Naegleria gruberi metabolism

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

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5-Bromodeoxyuridine-induced formation of virus-like particles in Naegleria gruberi EGs

Journal of Cell Science ◽

10.1242/jcs.26.1.359 ◽

1977 ◽

Vol 26 (1) ◽

pp. 359-371

Author(s):

F.L. Schuster ◽

J.S. Clemente

Keyword(s):

Food Source ◽

Virus Like Particles ◽

Culture Cells ◽

Mammalian Cell Lines ◽

Tissue Culture Cells ◽

Excess Thymidine ◽

Axenic Cultures ◽

Future Work ◽

Living Bacteria ◽

Naegleria Gruberi

Exposure of axenic cultures of the amoeboflagellate Naegleria gruberi EGs to the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) resulted in the induction of virus-like particles (VLP) and various structures associated with their development and presumed transmission. Previously, VLP induction could be accomplished only by growing amoebae in the presence of living bacteria as a food source. Addition of excess thymidine along with BrdU did not block induction of particles. This account demonstrates that the EGs-VLP system responded to BrdU as do a number of mammalian cell lines harbouring latent viruses, and provides the basis for future work on the infectivity of the VLPs for other amoebae as well as tissue culture cells.

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Hartmannella castellanii Naegleria gruberi, N. fowleri

Medizinische Parasitologie ◽

10.1007/978-3-662-08636-0_7 ◽

1975 ◽

pp. 43-47

Author(s):

Gerhard Piekarski

Keyword(s):

Naegleria Gruberi

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Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation

Nucleic Acids Research ◽

10.1093/nar/gkaa238 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5603-5615

Author(s):

Mihaela-Carmen Unciuleac ◽

Yehuda Goldgur ◽

Stewart Shuman

Keyword(s):

Crystal Structure ◽

Active Site ◽

Active Sites ◽

Enzyme Structure ◽

Rna Ligase ◽

Alanine Scan ◽

Unexpected Findings ◽

Metal Cofactors ◽

Conserved Amino Acids ◽

Naegleria Gruberi

Abstract Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3′-OH RNA strands in the context of 3′-OH/5′-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

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Plant-type mitochondrial RNA editing in the protist Naegleria gruberi

RNA ◽

10.1261/rna.02962911 ◽

2011 ◽

Vol 17 (12) ◽

pp. 2058-2062 ◽

Cited By ~ 30

Author(s):

M. Rudinger ◽

L. Fritz-Laylin ◽

M. Polsakiewicz ◽

V. Knoop

Keyword(s):

Rna Editing ◽

Mitochondrial Rna ◽

Plant Type ◽

Naegleria Gruberi

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New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates.

The Journal of Cell Biology ◽

10.1083/jcb.102.2.353 ◽

1986 ◽

Vol 102 (2) ◽

pp. 353-361 ◽

Cited By ~ 13

Author(s):

J Mar ◽

J H Lee ◽

D Shea ◽

C J Walsh

Keyword(s):

Blot Analysis ◽

Rna Synthesis ◽

In Vitro Translation ◽

Dot Blot ◽

Cross Hybridization ◽

Tubulin Mrna ◽

Dot Blot Analysis ◽

Rna Concentration ◽

Naegleria Gruberi

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).

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