Gene Paragraphs at TGD Wiki
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The PDD1 gene encodes Pdd1p, an abundant protein whoseexpression is limited to the sexual phase of theTetrahymena life cycle. Somatic knockout cells lackingPdd1p during the early stages of conjugation andmacronuclear development exhibit defects in a varietyof developmental processes, including programmed DNAelimination, macronuclear genome endoduplication, andnuclear resorption. While Pdd1p is not required forvegetative growth, exconjugants derived from matings ofsomatic knockout cells are inviable. Originallyidentified during a screen for proteins upregulatedduring macronuclear development (which also led to thecloning of PDD2 and PDD3), the gene encoding p65(Pdd1p) was cloned and shown to encode a novel proteincomposed of three chromodomains. Methylated histonebinding activity has been demonstrated in vitro for onechromodomain of Pdd1p, specifically to methylatedlysine-9 residues of histone H3. This histonemodification is required for programmed DNAelimination, and like Pdd1p, these modified histonescolocalize with chromatin containing the DNA sequencesdestined for elimination. Distribution of Pdd1p in thecell over time follows a remarkable pattern that issuggestive of its major role in programmed DNAelimination: Pdd1p is initially restricted to the oldmacronucleus, then relocalizes to the developingmacronucleus when it is formed. Studies have longsuggested an epigenetic contribution from the parentalmacronucleus that specifies the elimination of specificDNA sequences. The timing of its localization and itsability to bind chromatin suggests Pdd1p is directlyinvolved in communicating this information to the newmacronucleus.
A proposed model for the mechanism of programmed DNAelimination in Tetrahymena is based on the timing ofexpression, cellular distribution, mutant phenotypes,and predicted functions of the protein and RNAcomponents involved. In this model, both strands of themicronuclear genome (or perhaps only the portionscontaining internal eliminated sequences) aretranscribed early in conjugation to produce largenon-genic, double-stranded RNAs. This transcription islikely performed by RNA Polymerase II, based on thelocalization of its subunit Rpb3p to the micronucleusduring this time. These transcripts pass to thecytoplasm where they are processed into short (~28nucleotide) scan RNAs (scnRNA) by the dicer-likeprotein Dcl1p, similar to the production of the smallinhibitory RNAs (siRNA) central to the RNA interference(RNAi) pathway of other eukaryotes. The scnRNAs complexwith Twi1p, a member of the PPD (PAZ and Piwi Domain)protein family, whose members are commonly involved inRNAi and related processes. The scnRNA/Twi1p complexesenter the old macronucleus, where scnRNAs homologous toDNA sequences found there are degraded. The remainingscnRNAs, comprised of micronuclear-restrictedsequences, are transferred to the developingmacronucleus. There, histone H3 proteins (Hht1p, Hht2p)that are bound to sections of the genome sharingidentity to the scnRNAs are methylated on lysine-9.This modification, which is often associated with theformation of heterochromatin, is recognized by one ormore of the chromodomains belonging to Pdd1p and Pdd3p.Regions of DNA associated with these modified histonesare eliminated from the developing macronuclear genome.
Cyclin-dependent kinases (cdks) are a family ofserine-threonine kinases that are involved in cellcycle control and cell division in eukaryotes. Cdks arecatalytic subunits that are activated by associationwith proteins called cyclins, forming cyclin-cdkcomplexes. Cdk kinase activity is regulated by cyclinbinding, phosphorylation and dephosphorylation, proteindegradation, protein-protein interactions with cdkinhibitors, and subcellular localization.
Tetrahymena thermophila Cdk1p shares homology with cdkhomologs from other eukaryotes. It contains 11catalytic domains characteristic of protein kinases,conserves all of the regulatory phosphorylation sitesfound in cdks, and has a slightly modifiedcyclin-binding PSTAIRE motif that is a hallmark ofcdks. The Tetrahymena thermophila Cdk1p was also foundto bind Saccharomyces cerevisiae p13suc1, a yeastcyclin.
The level of Cdk1p fluctuates over the vegetative cellcycle, correlating with its histone H1 kinase activity.Cdk1p is associated with the basal bodies of theciliary rows of the cell cortex and the oral apparatus.This localization, along with the phenotype of apartial CDK1 knockout phenotype of bent and buckledciliary rows, suggests that Cdk1p is involved incortical morphogenesis.
HEH2 is expressed during vegetative growth of T.thermophila and its protein product has been localizedto basal bodies. Interestingly, its protein product wasfound to have high sequence similarity to the humandisease gene KIAA1279. Human KIAA1279 was also found tohave homologs in fruit fly, frog, rat, mouse, bee,chicken, and Japanese puffer fish, but none inSaccharomyces cerevisiae. Although the function ofKIAA1279 is not yet known, evidence suggests thatKIAA1279 is important in the development of the entericand central nervous system (CNS). KIAA1279 wasexpressed in different parts of the adult CNS, andmutations in KIAA1279 were associated withGoldberg-Shprintzen syndrome (OMIM).
HEH2 appears to be located on the right arm ofmicronuclear chromosome 2 based on mapping REP6, alocus upstream of HEH2.
The HHO1 gene encodes the macronuclear linker histoneH1 protein; the MLH1 gene encodes a polyproteincomprising a set of four micronuclear linker histoneproteins (alpha, beta, gamma, and delta) unrelated toHho1p. Histone H1 and the MLH proteins are chromatinproteins that associate with the inter-nucleosomal(linker) DNA. T. thermophila has two nuclei, one ofwhich is transcriptionally active (the macronucleus)and one that is silent during most of the life cycle(the micronucleus). Furthermore, the macronucleusundergoes amitosis, whereas the micronucleus undergoestypical mitosis. The fact that Hho1p and MLH proteinsare found exclusively in the macronucleus andmicronucleus, respectively, has led to studies of theirfunction, or lack of function, in transcriptionregulation, mitosis, and amitosis. Surprisingly, anHHO1 knockout showed this gene to be non-essential; itsmain observable phenotype was an overall decondensationof macronuclear chromatin. MLH1 knockouts, which arealso viable, showed a similar phenotype in themicronucleus.
HHO1 knockouts show no global increase or decrease inthe amount of transcription in the cell; however, thesesame knockouts also show that Hho1p is important forthe transcriptional regulation of individual genes inresponse to stimuli, such as starvation. Thedifferential regulation of Hho1p by phosphorylationunder vegetative growth and starvation conditions hasbeen well studied. During vegetative growth, Hho1p isphosphorylated on five closely spaced residues,preventing it from interacting with chromatin, likelyby interfering with its ability to bind DNA. Underthese conditions, expression is increased for CDC2, ahomolog of the cyclin dependent kinases responsible forhistone H1 phosphorylation, possibly creating apositive feedback loop that promotes the cell cycle.During starvation conditions, Hho1p isdephosphorylated, allowing it to bind to chromatin.This stimulates the expression of some genes, includingngoA, and protease genes such as CYP1, while inhibitingexpression of other genes, such as CDC2. This decreasein CDC2 expression may be responsible for cell cyclearrest during starvation.
Proteins of the myosin superfamily are ATP-dependentmolecular motors that travel unidirectionally alongactin filaments. The myosin heavy chain proteins arecomprised of three domains: a head (motor) domainresponsible for ATP hydrolysis at the N-terminus, aneck (lever arm) region, and a C-terminal tail region.Thirteen predicted myosin heavy chain genes have beenidentified in the Tetrahymena genome and namedMYO1-MYO13. A phylogenetic analysis comparing thesepredicted proteins with the 19 previously identifiedmyosin classes suggests that Myo1p-Myo12p belong to apreviously undescribed class of myosins. This newfamily, designated Class XX, does not include Myo13p,which did not branch with this class or any of theother classes in the analysis. The neck and tailregions of the Tetrahymena myosins include a variety ofdomains characterized in other myosin classes, witheach protein containing one or more of the followingdomains: coiled-coil (which may support dimerization);IQ motif (binding sites for calmodulin orcalmodulin-like proteins); FERM domain (Four-point-oneprotein, Ezrin, Radixin, Moesin homology); and MyTH4domain (Myosin Tail Homology 4).
A recessive gene determining temperature-sensitivefission arrest was described in 1976 under the name"mo1". Around 1979, following the (then) newnomenclatural rules, it was re-named cdaA1 (CDA="celldivision arrest"). In the early 1980's, Y. Watanabe andhis associates made some remarkable findings reportedin 1986. Using 2D-gel electrophoresis, they found aprotein, which they called p85 (later renamed Cmb1p),which was localized to the oral apparatus and also toan apical filament ring and to structures (which turnedout to be basal-body couplets) located just posteriorto the division furrow. The equatorial localization wasobserved in cdaA1 homozygotes at the permissivetemperature (when division took place), but not after ashift to the restrictive temperature (when the divisionfurrow failed to develop). Based on these studies itwas naturally assumed that p85 was the protein productof the cdaA gene, especially as p85 differed slightlyin mobility in 2D-gels made from wild-type and cdaA1cell extracts. However, in 1999 the gene encoding p85was cloned. This yielded a big surprise: "The cdaA1 p85cDNA contained one open reading frame and its deducedamino acid sequence, cdaA1 p85, was completelyidentical to that of wild-type p85" (p. 116). Therewere some differences in the 3'UTR and 5' UTRs, butthey "do not affect the transcription and translationof the p85 gene, because the amounts of transcribedmRNA and translated protein of cdaA1 p85 wereequivalent to those of wild type p85" (p. 118). Theauthors conclude the Results section as follows: "Thus,we suppose that the difference in molecular weightbetween cdaA1 and wild-type p85 was caused by adisorder of post-translational modification mechanismsof p85 in cdaA1 cells." (p. 116). These resultsdemonstrate that p85 is likely not the product of thecdaA1 gene, and that the gene mutated in the cdaA1strain is more likely to be a protein responsible forthe post-translational modification of p85, which isaltered in the cdaA1 mutant. (Contributed by J.Frankel, University of Iowa, 2005)
THD1, a homolog of the Saccharomyces cerevisiae Rpd3p,a class I histone deacetylase (HDAC), is localized tothe macronucleus during vegetative growth, anddistributed to developing new macronuclei early intheir differentiation. Thd1p deacetylates all four corehistones in vitro. Thd1p is a 52kDa polypeptide in anHDAC complex of approximately 160 kDa.
Tetrahymena cells with reduced Thd1p expressionexhibited phenotypes indicative of loss of chromatinintegrity, such as DNA fragmentation and extrusion ofchromatin from the macronucleus, variable macronuclearsize and shape, enlarged nucleoli, and reducedphosphorylation of histone H1 from bulk chromatin.Macronuclei in THD1 knockdown cells also contained moreDNA, suggesting Thd1p may play a role in regulatingmacronuclear DNA content. The THD1 gene could not becompletely replaced by a disruption construct,suggesting that THD1 is an essential gene.
A macronuclear chromosome containing a fusion gene wascloned from the spirotrichous ciliate Oxytrichatrifallax. The gene encodes a single polypeptidecontaining homologs of two proteins that catalyzesequential steps in the formaldehyde detoxificationpathway in Saccharomyces cerevisiae. These two proteinsare formaldehyde dehydrogenase (FALDH) andS-formylglutathione hydrolase (SFGH); the fusion geneis called FSF1 (FALDH/SFGH Fusion 1). A similar genewas identified in the Tetrahymena thermophila genomesequence, and a T. thermophila EST sequenced from bothends showed that the fusion gene is expressed in thisspecies in vivo. FSF1 has not yet been identified inother ciliates, but a fusion of these two genes hasbeen identified in another group of protists, thediatoms. An EST from Phaeodactylum tricornutum and agene from the genome sequence of Thalassiosirapseudonana both encode a fusion of these two genes, butin the opposite orientation of the ciliate genes. Indiatoms, the SFGH domain is found N-terminal to theFALDH domain, suggesting that these two fusion genesevolved independently in ciliates and diatoms. Thediatom genes were named SFF1 to highlight thesedifferences.
Spo11p induces DSBs and at the same time triggers theelongation of meiotic nuclei (crescents) via anATR-dependent response in Tetrahymena. The crescentresembles the conserved bouquet arrangement and thefission yeast horsetail nucleus. It promotes meioticchromosome pairing. Thus, by nuclear elongation and theensuing close juxtapositioning of homologous chromosomeregions within the tubular nucleus, Spo11p ensures thatDSBs formed by its activity can be repaired byhomologous recombination.
HOP2B is a homolog of budding yeast HOmologous Pairing2. It is essential for vegetative growth. HOP2B has ameiosis-specific paralog in Tetrahymena (HOP2, HOP2A,TTHERM_00794620) which is the yeast HOP2 ortholog.
Knockout prevents SPO11-dependent elongation of meioticnuclei. Involved in the signaling of meiotic DSBs andother DNA damage.
MBD1 is a gene fusion of two genes involved in themethionine salvage pathway:methylthioribulose-1-phosphate dehydratase -mtnB; and1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase -mtnD. These enzymes catalyze non-consecutive steps inthe pathway. Interestingly the gene that codes for theintervening enzyme in the pathway, mtnC, is missingfrom the genome of Tetrahymena. Complementation testsin yeast were used to show that MBD1 from Tetrahymenais able to do in one step what yeast does in three,since it can rescue yeast knockouts of mtnB, mtnC, ormtnD (Salim, Negritto and Cavalcanti 2009).
The phospholipid flippase family of genes inTetrahymena contains 20 members, FLP1-FLP20. Preliminary studies show that many of these genes aredifferentially regulated in response to temperatureand/or the presence of a polycyclic aromatichydrocarbon (pyrene).
LIA4 is expressed exclusively during conjugation andLia4p localizes to developing macronuclei. It isrequired for completion of conjugation, DNArearrangement, chromosome breakage, and Pdd1 foci(dumposome) formation (Horrell SA and Chalker DL,unpublished data).
ABC3 shares homology with ABC transporter proteins fromother eukaryotes. It contains an ATP-binding domainthat hydrolyzes ATP in to provide energy for theprotein to translocate various molecules across abiological membrane. Paragraph by undergraduates at the Keck Science Department, Pitzer College
Solute Transport Facilitator 1 (STF1) is a protein fromthe major facilitator superfamily (MFS), one of thelargest families of membrane transports known, found inarchaea, bacteria, and eukaryotes. This protein ispredicted to have a MFS1 domain (E-value 1e-08), and islikely to transport small solutes through the membranethrough either uniport, symport, or antiport. Paragraphby: Lisette Espinosa and Charles McGregor(undergraduates), Keck Science Department, ClaremontMcKenna College
NHX1 is an integral membrane protein that shareshomology with other proteins containing a sodiumhydrogen exchanger domain involved in transportingsodium and hydrogen ions across the concentrationgradient between a cell and its surroundings. Functioning as an antiporter for sodium and hydrogenions, the exchange function characteristic of thisdomain is highly dependent on pH. Although the exactmolecular mechanisms responsible for this behavior arenot well understood, a prominent current inference isthat these exchanger proteins use ATP to transport ionsacross membranes. Paragraph by: Samuel Rubin and OwenFoster (undergraduates) Keck Science Deparment, Claremont Colleges
NHX2 belongs in the family of sodium-hydrogenexchangers that act as antiporrters that maintain thepH of actively metabolizing cells by controlling thebalance of sodium. THe antiporters have 10-12 regionson the N-terminus and a large cytoplasmic region on theC-terminus. The 10-12 transmembrane regions contain 2highly conserved regions and most of the regions shareidentities within the family , while the largecytoplasmic region is noted to have little similaritywith other members in the family. Paragraph by: ChrisFang and Deanna Liou (undergraduates) Keck ScienceDepartment, The Claremont Colleges
GTP4 has only one known functional domain and is amember of the sugar transporter family, a subset of themajor facilitator superfamily (E-value: 5.4e-36). Thisprotein appears to be responsible for transportingsugar across the plasma membrane in response to changesin the electrochemical gradient, specifically in sugaruptake. Paragraph by: Kathleen Beardsworth and KristenKeller (undergraduates), Keck Science Department,Claremont Colleges.
ABC2 shares homology with members of the ABC familytransporter proteins. This protein appears to have twotypes of domains. It is thought that the two domainsfunction together to bind ATP to transport substancessuch as glutathione, glucuronate, and sulfate acrossthe membrane. Using energy from ATP hydrolysis, bothdomains of ABC transporters facilitate the transport ofa wide variety of materials out of the cell. Paragraphby undergraduates from the Keck Science Department atClaremont McKenna and Scripps Colleges
Tetrahymena thermophila MAF4 is homologous withproteins from the WD40 superfamily, mostly membrane andflagellar associated proteins. It contains twodomains; the first domain (E-value=1.95E-5), in theclathrin family, indicates that the protein could havea membrane spanning domain due to repeatedalpha-helices. The second domain (E-value=7.46E-3) isa kinase complex, which indicates possible movementfunction, and correlates with the homologs beingflagellar associated proteins. It is possible thatthis protein is an integral membrane protein that has afunction in flagellar movement. Paragraph by: LaurenMitten and Rebecca Dutta (undergraduates), Keck ScienceDepartment, Scripps College
PKC2 shares homology with a number of Protein Kinase Crelated proteins in other ciliated prokaryotes. PKC2contains only one single domain across its entire 517amino acids. It conserves most of the protein kinasedomain, and contains a 241 amino acid region withunknown function. PKC2 appears to play a role inamplifying the message of signal transduction pathwaysby phosphorylating serine and threonine. This inducesa conformational change in a targeted protein andsubsequently leads to a cellular response. Paragraphby: Jacqueline Kroll and Rachel Brunetti(undergraduates), Keck Science Department, TheClaremont Colleges.
Tetrahymena thermophilia PMR1 (potential mRNAregulator) is homologous to proteins from the LRR_RIdomain. The domain suggests that the protein is similarto Leucine rich repeat, ribonucleus inhibiter (Evalue =7.51 × 〖10〗^(-6)). The leucine rich protein mayform tight complexes with a certain ribonuclease, ormay be involved in other protein-protein interactions.It is possible that the PMR plays a role in regulatingthe lifetime of RNA like the ribonuclease inhibitor. Paragraph provided by undergraduates at the KeckScience Department of Claremont McKenna, Pitzer, andScripps Colleges.
Tetrahymena thermophile TIT1 is homologous withproteins from a cation channel family. It contains onedomain: the ion transport protein (Evalue: 1x10⁻⁸)whose function is to selectively transport ions throughthe membrane. It is possible that this protein is anintegral membrane protein that has a critical functionin facilitating ion transport. Transmembrane IonChannel Family proteins that are found in eukaryotestend to have up to four additional transmembranehelices. These additional helices help explain thephysical properties of the protein. We have determinedthat TIT1 has a sequence that consists of 604 aminoacids, a relatively lengthy sequence. Paragraph by:Kristiana Kim and Will Su(undergraduates), Keck ScienceDepartment, Scripps College, Claremont McKenna College
Tetrahymena thermophile TIT1 is homologous withproteins from a cation channel family. It contains onedomain: the ion transport protein (Evalue: 1x10⁻⁸)whose function is to selectively transport ions throughthe membrane. It is possible that this protein is anintegral membrane protein that has a critical functionin facilitating ion transport. Transmembrane IonChannel Family proteins that are found in eukaryotestend to have up to four additional transmembranehelices. These additional helices help explain thephysical properties of the protein. We have determinedthat TIT1 has a sequence that consists of 604 aminoacids, a relatively lengthy sequence. Paragraph by:Kristiana Kim and Will Su(undergraduates), Keck ScienceDepartment, Scripps College, Claremont McKennaCollege
Tetrahymena thermophila gene CUT1 (Common UnknownTrans-membrane) protein is a trans-membrane proteinwith unknown function. This gene is closely related toa similar gene in Ichthyophthirius multifiliis. Itcontains two CLPTM1 functional domains, one of which ismore strongly conserved than the other. When expressedin the human genome, this domain is known to be linkedto cleft lip and palate. This family (CLPTM 1) is oneof many eukaryotic, trans membrane protein sequencesthat are linked to cleft lip and palate; however,specific function is unknown. Paragraph provided byKristina Millar and Caroline Hays, undergraduates atthe Keck Science Department of Claremont McKenna,Pitzer, and Scripps Colleges.
Tetrahymena thermophila PKD1 (protein kinase domain) ishomologous with other protein kinase-like proteins thatare involved in catalytic functions and phosphorylationin cellular processes. PKD1 is located near theC-terminus of the protein appears to contain only oneknown domain (E-value= 1.2x10-42) in the protein kinasefamily although the substrate specificity of the PKD1is unknown. Paragraph by: Victoria Nguyen and JosephGrotts (undergraduates), Keck Science Department ofClaremont McKenna, Pitzer, and Scripps Colleges.
This protein is the in domain of sugar transporter withan E-value of 1.5X10-66, indicating that the sequencecontains strongly conserved amino acids typical of thedomain. . These types of transporters come from theMajor Facilitator Superfamily (MFS) that areresponsible for binding and then transporting severaldifferent molecules such as sugars, carbohydrates, andsmall, biological acids. While much is unknown aboutthis specific transporter, it most likely is involvedin the binding and transport of sugars across the cellmembrane. Written by Jessica Thomas and Paul Gonzalez,undergraduates at the Keck Science Department ofClaremont McKenna, Pitzer, and Scripps Colleges.
ATA2 from the eukaryote Tetrahymena thermophila issimilar to ATA homologs from other eukaryotes. Itcontains two of each of the two types of domains, anATP binding cassette (ABC) and a less conservedtransmembrane domain (TMD), totaling four domains. The3D structure of an ABC is a stubby L-shape with twodistinct arms. These transporters function as dimers.The purpose of the binding cassettes (approximately 200amino acid residues) is to bind and hydrolysis ATP,which releases energy that enables the transporters totransfer macromolecules and ions across cellularmembranes. This most commonly occurs in the transportof essential nutrients to bacteria, but also is relatedto diseases such as cystic fibrosis in humans. It isclear that ATA2 is important as we can see that it isstrongly conserved across many organisms from Homosapiens to Drosophila. Paragraph provided by AishSubramanian and Travis Tu, undergraduates at the KeckScience Department of Claremont McKenna, Pitzer, andScripps Colleges.
Tetrahymena thermophilia MSC1 has similarities withdifferent protein solute carriers from bacterialorganisms. It contains only one domain characteristicto UAA transporters, which has a specificity forUDP-N-acetylglucosamine. The protein is largely locatedon the membrane of the eukaryote. Investigation intothe family of UAA transporter proteins still remainslargely untouched. Paragraph provided by undergraduatesat the Keck Science Department of Claremont McKenna,Pitzer, and Scripps Colleges.
Tetrahymena thermophile ASH3 (Assistant for theprevention of Shock due to Heat) is homologous withTCP-1/cpn60 chaperonin family of heat shock proteins.This family plays a major role in cell growth byassisting with the folding of denatured or partiallydenatured polypeptides when heat shock occurs in thecell. Because they do not denature in a wide range oftemperatures, they are an important domain of heatshock proteins, which are mainly found in prokaryotes,chloroplasts, and mitochondria. Kate Jesse and AshleyGould, undergraduates at the Keck Science Department ofClaremont McKenna, Pitzer, and Scripps Colleges.
Tetrahymena thermophila gene CUT1 (Common UnknownTrans-membrane) protein is a trans-membrane proteinwith unknown function. This gene is closely related toa similar gene in Ichthyophthirius multifiliis. Itcontains two CLPTM1 functional domains, one of which ismore strongly conserved than the other. When expressedin the human genome, this domain is known to be linkedto cleft lip and palate. This family (CLPTM 1) is oneof many eukaryotic, trans membrane protein sequencesthat are linked to cleft lip and palate; however,specific function is unknown. Kristina Millar andCaroline Hays, undergraduates at the Keck ScienceDepartment of Claremont McKenna, Pitzer, and ScrippsColleges.
Two domains on Tetrahymena thermophila MTP1 suggestthat it belongs to the BT1 family. The proteins of thisfamily are transport proteins, suggesting that MTP1 isalso a transporter. Many proteins of this family arethought to be pteridine transporters, so it is possiblethat MTP1 also transports pteridine. By NicoleHohnstein and Emilie Fisher, undergraduates at the KeckScience Department of Claremont McKenna, Pitzer, andScripps Colleges.
APF1 (Assistant Protein Folder) is homologous withmembers of the TCP-1/cpn60 chaperonin family and isfound in abundance in prokaryotes, chloroplasts andmitochondria. It contains one domain (E-value=7.8x10-136) that is possibly used to stabilize andprotect disassembled polypeptides under heat shockconditions. In addition to its role as a heat shockprotein, they may also function to assist in amino acidchain folding into their three-dimensional proteinstructures. This paragraph was provided by Hannah Chiaand Jesse Honig, undergraduates at the Keck ScienceDepartment of Claremont McKenna, Pitzer, and ScrippsColleges.
Tetrahymena thermophila ICT1 is homologous to thenatural resistance-associated macrophage protein(NRAMP) family, composed of membrane proteins that aredivalent cation transporters. It contains one domain(E-value=3.7x10-108), which is located in the middle ofthe protein. Given the function of the homologousproteins, it is likely that ICT1 is an integralmembrane protein and functions as a cation transporter. Paragraph by: Alec Koh and Katherine Tully(undergraduates), Keck Science Department, ClaremontMcKenna College and Scripps College.
Tetrahymena thermophila STD1 (Sugar Transport andDistribution) is homologous to proteins from the majorfacilitator/general substrate transporter superfamilyof proteins. It contains a single domain (E-value = 9.6* 10-42) categorized as “sugar_tr,” which isresponsible for sugar and other solute transportationacross a membrane. This implies that STD1 integralmembrane protein may be involved in the transport ofsugars across the T. thermophila membrane. Paragraph byDaivik Vyas and Katie Liu, Keck Science Department,Claremont McKenna College and Scripps College.
ATA2 from the eukaryote Tetrahymena thermophila issimilar to ATA homologs from other eukaryotes. Itcontains two of each of the two types of domains, anATP binding cassette (ABC) and a less conservedtransmembrane domain (TMD), totaling four domains. The3D structure of an ABC is a stubby L-shape with twodistinct arms. These transporters function as dimers.The purpose of the binding cassettes (approximately 200amino acid residues) is to bind and hydrolyze ATP,which releases energy that enables the transporters totransfer macromolecules and ions across cellularmembranes. This most commonly occurs in the transportof essential nutrients to bacteria, but also is relatedto diseases such as cystic fibrosis in humans. It isclear that ATA2 is important as we can see that it isstrongly conserved across many organisms from Homosapiens to Drosophila. Paragraph provided by AishSubramanian and Travis Tu at the Keck ScienceDepartment of Claremont McKenna, Pitzer, and ScrippsColleges.
Tetrahymena thermophilia AKM1 (Advancer of K+ throughthe cell Membrane) contains one identified functionaldomain: the ion channel family (E-value 1.8 X 10〖10〗^(-12)). It is most closely related toIchthyophthirius multifilis EGR28840.1, asmall-conductance, calcium-activated potassium channelprotein. It is likely that AKM1 has a similarconformation as this protein. AKM1 is expected to be atetrameric potassium channel, located in thephospholipid bilayer and contains a “loop” which isinvolved in the selectivity of ions that may passthrough the channel. Paragraph by: Makari Krause andAmanda McQuade (undergraduates), Keck ScienceDepartment, Claremont McKenna, Pitzer, and ScrippsColleges.
The gene model is incorrect. The cDNA sequence, asdetermined by RT-PCR,is: ATGAGTTTAGTTTAGAGAACAATATAGGCTTATGAGAAGGATGAAAACAAAAACTTCGAAGAGTTCATTGAAAAAAGTTTAAAAGCATTTAGAGAAGAAGGTATGAAATTCGAGTAGTAAAAGGAGTGCAATTCGTAATAAATGTCTGATAACTAAAGAAACGAATGGGAAGAAAAAATAGCTAGTTTGGAAAGTCTTTTTAAAATGTTTTGTGTGCTTAAAGGTTAAAAGAGAAAGAAATCAAGAGTCATGTATAACATTTGTGAGCATATTTATGGAAAGAATTTACTAAAAAGAACATTTTGGTGCTGGAAAAGCCACCAGAAGAATGAAGAATATCTGCGTTAGATGGAAGAGTAAGCAGATGTATTTTATAACAGAAGGACACTAACAAAAATAATGAGAAGTTGGTAAGATGTTGTAATTGATGAGAATAAAACAATAGTTAAAAACACTGCCTTAAAGAAAACTGAGTTAGAATTGCAAAAAAATCAAAAGGAGTTTGAAAACCAAATTAAGAGTTTGGAAATTTTGCTATAATAAAAAATATTAACACTGAGACATGAAGAATAACAATACAACATTTTATTCTAAAAATAATAGCTCCTTTCATAAAATTAAAAAATTGAATTTGATTGA Thecorresponding protein sequenceis: MSLVQRTIQAYEKDENKNFEEFIEKSLKAFREEGMKFEQQKECNSQQMSDNQRNEWEEKIASLESLFKMFCVLKGQKRKKSRVMYNICEHIYGKNLLKRTFWCWKSHQKNEEYLRQMEEQADVFYNRRTLTKIMRSWQDVVIDENKTIVKNTALKKTELELQKNQKEFENQIKSLEILLQQKILTLRHEEQQYNILFQKQQLLSQNQKIEFD
Tetrahymena thermophilia CRP1 (calcium regulatorprotein 1) appears to be a member of the sodium/calciumexchanger protein family. It contains one putativesodium/calcium exchanger domain, which has an E-valueof 3.2*10^-13, indicating that it is reasonably relatedto this domain sequence. It is possible that thisprotein regulates Ca2+ cation concentration within thecell, where the movement of Ca2+ in or out of thecytoplasm is contingent upon the concentration of Na+in the cell. M. Huen and M. Greeley
In yeast, DNA-dependent ATPase that stimulates strandexchange; modifies the topology of double-stranded DNA;involved in the recombinational repair of double-strandbreaks in DNA during vegetative growth and meiosis
Hop2 and Mnd1 are meiosis-specific proteins thatfunction in a complex in budding yeast. Their generalarchitecture is strikingly similar, and therefore theyare potentially homologous protein families. TheHop2-Mnd1 system seems to have undergone duplication inthe evolutionary history of Tetrahymena, because bothprotein families are represented by two homologs withdistinct expression patterns in this species. Just asfor HOP2 (meiotic) and HOPP2 (ubiquitous), there is ameiotic (MND1)and a ubiquitously expressed (MNDP1)version, which raises the possibility that a meioticand a ubiquitous Mnd1p-Hop2p complex exists.
Named RPL8 in Klinge et al. Science 2011
Gene Model error: The open reading frame for the majorprotein product starts at an upstream in-frame ATG,adding 17 amino acids to the N-terminus of thepredicted protein (Couvillion et al., Mol. Cell, 2012).
Gene Information: TTHERM_00865050 is a homolog ofannotated and functionally characterized Orc1 in otherexperimental organisms (24% amino acid sequenceidentity, 49% similarity to human Orc1). Orc1 is acomponent of the heterohexameric origin recognitioncomplex (ORC) found to be involved in initiation of DNAreplication. TtOrc1 has canonical Walker A and B motifs(involved in ATP binding and hydrolysis). TtORC isunusual in that it contains an integral RNA subunit(26T RNA) that binds to its cognate DNA target in theribosomal DNA (rDNA) replication origin. Identifiedin: Tetrahymena ORC contains a ribosomal RNAfragment that participates in rDNA originrecognition Mohammad M Mohammad,1,*† Taraka RDonti,1,* J Sebastian Yakisich,1 Aaron G Smith,2 andGeoffrey M Kapler1,2,a PMID: 2140106 Characterizationof a novel origin recognition complex-like complex:implications for DNA recognition, cell cycle control,and locus-specific gene amplification. Mohammad M, YorkRD, Hommel J, Kapler GM. Mol Cell Biol. 2003Jul;23(14):5005-17. PMID:12832485 Differentialtargeting of Tetrahymena ORC to ribosomal DNA andnon-rDNA replication origins. Donti TR, Datta S,Sandoval PY, Kapler GM. PMID: 19153611 Nucleic AcidInteractions (with the ORC complex): 26T RNA(5’-AUGUCUAAGUGUGAUGAUAAACGAAAAAAAAUAAAAAUUAA-3’). Type I element T-rich strand: essential cis-actingreplication determinant in ribosomal DNA origin ofreplication. (Location 5’ non-transcribed spacer) (C3rDNA type IB element T-rich strand, T51:5’-CTCAAAAGTTGCAAAAGTTCGGAAGGTTTACTATTTTTGTTTTTTTTTT-3’). – requires 26T RNA and ATP dsDNA – non rDNAchromosomes Protein Interaction Partners: TtOrc2(Co-migration on a native gel, detection by WB). Biochemical Activities (of ORC complex): ATPbinding, likely ATP hydrolysis DNA binding - typeIelement T-rich strand DNA RNA binding – 26TRNA Regulation & Expression: mRNA and protein: Cellcycle regulated Maximal protein expression at G1/Sborder, degraded in S phase
Gene Information: TTHERM_00865050 is a homolog ofannotated and functionally characterized Orc1 in otherexperimental organisms (24% amino acid sequenceidentity, 49% similarity to human Orc1). Orc1 is acomponent of the heterohexameric origin recognitioncomplex (ORC) found to be involved in initiation of DNAreplication. TtOrc1 has canonical Walker A and B motifs(involved in ATP binding and hydrolysis). TtORC isunusual in that it contains an integral RNA subunit(26T RNA) that binds to its cognate DNA target in theribosomal DNA (rDNA) replication origin.
Nucleic Acid Interactions (with the ORC complex): 26TRNA(5’-AUGUCUAAGUGUGAUGAUAAACGAAAAAAAAUAAAAAUUAA-3’). Type I element T-rich strand: essential cis-actingreplication determinant in ribosomal DNA origin ofreplication. (Location 5’ non-transcribed spacer) (C3rDNA type IB element T-rich strand, T51:5’-CTCAAAAGTTGCAAAAGTTCGGAAGGTTTACTATTTTTGTTTTTTTTTT-3’). – requires 26T RNA and ATP dsDNA – non rDNAchromosomes
Protein Interaction Partners: TtOrc2 (Co-migration on anative gel, detection by WB).
Biochemical Activities (of ORC complex): ATP binding,likely ATP hydrolysis DNA binding - typeI elementT-rich strand DNA RNA binding – 26T RNA
Regulation & Expression: mRNA and protein: Cell cycleregulated Maximal protein expression at G1/S border,degraded in S phase
Gene Information: TTHERM_00684560 against human ORCsubunit 2 protein e-value= 8e-16 Reverse BLAST ofNP_006181.1 against TGD: TTHERM_00684560 hypotheticalprotein e-value= 8e-15.
The conserved Origin Recognition Complex (ORC)determines the sites for replication initiation ineukaryotic chromosomes and serves as a scaffold forpre-replicative complex (pre-RC) assembly. Although ORCsubunits are conserved in eukaryotes, the cis-actingDNA sequence requirements for replicator function arenot.
Nucleic Acid Interactions: Orc2p bound to streptavidinsepharose in 26T RNA aptamer-tagged strains (TD152 andMM202, respectively)
Protein Interaction Partners: Tetrahymena thermophilaORC physically associates with Orc1p in westernblotting (native gel electrophoresis andimmunoprecipitation analyses. Western blotting wassimilarly used to monitor the migration ofnuclease-treated ORC complexes under native EMSA gelconditions. The mobility of Orc1p and Orc2p increasedfollowing MNase and RNase A treatment, but wasunaltered by DNase I (Figure 3D). Orc1p and Orc2pco-migrated under all conditions, suggesting that theyremain associated after the RNA is destroyed.
Biochemical Activities: Consistent with previousanalyses of Orc2p/Tt-p69 and histone H3 (Mohammad etal, 2003), ∼50% of Orc2 was rendered soluble by DNaseI. TtORC also crossreacts with a rabbit polyclonalantibodies raised against Xenopus laevis / Orc2p.
Regulation & Expression: Orc2p crossreactive subunit,Tt-p69, localizes to the macronucleus during vegetativeS phase. Cell cycle regulated Maximal proteinexpression at G1/S border
Gene Information: MCM6 is a gene coding for a proteinproduct associated with replicative origins in G1 phaseduring pre-replicative complex assembly. ORC recruitsMCM6p on non-rDNA chromosomes. Mcm6p ChIP analysis wasperformed with affinity-purified rabbit antibodiesdirected against amino acids 34–51(GKKIKYYREKALLLKIYE) of the T. thermophila MCM6 protein(Tetrahymena Genome Database gene prediction:TTHERM-00448570, e value versus human MCM6(NP_005906.2): 1.0e-172. Human MCM6 (NP_005906.2)versus TGD TTHERM-0048570 e value : 1.0e-178.
Nucleic Acid Interactions: Non-rDNA and rDNA origin incells synchronized at the G1/S border.
Protein Interaction Partners: Not assessed
Biochemical Activities: Predicted helicase activity fordsDNA as a component of MCM2-7 complex
Gene Information: Tif1 is a non-ORC Type-I bindingfactor associated with ssDNA, particularly rDNAassociated with replication initiation. Essentialcis-acting replication determinant in ribosomal DNAorigin of replication. (Location 5’ non-transcribedspacer) Limited homology to Whirly family proteins inplants (transcription factors that bind to singlestrand DNA target sequences), but this homology isrestricted to the DNA binding domain.
Nucleic Acid Interactions: Tif1 assocates with theA-rich strand of rDNA in vivo, and can be purified toassociate with the T-rich strand in vitro.
Biochemical Activities: Regulates timing of rDNA originactivation. Involved in the S phase DNA damagecheckpoint response. Involved in unique ssDNA origin ofreplication recognition. TIF1 disruption mutants arehypersensitive to hydroxyurea andmethylmethanesulfonate, inducers of DNA damage in allexamined eukaryotes.
Regulation & Expression: TIF1 interacts with A-richstrand at the rDNA origins and the T-rich strand at therDNA promoter. TIF1p localization is dynamicallyregulated as it moves into the micro- and macronucleusduring the respective S phases.
Gene Information: The phosphatidylinositol 3-kinase(PI3K)-related sensor kinase ATR is a key player in thesignaling of induced DNA damage and self-inflicted DNAcuts in vegetative and meiotic cells (Richardson etal., 2004; Bassing and Alt, 2004; Hunter, 2008). Inhigher eukaryotes ATR is recruited by the MRX/MRNcomplex and possibly other unknown factors to the sitesof damage and phosphorylates a host of target proteinsto arrest replication forks and prevent new originsfrom firing. (Kurz and Lees-Miller,2004). TTHERM_01008650 BLAST against human ATR1NP_001175.2 e value : 6.0 e-5. Reverse BLAST of humanATR1 (NP_001175.2) against TTHERM_01008650 e value :3e-48
Nucleic Acid Interactions: Involved in ssDNA binding atrDNA origin and promoter. Sensing and repairmechanisms unknown.
Protein Interaction Partners: Not determined
Biochemical Activities: Ability to arrest cell cycle isinhibited by caffeine.
Regulation & Expression: Activated / Induced by DNAdamage
Gene Information: ASI2 is a gene regulatingendocycling in Tetrahymena thermophila. Thoughnonessential for vegative growth, it is upregulatedafter meiosis and is involved in the creation of a newMAC. Introduced via transduction with ASI2-GFP plasmidto establish parental cells that showed transcriptionof ASI2 occurs both in MAC and parental cells atconjugation.
Nucleic Acid Interactions: ASI2p independent of ssRNAsynthesis/accumulation
Regulation & Expression: The Tetrahymena gene ASI1(anlagen stage induced 1) was isolated from a cDNAlibrary of genes that are up-regulated duringdevelopment of the macronuclear anlagen. As its nameimplies, the abundance of ASI2 mRNA peaks at 9 h ofmating, early in macronuclear anlagen development.
Tetrahymena thermophila’s OGL1 protein is homologousto proteins from the HhH-GPD and OGG-N superfamilies,which are associated with DNA repair. It contains twodomains; the first domain (E-value= 3.0 -17), in theHhH-GPD family, is a Helix-hairpin-helix and Gly/Prorich loop, found on proteins that remove base lesions.The second domain (E-value=6.9-13), is common tooxoguanine gylcosylases, also known as OGG1, whichremoves 8-oxoG lesions. The closest homologs are inScheffersomyces stipitis, a negative Crabtree yeast,and in Taphrina deformans, a fungi/plant pathogen, with46% identical residues. OGL1 may be an essentialprotein in preventing mutations in DNA that lead to ashort lifespan, early aging, and cancer. Paragraph by:Lillian Horin, Maite Cortes Garcia, Francis Ryu,Forrest Fulgenzi, Keck Science Center, Pitzer College
The first 2769 bp and later 3243 bp of TTHERM_00530720(6012 bp) were expressed for MicNup98B and Nup96,respectively.
MacNup98A coding sequence is split in TTHERM_00071070and TTHERM_00071080
APAT1 contains an Asp domain (E-value 3.9e-32). Proteins that contain this domains typically catalyzethe transfer of acetyl groups and include pepsin-likeaspartate proteases. APAT1 is likely to cut peptidebonds, take out the aspartyl group, and substitute itwith an acetyl group. Paragraph by: Anna Cechony andMarzia Zendali (undergraduates), Keck ScienceDepartment, Scripps College.