Double Mutant Cells Research Articles

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.

The Oncogenic Cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development

The LIM domain protein rbtn2 is associated with T cell acute leukemias. We demonstrate that rbtn2 is a nuclear protein expressed in the erythroid lineage in vivo, and using homologous recombination, we show that it is essential for erythroid development in mice. The homozygous rbtn2 null mutation leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5. Moreover, in vitro differentiation of yolk sac tissue from homozygous mutant mice and sequentially targeted double-mutant ES cells demonstrates a block to erythroid development. This shows a pivotal role for a LIM domain protein in lineage specification during mammalian development and suggests that RBTN2 and GATA-1 are critical at similar stages of erythroid differentiation.

A Cell Biosensor Specific for Formaldehyde Based on pH-Sensitive Transistors Coupled to Methylotrophic Yeast Cells with Genetically Adjusted Metabolism

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded, The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor′s response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

Development and maintenance of a simple reflex circuit in small-patch mosaics of Drosophila: effects of altered neuronal function and developmental arrest.

A combined genetic, anatomical, and behavioral approach has been undertaken to study the developmental and functional plasticity of identified bristle mechanosensory neurons in Drosophila. A stereotyped grooming reflex in decapitated flies enabled simple but reliable assessments of the functional output of individual bristle sensory cells to correlate with their axonal projections and terminal arbors revealed by the cobalt backfill technique. Construction of small-patch mosaics that contain only a single mutant bristle allowed functional perturbation of individual neurons within an otherwise normal environment. Mutations that affect nerve excitability and membrane recycling have been used to examine their effects on neuronal pathfinding, arborization, and the initiation and maintenance of functional connections. Previous studies (Burg and Wu, 1986, J. Neurosci. 6:2968-2976; 1989, Dev. Biol. 131:505-514) have demonstrated that para(ts)nap(ts) double-mutant sensory neurons, in which action potentials are unconditionally blocked by defects in sodium currents, and eag Sh double-mutant sensory cells, in which membrane excitability is increased by alterations in potassium currents, can establish and maintain central projections that are indistinguishable from their functionally normal counterparts. Mutations of the shi(ts) gene cause a temperature-sensitive, reversible block of the membrane recycling process, resulting in arrest of neuronal growth in culture (Kim and Wu, 1987, J. Neurosci. 7:3245-3255) and depletion of synaptic vesicles that leads to transmission blockade at established synapses (Ikeda, Ozawa, and Hagiwara, 1976, Nature 259:489-491; Koenig and Ikeda, 1983, J. Neurobiol. 14:411-419; 1989, J. Neurosci. 9:3844-3860). Prolonged heat treatments (up to 16% of total development time) of small-patch shi(ts) mosaics at different pupal stages did not prevent the establishment of central projections characteristic of the various sensory cell types. However, none of the shi(ts) sensory neurons heat-pulsed during the initial or the final 16% of pupal development were able to initiate the reflex behavior, although a proportion of those treated in other periods apparently established functional contacts with appropriate targets to support the characteristic cleaning reflex. The possibility exists that the membrane recycling process blocked in shi(ts) cells provides a crucial mechanism for cell-cell interactions taking place during initial differentiation and final synaptic stabilization, and possibly competition, in the developing sensory neuron. Heat treatments of adult shi(ts) mosaics blocked the reflex initiated by the mutant (but not the surrounding normal) bristles, as expected from the effect of synaptic block.(ABSTRACT TRUNCATED AT 400 WORDS)

Mitochondrial DNA loss by yeast reentry-mutant cells conditionally unable to proliferate from stationary phase.

Double-mutant cells of the budding yeast Saccharomyces cerevisiae harboring the gcs1-1 and sed1-1 mutations are conditionally defective (cold-sensitive) only for reentry into the mitotic cycle from stationary phase. If already proliferating at the permissive temperature (29 degrees C), these reentry-mutant cells continue to proliferate when transferred to the restrictive temperature of 14 degrees C, but under these conditions reentry-mutant cells lose mitochondrial DNA (mtDNA). In addition, upon exhaustion of the nutrient supply at 14 degrees C, these reentry-mutant cells entered stationary phase at a decreased cell concentration and did not accumulate the reserve carbohydrates trehalose and glycogen. Both of these deficiencies were due to the loss of mtDNA, as shown by the responses of wild-type cells also lacking mtDNA. Mitochondrial status did not affect other aspects of the reentry-mutant phenotype. Although mitochondrial activity and the accumulation of carbohydrate reserves are typical features of cells in stationary phase, the reentry-mutant phenotype reveals that neither entry into nor exit from stationary phase need involve mitochondrial function.

Sensitization of Escherichia coli cells to oxidative stress by deletion of the rpoH gene, which encodes the heat shock sigma factor

A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.

Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation.

Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Regulation of cell division in Escherichia coli K-12: probable interactions among proteins FtsQ, FtsA, and FtsZ.

In Escherichia coli, the FtsQ, FtsA, and FtsZ proteins are believed to play essential roles in the regulation of cell division. Of the three proteins, FtsZ has received the most attention, particularly because of its interactions with SfiA. Double mutants which carry mutations located in the ftsQ, ftsA, or ftsZ gene in combination with the lon-1 mutation were constructed. In the presence of the lon-1 mutation, which is known to stabilize SfiA, the ftsQ1 mutant cells were not capable of forming colonies on a rich agar medium, whereas mutant cells harboring either one of the mutations grew well on this medium. Examination of lon-1 fts double-mutant cells for sensitivity to UV light revealed that those carrying the ftsA10 allele were resistant. It was also observed that in the presence of a multicopy plasmid containing a wild-type ftsZ gene, the ftsQ1 mutant filamented markedly following a nutritional shift-up and that the division rate of ftsZ84 mutant cells was slightly reduced when they harbored a wild-type ftsQ-containing plasmid. The possibility that the Fts proteins are interacting with one another and forming a molecular complex is discussed.

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species

A double-mutant cell line, which was unable to grow in a medium with NO 3 (-) as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179-183) supplemented with 50 mM chlorate and 229 μM 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO 3 (-) medium and then by transfer to the NO 3 (-) medium supplemented with 229 μM 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO 3 (-) medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.

Differentiation and central projections of peripheral sensory cells with action-potential block in Drosophila mosaics

The ultrastructural differentiation and central projection of identified bristle mechanosensory neurons were examined in Drosophila mutants lacking action potentials. Two mutations, parats1 and napts, are known to block axonal conduction in centrally located neurons at high temperatures. Their effects on epithelial sensory cells, which are derived from imaginal disks during pupation, have not been determined. Furthermore, the parats1 napts double-mutant flies are lethal at all temperatures; thus the synergistic effect of these mutations on neurons has not yet been studied. It is possible to examine the above questions in genetic mosaics. By monitoring a reflex response involving identified bristle sensory cells, we found that the 2 mutations exert similar effects on these epithelial sensory cells as seen in central neurons. This also indicates that the action potential mechanisms in both epithelial sensory cells and central neurons are under similar genetic control. The parats1 napts double-mutant sensory cells in mosaics are nonfunctional at all temperatures, providing an opportunity to examine, at the single cell level, the development of neurons with activity block. Ultrastructural specializations typical of epithelial sensory cells were found in the double-mutant cells. Cobalt backfilling experiments showed that central projections of these nonfunctional sensory cells were not altered, as compared with the active contralateral sensory cells. Therefore, blockage of the action potential mechanism in individual sensory cells has no effect on their pathfinding and arborization.

Fusion of Plasmacytoma and Host Cells In Vivo: Selection of Proliferating and Nonproliferating Cultures<xref ref-type="fn" rid="FN2">2</xref>

The double-mutant cell line 4T00.1 is derived from a plasmacytoma of a BALB/c mouse and is resistant to 6-thioguanine and ouabain. These cells were inoculated into (BALB/c X C57BL)F1 mice by different routes--sc, ip, intrasplenically, and intrathymically. The degree of tumorigenicity and pattern of tumor development were site-dependent. Intrasplenic inoculation of 10(3)-10(4) 4T00.1 cells resulted in development of large omental tumors accompanied by marked ascites. Tenfold to a thousandfold more 4T00.1 cells were required to obtain tumors by other routes. From all solid tumors and ascites and from various organs of tumor-bearing mice, which were explanted into culture in double selective medium containing hypoxanthine, aminopterin, and thymidine plus ouabain (in which only hybrids between 4T00.1 and normal cells can survive), proliferating and nonproliferating cultures were obtained. Of the 14 proliferating cultures, 9 proved to be hybrids by chromosome and H-2 isoantigen analyses and were tumorigenic when 10(6) cells were inoculated sc into syngeneic F1 mice.

Epigenetic modulation of the mouse HGPRT gene in interspecific reconstituted cells and cybrids

basic problems in research on eukaryotic cells. The enucleation technique with cytochalasin B (CB) makes it possible to isolate viable reconstituted cells or cytoplasmic hybrids (cybrids) by fusing karyoplasts (or intact cells) to cytoplasts. Chloramphenicol-resistant (CAP’) cytoplasts from the double-mutant cells which are sensitive to a medium containing hypoxanthine, aminopterin and thymidine (HAT medium) and resist chloramphenicol (CAP) can be fused with HAT-resistant, CAP-sensitive cells (or karyoplasts). Such fusions have led to the isolation of CAP’-reconstituted cells or cybrids essentially free of contaminating parental cells that were eliminated by selection with both HAT and CAP [l]. We have thus isolated many CAP’-interspecific reconstituted cells and cybrids by fusing karyoplasts (or intact cells) of HAT-resistant, CAP-sensitive mouse melanoma Bl6 cells with cytoplasts of HAT-sensitive, CAP-resistant rat L,jTG l CAP’ cells. Expression of HGPRT activity and CAP-resistance are essential for the survival of cells in this selection medium. This paper describes the epigenetic modulation of the mouse HGPRT gene in cells reconstituted by fusing rat cytoplasts with mouse karyoplasts. By gel electrophoretic analysis, we found 2 components of HGPRT, a main and a minor component, in several mouse cell lines. Epigenetic modulation of the mouse HGPRT gene occurred in some of the reconstituted cells or cybrids isolated and grown as clones. In these clones the main band of HGPRT migrated much faster than the HGPRT of the parental Bl6 cells.

CHO cells selected for phytohemagglutinin and con A resistance are defective in both early and late stages of protein glycosylation.

The lipid-linked and asparagine-linked oligosaccharides of two lectin-resistant and one parental Chinese hamster ovary (CHO) cell line have been compared by glycosidase digestion and gel filtration analysis of radiolabeled glycopeptides and oligosaccharides. The additional glycosylation defect in a double mutant cell line (CHO-PhaRConAR) selected from a phytohemagglutinin-resistant single mutant cell line (CHO-PhaR) for resistance to concanavalin A has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor, resulting in a structure with seven instead of the normal nine mannose units. Both the CHO-PhaRConR and CHO-PhaR cells were completely blocked in the synthesis of complex, acidic type oligosaccharides because of a previously demonstrated deficiency in a particular N--acetylglucosamine transferase activity. In addition, an altered collection of neutral type oligosaccharides (Man4-7GlcNAc2) accumulated in the glycoproteins of the double mutant.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

Second-site rho mutation: Genetic linkage and polyC-dependent ATPase

Rho has been purified to homogeneity from Escherichia coli double mutant rho-115 sur-38 cells, and from rho6 and rho-115 cells. The sur-38 mutation suppresses the original rho-115 phenotype. We observe that the polyC-dependent ATPases of these three rho preparations have the same specific activities. However, the ATPase of rho from the double rho-115 sur-38 mutant is extremely heat labile, while that from rho-115 shows a heat lability intermediate between the wild type and the double mutant. Transduction analysis suggests that sur-38 is closely linked to rho-115 in the order ilv--sur-38--rho-115--metE. These data are consistent with the model that the sur-38 mutation affects the structural gene for rho.

Heptose-deficient mutants of Escherichia coli K12 deficient in up to three major outer membrane proteins.

Mutants of Escherichia coli K12, deficient in up to three major outer membrane proteins b, c and d have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in protein b. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteins c and/or d have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell envelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.

Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I.

We have isolated a strain of Escherichia coli K-12 carrying a mutation, polA12, that results in the synthesis of a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I. The double mutants polA12 recA56 and polA12 recB21, constructed at 30 C, are inviable at 42 C. About 90% of the cells of both double mutants die after 2 hr of incubation at 42 C. Both double mutants filament at 42 C and show a dependence on high cell density for growth at 30 C. In polA12 recB21 cells at 42 C, DNA and protein synthesis gradually stop in parallel. In polA12 recA56 cells, DNA synthesis continues for at least 1 hr at 42 C, and there is extensive DNA degradation. The results suggest that the primary lesion in these double mutants is not in DNA replication per se.