A Mutation in the Flanking 5′-TA-3′ Dinucleotide Prevents Excision of an Internal Eliminated Sequence From the Paramecium tetraurelia Genome

Abstract The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5′-TA-3′ dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5′-TA-3′ and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5′-TA-3′ dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.

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A Mutation in Paramecium tetraurelia Reveals Functional and Structural Features of Developmentally Excised DNA Elements

Genetics ◽

10.1093/genetics/148.1.139 ◽

1998 ◽

Vol 148 (1) ◽

pp. 139-149 ◽

Cited By ~ 4

Author(s):

Kimberly M Mayer ◽

Kazuyuki Mikami ◽

James D Forney

Keyword(s):

Mutant Cell ◽

Consensus Sequence ◽

Surface Protein ◽

Structural Features ◽

Inverted Terminal Repeat ◽

Paramecium Tetraurelia ◽

Coding Region ◽

Conserved Sequence ◽

Variable Surface ◽

Dna Elements

Abstract The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28–882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tc1 transposons (Klobutcher and Herrick 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES2591 has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its wild-type micronuclear copy through multiple sexual generations.

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Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1133 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1133-1137 ◽

Cited By ~ 22

Author(s):

Y You ◽

K Aufderheide ◽

J Morand ◽

K Rodkey ◽

J Forney

Keyword(s):

Cell Line ◽

Dna Sequences ◽

Mutant Cell ◽

Restriction Pattern ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Coding Region ◽

Cytoplasmic Factor ◽

In The Wild ◽

Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

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Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1133-1137.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1133-1137

Author(s):

Y You ◽

K Aufderheide ◽

J Morand ◽

K Rodkey ◽

J Forney

Keyword(s):

Cell Line ◽

Dna Sequences ◽

Mutant Cell ◽

Restriction Pattern ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Coding Region ◽

Cytoplasmic Factor ◽

In The Wild ◽

Dna Restriction

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A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies

Journal of Cell Science ◽

10.1242/jcs.90.1.59 ◽

1988 ◽

Vol 90 (1) ◽

pp. 59-71

Author(s):

M. Schleicher ◽

A. Noegel ◽

T. Schwarz ◽

E. Wallraff ◽

M. Brink ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Polypeptide Chain ◽

Mutant Cell ◽

Polyacrylamide Gel Electrophoresis ◽

Cell Extract ◽

Binding Activity ◽

Partial Purification ◽

Wild Type ◽

Coding Region ◽

In The Wild

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3′-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 × 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 × 10(3) Mr and 88 × 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 × 10(3) Mr polypeptide only. The 88 × 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 × 10(3) Mr and 88 × 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.

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The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽

10.1093/genetics/155.3.1105 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1105-1117 ◽

Cited By ~ 4

Author(s):

W John Haynes ◽

Kit-Yin Ling ◽

Robin R Preston ◽

Yoshiro Saimi ◽

Ching Kung

Keyword(s):

Genomic Library ◽

Open Reading Frame ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Rt Pcr ◽

Reading Frame ◽

Close Proximity ◽

In The Wild ◽

Dna Fragment ◽

Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

The Journal of Cell Biology ◽

10.1083/jcb.135.3.647 ◽

1996 ◽

Vol 135 (3) ◽

pp. 647-660 ◽

Cited By ~ 150

Author(s):

G A Smith ◽

J A Theriot ◽

D A Portnoy

Keyword(s):

Listeria Monocytogenes ◽

Actin Polymerization ◽

Consensus Sequence ◽

Wild Type ◽

Movement Rate ◽

Bacterial Surface ◽

Rate Dependent ◽

Repeat Domain ◽

Low Levels ◽

Rate Of Movement

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

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Phenotypic and Genetic Analysis of “Chameleon,” a Paramecium Mutant With an Enhanced Sensitivity to Magnesium

Genetics ◽

10.1093/genetics/146.3.871 ◽

1997 ◽

Vol 146 (3) ◽

pp. 871-880

Author(s):

Robin R Preston ◽

Jocelyn A Hammond

Keyword(s):

Genetic Analysis ◽

Single Locus ◽

Behavioral Analysis ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Ion Conductance ◽

Membrane Excitation ◽

Enhanced Sensitivity ◽

Electrophysiological Analysis ◽

Mutant Strains

Three mutant strains of Paramecium tetraurelia with an enhanced sensitivity to magnesium have been isolated. These new “Chameleon” mutants result from partial- or codominant mutations at a single locus, Cha. Whereas the wild type responded to 5 mm Mg2+ by swimming backward for 10–15 sec, Cha mutants responded with ∼30 sec backward swimming. Electrophysiological analysis suggested that this behavior may be caused by slowing in the rate at which a Mg2+-specific ion conductance deactivates following membrane excitation. This would be consistent with an observed increase in the sensitivity of Cha mutants to nickel poisoning, since Ni2+ is also able to enter the cell via this pathway. More extensive behavioral analysis showed that Cha cells also overresponded to Na+, but there was no evidence for a defect in intracellular Ca2+ homeostasis that might account for a simultaneous enhancement of both the Mg2+ and Na+ conductances. The possibility that the Cha locus may encode a specific regulator of the Mg2+- and Na+-permeabilities is considered.

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Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

The Journal of Cell Biology ◽

10.1083/jcb.137.6.1265 ◽

1997 ◽

Vol 137 (6) ◽

pp. 1265-1278 ◽

Cited By ~ 102

Author(s):

Gary A. Eitzen ◽

Rachel K. Szilard ◽

Richard A. Rachubinski

Keyword(s):

Oleic Acid ◽

Yarrowia Lipolytica ◽

Consensus Sequence ◽

Wild Type ◽

Gene Encoding ◽

Peroxisomal Membrane ◽

Peripheral Protein ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

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ADP-Ribosylation of Wild-Type p53in Vitro:Binding of p53 Protein to Specific p53 Consensus Sequence Prevents Its Modification

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1996.0990 ◽

1996 ◽

Vol 224 (1) ◽

pp. 96-102 ◽

Cited By ~ 49

Author(s):

Józefa Węsierska-Gądek ◽

Gerald Schmid ◽

Christa Cerni

Keyword(s):

P53 Protein ◽

Consensus Sequence ◽

Wild Type ◽

Adp Ribosylation

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Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein

Journal of Virology ◽

10.1128/jvi.78.21.11879-11889.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11879-11889 ◽

Cited By ~ 89

Author(s):

Walter Fuchs ◽

Barbara G. Klupp ◽

Harald Granzow ◽

Thomas C. Mettenleiter

Keyword(s):

Gene Product ◽

Pseudorabies Virus ◽

Rabbit Kidney ◽

Tegument Protein ◽

Wild Type ◽

Coding Region ◽

Ordered Structures ◽

Ca 50 ◽

Essential Function ◽

Full Length Clone

ABSTRACT The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-ΔUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-ΔUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-ΔUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-ΔUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-ΔUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-ΔUL36F were found in large ordered structures similar to those which had previously been observed with PrV-ΔUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.

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