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

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

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.

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

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

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.

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

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.

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

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 597-604 ◽  
Author(s):  
Kimberly M Mayer ◽  
James D Forney
Keyword(s):  
Inverted Repeat ◽  
Mutant Cell ◽  
Consensus Sequence ◽  
Terminal Inverted Repeat ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Coding Region ◽  
Discrete Elements ◽  
Additional Information ◽  
Gene Excision

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.

scholarly journals Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines.

1994 ◽  
Vol 125 (6) ◽  
pp. 1225-1237 ◽  
Author(s):  
J M Riberdy ◽  
R R Avva ◽  
H J Geuze ◽  
P Cresswell
Keyword(s):  
Cell Line ◽  
Mhc Class Ii ◽  
Antigen Processing ◽  
Mutant Cell ◽  
Class Ii ◽  
Invariant Chain ◽  
Wild Type ◽  
Mutant Cell Line ◽  
In The Wild ◽  
Hla Dr3

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II-associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild-type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.

scholarly journals Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 727-734
Author(s):  
H Jessop-Murray ◽  
L D Martin ◽  
D Gilley ◽  
J R Preer ◽  
B Polisky
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Large Deletion ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Cytoplasmic Factor ◽  
Gene Copies ◽  
Unusual Phenomenon ◽  
Small Stable Rna

Abstract The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

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

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
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.

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

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 139-149 ◽  
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.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism

1999 ◽  
Vol 341 (1) ◽  
pp. 89-93 ◽  
Author(s):  
Gianluca TELL ◽  
Lucia PELLIZZARI ◽  
Gennaro ESPOSITO ◽  
Carlo PUCILLO ◽  
Paolo Emidio MACCHIA ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Congenital Hypothyroidism ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Structural Defects ◽  
Molecular Defect ◽  
Wild Type ◽  
Coding Region ◽  
Induced Fit ◽  
Helical Content

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in α-helical content upon interaction with DNA (‘induced fit’). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low α-helical content; however, in the Leu62Arg mutant, the gain in α-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia

2000 ◽  
Vol 203 (6) ◽  
pp. 1059-1070 ◽  
Author(s):  
U. Nagel ◽  
H. Machemer
Keyword(s):  
Swimming Speed ◽  
Sedimentation Rates ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Centre Of Gravity ◽  
In The Wild ◽  
Type Population ◽  
G Value ◽  
Mutant Cells

Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity.

Sign in / Sign up

Export Citation Format

Share Document