Biomedical subjects

James D Forney

Publications and source records attributed to James D Forney.

6 recordsLinked to original sources

PubMed2006

Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.

We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance.

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PubMed2006

The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

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PubMed2005

The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions.

Eukaryotic organisms with cilia or flagella typically express two non-axonemal or "cytoplasmic" dyneins, dynein-1 and dynein-2. Interestingly, we find that Leishmania mexicana is unusual and contains two distinct cytoplasmic dynein-2 heavy chain genes (designated LmxDHC2.1 and LmxDHC2.2) along with a single dynein-1 heavy chain (LmxDHC1). Disruption of LmxDHC2.2 resulted in immotile parasites that had a rounded cell body. Although they assume amastigote morphology, immunoblot analysis of these cells demonstrates protein expression consistent with the promastigote stage. Ultrastructural analysis revealed non-emergent flagella that lacked the paraflagellar rod and an axoneme with deficiencies in several components. We confirmed the absence of paraflagellar rod proteins PFR1 and PFR2. These results show that LmxDHC2.2 is required for flagellar assembly and also participates in the maintenance of promastigote cell shape. In contrast to the results with LmxDHC2.2, we were unable to generate homologous disruptions of LmxDHC2.1. This result suggests that, unlike LmxDHC2.2, LmxDHC2.1 is an essential gene in Leishmania. Together, these findings demonstrate that the two dynein-2 heavy chain isoforms in Leishmania perform distinct functions. The observation that the genomes of Leishmania major, Leishmania infantum and Trypanosoma brucei also contain two dynein-2 isoforms suggests that this unusual aspect of cytoplasmic dynein is a conserved feature of the kinetoplastids.

Amino Acid Sequence↗

PubMed2005

Analysis of Paramecium tetraurelia A-51 surface antigen gene mutants reveals positive-feedback mechanisms for maintenance of expression and temperature-induced activation.

In Paramecium tetraurelia, variable surface antigen loci show mutually exclusive expression which is controlled primarily at the transcriptional level. Clonally stable expression of a single antigen has attracted models involving self-regulation by their gene products. However, direct demonstration of self-feedback at the molecular level has been complicated due to the inability to separate the functional gene from its product as well as copy number effects associated with injected extrachromosomal DNA in the polygenomic somatic nucleus. In this study, we exploited several germ line termination and frameshift mutations in the A-51 surface antigen gene to analyze variable surface antigen expression. These mutant alleles have the same copy number as the wild-type allele and therefore eliminate possible copy number effects. The mutant alleles were not transcribed at 27 degrees C, consistent with positive-feedback models for gene expression. However, further analysis showed that high temperatures (34 degrees C) induced transcription of the mutant A genes even in the presence of a different antigen on the cell surface. Thus, transcription was temperature dependent. Unlike wild-type cells, transcription of the mutant A genes at high temperatures was not maintained after temperature shift back to 27 degrees C in homozygous mutant cells. Importantly, transcription of the mutant allele was maintained at 27 degrees C in heterozygous cells with one copy of the wild-type allele. These results indicate that expression of the wild-type gene is required to stabilize its own transcriptional state at 27 degrees C.

Amino Acid Sequence↗

PubMed2004

Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila.

Protein synthesis elongation factor 2 (eEF2) catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome. Most organisms encode a single EF2 protein and its activity is regulated by phosphorylation. We have identified a family of genes in Tetrahymena thermophila that encode proteins homologous to eEF2, yet are expressed only during sexual reproduction. These genes have been designated EFR for Elongation Factor 2 Related. EFR transcripts were not detected in vegetative cell cultures but rapidly increased about 6 h after the start of conjugation (mating). For comparison, we cloned, sequenced and analyzed the expression of the standard eEF2 gene from T. thermophila. Unlike EFR, transcripts from eEF2 were detected in vegetative cells but were present at lower concentrations during conjugation. Despite the high sequence identity between EFR and eEF2 from other organisms (about 42% at the amino acid level), key regulatory sequences that are involved in the regulation of eEF2 are altered in EFR. The sequence and expression data suggest that EFR is an eEF2 variant involved in a major translation regulatory mechanism that occurs during the formation of the macronuclear genome in conjugating cells.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia.

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.

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