- Anderson, Stephen
FAX - 235-4850
CABM - Room 206
679 Hoes Lane Piscataway, NJ 08854
protein folding, molecular recognition, proteases and their inhibitors, protein engineering, Alzheimer's disease
Our laboratory is focusing on the study of protein folding and molecular recognition and we are particularly interested in the role these processes may play in the pathophysiology of Alzheimer's disease. We are using bovine pancreatic trypsin inhibitor (BPTI) as a model system for the protein folding studies. BPTI is a small, globular, 58 amino acid polypeptide and our approach has been to express a recombinant gene for this inhibitor in E. coli and produce, via site-directed mutagenesis, mutants of BPTI having perturbed folding. The folding of these mutants is then characterized in vitro in terms of the thermodynamics and kinetics of the process.
Our work on molecular recognition has centered on the well-characterized protease-protease inhibitor interaction. The specific model system we have employed is the interaction of avian ovomucoid third domains (OM3D) with members of the trypsin family of serine proteases. Our approach is similar to that described above with BPTI, namely the expression of recombinant OM3D in E. coli and specific mutagenesis of reactive site residues. Mutant inhibitors are then tested for binding affinity with a panel of serine proteases. Investigations of the physiological roles that such inhibitors play in vivo are also ongoing.
The Alzheimer's b amyloid precursor protein (APP) is a large, membrane-bound glycoprotein that is expressed in the body from several different alternatively-spliced mRNAs. Contained within this precursor is the amyloid b peptide which has been strongly implicated as a causal agent in Alzheimer's disease. Our laboratory is currently using a protein engineering strategy to dissect the structure and function of the APP molecule as well as its component domains. We are also seeking to identify and isolate other proteins in the body that bind to APP. By characterizing these features of APP biology we hope to illuminate its role in both normal and diseased tissue.
- Marks, C.B., Naderi, H., Kosen, P., Kuntz, I.D., Anderson, S. (1987). Mutants of bovine pancreatic trypsin inhibitor are able to fold properly. Science 235:1370-1373.
- Hurle, M.R., Marks, C.B., Kosen, P.A., Anderson, S., Kuntz, I.D. (1990). The denaturant dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds. Biochemistry, 29:4410-4419.
- Castro, M., Marks, C.B., Nilsson, B., and Anderson, S. (1990). Does the Kunitz domain from the Alzheimer's amyloid b protein precursor inhibit a kallikrein responsible for post-translational processing of nerve growth factor precursor? FEBS Lett. 267:207-212.
- Higgins, D.L., Young, S. L., Powers, D., and Anderson, S. (1992). A zymogenic tissue plasminogen activator variant: The Phe305->His mutation suppresses fibrin(ogen)-stimulated plasminogen activation by one-chain tPA. Fibrinolysis 6:161-166.
- Lu, W., Zhang, W., Molloy, S.S., Thomas, G., Ryan, K., Chiang, Y.W., Anderson, S., and Laskowski, M. Jr. (1993). Arg15, Lys17, Arg18 turkey ovomucoid third domain inhibits human furin. J. Biol. Chem. 268:14583-14585.
- Brill, Steven J.
Dept. of Molecular Biology and Biochemistry
CABM - Room 304
679 Hoes Lane Piscataway, NJ 08854
yeast, DNA replication, ssDNA binding protein, DNA helicase, protein purification, mutational analysis
My laboratory uses the yeast S. cerevisiae as a model system to study the mechanism and regulation of eukaryotic DNA replication. The yeast system is favorable because it is the only eukaryotic species from which a cellular replication origin has been identified and cloned. Our approach is to biochemically identify proteins involved in the initiation of DNA replication and then use genetics to verify its function. Using an assay that detects one of the earliest steps in the initiation reaction - unwinding of the DNA duplex - a three-subunit protein complex (Replication Protein A; RPA) has been purified and cloned. RPA, a ssDNA binding protein, is conserved from yeast to man and probably also functions in the processes of DNA repair and recombination. Genetic and biochemical approaches are being used to understand the role of RPA in these processes and to identify factors that interact with the RPA complex during DNA replication. We have shown previously that each of the three RPA genes is essential for viability and are now using in vitro mutagenesis to generate conditional-lethal alleles of these genes. A key factor required for the unwinding of the DNA duplex is a DNA helicase. We are fractionating yeast extracts to purify DNA helicases and are using protein-affinity chromatography to identify enzymes the interact with RPA. Ultimately it should be possible to reconstitute the initiation reaction in vitro to learn more about the regulation of cellular DNA replication.
- Van Dyck, E., Foury, F., Stillman, B., and Brill, S. J. (1992). A single-strand DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. EMBO. J. 11, 3421-3430.
- Brill, S. J., and Stillman, B. (1991). Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes and Dev. 5, 1589-1600.
- Brill, S. J., and Stillman, B. (1989). Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature 342, 92-95.
- Brill, S. J., and Sternglanz, R. (1988). Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell 54, 403-411.
- Champe, Sewell P.
Waksman Institute - Room 234
Rutgers University Piscataway, NJ 08854-0759
fungal sporulation, fungal hormones, hydroxy fatty acids
The formation of metabolically dormant spores from metabolically active cells is a primitive example of cellular differentiation. We are studying the biochemical and genetic factors that trigger the onset of sporulation in the fungus Aspergillus nidulans. Sporulation in this organism is accompanied by the switching on of some 1000 genes, the functions of which are almost entirely unknown. We have identified several sporulation genes that appear to act very early in the sporulation process and which may be part of the triggering mechanism. Ongoing studies, including molecular cloning, are directed at revealing the function of these genes. In addition, we have isolated and identified an endogenous chemical inducer of sporulation, called psi factor, which is a novel C18 dihydroxy fatty acid. This compound has structural similarities to the leukotrienes which act as localized hormones mediating inflammatory responses in vertebrates. The enzymology of psi biosynthesis is being studied to reveal possible evolutionary commonalities with leukotriene biosynthesis.
- Champe, S.P. and El-Zayat, A.A. (1989). Isolation of a sexual sporulation hormone from Aspergillus nidulans. J. Bact. 171:3982-3988.
- Mazur, P., Meyers, H.V., Nakanishi, K., El-Zayat, A.A., Champe. S.P. (1990). Structural elucidation of sporogenic fatty acid metabolites from Aspergillus nidulans. Tetrahedron Lett. 131:3837-3840.
- Deis, Frank H.
email@example.com / firstname.lastname@example.org
Department of Molecular Biology and Biochemistry
Nelson Labs - Room A-311
Piscataway, NJ 08854
Teaching, Administration, Advising, and Computing, including HTML and other areas.
- Driscoll, Monica
Dept. of Molecular Biology and Biochemistry
Rutgers University CABM Building - Room 307
Piscataway, N.J. 08854
developmental neurogenetics, molecular genetics of inherited neurodegeneration, mechanosensory ion channels
Rare dominant mutations (d) in two C. elegans genes, mec-4 and deg-1, induce swelling and degeneration of different, but specific, groups of neurons in this nematode. Analysis of cloned mec-4 and deg-1 cDNAs has revealed that mec-4 and deg-1 are members of a novel gene family that have been postulated to be subunits of mechanosensory or volume-regulatory ion channels. We are interested in characterizing the normal activity of these proteins and in deciphering the molecular and cellular details of mec-4(d)-induced degeneration. We have undertaken an extensive structure/function analysis of MEC-4, testing mutant alleles in vivo for function. Complementary electrophysiological studies are getting underway in oocyte and cell culture systems. We are also identifying new genes that are required for mec-4(d)-induced deaths through genetic reversion analyses. Extragenic mutations that prevent mec-4(d)-induced deaths are expected to identify other channel subunits and proteins that interact with the channel as well as additional cellular components that participate in degeneration. Other ongoing projects include the characterization of cellular disruption that occurs during degeneration using light microscopy, electron microscopy and immunocytochemistry; determination of the sub-cellular location of the MEC-4 protein; and expression of the death-inducing MEC-4 protein in heterologous cell types. Recently it has been found that members of the mec-4/deg-1 gene family, called degenerins, are encoded in mammalian genomes. Since these genes might also be capable of mutation to aberrant forms that induce neuronal degeneration, they are logical candidates for involvement in degenerative conditions in higher organisms. In another line of experiments we are conducting searches for members of the mec-4/deg-1 gene family from other species in order to test this hypothesis. In summary, our work will serve to characterize a novel class of channels and will provide the first molecularly detailed description of an inherited neurodegenerative condition.
- Hong, K. and Driscoll, D.(1994). Residues in a transmembrane domain of the C. elegans MEC-4 predicted to line a channel pore influence mechanotransduction and neurodegeneration. Nature, 367:470-473.
- Mitani, S., Du, H., Hall, D., Driscoll, D. and Chalfie, M. Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans. Development 119:773-783.
- Chalfie, M, Driscoll, M. and Huang, M. (1993). Degenerin similarities. Nature 361:504.
- Driscoll, M. (1992). Molecular genetics of cell death in C. elegans. J. Neurobiol. 23:1327-1351.
- Driscoll, M. and Chalfie, M. (1992). Cell death genes in Caenorhabditis elegans. Trends Neurosci. 15:15-19.
- Edery, Isaac
Department of Molecular Biology and Biochemistry CABM Room 337 679 Hoes Lane Piscataway, NJ 08854
biological clocks, photic signal transduction, genes controlling Drosophila behavior, protein-protein interactions
The main goal of our laboratory is to understand the molecular and biochemical bases of biological clocks. To achieve this goal, we are using the powerful genetics available in Drosophila in combination with biochemical, molecular and histochemical approaches. Daily fluctuations in biochemical, physiological and behavioral phenomena are governed by one or a few endogenous circadian (~24 hour) clocks that adapt to external time cues. This adaptive response of a clock is likely its most significant role, enabling organisms to anticipate changes in environmental conditions. Thus, to better understand how biological clocks function, it is important to elucidate the time-keeping mechanism and determine how external stimuli ultimately perturb the oscillatory process. A genetic approach in Drosophila has led to the identification of a candidate clock gene period (per). Mutations in this gene can shorten, lengthen or abolish the periodicity of several behavioral rhythms. The per protein (PER) is the only characterized "clock protein" in any organism. However, the biochemical function of PER has not been established. Research in this laboratory is geared towards determining how PER activity contributes to the mechanism that measures or tells time. We are also interested in determining the signal transduction pathways involved in communicating environmental signals to the clock. Recent biochemical strategies have led to several interesting findings. For example, PER undergoes dramatic daily changes in abundance, phosphorylation and subcellular distribution. Furthermore, PER contains a novel dimerization motif that is shared by several transcription factors. These results suggest an attractive model for the role of PER in the manifestation of circadian rhythms: We propose that temporal changes in the phosphorylation state of PER may affect its ability to enter the nucleus and/or form heterotypic interactions with transcription factors, leading to changes in transcriptional programs as a function of time.
To test this model we are taking a multi-faceted approach. For example, phosphorylation sites on PER will be identified and mutagenized, and transgenic animals will be tested for altered clock function. Furthermore, we are analyzing circadian rhythms in flies bearing mutations in kinases. To identify the integral components required to assemble biological time-keeping devices we are generating flies expressing modified versions of PER. Studies currently in progress are also aimed at determining how the phosphorylation status and multimeric size of PER change in response to photic stimuli that cause the clock to shift phase.
In related studies we are characterizing the tissue specific regulatory features of per expression. PER is expressed in neuronal and non-neuronal tissues in the adult fly head. We created flies that express PER in a heat-inducible manner. The studies suggest that the stability of PER is tissue-dependent. We are interested in using these heat-inducible clock systems to investigate the tissue-specific pathways controlling the spatial distribution of PER. We are also interested in biophysical studies aimed at addressing the relationship between the structure and function of the PER dimerization motif, termed PAS. Related studies suggest that this motif can act as a "thermal switch" to protect the timing mechanism against fluctuations in temperature.
Since mounting evidence indicates that circadian clocks are governed by similar rules, knowledge of the time-keeping mechanism in Drosophila should apply to other organisms. As a result, these studies may help in developing more efficient treatments for several human disorders associated with altered clock function, such as manic depression, seasonal affective disorders (winter depression), jet-lag and chronic sleep problems. Elucidation of the time-keeping mechanism should also benefit the development of clinically relevant cells that can act as biological "timed-release capsules".
- Huang, J.H., Edery, I., Rosbash, M. (1993). PAS is a dimerization domain common to Drosophila Period and several transcription factors. Nature. 364: 259-262.
- Edery, I., Rutila, J.E. and Rosbash, M. (1994). Phase shifting of the circadian clock by induction of the Drosophila period protein. Science 263: 237-240.
- Edery, I., Zwiebel, L.J., Dembinska, M.E. and Rosbash, M. (1994). Temporal phosphorylation of the Drosophila period protein. Proc. Natl. Acad. Sci. USA. 91: 2260-2264.
- Gabriel, Abram
Dept. Molecular Biology and Biochemistry
CABM - Room 306 679 Hoes Lane Piscataway, NJ 08854
yeast, trypanosomes, retrotransposons, reverse transcriptase, fidelity
The overall goal of the research program in my laboratory is to understand mechanisms involved in eukaryotic retrotransposition; i.e., to study the enzymatic activities and complex molecular processes that are required for RNA-mediated transposable elements to move from one genomic location to another. We are focusing our efforts on two classes of elements: 1) The Ty1 transposon from Saccharomyces cerevisiae represents a genetically tractable, intracellular system whose replication strategies closely resemble those of retroviruses. Using the Ty1 reverse transcriptase (RT) as a model enzyme, we are examining the determinants of RT fidelity, with the objective of identifying both Ty-encoded and host-encoded factors responsible for the observed in vivo error rates during replication. 2) CRE1 from the trypanosome Crithidia fasciculata is a rapidly rearranging multicopy site-specific insertion sequence which encodes a novel RT and resembles mammalian LINE elements. We are interested in demonstrating transposition of this element, characterizing the properties of the RT, and identifying additional enzymatic activities associated with the element; e.g., a site-specific endonuclease.
- Teng, S.C., Kim, B., and Gabriel, A. Retrotransposon reverse transcriptase
-mediated repair of chromosomal breaks in Saccharomyces cerevisiae
, Nature, accepted.
- Gabriel, A., Willems, M., Mules, E. H., and Boeke, J. D. Replication
infidelity during a single cycle of Ty1 retrotransposition, Proceedings of
the National Academy of Science, USA, 93 7767-7771, 1996. (Subject of
commentary in same issue of the journal - Preston, B. D., Error-prone
retrotransposition: Rime of the ancient mutators, PNAS, USA, 93, 7427-
- Teng, S.C., Wang, X., and Gabriel, A. A new non-LTR retrotransposon
provides evidence for multiple distinct site-specific elements in Crithidia fasciculata miniexon arrays, Nucleic Acids Research, 2929-2936
- Gabriel, A., Transposons in the Human Genome, in Molecular Biology and Biotechnology: A Comprehensive Desk Reference, ed. Meyers, R.A., VCH
Publishers, 924-928, 1995.
- Gabriel, A. and Boeke, J.D. (1993). Retrotransposon Reverse Transcription, in Reverse Transcriptase eds. Skalka, A.M. and Goff, S.P. Cold Spring Harbor Laboratory Press, 275-328.
- Mathias, S., Scott, A., Kazazian, HJ., Boeke, J.D. and Gabriel, A. (1991). Reverse Transcriptase Encoded by a Human Transposable Element. Science, 254: 1808.
- Gabriel, A. and Boeke, J.D. (1991). Reverse Transcriptase Encoded by a Retrotransposon from the Trypanosomatid Crithidia fasciculata. Proc. Natl. Acad. Sci. U.S.A. 88: 9794.
- Gabriel, A., Yen, T., Schwartz, D., Smith, C., Boeke, J., Sollner-Webb, B, Cleveland, D.A..(1990). Rapidly Rearranging Retrotransposon within the Mini-exon Gene Locus of Crithidia fasciculata. Mol. Cell. Biol. 10:615.
- Gabriel, A., Sisodia, S., Cleveland, D.(1987). Evidence of Discontinuous Transcription in the Trypanosomatid Crithidia fasciculata. J. Biol. Chem. 262:16192.
- Huibregtse, Jon M.
Department of Molecular Biology and Biochemistry Nelson Hall A309, Busch Campus Rutgers University Piscataway, NJ 08854
human papillomaviruses in carcinogenesis, the HPV E6 protein and p53, the ubiquitin-dependent proteolysis system
Experimental and epidemiologic data have implied a causative role for human papillomaviruses (HPVs) in the development of cancers of the uterine cervix. Several lines of genetic evidence suggest that the viral E6 and E7 genes confer the oncogenic or immortalizing activities of these viruses, while biochemical studies have shown that these proteins complex with and disrupt the functions of the p53 and pRB (retinoblastoma) proteins, respectively. This suggests that the oncogenic activities of the E6 and E7 proteins are in part a consequence of their interaction with tumor suppressor proteins.
We are particularly interested in the mechanism by which the HPV E6 protein inactivates p53. The interaction of E6 with p53 leads to the targeted degradation of p53 through the ubiquitin proteolysis system. We found that the interaction of E6 with p53 requires an additional cellular protein, E6-AP (E6-Associated Protein), which we have shown is also directly involved in catalyzing the ubiquitination of p53. By several criteria, we determined that E6-AP can be classified as a novel type of protein known as an E3 ubiquitin protein ligase.
E3 ubiquitin protein ligases are probably the most important component of the ubiquitin system in terms of determining substrate specificity, however they are also the most poorly characterized component of the system. Biochemical analyses of E6-AP and the definition of structure/function relationships has led to the most complete characterization of an E3 ubiquitin protein ligase to date. The cloning of the cDNA for E6-AP has revealed that a group of proteins of previously unknown function are related to E6-AP, and initial biochemical analyses suggest that the E6-AP-related proteins comprise an entire family of E3 ubiquitin protein ligases.
Our future research will continue to explore the role of E6 in HPV-associated cancers, especially with regard to additional cellular proteins that the E6/E6-AP complex might target for degradation. We are also exploring ways in which E6-AP activities are regulated and how substrate specificities of the E6-AP-related E3 ubiquitin protein ligases are determined. As several of the E6-AP-related E3 proteins have been identified in genetically tractable organisms, including yeast, we hope to take combined genetic and biochemical approaches to addressing these questions.
- Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Cloning and expression of the cDNA for E6-AP: a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13:775-784.
- Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Localization of the E6-AP regions that direct HPV E6 binding, association with p53, and ubiquitination of associated proteins. Mol. and Cell. Biol. 13:4918-4927.
- Scheffner, M., Huibregtse, J. M., Vierstra, R. D., and Howley, P. M. (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:494-505.
- Scheffner, M., Nuber, U., and Huibregtse, J. M. (1995) Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373:81-83.
- Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) A class of proteins structurally and functionally related to the E6-AP ubiquitin protein ligase. Proc. Natl. Acad. Sci. U.S.A. 92:2563-2567.
- Irvine, Kenneth
Waksman Institute - Room 135
Rutgers University Busch Campus
Piscataway, NJ 08854
cell-signaling, pattern formation, growth control, cell movement
My research is concentrated in two areas: 1) patterning and growth during development, focusing on fringe-dependent cell signaling, and 2) directed cell rearrangements. My work takes advantage of the powerful genetic, molecular and cellular techniques available in Drosophila, which facilitate both gene discovery and the analysis of gene function.
I identified fringe because of its role in growth control in the Drosophila wing. fringe is expressed in dorsal wing cells and encodes a novel, putatively secreted, protein. Both cell proliferation and the specification of specialized cells at the dorsal-ventral wing boundary, the wing margin, are induced by the juxtaposition of cells with and cells without;fringe expression. fringe thus appears to encode a cell-signaling protein. A collaborative effort led to the identification of two different fringe-related genes in both humans and mice, suggesting that fringe in fact defines a new family of cell-signaling molecules. My studies on Drosophila fringe have also revealed that fringe acts in a unique way, as a boundary-specific signal, and I am investigating the cell biological nature of this unusual property. A second collaboration established that fringe expression boundaries are both necessary and sufficient to induce the expression of other key wing patterning genes including wingless, vestigial, and Serrate. We are thus beginning to establish the molecular pathway that leads from fringe to wing growth, although clearly other players, such as the fringe receptor, remain to be identified. Investigations are also in progress on fringe function in other tissues.
Oriented cell rearrangements occur during the development of man;y animals, but the mechanism and regulation of these rearrangements is not understood. In Drosophila, I was able to show that cell rearrangements can be regionally autonomous and dependent upon the striped expression of certain genes involved in embryonic segmentation. I am involved in genetic approaches to identify other genes involved in the cellular interactions which must accompany cell rearrangement. These genetic studies will ultimately be followed up by molecular and cell biological analyses.
- Kim, J., Irvine, K.D., and Carroll, S. B. (1995) Cell recognition, signal induction, and symmetrical gene activation at the dorsal/ventral boundary of the developing Drosophila wing. Cell. (in press)
- Irvine, K.D. and Wieschaus, E. (1994b) fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79:595-606.
- Irvine, K. D. and Wieschaus, E. (1994a) Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes. Development 120:827-841.
- Klessig, Daniel F.
Waksman Institute Rutgers University Piscataway, NJ 08854-0759
signal transduction in plant-pathogen interaction
The major focus of our research is to help understand, at molecular and cellular levels, how plants protect themselves against microbial pathogens. Two systems are being studied - the response of tobacco to infection by tobacco mosaic virus (TMV) and the interaction of Arabidopsis with turnip crinkle virus (TCV). Our major goal is to decipher the signal transduction pathway(s) which leads to induction of defense-related genes, such as the pathogenesis-related (PR) genes, that appear to underlie disease resistance. In particular, the role of salicylic acid (SA) in this pathway(s) is being studied. During the past several years, we have helped establish that a key component of this pathway is SA, which can be reversibly inactivated and activated by conjugation and deconjugation to glucose, respectively. We have identified SA's receptor, which is catalase. One of SA's mechanisms of action is to inhibit catalase's enzymatic activity, which results in increased H2O2 levels. The elevated levels of H2O2, or other active oxygen species derived from it, are thought to induce certain defense responses such as PR gene expression, perhaps by acting as a second messenger. Catalase and its encoding gene family are being analyzed for patterns of gene expression, sensitivity to SA, and location of the SA-binding site. Other components of the signal transduction pathway are being identified by: i) defining the cis-acting element and trans-acting factor involved in SA induction of the PR-2 gene promoter, ii) isolating new genes that are rapidly induced by SA or pathogen infection, and iii) obtaining mutants in this pathway using Arabidopsis.
- Chen, Z., Silva, H., and Klessig, D.F. (1993). Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262:1883-1886.
- Chen, Z., Ricigliano, J., and Klessig, D.F. (1993). Purification and characterization of a soluble salicylic acid binding protein from tobacco. Proc. Natl. Acad. Sci. USA. 90:9533-9537.
- Hennig, J., Malamy, J., Grynkiewicz, G., Indulski, J., and Klessig, D.F. (1993). Interconversion of the salicylic acid signal and its glucoside in tobacco. Plant Journal 4:593-600.
- Dempsey, D.A., Wobbe, K.K., and Klessig, D.F. (1993). Resistance and susceptible responses of Arabidopsis thaliana to turnip crinkle virus. Phytopathology 83:1021-1029.
- Greenberg, J.T., Guo, A., Klessig, D.F., and Ausubel, F.M. (1994). Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defense functions. Cell. 77:551-563.
- Krug, Robert M.
Dept. of MBB CABM - Room 305 Busch Campus Piscataway, NJ 08854
pre-mRNA splicing, nucleocytoplasmic transport of mRNA, virus gene expression, protein-RNA interactions
The major focus of our laboratory is the mechanism of the control of splicing and of nucleocytoplasmic transport of eukaryotic messenger RNAs (mRNAs). Most of our recent work has been directed at the role of the influenza virus NS1 protein in these processes. We have shown that this viral protein has dual functions in the regulation of mRNA processing. First, it inhibits the nuclear export of spliced mRNAs. Mutational analysis identified two functional domains in the protein; an RNA-binding domain and an effector domain that presumably interacts with host cell nuclear proteins. These two types of functional domains are also found in the Rev protein of human immunodeficiency virus 1 (HIV-1), which facilitates rather than inhibits nuclear export of mRNA. However, the NS1 effector domain differs in several significant ways from the effector domain of the HIV Rev protein, which likely explains the opposite effects of these two proteins on nuclear mRNA export. Gel shift assays demonstrated that the NS1 protein recognizes and binds to the 3' poly A sequence on mRNAs. Consistent with this, the NS1 protein inhibits the nuclear export of poly A-containing mRNAs but not the export of mRNAs lacking poly A. Secondly, the NS1 protein inhibits the splicing of pre-mRNAs whether or not they contain poly A. The mode by which this inhibition occurs is novel. Unlike other proteins that regulate splicing, the NS1 protein binds to a specific site on a key spliceosomal RNA, U6 small nuclear RNA (sn RNA), leading to the inhibition of splicing, followed by the inhibition of spliceosome formation at higher levels of the protein. The NS1 protein binds to a purine-containing bulge imbedded in a U6 snRNA stem structure. The binding of the NS1 protein to this U6 snRNA sequence inhibits the interactions of U6 snRNA with two other UsnRNAs, U4 and U2, that occur during splicing. Thus, the RNA binding specificities of the NS1 protein-poly A and a U6 snRNA sequence, mediate the dual functions of this protein in mRNA processing-transport inhibition and splicing inhibition, respectively.
- Alonso-Caplen, F.V., Nemeroff, M.E., Yun, Q. and Krug, R.M. (1992). Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes and Development 6:255-267.
- Qiu, Y. and Krug, R.M. (1994). The influenza virus NS1 protein is a poly A-binding protein that inhibits nuclear export of mRNAs containing poly A, J. Virol 68:2425-2432.
- Qian, X-Y, Alonso-Caplen, F.V., and Krug, R.M. (1994). Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA, J. Virol. 68:2433-2441.
- Lu, Y., Qian, X-Y., and Krug, R.M. (1994). Inhibition of pre-mRNA splicing by the influenza virus NS1 protein. Genes and Development 8:1817-1828.
- Matsumura, Fumio
Dept. Molecular Biology & Biochemistry
Nelson Hall - Room A-321
Busch Campus, Rutgers University
Piscataway, NJ 08854
Control of cell division, microfilaments, cytokinesis, protein phosphorylation, cell transformation.
Cell division is central to the life of all multicellular organisms. Our
research is aimed at understanding how cells divide, in particular, what
controls microfilament reorganization during mitosis of cultured cells.
Microfilaments are responsible for the profound changes in cell shape and
structure (such as cytokinesis) the causes of which are almost entirely
obscure. We have focused on two microfilament-associated proteins,
caldesmon and the regulatory light chain of myosin II (MLC). Both proteins
regulate, in a complementary way, the contractility of actomyosin that controls
microfilament organization as well as cell shape and motility. Two recent
findings relate specific cell cycle dependent phosphorylations to the changes
in activities of these two proteins. First, we have found that cdc2 kinase
phosphorylates caldesmon causing its dissociation from microfilaments during
mitosis. Because cdc2 kinase, along with cyclins, initiates the entire
sequence of mitotic events including the morphological changes, the direct
phosphorylation of caldesmonby cdc2 kinase indicates that caldesmon is a key
protein in the mitotic pathway. In this context, our recent observations
indicate that the expression of a mutant non-phosphorylated caldesmon not only
stabilizes microfilaments but also retards cell growth. Second, we have
demonstrated that phosphorylation of MLC is switched from Ser 1,2 to Ser 19
during cell division, a critical alteration that may serve to activate
cytokinesis. Our finding thus poses a new question for study: how does this
cell cycle control element regulate the massive microfilament reorganization
seen during mitosis? We hope that our studies will help us understand not
only how cells divide, but also why cancer cells lose control of cell
- Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990). Mitosis-specific phosphorylation causes 83kDa nonmuscle caldesmon to dissociate from microfilaments. Nature 344:675-678.
- Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991). Phosphorylation of nonmuscle caldesmon by p34 cdc2 kinase during mitosis. Nature 349:169-172.
- Hosoya, H., Yamashiro, S., and Matsumura, F. (1991). Mitosis-specific phosphorylation of myosin light chain kinase. J. Biol. Chem. 266:22173-22178.
- Hosoya, N., Hosoya, H., Yamashiro, S., Mohri, H., and Matsumura, F. (1993). Localization of caldesmon and its dephosphorylation during cell division. J. Cell Biol. 121:1075-1082.
- Yamakita, Y., Yamashiro, S., and Matsumura, F. (1994). In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells. J. Cell Bio. 124:129-137.
- Montelione, Gaetano
FAX 235- 4850
CABM Room 205
679 Hoes Lane Piscataway, N.J. 08854
new NMR methods development, molecular recognition, growth factors, protein: nucleic acid complexes, DNA and RNA structure, molecular dynamics, protein design
The general aim of our research is to use NMR spectroscopy as a tool for protein engineering and rational drug design. We develop new methods for protein solution structure determination and apply these techniques to proteins of pharmaceutical or medical interest. The combined methods of site-directed mutagenesis, NMR spectroscopy, and conformational energy calculations are being used to (1) determine three dimensional structures of small proteins in solution, (2) determine the structures of protein-protein, protein-receptor, and protein-nucleic acid complexes, (3) characterize effects of amino acid substitutions on protein structure, stability, and dynamics, (4) direct efforts to design and engineer proteins and provide information for rational drug design, and (5) study the molecular mechanisms by which proteins fold into their biologically-active conformations. We are currently working on structure determination and refinement of several protein growth factors including epidermal and type-a transforming growth factors, insulin-like growth factors, and heparin-binding growth factor. Several DNA-and RNA-binding proteins are also being studied by NMR. We have recently determined solution structures of an IgG-binding domain of staphylococcal Protein A and of an RNA-binding protein from E.coli which is overproduced in response to cold shock (Cold Shock Protein A). We have characterized structural changes in these molecules which are required for binding to IgG proteins or to nucleic acids. Nuclear relaxation time measurements are used to characterize intramolecular motion in these small proteins. This research has important implications in the fields of protein physical chemistry, molecular design, receptor-ligand interactions, and oncogenesis.
- Montelione, G.T.; WŁthrich, K.; Burgess, A.W.; Nice, E.C.; Wagner, G.; Scheraga, H.A. (1992). Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization. Biochemistry 31:236-249.
- Montelione, G.T.; Lyons, B.A.; Emerson, S.D.; Tashiro, M. (1993). An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically enriched proteins. J. Amer Chem. Soc. 114:10974-10975.
- Moy, F.J.; Li, Y.-C.;Winkler, M.E.; Scheraga, H.A.; Montelione, G.T. (1993). Solution structure of human type-a transforming growth factor determined by heteronuclear NMR spectroscopy and refined by energy minimization. Biochemistry 32:7334-7353.
- Lyons, B.A.; Tashiro, M; Cedergren, L., Nilsson, B.; Montelione, G.T. (1993). An improved strategy for determining sequence-specific resonance assignments of isotopically enriched proteins and its applications on an engineered domain of staphylococcal Protein A. Biochemistry 32: 7839-7845.
- Newkirk, K.; Feng, W.; Jiang, W.; Tejero, R.; Emerson, S.D.; Inouye, M.; Montelione, G.T. (1994). Solution NMR structure of the major cold-shock protein (CspA) from Escherichia coli: Identification of a binding epitope for single-stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 91:5114-5118.
- Neigeborn, Lenore
Waksman Institute - Room 244
Rutgers University Piscataway, NJ 08854-0759
regulation of meiotic gene expression
Our research focuses on how independent responses to discrete regulatory signals are integrated to direct gene expression. An understanding of these control mechanisms is especially relevant to developmental switches which often rely on multiple regulatory signals to initiate and sustain a program of coordinated gene expression. We have chosen to focus on a simple developmental model system: regulation of meiosis and spore formation in the yeast Saccharomyces cerevisiae. We have found that the protein kinase, MCK1 (Meiotic and Centromere Regulatory Kinase), governs the decision to initiate the sporulation pathway and plays a role in mediating chromosome segregation during cell division. MCK1p is a member of a new class of protein kinase, the dual-specificity kinases, characterized by the novel ability to phosphorylate target proteins on serine, threonine, and tyrosine residues.
The MCK1 gene product mediates the decision to enter the sporulation pathway by promoting expression of the sporulation activator, IME1, presumably by phosphorylating a positive transcription factor required for IME1 expression. Our present objective is to determine how the MCK1 gene product is integrated into the regulatory circuitry governing IME1 expression. To understand its role in the promotion of meiotic gene expression we must analyze the nature of regulation of MCK1p activity, characterize the targets of MCK1p phosphorylation, and identify additional cellular factors also functioning to control entry into the meiotic pathway. We have taken a multidisciplinary approach utilizing genetic, molecular, and biochemical techniques:
Genetic Approach: We have identified suppressor loci capable of bypassing the requirement for MCK1 in the stimulation of IME1 expression. These suppressors define several, previously unidentified, yeast genes. We are currently investigating the roles played by these genes in the regulation of the sporulation pathway.
Molecular Approach: We have utilized DNA sequence information derived from the genes functioning along with MCK1 to control IME1 expression to create testable models for the control of entry into the meiotic pathway. In addition, we are utilizing a 2-Hybrid Interaction Trap to identify gene products which physically interact with MCK1p and other meiotic regulators.
Biochemical Approach: We have undertaken an extensive structure/function analysis of MCK1p to characterize it's biochemical properties (substrate specificity, regulation of activity, etc.).
Given the universal role played by protein kinases in eukaryotic control pathways, we believe these studies can provide valuable insight into the ways cells accomplish signal transduction, development and differentiation.
- Neigeborn, L. and Mitchell, A. P. (1991) The yeast MCK1 gene encodes a protein kinase homolog that activates early meiotic gene expression. Genes and Development, 5: 533-48.
- Smith, H. E., Su, S. S., Neigeborn, L., Driscoll, S. E., Mitchell, A. P. (1990). Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae. Molecular and Cellular Biology, 10: 6103-6113.
- Niederman, Robert A.
Molecular Biology and Biochemistry
Nelson Hall - Busch Campus, A317
Rutgers University Piscataway, NJ 08854
structure, function, and assembly of photosynthetic membranes
A variety of integrated biochemical, biophysical and molecular genetic approaches are used to investigate the structural organization and assembly of photosynthetic membranes in Rhodobacter sphaeroides, an experimentally accessible prokaryotic model system. These membrane structures contain integral light-harvesting (antenna) complexes which transfer excitations to photochemical reaction center. This initiates cyclic electron transfer through a mitochondrial-type cytochrome bc1 complex, resulting in re-reduction of the photooxidized reaction center and conservation of the light energy as an electrochemical proton gradient coupled to the synthesis of ATP.
The role which noncatalytic subunits play in electron transport complexes is being examined in the R. sphaeroides cytochrome bc1 complex, which in addition to highly conserved cytochrome and iron-sulfur protein components, contains a unique polypeptide (subunit IV) lacking a prosthetic group. The membrane topology of subunit IV is being eluciated by newly developed proteolytic digestion procedures. We are also constructing strains from which the subunit IV structural gene will be deleted, to determine whether subunit IV is functionally essential, or has important structural and assembly roles. A second area of investigation involves elucidation of the structural basis for rapid and efficient excitation energy transfer by the light-harvesting complexes of R. sphaeroides. The questions currently under investigation include: the molecular nature of the interactions between the peripheral and core antenna complexes and the core antenna and reaction center; the structural basis for the organization of core antenna bacteriochlorophylls into an arrangement that facilitates directed energy transfer to the reaction center; and the role which an open reading frame (pufX), located immediately downstream from genes encoding core antenna and reaction center polypeptides, plays in the organization of photosynthetic units.
- Olivera, L.M. and Niederman, R.A. (1993). Effect of phospholipase A2 on the carotenold and bacterloclorophyll components of the light-harvesting complexes in Rhodobacter sphaeroides chromatophores. Biochemistry 32:858-866.
- Westerhuis, W.H.J., Farchaus, J.W. and Niederman, R.A. (1993). Altered spectral properties of the B875 light-harvesting pigment-protein complex in a Rhodobacter sphaeroides mutant lacking pufX. Photochem. Photobiol. 58:460-463.
- Olivera, L.M., Westerhuis, W.H.J. and Niederman, R.A. (1994). Spectral alterations in Rhodobacter capsulatus mutants with site-directed changes in the bacteriochlorophyll-binding site of the B880 light-harvesting complex. Biochem. Biophys. Acta 1185:318-326.
- Wu, J. and Niederman, R.A. (1995) Topological organization of the Rieske iron-sulphur protein and subunit IV in the cytochrome bc1 complex of Rhodobacter sphaeroides. Biochem. J. (In press).
- Norris, David
Waksman Institute Room 267
Busch Campus Piscataway, NJ 08854
chromosome structure, genetic recombination, enzymology
The intracellular process of genetic recombination moves genetic information from one genomic location to another. The DNA intermediates in this reaction are well understood. In our laboratory, in vitro model reactions are being used to purify proteins from the yeast Saccharomyces cerevisiae that catalyze subreactions of genetic recombination. In particular, the laboratory is purifying two types of enzyme: strand exchange proteins, which promote the formation of the central reaction intermediate of genetic recombination, and double-stranded DNA exonucleases, which process recombination intermediates. In addition, mutant strains lacking these proteins are being constructed to determine how they function in vivo in genetic recombination.
Chromosomal DNA inside eucaryotic cells is found complexed to many structural proteins. Chief among these are histones and HMG proteins. In an effort to understand the biology of these proteins, the laboratory is purifying novel histones and HMG proteins from yeast. In addition, mutant strains lacking these proteins are being isolated to determine how the proteins regulate chromosome structure in vivo.
- Norris, D., and Osley., M.A. (1987). The two gene pairs encoding H2A and H2B play different roles in the Saccharomyces cerevisiae life cycle. Mol. Cell. Biol. 7:3473-3481.
- Clark-Adams, C. Norris, D., . Osley, M.A., Fassler, J.S. and Winston, F.. (1988). Changes in histone gene dosage alter transcription in yeast. Genes and Development 2:150-159.
- Norris, D. B. Dunn, B. and . Osley., M.A. (1988). The effect of histone gene deletions on chromatin structure in Saccharomyces cerevisiae. Science 242:759-761.
- Norris, D. and R. Kolodner. (1990). Purification of a strand exchange stimulatory factor from Saccharomyces cerevisae. Biochemistry 29: 7903-7911.5.
- Norris, D. and R. Kolodner. (1990). Interaction of a Saccharomyces cerevisiae strand exchange stimulatory factor with DNA. Biochemistry 29: 7911-7917.
- Padgett, Richard W.
Waksman Institute Rutgers University Piscataway, NJ 08854-0759
signal transduction, molecular biology, developmental biology & genetics, Drosophila and C. elegans
The work in my laboratory focuses on the transforming growth factor-b-like (TGF-b -like) molecules and their signal transduction pathway. This growth factor family is found in vertebrates and invertebrates, but little is known about how they transduce their signal to the nucleus. Because of the powerful experimental approaches available in Drosophila and C. elegans, we are using them as model systems to dissect this signal transduction pathway. We are currently studying two aspects of the TGF-b-like molecules. First, we are studying upstream genes that regulate the activity of the growth factor, such as the tolloid and tolkin genes. Secondly, we are studying genes that encode receptors for the Drosophila TGF-b-like genes, with the aim of understanding how the signal is transferred from the receptor to other cytoplasmic molecules. In order to study these processes, we are using molecular genetic and biochemical methods to characterize these genes and to determine how their signals are transduced. As a complement to our Drosophila work, we are characterizing components of this signal transduction pathway in the nematode, C. elegans. Since these growth factors are involved in regulating the growth of many tissues, understanding how they function has important implications in development.
- Xie, T.A., Finelli, A.L. and Padgett, R.W. (1994). The Drosophila saxophone Gene Encodes a Serine-threonine Kinase Receptor of the TGF-b Superfamily. Science 263:1756-1759.
- Finelli, A.L., Bossie, C.A., Xie , T. and Padgett, R.W. (1994). Antimorphic Alleles of the Drosophila tolloid Gene Contain Amino Acid Substitutions in the Protease Domain. Development 120:861-870 .
- Padgett, R.W., Wozney, J and Gelbart, W.M. (1993). Human BMP Sequences Can Confer Normal Dorsal-Ventral Pattering in the Drosophila Embryo. Proc. Natl. Acad. Sci. USA 90:2905-2909.
- Hursh, D.A., Padgett, R.W..and Gelbart, W.M. (1993). Cross Regulation of decapentaplegic and Ultrabithorax transcription in Embryonic Visceral Mesoderm Mediates Gut Morphogenesis in Drosophila. Development 117:1211-1222.
- St. Johnston, R. Daniel, F., Hoffmann, M., Blackman, D., Segal, D., Grimaila, R., Padgett, R.W., Irick, H. and Gelbart, W.M. (1990). The Molecular Organization of the decapentaplegic Gene in Drosophila melanogaster. Genes and Devel. 4:1114-1127.
- Pietruszko, Regina
Center of Alcohol Studies Busch Campus Rutgers University Piscataway, NJ 08855-0969
alcohol metabolism, human aldehyde dehydrogenase isozymes - purification, characterization, and enzyme mechanism
My research is in the area of alcohol metabolism and the adverse effects of alcoholism. The major thrust is towards characterization of human aldehyde dehydrogenases (ALDH) which catalyze acetaldehyde metabolism. ALDHs occur in multiple molecular forms. The procedures employed include purification to homogeneity and characterization of individual isozymes. The characterization consists of: determination of native MW, subunit MW, substrate specificity in terms of Km and Vmax with various aldehydes and coenzymes, amino acid composition, amino terminal sequence. The characterization frequently also includes peptide mapping to isolate peptides to be submitted to an outside laboratory for the amino acid sequence determination to obtain information for development of nucleotide probes for cloning. The primary structure is then determined from the sequence of cloned cDNA. Some ALDHs which have been already characterized (and occur in large amounts in human post-mortem liver) are purified to homogeneity and are then used for identification of residues involved in catalysis by chemical modification by active-site-directed reagents. This work involves characterization of reagents to determine conditions in which they are active-site-directed. This is followed by labeling of the active site residues with radioactive reagents, peptide mapping, isolation of radioactive peptides, , their identification by amino acid sequence. Position of radioactive label in the amino acid sequence is determined by scintillation counting of the sequencing cycles. Some recent work has been concerned with mode of ALDH inactivation by nitrate esters and identity of the covalent reaction intermediate. Site-directed mutagenesis of cloned cDNA is planned in the near future.
- Mukerjee, N., and Pietruszko, R. (1994). Inactivation of human aldehyde dehydrogenase by isosorbide dinitrate. J. Biol. Chem. 269:21664-21669.
- Kurys, G., Shah, P.C., Kikonyogo, A., Reed, D., Ambroziak, W. and Pietruszko, R. (1993). Human aldehyde dehydrogenase cDNA cloning and primary structure of the enzyme that catalyzes dehydrogenation of 4- aminobutyraldehyde. Eur. J. Biochem. 218:311-320.
- Blatter, E.E., Abriola, D.P., and Pietruszko, R. (1992). Aldehyde dehydrogenase covalent intermediate in aldehyde dehydrogenation and ester hydrolysis. Biochem. J. 282:353-360.
- Abriola, D.P., Fields, R., Stein, S., MacKerell, A.D., Jr., and Pietruszko, R. (1987). Active site of human aldehyde dehydrogenase. Biochemistry U.S.A. 26:5679-5684.
- Sofer, William H.
Waksman Institute Rutgers University Piscataway, NJ 08854-0759
genetic algorithms and genetic programming, educational software
Genetic algorithms are search and classification methods that are loosely based on evolution, natural selection, genetic recombination and mutation. As implemented in computer programs, they are procedures that find solutions to difficult problems by having the computer develop rules by discarding poor solutions and rewarding good ones.
We are presently writing a program to predict secondary structure of proteins based primarily on their sequence. Most current methods do at best only about twice as well as random guesses in assigning secondary structure (alpha helix, beta strands and coils) to proteins that have little similarity to others in the database. We are investigating whether methods based on genetic algorithms will farebetter.
- Friedman, R., Hotaling, E., Borack, L., and Sofer, W. (1994). Interactions between the regulatory regions of two Adh alleles. Genetica. In press.
- Sofer, W. and Tompkins, L. (1994). Genetics in the Classroom. Genetics 136:417-422.
- Shen, N. L. L., Subrahmanyam, G., Clark, W., Martin, P., Sofer, W. (1989). Analysis of Adh gene regulation in Drosophila: studies using somatic transformation. Developmental Genetics 10:210-219.
- Sofer, W. and Martin, P. F. (1987). Analysis of alcohol dehydrogenase gene expression in Drosophila. Ann. Rev. Gen. 21:203-25.
- Steward, Ruth
Waksman Institute Rutgers University Busch Campus Piscataway, NJ 08854
pattern formation in Drosophila
The dorsal gene encodes the last step in the maternal signal-transduction pathway that results in dorsal-ventral polarity. Dorsal is the ventral morphogen that instructs ventral and lateral cells as to their identity, and is a member of the Rel family of transcription factors that also includes the lymphocyte transcription factor NF-kB, the viral oncogene v-rel, and v-rel's cellular homology, c-rel. The activity of dorsal is regulated postranscriptionally. It is found in inactive form in the cytoplasm of early embryos and is present in a ventral-to-dorsal nuclear gradient in blastoderm stage embryos. Once in the nucleus, dorsal protein acts as a transcriptional activator as well as a repressor of different zygotic genes within the same nucleus. Our work on dorsal concentrates on two major questions. How is the dorsal nuclear protein gradient established, and how does dorsal protein function once it is in the nucleus?
Genetic studies show that the maternal Bicaudal-D gene product is involved in localizing determinants at the posterior end of the early embryo and that it is also essential for the differentiation of the oocyte early in oogenesis, most likely by localizing oocyte determinants as RNAs in the prospective oocyte. We have shown that both the Bicaudal-D RNA and p[protein accumulate in the prospective oocyte and that the localization of the Bicaudal-D and other RNAs in the oocyte is dependent on the accumulation of Bicaudal-D protein. The Bicaudal-D protein shows similarity to myosin heavy chain tails, paramyosin, and kinesin, and so may be involved in transporting RNAs to specific domains of the oocyte. We are investigating these possibilities by identifying proteins associated with Bicaudal-D genetically and biochemically. We are also determining the function of the localization of the RNA and protein, and studying which sequences are necessary for the localization.
- Suter, B. and Steward, R. (1991). Requirement for phosphorylation and localization of the Bicaudal-D protein in Drosophila oocyte differentiation. Cell 67:917-926.
- Govind, S., Whalen, A. M., and Steward, R. (1992). In vivo self-association of the Drosophila rel protein dorsal. Proc. Natl. Acad. Sci. US 89:6761-6765.
- Govind, S., Brennan, L., and Steward, R. (1993). Homeostatic balance between dorsal and cactus proteins in the Drosophila embryo. Development 117:135-148.
- Whalen, A. and Steward, R. (1993) Dissociation of the dorsal-cactus complex and phosphylation of the dorsal protein correlate with the nuclear localization of dorsal. J. Cell. Biol. 123:523-534.
- Vershon, Andrew K.
Waksman Institute Rutgers University Piscataway, NJ 08854-0759
transcriptional regulation in eukaryotes
The focus of our research is on the regulation of transcription in eukaryotes. Specifically, we are investigating how different regulatory proteins interact to control gene expression, and how, in some cases, these interactions change the apparent regulatory function of the protein. We have chosen two systems in the yeast Saccharomyces cerevisiae to study the regulation of eukaryotic transcription. The first project investigates the protein-protein interactions and the structure/function relationship of the yeast MAT a2 protein- a cell-type specific repressor of mating type. We are currently examining how a2, a member of the homeodomain family of DNA-binding proteins, specifically binds to its DNA sites and how it interacts with a second protein, MCM1. MCM1 has significant sequence similarity to the mammalian serum response factor, SRF, and we are also investigating how the MCM1 protein specifically recognizes its DNA site.
The second project investigates the components involved in the regulation of the transcription of HOP1, a yeast gene that is expressed only during the early stages of meiosis. We are presently using biochemical and genetic strategies to isolate the proteins that bind to regulatory sites that we have identified in the HOP1 promoter. Once we have identified these proteins we plan to study how signals in the meiotic pathway control the function of these proteins to regulate HOP1 transcription.
- Wolberger, C., Pabo, C.O., Vershon, A.K., and Johnson, A.D. (1991). Crystallization and Preliminary X-ray Diffraction Studies of a MAT a2- DNA Complex. J. Mol. Biol. 217:11-13.
- Phillips, C.L., Vershon, A.K., Johnson, A.D., and Dahlquist, F.W. (1991). Secondary Structure of the Homeodomain of Yeast a2 repressor by NMR. Genes & Dev. 5:764-772.
- Wolberger, C., Vershon, A.K., Liu, B., Johnson, A.D., and Pabo, C.O. (1991). Crystal Structure of a MAT a2 Homeodomain-Operator Complex Suggests a General Model for Homeodomain-DNA Interactions. Cell 67: 517-528.
- Vershon, A. K., Hollingsworth, N.M., and Johnson, A.D. (1992). Meiotic Induction of the Yeast HOP1 Gene is Controlled by Positive and Negative Regulatory Elements. Mol. Cell. Biol. 12:3706-3714.
- Vershon, A.K. and Johnson, A.D. (1993). A Short, Disordered Protein Region Mediates Interactions between the Homeodomain of the Yeast a2 Protein and the MCM1 Protein. Cell 72:105-112.
- Vershon, A. K., Jin, Y., and Johnson, A. D. (1995). A homeodomain protein
lacking specific side chains of Helix 3 can still bind DNA and direct transcriptional repression. Genes & Dev. 9: 182-192.
- Jin, Y., Mead, J., Li, T., Wolberger, C., and Vershon, A. K. (1995). Altered DNA recognition and bending by insertions in the a2 tail of the yeast a1/a2
homeodomain heterodimer. Science 270: 290-293.
- Mead, J. Zhong, H., Acton, T. B., and Vershon, A. K. (1996). Yeast a2 and
Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes. Mol. Cell. Biol. 16: 2135-2143.
- Gailus-Durner, V., Xie, J., Chintamaneni, C., and Vershon, A. K. (1996).
ABF1 regulates the expression of the meiosis-specific gene HOP1. Mol. Cell. Biol. 16: 2777-2786.
- Yamashiro, Shigeko
Control of cell division, microfilaments, cytokinesis, protein phosphorylation, cell transformation.
Dr. Yamashiro collaborates closely with Dr. Matsumura - please see the
description of his research and publications for more details.