scholarly journals Microinjection of cytoplasm as a test of complementation in Paramecium.

1982 ◽  
Vol 92 (2) ◽  
pp. 559-564 ◽  
Author(s):  
N Haga ◽  
M Forte ◽  
Y Saimi ◽  
C Kung
Keyword(s):  
Action Potentials ◽  
Complementation Group ◽  
Genetic Complementation ◽  
Complementation Test ◽  
Paramecium Tetraurelia ◽  
Active Components ◽  
Cell Homogenate ◽  
A Cell ◽  
Complementation Groups ◽  
Favorable Conditions

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.

scholarly journals Genetic analysis of axonemal mutants in Paramecium tetraurelia defective in their response to calcium

1984 ◽  
Vol 43 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Robert D. Hinrichsen ◽  
Ching Kung
Keyword(s):  
Genetic Analysis ◽  
Elevated Temperatures ◽  
Complementation Group ◽  
Paramecium Tetraurelia ◽  
Genetic Analyses ◽  
Complementation Groups

SUMMARYSix axonemal mutants of Paramecium tetraurelia have been isolated that are unable to respond properly to calcium. The mutants, designated atalantas, cannot swim backward when stimulated by ions or heat. Genetic analyses reveal that all six mutants are recessive and fall into four complementation groups. Three of the mutants in one complementation group are phenotypically non-leaky, one is leaky and two are extremely leaky, only displaying their phenotypes at elevated temperatures. The complete mutants, ataA, are also abnormal in their forward swimming. This abnormality co-segregates with the inability to swim backward. ataA1 is not allelic to several membrane mutants of P. tetraurelia.

scholarly journals amontillado, the Drosophila Homolog of the Prohormone Processing Protease PC2, Is Required During Embryogenesis and Early Larval Development

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 227-237 ◽  
Author(s):  
Lowell Y M Rayburn ◽  
Holly C Gooding ◽  
Semil P Choksi ◽  
Dhea Maloney ◽  
Ambrose R Kidd ◽  
...  
Keyword(s):  
Larval Development ◽  
Secretory Pathway ◽  
Larval Growth ◽  
Peptide Hormones ◽  
Complementation Group ◽  
Prohormone Processing ◽  
Complementation Groups ◽  
Regulated Secretory Pathway ◽  
Processing Protease ◽  
Larval Molt

Abstract Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.

scholarly journals A Yeast taf17 Mutant Requires the Swi6 Transcriptional Activator for Viability and Shows Defects in Cell Cycle-Regulated Transcription

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1561-1576
Author(s):  
Neil Macpherson ◽  
Vivien Measday ◽  
Lynda Moore ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
A Cell ◽  
Allelism Tests ◽  
Complementation Groups

Abstract In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

Fanconi's anaemia: correlation of genetic complementation group with psoralen/UVA response

1988 ◽  
Vol 78 (1) ◽  
pp. 51-54 ◽  
Author(s):  
Martin Digweed ◽  
Sabine Zakrzewski-L�dcke ◽  
Karl Sperling
Keyword(s):  
Complementation Group ◽  
Genetic Complementation

scholarly journals Overcoming the Challenges for a Mass Manufacturing Machine for the Assembly of PEMFC Stacks

Machines ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 66 ◽  
Author(s):  
Porstmann ◽  
Wannemacher ◽  
Richter
Keyword(s):  
Fuel Cell ◽  
Economies Of Scale ◽  
Polymer Electrolyte Membrane ◽  
Cell System ◽  
Active Components ◽  
Fuel Cell Stack ◽  
Electrolyte Membrane ◽  
Modular Concept ◽  
A Cell ◽  
Manufacturing Machine

One of the major obstacles standing in the way of a break-through in fuel cell technology is its relatively high costs compared to well established fossil-based technologies. The reasons for these high costs predominantly lie in the use of non-standardized components, complex system components, and non-automated production of fuel cells. This problem can be identified at multiple levels, for example, the electrochemically active components of the fuel cell stack, peripheral components of the fuel cell system, and eventually on the level of stack and system assembly. This article focused on the industrialization of polymer electrolyte membrane fuel cell (PEMFC) stack components and assembly. To achieve this, the first step is the formulation of the requirement specifications for the automated PEMFC stack production. The developed mass manufacturing machine (MMM) enables a reduction of the assembly time of a cell fuel cell stack to 15 minutes. Furthermore the targeted automation level is theoretically capable of producing up to 10,000 fuel cell stacks per year. This will result in a ~50% stack cost reduction through economies of scale and increased automation. The modular concept is scalable to meet increasing future demand which is essential for the market ramp-up and success of this technology.

scholarly journals AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

1989 ◽  
Vol 9 (8) ◽  
pp. 3155-3165 ◽  
Author(s):  
P N Lipke ◽  
D Wojciechowicz ◽  
J Kurjan
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Structural Gene ◽  
Signal Sequence ◽  
Positive Control ◽  
Complementation Group ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Amino Terminal ◽  
A Cell

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.

scholarly journals Episomic suppression of phenotype in Salmonella

1964 ◽  
Vol 5 (2) ◽  
pp. 269-281 ◽  
Author(s):  
P. F. Smith-Keary ◽  
G. W. P. Dawson
Keyword(s):  
Gene Expression ◽  
Salmonella Typhimurium ◽  
Uv Irradiation ◽  
Complementation Group ◽  
Wild Type ◽  
Complementation Groups

1. An auxotroph of Salmonella typhimurium, pro-401, was isolated in a strain that was unstable at the su-leuA locus. The auxotrophy of pro-401 is probably due to the attachment of a controlling episome to the proline region of the genome where it suppresses gene expression.2. The controlling episome frequently transposes over short distances so that all clones consist of cells, mixed for the site at which the controlling episome is attached; homologous transductions yield prototrophs.3. The controlling episome can transpose to a different complementation group; homologous transductions yield abortive transductants; syntrophism occurs between cells that are ‘mutant’ in different complementation groups to give reversions consisting entirely of auxotrophic cells which are called auxotrophic reversions.4. The controlling episome transposes over very short distances and never to beyond the limits of this proline region of the genome; no wild-type reversions were found.5. The controlling episome can be located at relatively distant proline sites in different clones; prototrophs from transductions between clones that are separated by many subculturings can be 100 times more frequent than from homologous transductions.6. The controlling episome has its frequency of transposition to different complementation groups increased by UV; irradiation increases the frequency of auxotrophic reversions.7. The controlling episome continues to transpose in stored cells.8. The pattern of reversions of pro-401 is different in these studies from its pattern two years previously. This is discussed.

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Polymorphism of t-complex genes in European wild mice

1984 ◽  
Vol 44 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Jan Klein ◽  
Peter Sipos ◽  
Felipe Figueroa
Keyword(s):  
Soviet Union ◽  
North America ◽  
Common Ancestor ◽  
Complementation Test ◽  
Homozygous State ◽  
Wild Mice ◽  
T Complex ◽  
The Soviet Union ◽  
Complementation Groups ◽  
The Individual

SummaryThirty-two t haplotypes were extracted from wild mice captured in Central Europe, Spain, the Soviet Union, Israel, Egypt, the Orkneys and South and North America, and tested for lethality in the homozygous state. Twenty-two proved to be homozygous lethals, 8 semilethals and 2 viables. The lethal t haplotypes were then tested by the genetic complementation test for identity with representatives of known complementation groups and with each other. Five of the 22 haplotypes proved to carry previously identified lethality factors (tw5, tw73, and tLub-1), while the rest carried new factors. The 17 haplotypes fell into 8 new complementation groups. Two of the new groups are partially overlapping in that they seem to share some lethality factors and differ in others. These tests raise the total number of known complementation groups to 16. The distribution of the individual t haplotypes among wild mice populations seems to reflect their differentiation from a common ancestor haplotype.

scholarly journals Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

1996 ◽  
Vol 16 (5) ◽  
pp. 2025-2036 ◽  
Author(s):  
M A Kenna ◽  
J G Petranka ◽  
J L Reilly ◽  
L I Davis
Keyword(s):  
Nuclear Pore Complex ◽  
Complementation Group ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Localization Sequence ◽  
Complementation Groups ◽  
Amino Terminal Domain ◽  
Yeast Homolog

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.

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