scholarly journals Centromeric Histone H3 Is Essential for Vegetative Cell Division and for DNA Elimination during Conjugation in Tetrahymena thermophila

2006 ◽  
Vol 26 (12) ◽  
pp. 4499-4510 ◽  
Author(s):  
Bowen Cui ◽  
Martin A. Gorovsky
Keyword(s):  
Vegetative Cell ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
S Phase ◽  
Dna Elimination ◽  
Spherical Structures ◽  
Dna Loss ◽  
Green Fluorescent ◽  
And Behavior

ABSTRACT The Tetrahymena thermophila CNA1 gene encodes the centromeric H3, Cna1p. Green fluorescent protein (GFP)-tagged Cna1p localizes in micronuclei in dots whose number and behavior during mitosis and conjugation are consistent with centromeres. During interphase, Cna1p-GFP localizes in peripheral dots, suggesting centromeres are associated with the nuclear envelope. Newly synthesized Cna1p-GFP enters micronuclei in mitosis and accumulates in the nucleoplasm. Its deposition at centromeres starts at early S phase and continues through most of S phase. CNA1 is required for vegetative cell growth. Knockdown of CNA1 genes in the somatic macronucleus results in micronuclear DNA loss and delayed chromosome segregation during mitosis. During conjugation, Cna1p-GFP disappears from the centromeres in the developing macronucleus, consistent with centromeric sequences being internal eliminated sequences. Surprisingly, zygotic CNA1 is required for efficient elimination of germ line-specific sequences during development of the new macronuclei but not for the RNA interference pathway, through which sequences are targeted for elimination. Zygotically expressed Cna1p localizes in the spherical structures in which the later stages of DNA elimination occur, and these structures cannot be formed in the absence of zygotic CNA1, suggesting that, in addition to functioning in centromeres, Cna1p may also play a role in organizing the formation of the DNA elimination structures.

scholarly journals Deposition and Function of Histone H3 Variants in Tetrahymena thermophila

2006 ◽  
Vol 26 (20) ◽  
pp. 7719-7730 ◽  
Author(s):  
Bowen Cui ◽  
Yifan Liu ◽  
Martin A. Gorovsky
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Histone H3 ◽  
Repair Synthesis ◽  
Nucleosome Density ◽  
Sexual Progeny ◽  
Green Fluorescent ◽  
Dna Repair Synthesis ◽  
And Function

ABSTRACT In Tetrahymena, HHT1 and HHT2 genes encode the same major histone H3; HHT3 and HHT4 encode similar minor H3 variants (H3s), H3.3 and H3.4. Green fluorescent protein (GFP)-tagged H3 is deposited onto chromatin through a DNA replication-coupled (RC) pathway. GFP-tagged H3.3 and H3.4 can be deposited both by a transcription-associated, replication-independent (RI) pathway and also weakly by an RC pathway. Although both types of H3s can be deposited by the RC pathway, DNA repair synthesis associated with meiotic recombination utilizes H3 specifically. The regions distinguishing H3 and H3.3 for their deposition pathways were identified. RC major H3 is not essential. Cells can grow without major H3 if the minor H3s are expressed at high levels. Surprisingly, cells lacking RI H3s are also viable and maintain normal nucleosome density at a highly transcribed region. The RC H3 is not detectably deposited by the RI pathway, even when there are no RI H3s available, indicating that transcription-associated RI H3 deposition is not essential for transcription. Minor H3s are also required to produce viable sexual progeny and play an unexpected role in the germ line micronuclei late in conjugation that is unrelated to transcription.

scholarly journals Lia1p, a Novel Protein Required during Nuclear Differentiation for Genome-Wide DNA Rearrangements in Tetrahymena thermophila

2007 ◽  
Vol 6 (8) ◽  
pp. 1320-1329 ◽  
Author(s):  
Charles H. Rexer ◽  
Douglas L. Chalker
Keyword(s):  
Genome Rearrangement ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Chromatin Modification ◽  
Striking Similarity ◽  
Macronuclear Development ◽  
Genome Wide ◽  
Green Fluorescent ◽  
Novel Protein

ABSTRACT Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (ΔLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome.

scholarly journals Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila

2015 ◽  
Vol 14 (12) ◽  
pp. 1240-1252 ◽  
Author(s):  
Qianyi Yang ◽  
Amjad M. Nasir ◽  
Robert S. Coyne ◽  
James D. Forney
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Fusion Gene ◽  
Life Cycles ◽  
Sexual Life ◽  
Proper Function ◽  
Content Type ◽  
Macronuclear Development ◽  
Green Fluorescent

ABSTRACT Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila , the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl 2 -dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena . Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing macronucleus.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 373
Author(s):  
M. Reichenbach ◽  
F. A. Habermann ◽  
H. D. Reichenbach ◽  
T. Guengoer ◽  
F. Weber ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Phosphoglycerate Kinase ◽  
Healthy Male ◽  
Transgenic Founder ◽  
Green Fluorescent

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

scholarly journals Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 445-453 ◽  
Author(s):  
Jiří Kalina ◽  
Filip Šenigl ◽  
Alena Mičáková ◽  
Jitka Mucksová ◽  
Jana Blažková ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Retroviral Vector ◽  
Male Germ Line ◽  
Testicular Cells ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Transgenic Chickens

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated γ-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.

scholarly journals Vacuolar Protein Sorting Protein 13A, TtVPS13A, Localizes to the Tetrahymena thermophila Phagosome Membrane and Is Required for Efficient Phagocytosis

2011 ◽  
Vol 10 (9) ◽  
pp. 1207-1218 ◽  
Author(s):  
Haresha S. Samaranayake ◽  
Ann E. Cowan ◽  
Lawrence A. Klobutcher
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Genetic Disorders ◽  
Protein Sorting ◽  
Mass Spectrometry Analysis ◽  
Vacuolar Protein Sorting ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Green Fluorescent ◽  
Vacuolar Protein

ABSTRACT Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990–2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.

scholarly journals ZFP100, a Component of the Active U7 snRNP Limiting for Histone Pre-mRNA Processing, Is Required for Entry into S Phase

2006 ◽  
Vol 26 (17) ◽  
pp. 6702-6712 ◽  
Author(s):  
Eric J. Wagner ◽  
William F. Marzluff
Keyword(s):  
Fluorescent Protein ◽  
S Phase ◽  
Mrna Processing ◽  
Limiting Factor ◽  
Stem Loop ◽  
Histone Mrnas ◽  
U7 Snrnp ◽  
Eukaryotic Mrnas ◽  
Green Fluorescent

ABSTRACT Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3′ end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3′ end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3′ end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G1, suggesting that active U7 snRNP is necessary to allow progression through G1 phase to S phase.

scholarly journals Two GW Repeat Proteins Interact with Tetrahymena thermophila Argonaute and Promote Genome Rearrangement

2009 ◽  
Vol 29 (18) ◽  
pp. 5020-5030 ◽  
Author(s):  
Janna Bednenko ◽  
Tomoko Noto ◽  
Leroi V. DeSouza ◽  
K. W. Michael Siu ◽  
Ronald E. Pearlman ◽  
...  
Keyword(s):  
Genome Rearrangement ◽  
Developmental Stages ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Nuclear Bodies ◽  
Interacting Proteins ◽  
Heterochromatin Formation ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Argonaute Family

ABSTRACT In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, ∼28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of ΔWAG1, ΔCnjB, and double ΔWAG1 ΔCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.

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