, by a receptor related to but distinct from the PTH receptor, and 3) suggest that PTHrP functions in the islet as an autocrine or paracrine factor. Further studies are required to determine the physiological consequences of PTHrP expression by the pancreatic islet.

Retrovirally Transduced Antisense Sequences Stably Suppress P210BCR-ABL Expression and Inhibit the Proliferation of BCR/ABL-Containing Cell Lines

AbstractThere is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.

Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines

There is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.