Progress 10/01/09 to 09/30/14
Outputs
Target Audience: During this reporting period the primary audience has been the scientific community. Two posters were presented at the Seventh International Conference SUMO, Ubiquitin, UBL Proteins: Implications for Human Disease in Shanghai, China, May 10-13, 2014. One of the posters entitled "Depletion of UBC9 causes defects in somatic and germline nuclei in Tetrahymena thermophila." presented by Qianyi Yang (graduate student) was selected for a best poster award. This conference hosted over 250 international scientists who are investigating fundamental and clincal aspects of SUMOylation and ubiquitination. On May 14th, 2014 the PI presented a seminar at Northwest Agriculture and Foresty University entitled "Multiple Roles for SUMOylation in Ciliated Protozoa". Dr. Forney met with student and faculty to discuss research and educational goals. In addition to the international presentations, 4 undergraduate students were taught by participating in the laboratory research, attending labortory group meeting and writing their results in the form of a paper at the end of each semester. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students have been trained during the project period and they are currently preparing and submitting manuscripts for publication. A total of four undergraduates recieved laboratory training in sterile techniques, recombinant DNA techniques, protein isolation and immunoblotting. Each student prepared a final report written in the form of a journal paper at the end of each semester. One of the undergraduate students was from Wheaton College and part of an NSF Research Experiences for Undergraduates (REU) summer pogram awarded to the Department of Biochemistry. One faculty member from St. Olaf College in Minnesota worked on the project for 9 months during a sabbatical leave. The faculty member learned the details of performing molecular biology procedures with Tetrahymena so she could use the organisms for educational and research purposes at St. Olaf. How have the results been disseminated to communities of interest? As previously mentioned in the "target audience" section the results have been presented at an international meeting on SUMOylation and a seminar at Northwest Agriculture Unviersity in China. As described above in "accomplishments" one manuscript has been submitted and another is in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project continued to focus on SUMOylation and not pursue histone demethylases. We have submitted a manuscript describing our initial results investigating sumoylation in Tetrahymena. Using a custom made SUMO antibody we show that there is a four fold increase in SUMOylated proteins during sexual reproduction in Tetrahymena thermophila. We showed that the SUMOylated proteins accumulate in the developing macronucleus suggesting a role in the genome reorganization that occurs in this stage. During vegetative cell growth SUMO protein and UBA2 (SUMO activating protein) are primarily located in the transcriptionally active macronucleus of Tetrahymena thermophila but not in the non-transcribed micronucleus. The genes encoding both SUMO and UBA2 are essential; complete deletions of either gene results in death. This is consistent with findings in most other species. Depletion (but not complete elimination) of either SUMO or UBA2 protein results in reduced growth, DNA damage sensitivity and failure of cell pairing during sexual reproduction. Although we believe SUMOylation is critical for genome reorganization the inability of cells to pair during sexual reproduction prevents us from examing that stage of the sexual cycle with our current approach. A second manuscript is in preparation describing the role of UBC9 the protein that attaches SUMO to its protein targets. As found with UBA2, UBC9 is located in the transcriptionally active macronucleus but not the micronucleus. UBC9 accumulates in the developing macronucleus during sexual reproduction. The UBC9 gene is essential and depletion of UBC9 protein results in reduced cell growth, DNA damage sensitivity and the inability of cells to pair during mating. Unlike SUMO the depletion of UBC9 also causes defects in mitosis, a process that only occurs in the micronucleus. Depletion of UBC9 results in the loss of micronuclei. In contrast the over expression of a dominant negative UBC9 mutant results in extra micronuclei. Expression of the dominant negative during sexual reproduction reduces the number of progeny from genetic crosses. Further investigations will test possible reasons for the defects during sexual reproduction. Our investigations show that Tetrahymena has an unusual and highly regulated system of SUMOylation during sexual reproduction that is connected to the formation of a new nucleus. This provides an opportunity for detailed studies of SUMOylation during a developmentally regulated series of events connected with changes in the genome.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Dr. Forney led his third Faculty Learning Community to facilitate changes in classroom pedagogy. The goal of this effort is to increase the exposure of students to statistical concepts within life science courses. In 2013, seven Purdue instructors and two graduate students were members of the Faculty Learning Community. Dr. Forney directed a 10-week summer research program for nine undergraduates on the Purdue campus. This NSF funded Research Experience for Undergraduates (REU) provided mentored research experiences focused on protein biochemistry. In 2013 five of the participating students were from underrepresented groups. Dr. Forney hosted one of these students in his laboratory during the summer. Changes/Problems: We will extend our studies of SUMOylation to additional stages of the Tetrahymena life cycle. This will leverage our expertise in affinity purification of SUMOylated proteins developed during the past two years. To compensate for this effort we will delay studies of histone methylation during meiosis. This is not the result of a technical problem, rather it is a strategic decision to gain a deeper understanding of SUMO substrates and dynamics of SUMOylation. We believe opportunities for the identification of novel substrates and regulatory mechanisms are present in Tetrahymena. What opportunities for training and professional development has the project provided? Dr. Forney served as research advisor for two graduate students in 2012-2013. Four undergraduates received training in laboratory research, currently one is enrolled in a veterinary technology program, one is attending veterinary school and one is enrolled in graduate school. The remaining student is still working on his B.S. degree. During the 10 week NSF REU program the Forney lab hosted one student from the University of Puerto Rico. She presented her results at a university wide poster session and a short talk in the Department of Biochemistry at the end of the 10-week period. Forney facilitated weekly group meetings for the nine NSF REU students. Topics included ethical conduct in research, reading primary literature, applying and interviewing for graduate school, plus guests who spoke about their careers related to biochemistry and biotechnology. How have the results been disseminated to communities of interest? We presented our SUMOylation results at two meetings during the past year. In April of 2013, Amjad Nasir presented a poster entitled “Developmental Regulation of SUMOylation During Conjugation in Tetrahymena” at the Midwest Protozoology Society Meeting at Bradley University in Peoria IL. He presented an updated version of the same poster in July at the FASEB Ciliate Molecular Biology Meeting in Steamboat Springs, Colorado. What do you plan to do during the next reporting period to accomplish the goals? As explained in the project change section we will focus on SUMOylation and delay investigations of histone methylation. First, we will refine our procedure for the affinity purification of sumoylated proteins in Tetrahymena to identify a larger subset of SUMOylated proteins. Second, we will construct plasmids to silence expression of genes in the SUMOylation pathway using RNA interference (RNAi). This will enhance our study of phenotypes resulting from SUMOylation deficiency during conjugation. Third, we will begin functional analysis of Tetrahymena SUMOylated proteins.
Impacts
What was accomplished under these goals?
During the 2012 reporting period we constructed Tetrahymena cells lines that conditionally express Ubc9p (SUMO conjugating enzyme) and Uba2p (SUMO activating enzyme). Studies with these lines showed that Ubc9p or Uba2p deficient cells have reduced growth rates and greater sensitivity to DNA damaging agents than wild type cells. When expression of Ubc9p or Uba2p is increased in the same cell lines growth rates and DNA damage sensitivity returns to nearly wild type levels, consistent with direct effects from limiting amounts of these proteins. In addition, Ubc9 deficient cell lines showed defects in mitotic divisions of the micronucleus resulting in loss of micronuclei. We have used tagged versions of SUMO protein to affinity purify targets of sumoylation from cells during vegetative growth and now our attention has turned to purification during the sexual cycle including meiosis.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The PI attended the Midwest Protozoology meeting (Peoria, IL) with two graduate students on April 21, 2012. Qianyi Yang (graduate student) presented a talk entitled "Analysis of SUMO-activating Uba2p and SUMO-conjugating Ubc9p in Tetrahymena". During the past year we continued our experimental analysis of SUMOylation in the ciliate Tetrahymena thermophila. Efforts were focused on phenotypic analysis of mutant cell lines deficient in proteins required for the SUMOylation pathway and affinity purification of the same proteins from Tetrahymena. Research Education Outputs Dr. Forney served as research advisor for two PhD graduate students in 2012 and was a member on five other graduate thesis advisory committees. Five undergraduate students were provided mentored research projects in Forney's laboratory during 2012. Forney directed a NSF sponsored research experience for eight undergraduates (REU) program in the summer of 2012. The students received 10 weeks of training in the biochemical analysis of proteins in the Department of Biochemistry at Purdue University. Most of the training was accomplished through mentored research projects in individual faculty laboratories. Weekly career development activities included ethical conduct in research, entrepreneurship, applying to graduate school and scientific communication. As a co-PI of a Howard Hughes Medical Institute education award Dr. Forney led a second cohort of faculty and postdocs through a Faculty Learning Community (FLC) focused on integrating statistics into the life science curriculum. The group met on a biweekly basis during the 2011-2012 academic year to discuss educational research, pedagogy, and statistical concepts. In April 2012 the FLC offered a campus workshop on active learning strategies in life science courses featuring Professor Robin Wright from the University of Minnesota. Approximately 30 faculty members and graduate students attended the workshop. Dr. Forney developed and offered a new biochemistry course entitled "Experimental Design Seminar". The goal of this one credit course is to introduce undergraduates to key concepts in the design of biochemical experiments. Emphasis is placed on identifying proper controls, quantitative results, reproducibility, and the importance of statistics. These concepts are embedded within topics of contemporary interest such as the role of dietary fiber in colon cancer and the function of the anti-cancer drug cisplatin. PARTICIPANTS: Professor Doug Chalker at Washington University at St. Louis, Department of Biology is a collaborator on our study of SUMOylation in Tetrahymena. He provided materials and reagents early in the project and continues to offer experimental advice. Amjad Nasir and Qianyi Yang are biochemistry graduate students who joined the project in 2009. Professor Mark Hall (Purdue, Biochemistry) has become a consultant for our efforts on protein purification. TARGET AUDIENCES: The Faculty Learning Community led by Dr. Forney targeted university instructors to facilitate changes in classroom pedagogy. The goal is to increase active learning in the classroom and increase the exposure of students to statistical concepts within life science courses. In 2012, eight instructors and two postdoctoral researchers were members of the Faculty Learning Community. The course "Experimental Design Seminar" is targeted to undergraduate biochemistry majors, although it could serve any life science major if expanded to a larger audience. In 2012, 30 students completed the course. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Sexual reproduction in ciliated protozoa involves extensive genome reorganization within the developing macronuclei. Events include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Understanding how the process of DNA elimination is regulated could lead to advances in the biotechnology of genome manipulation or improved genome stability. We previously demonstrated that silencing Paramecium genes encoding proteins in the SUMOylation pathway resulted in normal amplification of DNA in developing macronuclei but a failure to excise IESs. Our hypothesis is that SUMOylation is a key regulatory mechanism of ciliate genome rearrangements. We have begun studies of SUMOylation in Tetrahymena thermophila because this ciliate shares the same features of genome rearrangements during formation of the macronucleus but has more advanced tools for experimental analysis. We constructed germline (micronuclear) knockout strains for SUMO, UBA2 and UBC9 genes. In each case these knockouts display a lethal phenotype during vegetative cell culture, consistent with other eukaryotic organisms. We are now manipulating these cell lines to generate conditional expression from cells that will be deficient for the corresponding proteins.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The PI and two graduate students attended the Midwest Protozoology meeting at Bradley University, Peoria IL in April 2011. Amjad Nasir (graduate student), Qianyi Yang (graduate student) Jim Forney (PI) and Doug Chalker (PI, Washington University, St. Louis) were authors on an abstract entitled "SUMO pathway components in Tetrahymena thermophila are essential and nuclear". Amjad Nasir presented the talk describing Tetrahymena cell lines with germline deletions of two genes required for the SUMO modification pathway. These mutant lines are now being analyzed and they will be used for future studies on the developmental regulation of sumoylation. In July 2011 Dr. Forney attended the Federation of American Societies of Experimental Biology (FASEB) summer conference "Ciliate Molecular Biology" at the Orthodox Academy in Crete, Greece where he presented a talk entitled "SUMO pathway components in Tetrahymena thermophila are essential and nuclear". Dr. Forney served as academic advisor for two graduate students and 8 undergraduates during the past year. Forney served on the thesis advisory committees for 4 PhD students in 2011. During the 2010-11 academic year Dr. Forney led a faculty learning community on the Purdue campus that included faculty from Biochemistry, Biological Sciences, Biomedical Engineering and Statistics. The group met on a regular basis to discuss pedagogical methods and educational research focused on the STEM disciplines. In April 2011 the group sponsored an open workshop for faculty, instructors and graduate students entitled "Statistics in the life sciences classroom: don't leave learning to chance!". Over 50 participants attended. The program is funded as part of a Howard Hughes Medical Institute grant to Purdue University. PARTICIPANTS: Professor Doug Chalker at Washington University at St. Louis, Department of Biology is a collaborator on our study of SUMOylation in Tetrahymena. He provided materials and reagents early in the project and continues to offer experimental advice. We collaborated with Dr. Mireille Betermier, Centre de Genetique Moleculaire, Gif-sur-Yvette, France on studies of Paramecium developmentally regulated DNA elimination and the role of ligase IV. Amjad Nasir, a biochemistry graduate student, joined the project in May of 2009. Qianyi Yang, biochemistry graduate program in August 2009. She is supported in part through a China Scholarship that will continue for a total of four years. TARGET AUDIENCES: The target audience includes research biochemists, microbiologists, university course instructors. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Sexual reproduction in ciliated protozoa involves extensive genome reorganization within the developing macronuclei. Events include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Some of the proteins required for these events are related to those found in many eukaryotes. In collaboration with the laboratory of Dr. Mireille Betermier (Centre de Recherches, Gif-sur-Yvette, France) we demonstrated that the Paramecium homologs of ligase IV and XRCC4 are required to join the broken DNA ends after excision of short IESs. Disruption of ligase IV function by RNAi-mediated silencing resulted in the failure of developing macronuclei to differentiate into new macronuclei. Broken ends persist in these cells and they arrest during sexual reproduction. The results support the hypothesis that the non-homologous end-joining pathway (NHEJ), used to repair double-strand DNA breaks in mammalian cells, is utilized in ciliates for precise and efficient repair of genome rearrangements. These data have stimulated experiments that will test other proteins in the NHEJ pathway for their role in ciliate DNA elimination. The involvement of XRCC4 provides a potential link between DNA elimination and the previously observed requirement for the SUMO pathway because Xrcc4p is SUMOylated in mammalian cells. A potential regulatory role for SUMOylation of Tetrahymena Xrcc4 protein will be tested directly. The findings will contribute to efforts by many investigators to elucidate the molecular mechanism of the precise and efficient DNA rearrangements in ciliates.
Publications
- Kapusta A., Matsuda A., Ku M., Marmignon A., Silve A., Meyer E., Forney J.D., Malinsky S., Betermier M. 2011. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end-joining. PLOS Genetics 7: e1002049.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The PI and two graduate students attended the Midwest Protozoology meeting at Bradley University, Peoria IL in April 2010. Amjad Nasir (graduate student), Qianyi Yang (graduate student) Jim Forney (PI) and Doug Chalker (PI, Washington University, St. Louis) were authors on a poster entitled "Analysis of SUMO pathway genes n Tetrahymena thermophila". The PI completed his sabbatical in December of 2009 at Washington University in St. Louis. The work generated Tetrahymena cell lines with germline deletions of two genes required for the SUMO modification pathway. These mutant lines are now being analyzed and they will be used for future studies on the developmental regulation of sumoylation. Dr. Forney served as academic advisor for two graduate students and 10 undergraduates during the past year. One undergraduate, Brian Zedalis started a M.S. program in Biochemistry and Biotechnology at Georgetown University. Forney serves on the thesis advisory committees for 5 PhD students. In September of 2010 Dr. Forney initiated a faculty learning community on the Purdue campus that includes faculty from Biochemistry, Biological Sciences, Biomedical Engineering and Statistics. The group meets on a regular basis to discuss pedagogical methods and educational research focused on the STEM disciplines. The program is funded as part of a Howard Hughes Medical Institute grant to Purdue University. PARTICIPANTS: Professor Doug Chalker at Washington University at St. Louis, Department of Biology has been a collaborator on one previous project and offered training in Tetrahymena molecular genetics during Professor Forney's sabbatical. Two graduate students worked on the project during this period. Amjad Nasir started the biochemistry graduate program in 2008 and joined the project in May of 2009. Qianyi Yang entered the biochemistry graduate program in August 2009 and joined the Forney lab directly. She is supported in part through a China Scholarship that will continue for a period of four years. TARGET AUDIENCES: Biochemists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Sexual reproduction in ciliated protozoa involves extensive genome reorganization within the developing macronuclei. Events include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Some of the proteins required for these events are related to those found in many eukaryotes, but others appear to be unique to ciliates. We identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Disruption of PtDie5p function by RNAi-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new macronuclei. Tetrahymena cells lacking the DIE5 gene arrested late in development prior to genome amplification whereas Paramecium cells deficient in DIE5 transcripts amplified new macronuclear DNA before the failure in differentiation. IES excision and chromosome fragmentation failed to occur in both ciliates, which strongly supports that Die5p is a critical protein in these processes. In Tetrahymena loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Our data establish DIE5 has part of a conserved mechanism for DNA rearrangements within diverse ciliates. The findings will contribute to efforts by many investigators to elucidate the molecular mechanism of the precise and efficient DNA rearrangements in ciliates.
Publications
- Matsuda, A., Shieh, A.W.Y., Chalker, D. and Forney JD. 2010. The conjugation specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena. Eukaryotic Cell, 9, 1087-1099.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Research findings of current projects were presented at the Ciliate Molecular Biology FASEB Summer Conference at Saxtons River Vermont, an international meeting attracting approximately 150 scientists. The PI was an author on three abstracts "Using ohonlogs to search for transcriptional promoter elements in P. tetraurelia", James Forney. "Defective IES excision 5 (DIE5), a nuclear protein conserved between Paramecium tetraurelia and Tetrahymena thermophila, is required for somatic nuclear differentiation", A. Shieh, A. Matsuda, J. Forney, D. Chalker. "Programmed DNA elimination in Paramecium tetraurelia: molecular scenarios and actors", C. Baudry, S. Malinsky, A. Matsuda, M. Restituito, A. Kapusta, S. Rosa, J. Forney, E. Meyer, M. Betermier. The first two abstracts were presented as posters and the third as a talk by M. Betermier (Centre de Genetique Moleculaire, Gif-sur-Yvette, France). Experiments on the last two topics are coming to a close and manuscripts are in various stages of preparation. The investigator also mentored four undergraduate students in research during the spring semester. One is now attending graduate school and another is enrolled in medical school, a third is applying to doctor of pharmacy programs. Professor Forney was a member of the external review team for the 2009 USDA, CREES sponsored review of the Virginia Tech Department of Biochemistry. PARTICIPANTS: Professor Doug Chalker at Washington University at St. Louis, Department of Biology has been a collaborator on one previous project and offered training in Tetrahymena molecular genetics during Professor Forney's sabbatical. Two graduate students began work on the project during this period. Amjad Nasir started the biochemistry graduate program in 2008 and joined the project in May of 2009. Qianyi Yang entered the biochemistry graduate program in August 2009 and joined the Forney lab directly. She is supported in part through a China Scholarship that will continue for a period of four years. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The investigator started a six-month sabbatical at Washington University in St. Louis in July 2009. The goal was to generate Tetrahymena thermophila cell lines that have complete deletions of two genes in the SUMOylation pathway, SUMO and UBA2. SUMO (Small Ubiquitin-like MOdifier) is a small protein that can be covalently attached to specific target proteins to alter their function. UBA2 is an enzyme required for the first step in the SUMO pathway. The work is an effort to pursue detailed investigations of our observation in Paramecium tetraurelia that UBA2 and SUMO are required for formation of the somatic macronucleus but not for vegetative cell division. Cell lines with deletions of the macronuclear copies of each gene have been generated. In addition, cell lines with a micronuclear (germline) deletion of UBA2 have been constructed. These lines will be used to establish whether UBA2 and SUMO are essential for vegetative growth, and then to introduce tagged copies of UBA2 and SUMO for functional analysis in the next step of the project.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We have continued our research investigations in two different areas; the analysis of precise and efficient DNA splicing events that occur during the formation of the cilate macronuclear genome and the regulation of mRNA stability in Leshmania parasites. An antibody was developed against the newly discovered Die5p protein in Paramecium. This protein is required for the DNA splicing events that occur in the developing macronucleus. The antibody will be used to detect Die5p in future studies. The student who performed this work successfully defended her M.S. thesis in 2008. Other activities included mentoring four undergraduate students in independent research projects in my laboratory and providing academic advising for ten undergraduate biochemistry students. I presented work at the GRDE Paramecium Genomics meeting in Potzberg Germany "Comparative genomics within a single species: using ohnologs to search for transcriptional promoter elements in P. tetraurelia" James Forney. The audience was a group of approximately 40 international scientists who work on Paramecium. PARTICIPANTS: James Forney, PI; Stephanie Mowery, graduate student; Michael Ku, Research Assistant Four undergraduates have received mentoring on independent research projects during the reporting period; Emily Sturm, Kara Levell, Seung hee Choi, Stephany Suparno. TARGET AUDIENCES: Molecular biologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have expanded our work on differential mRNA stability in Leshmania to demonstrate that a previously discovered nine-nucleotide sequence in the 3' untranslated region of a specific mRNA is present in multiple other mRNAs. All of these mRNAs are more abundant in the promastigote (insect) stage and less abundant in the amastigote (mammalian) stage of the life cycle. We found the nine-nucleotide sequence was conserved in the corresponding mRNAs from different Leishmania species. These results are particularly significant because in Leishmania all genes synthesize (transcribe) mRNA at the same rate so all differences in mRNA abundance are the result of different post-transcriptional events such as degradation. These findings will aid the search for additional mRNAs and proteins that are required for the Leishmania life cycle.
Publications
- Holzer, T.R., Mishra, K.K., LeBowitz, J.H. and Forney, J.D. (2008). Coordinate regulation of a family of promastigote-enriched mRNAs by the 3' UTR PRE element in Leishmania mexicana. Molecular and Biochemical Parasitology 157: 54-64.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Research Presentation at the Ciliate Molecular Biology Meeting (FASEB Summer Conference at Tucson, AZ. July 21-26, 2007). "Identification of a Novel Nuclear Protein Required for DNA Elimination in Diverse Ciliates" Matsuda, A, Rexer C, Chalker D, and Forney J.
PARTICIPANTS: James Forney PI; Michael Ku, Research Assistant; Stephanie Mowery, graduate student
TARGET AUDIENCES: Life Scientists
PROJECT MODIFICATIONS: Plans to perform genome wide microarrys for analysis of gene expression have not been possible due to lack of funds. These experiments will be deferred. The data will be available after publication from investigators in Europe.
Impacts
We continued our analysis of the precise and efficient DNA splicing events that occur during the formation of the ciliate macronuclear genome. Paramecium micronuclear chromosomes contain thousands of internal eliminated sequences (IESs) that are removed as the macronuclear genome is formed during sexual reproduction. This process most likely evolved as a mechanism to protect the transcribed genome (macronucleus) from transposable elements and viruses that insert into the germline genome (micronucleus). Our previous studies have defined cis-acting sequences that are required for the excision of these DNA elements. New studies are focused on the identification of proteins and pathways that are required for IES elimination. Using differential mRNA display we identified a Paramecium gene we call Defective IES Excision 5 (DIE5). This gene encodes a 199 amino acid protein that localizes to the developing
macronucleus. RNAi silencing of the DIE5 gene has no effect on vegetative cell division but prevents formation of a functional macronucleus during mating. IES excision does not occur in the developing macronucleus, but DNA amplification is normal. In addition, we have shown the disrupting the DIE5 gene homolog in another ciliate, Tetrahymena thermophila, prevents IES excision also. These results suggest that DIE5 is an evolutionarily conserved gene that is required for the developmentally regulated DNA rearrangements in ciliates. Comparative studies between these two divergent ciliates should reveal common features of the DNA excision pathway.
Publications
- Matsuda A. and Forney J.D. 2006. The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia. Eukaryot. Cell 5(5), 805-815.
- Cohen-Freue G., Holzer T., Forney J.D. and McMaster W.R. 2007. Global gene expression in Leishmania. Int. J. Parasitol. 37: 1077-1086.
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Progress 10/01/05 to 09/30/06
Outputs
We have continued our analysis of the precise and efficient DNA splicing events that occur during the formation of the ciliate macronuclear genome. Paramecium micronuclear chromosomes contain thousands of internal eliminated sequences (IESs) that are removed as the macronuclear genome is formed during sexual reproduction. This process most likely evolved as a mechanism to protect the transcribed genome (macronucleus) from transposable elements and viruses that insert into the germline genome (micronucleus). Our previous studies have defined cis-acting sequences that are required for the excision of these DNA elements. New studies are focused on the identification of proteins and pathways that are required for IES elimination. Using differential mRNA display we identified a Paramecium gene encoding a homolog of ubiquitin activating enzyme 2 (UBA2) that is upregulated during macronuclear development. UBA2 is a component of the small ubiquitin-related modifier (SUMO)
pathway that modifies target proteins by covalent addition of SUMO, a small conserved peptide. Silencing the UBA2 gene using RNA interference (RNAi) had little effect on cell survival or fission rates during vegetative growth but prevented the formation of a functional macronucleus after mating. Detailed analysis revealed that disrupting UBA2 or SUMO gene expression prevents IES excision but allows normal amplification of the developing macronucleus. Further, we showed that UBA2 and SUMO are upregulated during macronuclear development in Tetrahymena thermophila. 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.
Impacts
Single-celled organisms called ciliates have a remarkable ability to reorganize their genome during sexual reproduction. This process most likely evolved as a mechanism to remove infectious DNA elements that insert into the genome of nearly all organisms including humans. Rather than suppressing the spread of DNA insertions, ciliates have perfected a method of making a second genome, called the macronucleus, which has been cleaned of mobile DNA elements. The mechanism that is used to identify and precisely remove the elements is not fully understood. We discovered that a conserved biochemical pathway called SUMO is required for elimination of these unwanted DNA elements from the macronuclear genome. Although the practical value of this information cannot be easily predicted, a complete understanding of the mechanism could be used to eliminate specific regions of DNA from a complex genome, a technique that could be useful in biotechnology or medicine.
Publications
- Matusda A. and Forney J.D. 2005. Analysis of Paramecium tetraurelia A-51 Surface Antigen Gene Mutants Reveals Positive-Feedback Mechanisms for Maintenance of Expression and Temperature-Induced Activation. Eukaryot. Cell 4(10):1613-1619.
- Adhiambo C., Forney J.D., Asai D.J. and LeBowitz J.H. 2005. The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions. Mol. Biochem. Parasitol. 143(2):216-225.
- Matsuda, A. and Forney, JD. 2006. The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia. Eukaryotic Cell 5:806- 815.
- Holzer T.R., McMaster W.R., Forney J.D. 2006. Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Mol. Biochem. Parasitol. 146(2):198-218.
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Progress 10/01/04 to 09/30/05
Outputs
The excision of short segments of DNA called 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 for 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 sequences from many IESs from P. tetraurelia reveals that there is a strong bias against particular nucelotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations and others correlate with unusual features of excision.
Impacts
The elimination of DNA from the developing macronucleus in ciliated protozoa most likely evolved as a mechanism to protect the transcriptionally active macronucleus from invading DNA including transposable elements and retro viruses. The results of our recent study suggests that little additional understanding will be gained by isolating mutant cells lines that are defective in IES excision. There are few single nucleotide mutations that prevent excision and they are clustered near the termini of the IES. A more productive approach might be a comparative sequence analysis of the same IES in different strains and species. This could reveal additional sequences in the excised region that are important for DNA removal.
Publications
- Matsuda, A, Mayer, KM and Forney JD. 2004. Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia. J. Eukaryotic Microbiology 51:664-669.
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Progress 10/01/03 to 09/29/04
Outputs
We have discovered that the (Small Ubiquitin-like Modifier)SUMO protein modification pathway is required for macronuclear development in the ciliated protozoan Paramecium. Our work has relied on the recent release of the complete DNA sequence of the Paramecium macronuclear genome. In addition, we utilized RNA interference (RNAi) as an effective method to inhibit gene expression in Paramecium. Differential mRNA display was used to compare mRNA samples from 8 hour conjugating cells, cells in log growth and starved cells. Several candidate genes showed increased expression during conjugation and one, a Paramecium homologue of yeast UBA2, was selected for further analysis. UBA2 is a SUMO (Small Ubiquitin-like Modifier) activating enzyme required for modification of proteins in the SUMO pathway. The mRNA transcript for Paramecium UBA2 is up-regulated eight hours after the start of conjugation. This is roughly the time that major DNA rearrangements occur during formation of
the macronuclear genome. A green fluorescent protein (GFP) fusion with UBA2 localizes to the developing macronucleus (anlagen) even though there are fragments of the old macronucleus located in the same cytoplasm. RNAi against Paramecium UBA2 prevents macronuclear development including excision of internal eliminated sequences (IESs). Cells that survive conjugation after RNAi treatment regenerate a macronucleus from the fragments of the old macronucleus. There is no effect (or only a minor negative effect) on vegetative cell growth. Despite the eventual failure of macronuclear development, DNA amplification appears normal in anlagen at the second cell division. Blast searches of the Paramecium genome revealed 3 genes encoding SUMO protein, each gene is greater than 95% identical to each other. Northern hybridization analysis showed that SUMO expression is regulated and peak mRNA concentrations occur four hours after the start of conjugation. RNAi against Paramecium SUMO gave the same
phenotype as RNAi against Uba2. We believe that the SUMO pathway is required to modify key proteins involved in IES excision, most likely by directing the proteins into the macronucleus. Our results provide an unusual example of developmental regulation of the SUMO pathway.
Impacts
We believe that many of the DNA rearrangements that occur during the formation of the ciliate macronuclear genome have evolved to protect the transcriptionally active nucleus from invading DNA including transposable elements and retro viruses. Our discovery that the SUMO pathway is activated suggests several approaches to identify important proteins (or RNAs) that are involved in these rearrangements. Analysis of the biochemistry of DNA elimination could lead to new tools for biotechnology that involve the precise manipulation of DNA in eukaryotic cells.
Publications
- Malave TM and Forney J. 2004. Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila. Gene 326, 97-105.
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Progress 10/01/02 to 09/30/03
Outputs
In addition to our studies on DNA rearrangements during formation of the macronuclear genome, we have started to investigate general biochemical pathways that may be altered in the cell during the formation of the macronucleus. Comparisons of messenger RNAs from vegetative and mating Tetrahymena thermophila, led to the discovery of a gene encoding a developmentally regulated translation elongation factor 2 (eEF2). 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 the ciliated protozoanTetrahymena 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 hours 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) there are differences in key regulatory regions of EFR. For example, the conserved threonine residue that is phosphorylated in eEF2 from yeast and other organisms is altered to a methionine in EFR. This change is predicted to result in a loss of regulation by phosphorylation in EFR. The results 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. Current research is focused on detailed studies of the EF2 and EFR proteins and their regulation.
Impacts
Protein translation is a fundamental process in all organisms. The regulation of translation has been studied in yeast and mammals, but our investigations suggest that different mechanisms may operate in Tetrahymena and other protozoa. The insights gained from future studies could impact agriculture and medicine since many protozoa are parasites that infect domesticated animals and humans.
Publications
- Free-living and parasitic ciliates. T. G. Clark and J.D. Forney. 2003. In Antigenic Variation eds. A. Craig and A. Scherf. Academic Press. London.
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Progress 10/01/01 to 09/30/02
Outputs
The ciliated protozoan Paramecium contains two functionally and morphologically distinct nuclei within a single cell. The non-transcribed micronuclei have a complete copy of the genome that is used to create the transcriptionally active macronucleus. During the formation of the macronuclear genome, more than 10,000 short DNA elements are eliminated during a 8-10 hour period. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a common feature of some types of DNA transposable elements. In fact, it has been proposed that IESs are evolutionarily related to transposable elements. Previously we developed an in vivo assay to analyze IES excision and found that DNA outside the element is required for IES excision (Ku, M., K. Mayer and J.D. Forney. 2000. Mol. Cell. Biol. 20:8390-8396). We have continued detailed analysis of these flanking sequences and find that nucleotides directly adjacent to the TA dinucleotides are critical as well as
those in more distant regions. These results are not consistent with known requirements of transposable element excision and therefore we believe that the mechanism for excision is substantially different for the two types of DNA elements. Information about functionally important sequences will be used to identify the enzymes required for IES excision.
Impacts
Understanding the details of highly efficient and accurate DNA excision could have important impacts in the area of biotechnology and human health.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
Protein translation is typically described by three basic steps called initiation, elongation and termination. Initiation is the most common regulated step, but some examples of alterations in elongation rates are known. Elongation factor-2 (EF-2) is required for the elongation step in all eukaryotic cells but usually there is a single EF-2 present in an organism and it is used under all conditions. We have cloned and sequenced a gene called EFR (elongation factor-2 related) that encodes a developmentally regulated EF-2 in Tetrahymena thermophila. The EFR mRNA is present only during sexual reproduction, when extensive DNA rearrangements occurr in the developing macronucleus. We have also cloned and sequenced the standard EF-2 that is expressed in vegetative cells. Comparison of EF-2 and EFR protein structure and function will be used to evaluate the role of elongation factor-2 and the function of EFR during sexual reproduction. We speculate that EFR regulates the
translation of proteins involved in genome alterations.
Impacts
Protein translation occurs in all living organisms and understanding translation elongation may have important impacts on human and animal health as well as biotechnology.
Publications
- No publications reported this period
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Progress 10/01/99 to 09/30/00
Outputs
Each Paramecium tetraurelia cell contains two non-transcribed diploid micronuclei and a transcriptionally active polyploid macronucleus. The micronuclear DNA contains more than 10,000 short DNA elements that are removed during the formation of the macronucleus. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA splicing events. This assay is being used to determine which sequences regulate DNA excision of normal IESs. Surprisingly, we have found that in addition to the normal IES another segment of DNA located in the plasmid is eliminated. This excision event is not dependent on TA dinucleotide repeats and unlike normal excision there is heterogeneity in the splice junction site. We are testing the possibility that placement of the normal IES adjacent to the plasmid sequences causes DNA elimination.
These data are suggesting new interpretations for the mechanism and role of DNA excision in ciliated protozoa.
Impacts
The project consists of basic research that may have important impacts on human and animal health as well as biotechnology.
Publications
- Ku, M. K. Mayer and J. D. Forney. 2000. Developmentally regulated excision of a 28 base pair sequence from the Paramecium genome requires flanking DNA. Mol. Cell. Biol. 20: 8390-8396.
- Thai, K. and J. D. Forney. 1999. Evidence of transcriptional self-regulation of variable surface antigens in Paramecium tetraurelia. Gene Expression 8: 263-272.
- Thai, K. and J. D. Forney. 2000. Analysis of the conserved cysteine periodicity of Paramecium variable surface antigens. J. Eukaryot. Microbiol. 47: 242-248.
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Progress 10/01/98 to 09/30/99
Outputs
Each Paramecium tetraurelia cell contains two non-transcribed diploid micronuclei and a transcriptionally active polyploid macronucleus. The micronuclear DNA contains more than 10,000 short DNA elements that are removed during the formation of the macronucleus. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA splicing events. The microinjection of a cloned IES into mating cells results in accurately spliced products and the transformed cells maintain the injected DNA as extrachromosomal molecules at high copy number. A series of deletions flanking one side of a 28 bp IES were constructed and analyzed with the in vivo assay. We find that removal of all wild type sequences adjacent to the TA results in complete failure of excision. In contrast, nucleotide mutations within the middle of the 28 bp IES
do not prevent excision. Based upon these results are developing a model in which perfect inverted repeats located directly adjacent to and flanking the IES play a functional role in DNA excision.
Impacts
The project consists of basic research that may have important impacts on human and animal health as well as biotechnology.
Publications
- Mayer, K.M. and J. D. Forney. 1999. A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome. Genetics 151: 597-604.
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Progress 10/01/97 to 09/30/98
Outputs
Our investigations of internal eliminated sequences (IESs) have continued. A total of four mutants that fail to excise a specific IES from the A surface antigen gene have been isolated. Each of the mutations is located within the inverted terminal repeat region of the IES and this supports the proposed evolutionary relationship between Paramecium IESs and the mariner/Tc1 class of transposable elements. The nucleotide changes completely eliminate any detectable IES excision in vivo. An important advance is our development an in vivo assay for Paramecium IES excision. We have already shown that the procedure correctly performs the normal DNA splicing reaction. This technique will allow us to identify the critical sequences required for excision of IESs.
Impacts
(N/A)
Publications
- Mayer, K. M. and Forney, J.D.: 1998. A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome. Genetics: in press.
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Progress 10/01/96 to 09/30/97
Outputs
Our investigations of internal eliminated sequences (IESs) have continued. Two mutant cell lines defective in IES removal have defined nucleotides functionally important for excision. These nucleotides are located within the inverted terminal repeat region of each IES and support the evolutionary relationship between Paramecium IESs and the mariner/Tc1 class of transposable elements. The nucleotide changes completely eliminate any detectable IES excision in vivo. In addition, the mutants have demonstrated that a small (28 or 29 bp) IES can be located inside of a larger IES. Screens for additional mutants as well as suppressors of our first mutation are now underway. Much of our effort now is directed toward developing in vivo or in vitro assays for Paramecium IES excision. Encouraging preliminary data have been obtained.
Impacts
(N/A)
Publications
- Mayer, K. K., Mikami, K. and Forney, J. D. 1998. A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements. Genetics 148, 1-11.
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Progress 10/01/95 to 09/30/96
Outputs
A single homozygous cell line of the protozoan Paramecium can express at least 11 different variable surface proteins, yet only a single type is found on the surface at any one time. The 51A and 51B surface protein genes are regulated at the level of transcription. The construction and analysis of chimeric genes containing portions of 51A and 51B genes has demonstrated that the upstream region is not sufficient to control mutually exclusive transcription. Surprisingly, a portion of the 5# coding region controls transcription. The transcription of most protein coding genes in eukaryotic cells is regulated by sequences upstream of the coding region. These results suggest that an unusual mechanism of transcriptional control may operate on these genes. During the past year we have shown that other parts of the coding region (e.g. the 3# end of the gene) do not affect transcription, and we have narrowed the DNA sequences required for transcription to the first 450 bp of
the coding region. The high sequence identity between the 51A and 51B genes in this region (ca. 80%) makes it likely that the regulatory sequences can be identified quickly.
Impacts
(N/A)
Publications
- Leeck, C., and Forney, J. 1996. The 5[ coding region of Paramecium surface antigen genes controls mutually exclusive transcription. J. Biol. Chem. Proc. Natl. Acad. Sci. 93:2838-2843.
- Forney, J.D., Yantiri, F., and Mikami, K. 1996. Developmentally controlled rearrangement of surface protein genes in Paramecium tetraurelia. J. Eukaryotic Microbiology 43:462-467.
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Progress 10/01/94 to 09/30/95
Outputs
A single homozygous cell line of the protozoan Paramecium can express at least 11 different variable surface proteins, yet only a single type is found on the surface at any one time. The 51A and 51B surface protein genes are regulated at the level of transcription. When the 51A and 51B genes are co-injected into an A-B- mutant, the 51A antigen is dominant at 27#C just as in wild type cells. We have utilized this cotransformation system to experimentally determine that 273 base pairs of DNA upstream of the 51A gene is sufficient to allow the dominant expression of 51A. The construction and analysis of chimeric genes containing proteins of 51A and 51B has demonstrated that the upstream region is not sufficient to control mutually exclusive transcription. Surprisingly, sequences within the 5# coding region control transcription. These results suggest that an unusual mechanism of transcriptional control may operate on these genes. The details of this regulatory pathway are
being investigated.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
All ciliated protozoans including Paramecium have a large transcriptionally active macronucleus and a non-transcribed, diploid micronucleus. During sexual reproduction the old macronucleus is destroyed and a new one is made from the DNA in the micronucleus. We previously showed that the correct incorporation of both the A and B type surface protein genes is dependent on copies of each gene in the old macronucleus. If the A gene is missing from the old macronucleus, then the new macronucleus forms a telomere at the 5' end of the A gene rather than 20 kb downstream of the gene. The molecular explanation for this effect is not known. During the past year we cloned and sequenced the micronuclear version of the B surface protein gene. Comparison with the macronuclear B gene sequence showed three micronuclear specific sequences called IESs (Internal Eliminated Sequences) that are excised during macronuclear development. Each excised sequence is bounded by the dinucleotide
5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A gene shows that the sequences in the IESs are not conserved. Using macronuclear transformation, we showed that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.
Impacts
(N/A)
Publications
- YOU, Y., SCOTT, J., and FORNEY, J. 1994. The role of macronuclear DNA sequences in the permanent rescue of a non-Mendelian mutation in Paramecium tetraurelia. Genetics 136:1319-1324.
- ASAI, D., BECKWITH, S.M., KANDL, K.A., KEATING, H.H., TJANDRA, H., and FORNEY, J.D. 1994. The dynein genes of Paramecium tetraurelia: Sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms.
- SCOTT, J., LEECK, C., MIKAMI, M., and FORNEY, J. 1994. Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia. Molecular and Cellular Biology 14:2479-2484.
- LEECK, C., and FORNEY, J. 1994. The upstream region is required but not sufficient to control mutually exclusive expression of Paramecium surface antigen genes. J. Biol. Chem., in press.
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Progress 10/01/92 to 09/30/93
Outputs
All ciliated protozoans including Paramecium have a large transcriptionally active macronucleus and a non-transcribed, diploid micronucleus. During sexual reproduction the old macronucleus is destroyed and a new one is made from the DNA in the micronucleus. We previously showed that the correct incorporation of the A type surface protein gene into the macronucleus of Paramecium is controlled by the A gene in the old macronucleus. For example, a strain that has a normal A gene in the micronucleus but a deletion of the macronuclear A gene will not include the A gene in the next macronucleus. To determine if this is a general feature that controls the processing of other genes in Paramecium, we constructed a strain that contains a normal micronucleus but has a deletion of the A gene and the B gene in the macronucleus. In this strain neither the A nor the B gene are incorporated into the next macronucleus. If the A gene is transformed into the macronucleus of this strain
it allows the correct incorporation of the A gene but not B into the next macronucleus. Conversely, transformation with the B gene rescues B but not A after formation of a new macronucleus. These results indicate that not only is the processing of the B gene controlled by the presence of the B gene in the old macronucleus but that the mechanism is gene specific. As part of our study of DNA rearrangements we have cloned and sequenced the micronuclear copy of the B gene.
Impacts
(N/A)
Publications
- FORNEY, J., and RODKEY, K. 1992. A repetitive DNA sequence in Paramecium macronuclei is related to the B subunit of G proteins. Nucleic Acids Res. 20:5397-5402.
- SCOTT, J., LEECK, C., and FORNEY, J. 1993. Molecular and genetic analyses of the B type surface protein gene from Paramecium tetraurelia. Genetics Society of America 133:189-198.
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Progress 10/01/91 to 09/30/92
Outputs
As part of our studies on the mutually exclusive expression of variable surface proteins in Paramecium, we have determined the entire sequence of the B type variable surface protein gene from Paramecium tetraurelia and a genetic analysis of three B- mutant cell lines has been completed. Using this information it has been possible to construct a recombinant gene that contains the 5' upstream region of the B gene attached to the coding region of the A type variable surface protein gene. This gene has been expressed in Paramecium and is now being analyzed for its effect on mutual exclusion. We previously showed that the DNA rearrangements which result in the formation of the A type surface protein gene in Paramecium are controlled by the macronuclear copy of the A gene. Our recent work suggests that a region of the macronuclear A gene that contains three tandem repeats of 210 bp is important in controlling this processing event. By marking this DNA with a unique
restriction site we have shown that the macronuclear DNA is not used directly as a template for the formation of the new macronuclear copy of the A gene.
Impacts
(N/A)
Publications
- YOU,Y, AUFDERHEDIED,K, MORAND,J, RODKEY,K, AND FORNEY,J. 1991. Macromolecular transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium. Mol. Cell. Biol. 11:1133-1137.
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Progress 10/01/90 to 09/30/91
Outputs
We have continued our studies on gene expression and DNA rearrangements in Paramecium. Our results during the past year can be summarized as follows: 1. We have cloned and partially sequenced the gene encoding the type B variable surface protein from Paramecium tetraurelia, stock 51. The B gene is located near the end of a macronuclear DNA fragment and as expected, the sequence of this gene is very similar to the A type surface protein. Due to the availability of B- mutant strains (J. Preer, Indiana University) it is now possible to construct and analyze a series of chimeric surface protein genes containing portions of both the A and B genes. These recombinant genes will be used to determine the mechanisms important in the mutually exclusive expression of variable surface proteins. 2. A DNA sequence with striking similarity to the beta subunit of trimeric G proteins has been identified in Paramaceium. This sequence is repeated throughout the Paramecium macronuclear
genome. A polyadenylated RNA homologous to these sequences has been identified, thus a protein is most likely produced from this sequence. Other proteins similar to the beta subunit of G proteins are involved in signal transduction and mRNA splicing. The role of the protein in Paramecium will be investigated.
Impacts
(N/A)
Publications
- YOU, Y., AUFDERHEDIED, K., MORAND J., RODKEY, K., and FORNEY, J. 1991. Macromolecular transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium. Mol. Cell. Biol. 11:1133-1137.
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Progress 10/01/89 to 09/30/90
Outputs
Our research on the control of DNA rearrangements in Paramecium has led to the identification of an 8.8 kb EcoRI fragment of DNA that can control the correct formation of the macronuclear DNA containing the A type variable surface protein gene. Previous research has demonstrated that this DNA rearrangement is controlled by a molecule that is made in the macronucleus and acts through the cytoplasm. Since the 8.8 kb DNA fragment does not contain the entire A surface protein gene, we are now searching for alternative gene products that could be made from these sequences. We have determined the complete sequence of the 8.8 kb EcoRI DNA fragement in order to facilitate this analysis and the subsequent manipulation of these sequences. The entire coding sequence of the C type variable surface protein gene has been determined. Comparison with the A surface protein gene indictes that despite the poor similarity between the primary sequences, the spacing of the cysteine
residues in the deduced amino acid sequence in strongly conserved. This sequence information will be used to construct chimeric surface protein genes that should be useful in understanding the control of variable surface protein expression in Paramecium.
Impacts
(N/A)
Publications
- PHAN, H.L., FORNEY, J. and BLACKBURN, E.H. 1989. Analysis of Paramecium macronuclear DNA using pulsed field gel electrophoresis. Journal of Protozoology 36:318-324.
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