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 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 Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2

1998 ◽  
Vol 66 (6) ◽  
pp. 2576-2586 ◽  
Author(s):  
Leigh Rice Washburn ◽  
Keith E. Weaver ◽  
Elizabeth J. Weaver ◽  
Wendy Donelan ◽  
Suhaila Al-Sheboul
Keyword(s):  
Amino Acid ◽  
Signal Peptide ◽  
Dna Sequences ◽  
Surface Protein ◽  
Carboxypeptidase Y ◽  
Coding Region ◽  
Intact Cells ◽  
Mycoplasma Arthritidis ◽  
E Coli ◽  
Variable Surface

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed inEscherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidisclonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed inE. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

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.

scholarly journals Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia

1994 ◽  
Vol 14 (4) ◽  
pp. 2479-2484
Author(s):  
J M Scott ◽  
K Mikami ◽  
C L Leeck ◽  
J D Forney
Keyword(s):  
Sexual Reproduction ◽  
Unique Feature ◽  
Protein Gene ◽  
Surface Protein ◽  
Mendelian Inheritance ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Variable Surface ◽  
Type Variable ◽  
Surface Protein Gene

Paramecium tetraurelia contains two types of nuclei, a diploid germinal micronucleus and a large transcriptionally active macronucleus. The macronuclear genome is formed from the micronuclear DNA during sexual reproduction. Previous studies have shown that the processing of the A-type variable surface protein gene during formation of a new macronucleus is dependent on the presence of the A gene in the old macronucleus. It is not clear if this is a general feature that controls the formation of the Paramecium macronuclear genome or a unique feature of the A locus. Using micronuclear transplantation, we have constructed a strain that has a wild-type micronucleus but has macronuclear deletions of the A- and B-type surface protein genes. Neither the A nor the B gene is incorporated into the new macronucleus after sexual reproduction. Macronuclear transformation of this strain with the B gene rescues the B-gene deletion after formation of the next macronucleus but has not effect on the A deletion. Similarly, transformation with the A gene shows gene-specific rescue for A but not B. The effect of the old macronucleus on the processing of the new macronucleus results in a pattern of non-Mendelian inheritance of both macronuclear deletions. Progeny from the wild-type exconjugant are all wild type, and progeny from the A- B- exconjugant are mutant. The features of this A- B- non-Mendelian mutant demonstrate that the regulation of macronuclear DNA processing is gene specific, and our results open the possibility that this type of regulation affects many regions of the Paramecium genome.

scholarly journals Molecular and genetic analyses of the B type surface protein gene from Paramecium tetraurelia.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 189-198 ◽  
Author(s):  
J Scott ◽  
C Leeck ◽  
J Forney
Keyword(s):  
Cell Lines ◽  
Tandem Repeats ◽  
Protein Gene ◽  
Surface Protein ◽  
Segregation Ratio ◽  
Paramecium Tetraurelia ◽  
Mendelian Segregation ◽  
Variable Surface ◽  
Surface Protein Gene ◽  
F2 Progeny

Abstract The gene encoding the B type variable surface protein from Paramecium tetraurelia stock 51 has been cloned and sequenced. The 7,182 nucleotide open reading frame contains no introns and encodes a cysteine-rich protein that has a periodic structure including three nearly perfect tandem repeats in the central region. Interestingly, the B gene is located near a macronuclear telomere as was shown previously for two other paramecium surface protein genes. In this paper, we characterize four independent mutants with complete macronuclear deletions of the B gene. Previous analysis of different macronuclear deletion mutants of the A surface protein gene demonstrated two types of inheritance: typical Mendelian segregation (as illustrated by d12) and cytoplasmic inheritance (shown by d48). F1 analysis of four B- mutants crossed with wild-type cells reveals heterozygous F1 cell lines derived from both parental cytoplasms contain approximately the same copy number of the B gene, as expected for a recessive Mendelian mutation. Analysis of F2 progeny from three of these four B- mutant crosses indicates that one of the three exhibits a Mendelian 1:1 segregation ratio of B+ and B- cell lines. The other two show a preponderance of B+ cells, but this is not correlated with the parental cytoplasmic type. In addition to having a large number of B+ individuals, the d12.144, A-, B- mutant produced some F2 progeny that stably maintain less than normal macronuclear amounts of the A gene and/or the B gene.

scholarly journals Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia.

1994 ◽  
Vol 14 (4) ◽  
pp. 2479-2484 ◽  
Author(s):  
J M Scott ◽  
K Mikami ◽  
C L Leeck ◽  
J D Forney
Keyword(s):  
Sexual Reproduction ◽  
Unique Feature ◽  
Protein Gene ◽  
Surface Protein ◽  
Mendelian Inheritance ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Variable Surface ◽  
Type Variable ◽  
Surface Protein Gene

Paramecium tetraurelia contains two types of nuclei, a diploid germinal micronucleus and a large transcriptionally active macronucleus. The macronuclear genome is formed from the micronuclear DNA during sexual reproduction. Previous studies have shown that the processing of the A-type variable surface protein gene during formation of a new macronucleus is dependent on the presence of the A gene in the old macronucleus. It is not clear if this is a general feature that controls the formation of the Paramecium macronuclear genome or a unique feature of the A locus. Using micronuclear transplantation, we have constructed a strain that has a wild-type micronucleus but has macronuclear deletions of the A- and B-type surface protein genes. Neither the A nor the B gene is incorporated into the new macronucleus after sexual reproduction. Macronuclear transformation of this strain with the B gene rescues the B-gene deletion after formation of the next macronucleus but has not effect on the A deletion. Similarly, transformation with the A gene shows gene-specific rescue for A but not B. The effect of the old macronucleus on the processing of the new macronucleus results in a pattern of non-Mendelian inheritance of both macronuclear deletions. Progeny from the wild-type exconjugant are all wild type, and progeny from the A- B- exconjugant are mutant. The features of this A- B- non-Mendelian mutant demonstrate that the regulation of macronuclear DNA processing is gene specific, and our results open the possibility that this type of regulation affects many regions of the Paramecium genome.

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, Anguilla japonica

2011 ◽  
Vol 6 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Malay Choudhury ◽  
Takahiro Oku ◽  
Shoji Yamada ◽  
Masaharu Komatsu ◽  
Keita Kudoh ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Structural Features ◽  
Lipid Binding ◽  
Japanese Eel ◽  
Amino Acid Sequences ◽  
Novel Genes ◽  
Isolation And Characterization

AbstractApolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

scholarly journals Genetic variation in West Nile virus from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection

2005 ◽  
Vol 86 (8) ◽  
pp. 2175-2183 ◽  
Author(s):  
Greta Jerzak ◽  
Kristen A. Bernard ◽  
Laura D. Kramer ◽  
Gregory D. Ebel
Keyword(s):  
Genetic Diversity ◽  
West Nile Virus ◽  
Consensus Sequence ◽  
Purifying Selection ◽  
Infected Mosquito ◽  
Structural Protein ◽  
Coding Region ◽  
West Nile ◽  
Transmission Cycle ◽  
The Mean

Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 3′ 1159 nt of the WNV envelope (E) coding region and the 5′ 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0·016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19·5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra- and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations.

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