Gretchen Edwalds-Gilbert

Associate Professor of Biology

Email: [email protected]
Office: Keck Science Center 202
Phone: 909-607-7068

Educational Background

B.A., Swarthmore College
Ph.D., Weill Medical College of Cornell University/Sloan Kettering Institute

Research Interests

Comparative genomics, genetic networks involved in response to environmental toxins Role termini of DEAH family members in protein function, specifically spliceosome binding, mechanism(s) that explain the phenothypes of certain alleles of PRP43 and PRP2

Thesis Topics

Cell and molecular biology
Gene expression
Pre-mRNA splicing in yeast

Selected Publications

1. Gretchen Edwalds-Gilbert. (2015). Location is Everything: an Educational Primer for Use with “Genetic analysis of the ribosome biogenesis factor Ltv1 of S. cerevisiae.” Genetics  199.
2. Kum-Loong Boon, Tatsiana Auchynnikava, Gretchen Edwalds-Gilbert, J. David Barrass, Alastair P. Droop, Christophe Dez, and Jean D. Beggs. (2006). Yeast Ntr1/Spp382 Mediates Prp43 Function in Postspliceosomes. Molecular and Cellular Biology 26: 6016-6023.
3. Edwalds-Gilbert, G., D.-H. Kim, E. Silverman, C. Cheng, and R.-J. Lin. (2004). Definition of a spliceosome interaction domain in yeast Prp2 ATPase. RNA 10: 210-220.
   Abstract: The Saccharomyces cerevisiae splicing factor Prp2 is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2Ts strain, suggesting that the C-domain interferes with the activity of the Prp2Ts protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.
   Article – https://rnajournal.cshlp.org/content/10/2/210.full.pdf+html?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexactfulltext=and&searchid=1098985553373_458&stored_search=&FIRSTINDEX=0&sortspec=relevance&volume=10&firstpage=210&resourcetype=1&journalcode=rna
4. Silverman, E., G. Edwalds-Gilbert, and R.-J. Lin. (2003). DExD/H-box proteins and their partners: helping RNA helicases unwind. Gene 312: 1-16.
   Article – URL not found
5. Edwalds-Gilbert, G., D.-H. Kim, S.-H. Kim, Y.-H. Tseng, Y Yu, and R.-J. Lin. (2000). Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins. RNA  6(8): 1106-19.
   Article – https://rnajournal.cshlp.org/content/6/8/1106.full.pdf+html?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexactfulltext=and&searchid=1098985186237_441&stored_search=&FIRSTINDEX=0&sortspec=relevance&volume=6&firstpage=1106&resourcetype=1&journalcode=rna
6. Veraldi, K. L., G. Edwalds-Gilbert, C. C. MacDonald, A. M. Wallace, and C. Milcarek. (2000). Isolation and characterization of polyadenylation complexes assembled in vitro. RNA   6: 768-777.
   Article – https://rnajournal.cshlp.org/content/6/5/768.full.pdf+html?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexactfulltext=and&searchid=1098985246992_448&stored_search=&FIRSTINDEX=0&sortspec=relevance&volume=6&firstpage=768&resourcetype=1&journalcode=rna
7. Kim, D.-H., G. Edwalds-Gilbert, C. Ren, and R.-J. Lin. (1999). A mutation in a methionine tRNA gene suppresses the prp2-1Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae. Genetics   153: 1105-1115.
   https://www.genetics.org/content/153/3/1105.fullArticle –
8. Martincic, K., R. Campbell, G. Edwalds-Gilbert, L. Souan, M. T. Lotze, and C. Milcarek. (1998). Increase in the 64-kDa subunit of the polyadenylation/cleavage stimulatory factor during the G0 to S phase transition. Proc Natl Acad Sci U S A   95: 11095-100.
9. Edwalds-Gilbert, G., K. L. Veraldi, and C. Milcarek. (1997). Alternative poly(A) site selection in complex transcription units: means to an end?. Nucleic Acids Res   25: 2547-61.
   Abstract: Many genes have been described and characterized which result in alternative polyadenylation site use at the 3′-end of their mRNAs based on the cellular environment. In this survey and summary article 95 genes are discussed in which alternative polyadenylation is a consequence of tandem arrays of poly(A) signals within a single 3′- untranslated region. An additional 31 genes are described in which polyadenylation at a promoter-proximal site competes with a splicing reaction to influence expression of multiple mRNAs. Some have a composite internal/terminal exon which can be differentially processed. Others contain alternative 3′-terminal exons, the first of which can be skipped in some cells. In some cases the mRNAs formed from these three classes of genes are differentially processed from the primary transcript during the cell cycle or in a tissue-specific or developmentally specific pattern. Immunoglobulin heavy chain genes have composite exons; regulated production of two different Ig mRNAs has been shown to involve B cell stage-specific changes in trans-acting factors involved in formation of the active polyadenylation complex. Changes in the activity of some of these same factors occur during viral infection and take-over of the cellular machinery, suggesting the potential applicability of at least some aspects of the Ig model. The differential expression of a number of genes that undergo alternative poly(A) site choice or polyadenylation/splicing competition could be regulated at the level of amounts and activities of either generic or tissue-specific polyadenylation factors and/or splicing factors.
   Article – page not found
10. Edwalds-Gilbert, G., and C. Milcarek. (1995). Regulation of poly(A) site use during mouse B-cell development involves a change in the binding of a general polyadenylation factor in a B-cell stage-specific manner. Mol Cell Biol 15: 6420-9.
    Abstract – During the development of mouse B cells there is a regulated shift from the production of membrane to the secretion-specific forms of immunoglobulin (Ig) mRNA, which predominate in the late-stage or plasma B cells. By DNA transfection experiments we have previously shown that there is an increase in polyadenylation efficiency accompanying the shift to secretion-specific forms of Ig mRNA (C. R. Lassman, S. Matis, B. L. Hall, D. L. Toppmeyer, and C. Milcarek, J. Immunol. 148:1251- 1260, 1992). When we look in vitro at nuclear extracts prepared from early or memory versus late-stage or plasma B cells, we see cell stage-specific differences in the proteins which are UV cross-linked to the input RNAs. We have characterized one of these proteins as the 64-kDa subunit of the general polyadenylation factor cleavage-stimulatory factor (CstF) by immunoprecipitation of UV-cross-linked material. The amount of 64-kDa protein and its mobility on two-dimensional gels do not vary between the B-cell stages. However, the activity of binding of the protein to both Ig and non-Ig substrates increases four- to eightfold in the late-stage or plasma cell lines relative to the binding seen in the early or memory B-cell lines. Therefore, the binding activity of a constitutive factor required for polyadenylation is altered in a B-cell-specific fashion. The increased binding of the 64-kDa protein may lead to a generalized increase in polyadenylation efficiency in plasma cells versus early or memory B cells which may be responsible for the increased use of the secretory poly(A) site seen in vivo.
    Article – https://mcb.asm.org/content/mcb/15/11/6420.full.pdf?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1099331142289_2495&stored_search=&FIRSTINDEX=0&volume=15&firstpage=6420&journalcode=mcb
11. Edwalds-Gilbert, G., and C. Milcarek. (1995). The binding of a subunit of the general polyadenylation factor cleavage- polyadenylation specificity factor (CPSF) to polyadenylation sites changes during B cell development. Nucleic Acids Symp Ser  33: 229-33.
12. Guarini, L., M. Temponi, G. M. Edwalds, J. R. Vita, P. B. Fisher, and S. Ferrone. (1989). In vitro differentiation and antigenic changes in human melanoma cell lines. Cancer Immunol Immunother 30: 262-8.
    Abstract – Malignant transformation of melanocytes may be associated with changes in the expression of HLA antigens and melanoma-associated antigens (MAA). To determine whether these changes reflect the differential expression of HLA antigens and MAA by melanocytes at different stages of differentiation, we have studied the effect of the reversible induction of differentiation by fibroblast interferon (interferon beta) and/or 12-O-tetradecanoyl-phorbol 13-acetate (TPA) on the expression of HLA antigens and MAA by the melanoma cell lines DU-2, FO-1 and HO-1. The three melanoma cell lines differed in their sensitivity to the differentiating and antiproliferative activity of these two compounds and displayed an increased growth suppression and induction of differentiation, when incubated with the combination of TPA and interferon beta. Incubation of the three melanoma cell lines with interferon beta, TPA or their combination resulted in a differential modulation of the expression of membrane-bound high-molecular-mass melanoma-associated antigen, 115-kDa MAA, 100-kDa MAA, intercellular adhesion molecule 1, HLA class I antigens and gene products of the HLA-D region. Each melanoma cell line displayed a unique pattern of antigenic modulation when exposed to the two differentiating agents alone or in combination. No direct relationship was found between the effects of interferon beta and/or TPA on the growth and differentiation of the three melanoma cell lines and the expression of HLA antigens or the MAA evaluated in the present study. These findings argue against a direct role of any of the antigens tested in the reversible induction of human melanoma cell differentiation in the in vitro system.
13. Vita, J. R., G. M. Edwalds, T. Gorey, E. M. Housepian, M. R. Fetell, L. Guarini, J. A. Langer, and P. B. Fisher. (1988). Enhanced in vitro growth suppression of human glioblastoma cultures treated with the combination of recombinant fibroblast and immune interferons. Anticancer Res 8: 297-302.
    Abstract – In the present study, we have evaluated the effect of recombinant human fibroblast, IFN-beta ser, and immune, IFN-gamma, interferon, alone and in combination, on the proliferation of fifteen early passage human glioblastoma cell cultures. Explant cultures were established from glioblastoma tumor tissue obtained at the time of surgery. After sufficient outgrowth, cultures were dispersed with trypsin/versene and maintained as independent cell lines. IFN-beta ser induced a greater than or equal to 50% reduction in the 7 day growth of 6 of the 15 cultures. The majority of cultures, 9 of 15, displayed less than or equal to 50% growth suppression in comparison with control cultures after 7 days exposure to 2000 Units/ml of IFN-beta ser. When treated with 2000 Units/ml of IFN-gamma, only 1 of the 15 glioblastoma cultures exhibited a greater than or equal to 50% reduction in growth. In contrast, when treated with the combination of IFN-beta ser plus IFN-gamma, 1000 Units/ml of each interferon preparation, 12 of 15 cultures were inhibited by greater than or equal to 50% after 7 days growth. The combination of interferons was effective in suppressing glioblastoma growth both in cultures displaying relative sensitivity and those exhibiting innate resistance to either or both types of interferon when employed alone. One glioblastoma culture, G-7, was studied through 45 passages and displayed the same sensitivity at different passages to growth inhibition when exposed to IFN-beta ser, IFN-gamma or both interferons. Based on previous clinical studies indicating that IFN-beta or IFN-gamma when administered alone to patients do not generally alter the clinical progression of malignant gliomas, the present results suggest that the combination of IFN-beta plus IFN-gamma may prove more effective than either agent alone in the clinical treatment of patients with glioblastoma multiforme.
14. Siegel, R. J., M. Fealy, G. Edwalds, H. Karagueuzian, and M. C. Fishbein. (1987). Immunohistochemical localization of procainamide in normal, ischemic, and necrotic canine myocardium during acute experimental myocardial infarction. Am Heart J   113: 1383-9.
    Abstract – This report represents the first application of immunohistochemical methods for localizing an exogenously administered drug. Intravenously administered procainamide was localized in normal, ischemic, and necrotic myocardium in 23 dogs. Rabbit antiprocainamide antibodies were used in an avidin-blotin-peroxidase complex staining method. Normal myocardium demonstrated diffusely positive immunostaining for procainamide, as did the cardiac conduction system and vascular endothelial cells. Necrotic myocardium demonstrated markedly reduced to absent immunostaining. By contrast, in regions of myocardial ischemia without necrosis, immunostaining for procainamide was similar to that in the normal myocardium. Procainamide myocardial tissue levels were reduced in necrotic and ischemic zones compared to normal (p < 0.05) only in those animals in which procainamide was administered after rather than before the onset of coronary occlusion. The demonstration of the absence of drug binding in the necrotic cells suggests that myocardial tissue levels or radiolabelled assessment of drug distribution can be misleading when nonhomogenous tissue is sampled. The immunohistochemical technique provides additional information about the regional and cellular distribution of procainamide that is complementary to the information obtainable by radiolabelling microspheres and from biochemical assays
15. Fisher, P. B., H. Hermo, Jr., W. E. Solowey, M. C. Dietrich, G. M. Edwalds, I. B. Weinstein, J. A. Langer, S. Pestka, P. Giacomini, M. Kusama, and et al. (1986). Effect of recombinant human fibroblast interferon and mezerein on growth, differentiation, immune interferon binding and tumor associated antigen expression in human melanoma cells. Anticancer Res   6: 765-74.
    Abstract – The combination of recombinant human fibroblast interferon (INF-delta) and the antileukemic compound mezerein (MEZ) results in a synergistic suppression in the growth of human melanoma cells and a concomitant increase in melanin synthesis. In the present study we have further analyzed this synergistic interaction and have also evaluated the effect of IFN-delta and MEZ, alone and in combination, on recombinant human gamma interferon (IFN-gamma) binding and Class I HLA and melanoma associated antigen (MAA) expression in the HO-1 human melanoma cell line. Single cell clones isolated from the HO-1 cell line varied in their sensitivity to the antiproliferative effects of IFN-delta and MEZ. With all twelve clones, however, the combination of IFN-delta plus MEZ was more growth inhibitory than either agent used alone, even in HO-1 subclones displaying relative resistance to IFN-delta. By continuous growth in gradually increasing concentrations of IFN-delta, a variant population of HO-1 cells, HO-1 delta R-D, was generated which was more resistant to the antigrowth effects of IFN-delta than the original HO-1 parental cell line. In the IFN delta R-D cell line the combination of IFN-delta plus MEZ synergistically suppressed growth. Exposure of HO-1 cells to 2500 units/ml IFN-delta or 50 ng/ml MEZ for 96 hr resulted in no change or an increase in the binding of labelled IFN-gamma to surface receptors, whereas the combination of IFN-delta plus MEZ increased IFN-gamma binding 2-to-4-fold in HO-1 cells. This increase was the result of an increase in the number of receptors on treated cells coupled with a protection against a decrease in receptors observed for confluent untreated cells. Changes in IFN-gamma binding resulting from treatment with IFN-delta plus MEZ were not associated with alterations in the binding affinity of INF-gamma to its receptor. Changes were also observed in the expression of HLA Class I antigens and MAAs following treatment of HO-1 cells with IFN-delta, MEZ or IFN-delta plus MEZ. IFN-delta and MEZ increased the expression of HLA Class I antigens a 96 kd MAA defined by MoAb CL203, a 100 kd MAA defined by MoAb 376.96 and a 115 kd MAA defined by MoAb 345.134 but decreased the expression of a high molecular weight-melanoma associated antigen (HMW-MAA) defined by MoAb 325.28S. abstract truncated at 400 words
16. Fishbein, M. C., D. Kulber, M. Stancl, and G. Edwalds. (1986). Distribution of fibrinogen and albumin in normal, ischaemic, and necrotic myocardium during the evolution of myocardial infarction: an immunohistochemical study. Cardiovasc Res 20: 36-41.
17. Siegel, R. J., K. Swan, G. Edwalds, and M. C. Fishbein. (1985). Limitations of postmortem assessment of human coronary artery size and luminal narrowing: differential effects of tissue fixation and processing on vessels with different degrees of atherosclerosis. J Am Coll Cardiol  5: 342-6.
18. Harold, J. G., R. J. Siegel, G. M. Edwalds, P. Satoh, and M. C. Fishbein. (1985). Immunohistochemical localization of 6-keto-PGF1-alpha in canine coronary vasculature. Prostaglandins   29: 19-23.
    Abstract – The localization of the prostacyclin metabolite, 6-keto-PGF2-alpha, in canine coronary vasculature was accomplished using immunohistochemical techniques (avidin-biotin method of immunoperoxidase staining). Six-keto-PGF1-alpha was localized to the intimal endothelial cell layer of epicardial and intramyocardial arteries and veins. No specific staining was seen in the media or adventitia of canine coronary vasculature, or in capillaries, or myocardial fibers. To our knowledge, these studies represent the first immunohitochemical demonstration of the endothelial cell localization of the prostacyclin metabolite, 6-keto-PGF1-alpha. The described technique allows the cellular localization of prostaglandin metabolites in histologic sections.
19. Herscher, L. L., R. J. Siegel, J. W. Said, G. M. Edwalds, M. M. Moran, and M. C. Fishbein. (1984). Distribution of LDH-1 in normal, ischemic, and necrotic myocardium. An immunoperoxidase study. Am J Clin Pathol  81: 198-203.
    Abstract – To study the distribution of lactate dehydrogenase (LDH-1) (H4) in normal, ischemic, and necrotic myocardium using the peroxidase-antiperoxidase technic, the authors studied formalin-fixed paraffin-embedded sections of human (n = 11) and canine (n = 28) myocardium. All normal control myocardium showed positive immunostaining for LDH-1 (H4). In infarcts 10 hours or more old, the histologically necrotic myocardium (by triphenyl tetrazolium chloride staining) (TTC) showed markedly diminished immunostaining. In 24-dogs ischemia was induced in a closed-chest model using a balloon-tipped catheter inflated in the left anterior descending coronary artery. In dogs with 3 hours or more of occlusion, myocardium that was necrotic by TTC staining, light and/or electron microscopy, showed diminished staining for LDH-1, while normal, control myocardium stained intensely. In four dogs, ischemia was induced by a controlled perfusion apparatus by which left main coronary flow was reduced by 50%. Ischemia without necrosis was documented by demonstration of glycogen loss with no light or electron microscopic evidence of necrosis. These ischemic fibers stained intensely for LDH-1, as did controls. Thus, by immunoperoxidase staining, LDH-1 can be demonstrated in normal human and canine myocardium. In experimental models of ischemia in dogs, tissue that was ischemic but not necrotic showed no diminished staining. LDH-1 loss can be detected in necrotic myocardium as early as 3 hours after coronary artery occlusion.
20. Edwalds, G. M., J. W. Said, M. I. Block, L. L. Herscher, R. J. Siegel, and M. C. Fishbein. (1984). Myocytolysis (vacuolar degeneration) of myocardium: immunohistochemical evidence of viability. Hum Pathol  15: 753-756.
    Abstract – Human myocardium with focal myocytolysis (vacuolar degeneration, colliquative myocytolysis) was examined by routine light microscopy and by immunoperoxidase staining techniques for creatine kinase (CK) M and B, myoglobin, lactate dehydrogenase (H4)(LDH-1), and aspartate aminotransferase (AST, GOT). Sections of myocardium were selected from autopsy and surgical specimens from patients with and without clinical morphologic evidence of ischemic heart disease. Areas of coagulation necrosis showed loss of enzyme staining, while both normal and myocytolytic cells stained darkly. These results indicate that fibers with myocytolysis retain enzymes and other proteins, indicating sarcolemmal integrity, which is not present in fibers with coagulation necrosis. The implication of these findings is that fibers with myocytolysis are viable; thus, myocytolysis may be a reversible form of myocardial alteration that does not necessarily lead to cell death and eventual myocardial fibrosis.
21. Siegel, R. J., G. Edwalds, R. Rej, and M. C. Fishbein. (1984). Distribution of cytosolic and mitochondrial aspartate aminotransferase in normal, ischemic, and necrotic myocardium. An immunohistochemical study. Lab Invest   51: 648-54.
    Abstract – We utilized immunoperoxidase methods to study the distribution of both cytosolic or soluble(s) and mitochondrial (m) aspartate aminotransferase (AspAT) in normal, ischemic, and necrotic myocardium. Human myocardium was obtained from autopsy (n = 9) and surgery (n = 6). Cardiac tissue from 26 dogs with experimental myocardial infarction induced by closed-chest balloon occlusion and four dogs with myocardial ischemia without necrosis induced by a 50% reduction in left main coronary artery flow for 3 hours were studied. Duration of occlusion was 45 minutes (n = 1), 3 hours (n = 11), 5 to 6 hours (n = 10), or 15 to 24 hours (n = 4). Highly purified m- and s-AspAT and specific antibodies were prepared in our laboratory. In all cases, control experiments were performed. Microscopically normal human and dog myocardium uniformly stained for m- and s-AspAT. Necrotic myocardium from patients with infarcts showed markedly reduced immunostaining. In those dogs with myocardial necrosis, all dogs with coronary occlusion of 5 to 24 hours, and eight of 11 dogs with 3-hour occlusions, immunostaining was significantly reduced for both s- and m-AspAT in regions confirmed to be necrotic by triphenyl tetrazolium chloride and hematoxylin and eosin staining. Myocardial necrosis was confirmed in the 3-hour infarcts by electron microscopy. In the four dogs with a 50% reduction in left main flow for 3 hours, and one dog with a 45-minute coronary occlusion, ischemia was demonstrated by glycogen loss in period acid-Schiff-stained sections but there was no evidence of necrosis by electron microscopy or triphenyl tetrazolium chloride staining and there was no loss of immunostaining evident for s- or m-AspAT. Thus, s- and m-AspAT were visualized in normal and ischemic myocardium with decreased staining in necrotic tissue using immunoperoxidase techniques. Loss of both s- and m-AspAT can be demonstrated in human myocardium and in experimental canine myocardium as early as 3 hours after coronary occlusion and appears to be specific for irreversible myocardial injury. No depletion of isoenzyme can be detected by immunohistochemical techniques in tissue that is ischemic but not necrotic. Furthermore, by these immunoperoxidase techniques, loss of s- and m-AspAT from necrotic myocardium appears to be simultaneous.