Transforming capacities of avian erythroblastosis virus mutants deleted in the erbA or erbB oncogenes

Mutants of avian erythroblastosis virus (AEV) were constructed by deleting large nucleotide segments in each of the viral oncogenes termed v-erbA and v-erbB. Mutants in erbA (erbA ^?B⁺) retained the ability to transform fibroblasts in vitro, and these cells exhibited most of the transformation characteristics that typify wild-type AEV-transformed fibroblasts. In addition, the mutants induced small erythroid colonies upon infection of bone marrow cells in culture. Chickens inoculated with erbA ^?B⁺ virus or with erbA ^?B⁺-transformed cells developed sarcomas or atypical erythroid leukemias. The erythroid cells transformed in vivo or in vitro by the erbA ^?B⁺ viruses appeared not to be as tightly blocked in differentiation as wild-type transformed cells. In contrast, fibroblasts infected with the erbA ⁺B^? mutant resembled normal cells in all transformation parameters tested, and no bone marrow cell transformation was observed with the mutant. The results indicate that the main transforming properties of AEV are encoded in erbB and that its effects are enhanced by erbA.     

Identification and characterization of the avian erythroblastosis virus erbB gene product as a membrane glycoprotein

Avian erythroblastosis virus causes erythroid leukemia and sarcomas in chickens. The viral oncogene responsible for these diseases, erb, is divided into two regions known as erbA and erbB, and recent evidence suggests that it is the erbB gene that is responsible for the transforming activity. From rats bearing avian erythroblastosis virus-induced sarcomas, we have obtained antisera which are specific for the erb gene products. Using such antisera, we have been able to characterize the erbB gene product as a 68,000 molecular weight protein. Pulse-chase and cell-free in vitro translation experiments show that the initial product is a 62,500 dalton protein which is initially modified to a 66,000 dalton protein, and then further modified to a 68,000 dalton form. These modifications could be shown to be associated with glycosylation and phosphorylation. Cell fractionation experiments revealed that the 66,000 and 68,000 dalton proteins were located in cell membrane fractions, and immunofluorescence results showed the erbB gene product to be expressed on the cell surface.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 2. Genetic polymorphism of lymphocyte cytosol 64K polypeptide

Summary We describe a genetic polymorphism of human lymphocyte cytosol major polypeptide with mol. wt. 64,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is predominent in peripheral blood lymphocytes, B-lymphoblastoid cells, T-lymphoblastoid cells, lymph node, and spleen. The polypeptide has not been detected in HeLa cells, fibroblasts, erythrocytes, serum, and cerebrum. Traces of the polypeptide exist in liver, kidney, and skeletal muscle. It is proposed that the polypeptide and its locus be temporarily designated lymphocyte cytosol 64K polypeptide (LC64K polypeptide) andLC64P, respectively. In a Japanese population, the gene frequencies ofLC64P ¹ andLC64P ² were 0.936 and 0.064, respectively. The data suggest thatLC64P is a new locus, product of which shows genetic polymorphism and is associated with the function and/or the structure of lymphocytes.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Identification of a human Ia antigen that is different from HLA-DR and DC antigens

A monoclonal antibody, MHM4, identified a cell surface antigen present on B cells and not resting T cells. It precipitated two polypeptide chains of 34 000 and 28 000 daltons from B lymphoblastoid cells. This antibody bound to all B-cell lines tested, except those homozygous for HLA-DR7. Saturation binding assays and Scatchard plots of MHM4 binding to cells that did not carry HLA-DR7 indicated that this antibody bound less than the total surface Ia antigen. When the antibody was competed with eight other HLA-D-specific antibodies, the epitope recognized was shown to be distinct. Two-dimensional gel analysis revealed that a simple pattern of spots was precipitated, unlike the complex patterns obtained with other HLA-D-specific antibodies. The α and β spots were different from those precipitated by HLA-DR- or DC-specific antibodies. It is argued that the MHM4 antigen is the product of an HLA locus that is distinct from HLA-DR and DC. Its relationship with HLA-SI3 is discussed.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 4. Genetic polymorphism of cytosol 100k polypeptide

Summary We describe a genetic polymorphism of a human cellular polypeptide with mol. wt. 100,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, and 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is one of the abundant polypeptides of B-lymphoblastoid cells, T-lymphoblastoid cells, fibroblasts, and HeLa cells. The data indicate that the cytosol polypeptide with mol.wt. 100,000 shows a genetic polymorphism determined by aew autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 100k polypeptide (C100k polypeptide) and C100P, respectively. In a Japanese population, the gene frequencies of C100P ¹ and C100P ² were 0.907 and 0.093, respectively.     

Transfer of dye among salivary gland cells is not affected by genetic variations of the period clock gene in Drosophila melanogaster

Larval salivary gland cells of Drosophila melanogaster were injected with a fluorescent dye to assess strengths of intercellular communication among such cells, as influenced by mutations at the period locus and by a per transgene. This clock gene had been reported to increase the extent of dye transfer when mutated such that it shortens the period of biological rhythms; the previous study also showed that a per-null mutant decreased the strength of transfer among salivary gland cells. Our re-examination of this feature of larval physiology—in observer-blind analyses, using the per ^s and per ^o mutants as well as two per-normal strains—revealed no appreciable differences in extents of dye transfer among these four genotypes. These results are discussed in the context of emerging findings which suggest that the period gene's product controls pacemaker functioning as an intracellularly acting entity.     

Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

Genetic dissection of functional domains within the avian erythroblastosis virus v-erbA oncogene.

The avian erythroblastosis virus v-erbA locus potentiates the oncogenic transformation of erythroid and fibroblast cells and is derived from a host cell gene encoding a thyroid hormone receptor. We report here the use of site-directed mutagenesis to identify and characterize functional domains within the v-erbA protein. Genetic lesions introduced into a putative hinge region or at the extreme C-terminus of the v-erbA coding domain had no significant effect on the biological activity of this polypeptide. In contrast, mutations introduced within the cysteine-lysine-arginine-rich center of the v-erbA coding region, a DNA-binding domain in the thyroid and steroid hormone receptors, abolished or severely compromised the ability of the viral protein to function. Our results suggest that the mechanism of action of the v-erbA protein in establishing the neoplastic phenotype is closely related to its ability to interact with DNA, presumably thereby altering expression of host target genes by either mimicking or interfering with the action of the normal c-erbA gene product.     

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [³H]glucosamine or [³H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M_r 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M_r 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [³H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.     

The distribution of DR5, MT2, and MB3 specificities on human Ia subsets

Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component α and β chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.     

Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.     

A Novel Serine/Threonine Protein Kinase Homologue of Pseudomonas aeruginosa Is Specifically Inducible within the Host Infection Site and Is Required for Full Virulence in Neutropenic Mice

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.     

Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of ≤10⁵ cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10⁶ cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.     

Appraising freeze-drying for storage of bacteria and their ready access in a rapid toxicity assessment assay

Direct toxicity assessment (DTA) techniques seek to measure the impact of toxic chemicals on biological materials resident in the environment. This study features the use of freeze-dried bacterial cells in combination with a rapid DTA analyser, SciTOX^?. The effects of three factors—cryoprotectant type, bacterial strain, and storage temperature—were tested in order to validate the shelf life of the freeze-dried cells. Three freeze-dried Gram-negative bacterial strains, Acinetobacter calcoaceticus, Escherichia coli and Pseudomonas putida, were tested by using the bacteria in the SciTox^? DTA assay and recording their responses to two standard toxicants: 2,4-dicholorophenol and 3,5-dichlorophenol. Each freeze-dried strain of bacteria was prepared in two forms—either pre-treatment with polyethylene glycol (PEG) or with sucrose/Tween 80—prior to storing at either 4 or ?20 °C for three different storage periods (1, 2 or 3 months). While the sucrose/Tween 80 pre-treated freeze-dried cells exhibited better cell viability, we concluded that PEG was a more suitable cryoprotectant for the bacteria used in the DTA assay because of EC₅₀ parity with fresh cell and zero-time freeze-dried cell assays. The results showed that freeze-dried cells, with appropriate materials and conditions, can give reproducible DTA results for up to 3 months. The availability of a biocomponent that can be activated by simple rehydration makes the deployment of this technology much easier for an end user.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.