Notre Dame NSF Biology REU 2003

Abstracts

 

GPCR Studies:
Characterization Of The Opsin Gene Family In The Malarial Vector, Anopheles gambiae

Perciliz Tan, Catherine Hill, Neil Lobo, Lucas Ton, & Frank Collins

G protein-coupled receptors (GPCRs) are a large family of proteins with various functions such as chemo- and photoreception, neurophysiological function, and hormone regulation. Consequently GPCRs are important drug and insecticide targets. Among the sensory and non-sensory GPCRs that we have identified in silico from the genome of the principal malarial vector mosquito, Anopheles gambiae, are a family of opsins thought to be involved in visual processes as part of phototransduction signaling cascades in the mosquito eye.

In Anopheles gambiae, crepuscular behaviors such as mating, feeding, host location, and the selection of oviposition sites that are dependent upon light and spectral conditions. At the present, the role of opsins in mosquito photoreception and vision related behaviors are not known. The opsins are of interest as they have undergone a gene expansion of the blue/green/violet sensitive receptors relative to Drosophila melanogaster. Therefore we have undertaken a detailed analysis of the opsin gene family and this gene expansion in A. gambiae. We have used a combination of bioinformatic approaches together with direct DNA sequencing, and quantitative RT-PCR to analyze opsin expression in A. gambiae heads and bodies and in male and female mosquitoes collected during the light and dusk photoperiods.

Preliminary results suggest that there are different expression levels of opsin transcripts in male and female A. gambiae at different times throughout the twenty-four hour photoperiod. The observed differences may result from sexual dimorphisms, photoreceptor membrane turnover, or other specialized roles that allow A. gambiae to be very active at low light intensities. This is the first study of the A. gambiae opsin gene family and is part of an ongoing effort to discover possible targets that may lead to the establishment of attractants, repellants, or novel methods for control of mosquitoes and other disease vectors.

 

GROWTH CHARACTERISTICS OF PLASMODIUM FALCIPARUM

Steuter, John, Reilly,Heather, Patel, Jigar, Deng, Bingbing, Ferdig, Michael

Biological Sciences, University of Notre Dame, Notre Dame, United States

The disease malaria affects 300 to 500 million people and accounts for an estimated 2 million deaths a year.  Of the malaria parasites, Plasmodium falciparum causes the most severe and deadly form of malaria.  An important determining factor of the parasites effects on humans is its proliferative capacity in the erythrocytic cycle.  Thus, the identification of factors that contribute to the overall growth rate of the parasite will result in a greater understanding of the disease.  In this research we have looked at the relative differences in cycle time of the parasites, cell cycle synchronization, and overall growth rate.  To score overall growth rate we used radiolabeled hypoxanthine (Hx) incorporation into proliferating progeny populations derived from a parental cross of HB3 and Dd2. The growth data was then analyzed using quantitative trait loci (QTL) mapping to identify chromosomal regions carrying genes controlling parasite growth.  Cycle time and synchronization were measured using blood smear data from the parents HB3 and Dd2. Knowledge of genetic mechanisms of growth rate, cycle time, and cell cycle synchronization will lead to an enhanced biological understanding of the disease.

 

Molecular Characterization of VDR Regulation in Kidney Cells

Erin C. Ward, Jennifer Wietzke, and JoEllen Welsh.  Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

   1,25 dihydroxyvitamin D (1,25D), the biologically active form of vitamin D, binds to the nuclear vitamin D receptor (VDR) inducing a cascade of events that activates genes involved in cell differentiation, apoptosis, and cell cycle.  In previous studies it has been shown that 1,25D acting through the VDR has inhibitory effects on breast cancer cells.  In addition, the effects of 1,25D are enhanced through co-treatment with trans-resveratrol (RES), a phytoestrogen found in red wine.  The mechanism by which RES enhances the actions of 1,25D in breast cancer cells involves up regulation of VDR gene expression through the exon 1c promoter.  In view of the development of vitamin D based therapies for breast cancer, it is important to characterize VDR regulation in other target tissues.  The focus of this study was to characterize the VDR in human kidney cells using HKC-8 cells, a human proximal tubule kidney cell line.  HKC-8 cells were incubated with 1,25D, RES, and estradiol (E2), as well as other treatments such as, forskolin (FSK) and parathyroid hormone fragments (PTH) and the activity of the VDR exon 1c promoter was measured.  It was found that 1,25D up regulated VDR promoter activity, while RES and E2 had no effect on VDR promoter activity, in HKC-8 cells.  Using western blot analysis, it was found that treatment with 1,25D increased the amount of VDR protein, while treatment with RES and E2 had no effect on the amount of VDR protein.  Other treatments (FSK, PTH) showed varying effects on VDR promoter activity and VDR protein expression in HKC-8 cells.  These results suggest that VDR regulation by estrogenic compounds occurs in breast cancer cells but not kidney cells.  Thus, co treatment of breast cancer with 1,25D and RES may not enhance vitamin D action in the kidney.  The cell type specific regulation of VDR by RES may therefore enhance vitamin D action in breast cancer cells with minimal side effects due to vitamin D action in kidney, such as hypercalcemia.  Further studies on the interactions of vitamin D and phytoestrogens in various tissues in vivo will be necessary to test this concept.

 

Investigation of incipient speciation between M and S molecular forms of Anopheles gambiae using X-linked microsatellite markers

Jennifer Shoener, Aram Stump, Nora Besansky

Anopheles gambiae, or the African malaria mosquito, is characterized by profound genetic plasticity, which is manifested as rapid specialization for humans and human-manipulated environments.  The mosquito’s exploitation of agriculturally generated water pools and rice fields resulted in its reproductive expansion into the dry season as well as into perpetually arid regions of West Africa, thus overcoming former seasonal and geographic barriers.  Countless malaria cases, of which A. gambiae is the primary vector, have been the consequence. 

A. gambiae has been sub-classified into M and S molecular forms by virtue of X-linked ribosomal DNA (rDNA) distinctions—an indicator of incipient speciation.  The M and S forms of A. gambiae also exhibit a strong pre-mating barrier and discrete ecological niches.  Two accounts of microsatellite allele frequency differences between the forms have previously been mapped to the X chromosome, and a fixed transposable element difference has been found in the vicinity of a pericentromeric X-linked gene.  However, previous studies have not found variation on distal regions of the X chromosome or elsewhere in the genome.  This information suggests the existence of differential selection in noncoding markers which should presumably evolve neutrally.  We hypothesize this phenomenon is occurring because genes linked to these markers are being targeted for selection.

 DNA was extracted from 480 male mosquitoes captured in Burkina Faso.  The species/form of each individual was identified and categorized as A. gambiae M, A. gambiae S, or A. arabiensis (a sibling species).  Fifty samples from each category were genotyped for microsatellite marker polymorphisms spanning the X chromosome.  The initial study included 16 microsatellite-flanking primers, 8 of which were novel.  Preliminary analysis of one sympatric population has indicated strong frequency differences between M and S microsatellite alleles occurring in the proximal region of X.  Localized microsatellite fixed or frequency differences will suggest selection occurring at the genes to which they are linked. 

The ultimate aim of this study is to reveal genes responsible for early stages of speciation between A. gambiae molecular forms.  Recent sequencing and microsatellite data suggest these genes may be linked to the X chromosome, specifically in the pericentromeric region.  Better comprehension of the mosquito adaptation process may provide information for the development of alternate approaches toward treatment and prevention of malaria.

 

Sequence analysis of the vH13 locus of Mayetiola destructor reveals six putative genes and synteny with the Anopheles but not Drosophila genome

Adam D. Will, Neil F. Lobo, and Frank H. Collins; Dept. of Biological Sciences, University of Notre Dame

  Mayetiola destructor, commonly known as the “Hessian” fly, is a major pest of wheat (Triticum sp.), but efforts to combat crop yield loss by the breeding of resistant strains of wheat are complicated by the virulence-avirulence interactions of Triticum and Mayetiola.  Results of previous investigations suggest that a gene-for-gene relationship underlies Hessian fly avirulence to wheat virulence; however, the specific gene involved in the wheat-fly H13-vH13 interaction has not yet been identified.  Markers specific to the vH13 locus have facilitated the screening of a Mayetiola destructor BAC library for genomic BAC8i18, which is associated with the avirulence locus.  In this investigation, BAC8i18 was subcloned, sequenced, and assembled.  Sequence analysis using available bioinformatics programs (FGENESH, GENSCAN, and geneid) revealed six putative genes, designated Mad1 through Mad6.  NCBI BLASTp (nr) and Ensembl tBLASTn (A. gambiae masked genomic sequence; D. melanogaster genomic sequence) were used to reveal possible homology and synteny with insect genomic sequences.  Mad1, Mad3, and Mad6, though predicted by all three gene-finding programs, do not have significant matches (each has an E-score>0.01) in the NCBI nr database or the Ensembl assemblies.  Genes Mad2, Mad4, and Mad5 show significant sequence identity with D.melanogaster genes encoding a-catenin-related protein, Toll-6, and GCN2, respectively, and uncharacterized predicted transcripts in the A. gambiae assembly.  Furthermore, Mad2, Mad4, and Mad5 all localize on the 3L chromosome of A. gambiae, suggesting evolutionary synteny by gene cluster conservation (but not through conservation of gene order); interestingly, this localization of Mad genes is not observed when D. melanogaster provides the reference genome.  This initial analysis not only provides important clues to the identity of the gene responsible for the vH13 trait in Mayetiola destructor, but also raises important questions regarding the selective pressures that resulted in synteny between Mayetiola and Anopheles but not Drosophila.

 

Abdominal pigmentation variations due to the yellow gene in the Drosophila polymorpha of Brazil

Nick Russell*, Mehgan Schaffner*, Shannon Gray*, Daniela DeToni*, Jennifer Brisson°, and Hope Hollocher*

*Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

Department of Biology, Washington University, St. Louis, MO 63130

Clare Boothe Luce Associate Professor, University of Notre Dame

Recently, scientists have been attempting to find a genetic basis behind evolutionary changes and to understand how changes at the molecular level can affect phenotypic diversity.  This is currently a popular research area because it can lead to a better understanding of natural selection pathways resulting in the divergence of phenotypes among a single species.  A major candidate gene that is thought to be associated with this phenotypic divergence is the yellow gene.  This project involves investigation of both environmental and genetic effects on the phenotype of abdominal pigmentation in Drosophila polymorpha.  This species is unique in that it exhibits a cline in abdominal pigmentation on mainland Brazil.  This cline is expressed from lighter abdominal pigmentation in the northern latitudes to darker abdominal pigmentation in the southern latitudes.  Nine Phenotypic scores of D. polymorpha were made; categories depended upon the percent of the abdomen that had pigmentation.  These were then compared to the divergence of the yellow gene across this cline.  This is to evaluate the hypothesis of environmental involvement in the correlation of divergence of the yellow gene to abdominal pigmentation in D. polymorpha.  To accurately account for the pigmentation cline and for different environmental habitats, individuals were collected from widespread locations across the Brazilian mainland and from the small island of Santa Catarina.  This research will eventually determine if the yellow gene plays the same role in a single species with gene flow, as it does with six isolated species with no gene flow, of the Caribbean Island seen in previous research done by the Hollocher lab, University of Notre Dame.

 

Characterization of Apoptotic Pathways in a Murine Mammary Tumor Cell Line

Anna Acosta1, Meggan Valrance2, and JoEllen Welsh2

1,25(OH)2D3 (1,25 D), the active metabolite of vitamin D3, produces anti-proliferative and pro-apoptotic effects in mammary epithelial tumor cells in vivo and in vitro. 1,25D exerts these effects through the vitamin D3 receptor (VDR). To better understand the role of the VDR, mammary tumor cell lines were created from VDR wild-type (WT) and VDR knockout (KO) mice. In these studies we have further characterized the apoptotic responses of one of these cell lines, WT145, to 1,25D and etoposide. Etoposide is a chemotherapeutic drug that triggers apoptosis secondary to DNA damage. WT145 cells were dose-dependently growth inhibited following treatment with 1,25D or etoposide, and cells exhibited apoptotic morphology. To assess the expression of caspases, cysteine protease enzymes activated during apoptosis, cells were treated with 0.5-3mM etoposide or 100 nM 1,25D. Cleavage of PARP, a caspase substrate, was observed following treatment suggesting the involvement of caspases in apoptosis induced by 1,25D or etoposide. The expression levels of the proforms of initiator caspases-9, 10 and 12 were decreased following etoposide or 1,25D treatments, indicating activation of the enzymes and initiation of the caspase cascade. This was also suggested by decreases in effector caspase-3 levels following etoposide or 1,25D treatment. No change was observed in procaspase-6 expression with treatment. Collectively, these data indicate that apoptosis in WT145 cells treatment with 1,25D or etoposide is triggered through activation of several caspases with the exception of caspase-6.

1: Santa Clara University, Santa Clara, CA 95050

2: University of Notre Dame, Notre Dame, IN 46556

 

Immunochemical and Functional Analysis of the Duffy Binding Protein of Plasmodium vivax  

Elitza Sevova and Dr. John Adams

            Invasion of erythrocytes by Plasmodium vivax merozoites requires the formation of a tight junction between the parasite and host erythrocyte.  This junction is dependent on the interaction of the Duffy Binding Protein (DBP) of P. vivax with the Duffy Antigen Receptor Chemokine (DARC) of red blood cells.  The ligand domain of the DBP is in the cysteine-rich region II (rII) with the critical binding domain between cysteines 4 and 7.  This area of the DBP also shows the highest rate of non-synonymous polymorphisms and hypervariability, which suggest immune selection by the host.  These mutations have no apparent effect on the binding affinity of the protein and it is thought that they are selected for their ability to change the antigenic character of the molecule.  Previous experiments in our lab have shown that individual polymorphisms are important to antibody recognition of DBP. These results were obtained with a cytoadherence assay using the DBPrII expressed on the surface of transiently transfected COS 7 cells.   The aim of this study is to provide an alternative method to confirm previous data. Polymorphic DBPrII coding sequences were cloned into an eukaryotic expression plasmid in order to express the DBP ligand as a soluble protein. Erythrocyte binding assays will be used to confirm the conservation of protein function. Following this, inhibition assays with select immune sera will be performed to analyze their inhibitory effect on DBP. In solution, the binding of these soluble rDBPs to the erythrocyte receptor are one to one and so weaker interactions can be measured.  This allows for a more sensitive method to confirm results from in vitro cytoadherence assays due to the lower minimum threshold for binding detection.  Further experiments to characterize polymorphic DBPs include antibody and affinity of binding assays, immunolocalization, and studies of protein structure through MALDI-TOF and LC-MS/MS.

 

Cloning of the HuC Promoter for Analysis of Retinal Repair in Zebrafish

Matt LaFave, Chris Burket, David Hyde

Zebrafish is a useful model system for studying neuronal regeneration from various types of retinal damage.  It was shown that constant exposure to intense light results in photoreceptor apoptosis in the zebrafish retina, but that a return to normal light conditions allows photoreceptor regeneration.  Recent findings present compelling evidence that Müller glial cells play a role in this regeneration process.  A DNA construct utilizing the Cre recombinase and lox P sites was proposed to specifically label Müller glial cells and trace their fate during retinal regeneration.  To test the feasibility of the Cre/lox system in the transgenic zebrafish retina, a preliminary construct involving the neuronal-specific HuC promoter was designed.  Genomic zebrafish DNA was used to PCR amplify this promoter as either a 3.1 kb or 2.5 kb fragment that contains the necessary neuronal-specific sequences.  These PCR products were ligated into the TOPO 2.1 vector and sequenced.  Preliminary sequence data confirmed the cloning of the expected promoter elements.  Both forms of the HuC promoter were ligated into the pT2KXIG poly transformation vector.  The next step will involve ligating the Cre recombinase gene into the HuC promoter-containing pT2KXIG vector.  Upon completion, the construct will be injected into zebrafish embryos, which will be crossed with wild-type fish upon maturation.  The F1 progeny of that cross will then be analyzed for Cre expression by immunoblots.  Those F1 fish that express Cre will then be crossed with fish containing a lox P-EGFP transgene to determine if the Cre/lox system can be used in future experiments with zebrafish.  Ultimately, the Cre/lox system will be used to identify the role of Müller cells in retinal regeneration.

 

MECHANISMS OF TRAIL-INDUCED APOPTOSIS IN OVARIAN CANCER CELL LINES

Chrissy Ratajczak & Dr. Alan Johnson

Ovarian cancers currently represent the fifth leading killer, and most common gynecologic cancer, among women.  Clinical treatment of ovarian cancers is complicated by a high incidence of developed resistance to conventional treatments such as irradiation, cisplatin, and taxol.  Many of such therapeutics directly promote nuclear or structural damage to cells, and as a result initiate apoptotic cell death through internal (intrinsic) cell death pathways.  Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) represents a potential novel therapeutic which, unlike conventional chemotherapeutics, promotes apoptosis by first binding to a membrane-associated death receptor and subsequently inducing activation of death receptor-mediated signaling (an extrinsic pathway). Recent studies have reported that TRAIL induces apoptosis in cancerous but not healthy cells, raising considerable interest in the use of TRAIL as a novel therapeutic.  Given distinct differences in the initial cellular pathways by which the two different therapies are predicted to act in transformed cells, combinational therapy combining TRAIL with conventional chemotherapeutics could minimize resistance and/or acquired desensitization to cancer therapy.  To date, however, little work has been accomplished to evaluate TRAIL-induced apoptosis, cellular mechanisms associated with TRAIL-induced apoptosis, or the potential for differential sensitivity to TRAIL treatment in ovarian cancer cell lines. In these studies, PA-1 and SK-OV-3 human ovarian cancer cell lines were chosen to evaluate cellular mechanisms associated with TRAIL-induced cell death, as well as the expression of TRAIL and a TRAIL receptor, DR5. Notably, SK-OV-3 cells (but not PA-1 cells) are deficient for the tumor suppressor gene, p53, and have previously been shown to display reduced sensitivity to irradiation and cisplatin treatment.  In initial studies, both cell lines were determined to express TRAIL mRNA following RT-PCR amplification. This is of interest in light of the potential for promoting TRAIL autocrine effects.  The two cell lines were next examined for the ability of TRAIL, irradiation (using UV light), cisplatin, and taxol to induce expression of RNA coding for the rapidly-induced pro-apoptotic protein, Noxa.  Significantly, Noxa RNA was found to be induced by UV irradiation, but not by taxol, cisplatin, or TRAIL.   Furthermore, initial studies in our lab noted differential sensitivity to TRAIL treatment between these two cell lines, with PA-1 cells being significantly more susceptible to TRAIL-induced cell death.  To evaluate whether such a difference in sensitivity is associated with a difference in the level of DR5 receptor expression, DR5 protein expression was compared between the two cell lines.  DR5 receptor was expressed at somewhat higher levels in TRAIL-sensitive PA-1 cell line than in SK-OV-3.  Finally, none of the factors previously implicated in regulating DR5 expression in non-ovarian cell types (interferon-g, cisplatin, taxol or TRAIL) was found to upregulate DR5 expression in either ovarian cancer cell line after 24 h of culture.  Accordingly, additional work is required to determine whether differences in TRAIL-induced cell death are related to DR5 expression, and if so, how increased sensitivity might be promoted via increased receptor expression.

 

Rab5 as a stimulatory molecule for the phagocytosis of mycobacterium avium

Lamont Barlow, Victoria Kelley, and Dr. Jeffrey Schorey

Phagocytosis is an essential first step for the ingestion and subsequent  killing of microbes by macrophages and other phagocytes.  Based on its vital role in endocytotic fusion events, it is thought that the small molecular weight GTPase Rab5 may play an essential role in the phagocytotic process.  Mycobacteria are known to undergo receptor-mediated phagocytosis by macrophages and have been shown to localize with Rab5 on their phagosomal membranes.  To analyze the phagocytotic effects of Rab5, we overexpressed Rab5 in J774 cells and infected the cells with Mycobacterium avium.  We found that overexpression of Rab5 caused a significant increase in complement opsinized M. avium uptake by the cells in comparison to cells transfected with an empty plasmid vector.  Experiments also showed that overexpression of both constitutively active and dominant negative Rab5, Rab5(Q79L) and Rab5(S34N) respectively, increased uptake levels to a similar extent as compared to the wildtype Rab5.  These findings indicate that Rab5 does not have an active function in complement mediated phagocytosis of mycobacteria, but the presence of Rab5 in the host cell does promotes uptake of M. avium, suggesting the possibility of Rab5 as a stimulatory molecule for phagocytosis.

 

Association of CBD1-4 SNP Polymorphism in AeIMUC1 gene with Aedes agypti Susceptibility to Plasmodium gallinaceum

K. Palomares, Y. M. Colton, I. Morlais, J.R. Schneider, D. Lovin, D.W. Severson

            An alternative strategy in controlling the transmission of malaria is genetic analysis of mosquito populations to reduce their competence as disease vectors.    AeIMUC1 was identified as an Aedes agypti candidate gene controlling susceptibility to Plasmodium gallinaceum.  This study sought to determine whether a polymorphism in AeIMUC1 is directly associated with vector competence.  Previous work identified a CBD1-4 substitution in a Red strain as a significant predictor of oocyst number.  The region of interest was amplified and digested, and individuals were scored at this locus in order to determine associations with phenotype.

 

Mitosis and Cytokinesis in Acentrosomal BSC-1 Cells

Christopher Mader, Emily Tribble, Edward Hinchcliffe

Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556

Centrosomes form the major component in the microtubule-organizing center (MTOC) of eukaryotic cells.  However, it is not fully known what parts of mitosis the centrosomes and MTOC play a critical role in.  The aim of this experiment was to determine what events during mitosis and cytokinesis involving spindle formation and organization of the various microtubules are dependent upon the centrosome.  BSC-1 African green monkey kidney cells were transfected with a green fluorescent protein (GFP)-a tubulin construct and a cell line constitutively expressing GFP-a tubulin was established (BSC-1a).  The BSC-1a cell line showed no significant difference in mitotic doubling time from the control BSC-1 cells.  BSC-1a cells showed an average cell cycle timing of 12.5 hours from the entrance into M phase to subsequent M phase under both normal conditions and when exposed to fluorescent light.  The microtubule network showed no observed defects in spindle formation due to the GFP labeled a-tubulin (Rusan, 2001).   The centrosomes and resulting MTOC were microsurgically removed from BSC-1a cells during interphase.  Cut BSC-1a cells healed the surgery point after an average of 2.5 hours (N=7).  The acentrosomal BSC-1a cells were imaged under fluorescent microscopy from the cut point through mitosis and cytokinesis.  The acentrosomal cells showed a non-bipolar spindle formation 75% of the time during M phase (N=4).  This failure to produce a bipolar spindle most likely indicates that the centrosome has a role in proper spindle formation.  In the non-bipolar spindles, astral microtubules appeared to be absent as the spindle showed sporadic movement inside the cell.   The same 75% of the cells failed to have proper chromosomal segregation and generated multiple cleavage furrows during cytokinesis.  The movement of the spindle within the cell during M phase confirms that astral microtubules nucleate from the MTOC.  Midbody formation was also observed in 75% of the cells during cytokinesis (N=4). The presence of the midbody in the acentrosomal cells may show that the midzone microtubules may not nucleate from the MTOC like normal microtubule networks.

 

Elucidating the role of ARF6 in the Raf/MEK/ERK signaling pathway during melanoma cell invasion.

Anne Marie Corgan, Vandhana Muralidharan and Crislyn D’Souza-Schorey, Department of Biological Sciences, University of Notre Dame

            The incidence rate of melanoma is increasing faster than any other form of cancer; it is rising at an epidemic rate of more than 4% annually.  Melanomas are highly invasive.  Understanding the mechanisms contributing to cell invasion could potentially lead to new therapies and treatments for melanomas.  ERK (Extracellular-signal-regulated kinase) is found to be upregulated in melanoma cells, tissues, and organs. However, there still exists considerable gaps in the understanding of molecular events that result in increased ERK activation during melanoma tumor progression. Previous studies done by our laboratory using invasive melanoma cell lines have suggested that ARF6 (ADP-ribosylation factor 6), a member of the Ras superfamily of small GTPases, may regulate invasion through the MEK/ERK signaling pathway. We have shown that activation of ARF6 is increased in response to physiological stimuli that provoke ERK activation, whereas the inhibition of ARF6 activity blocks ERK phosphorylation during melanoma cell invasion in vitro extracellular matrix degradation assays.  Our current efforts are directed at testing the hypothesis that ARF6-GTP promotes ERK activation by enhancing the activity of one or more kinases of the Raf-MEK-ERK pathway.  Our studies on the relationship between ARF6 and Raf-1 will be discussed.