My Background:

I graduated with a B.S. degree in Agricultural Biochemistry from Iowa State University in 1990.  I stayed at ISU to earn a Master's degree in the laboratory of Dr. Don Beitz in the Departments of Biochemistry and Animal Science.  I identified sequence changes in the mitochrondrial DNA of dairy cattle, which were later correlated to changes in milk production characteristics.

I earned my Ph.D. at the McArdle Laboratory for Cancer Research in the laboratory of Dr. Janet Mertz, studying the regulation of the late promoter of the virus SV40.  I found that the T Antigen protein provided by the virus can physically bind to one of at least four transcription factors provided by the host cell.  Genetic data indicates that this interaction is important for the ability of T Antigen to turn on RNA production from the SV40 late promoter.  In a second project, our laboratory identified the human estrogen related receptor a1 (hERRa1) as a protein made by the host cell that can turn off this promoter.  I determined the DNA sequence bound by hERRa1 and found that this protein forms a complex with the human estrogen receptor.

In the summer of 1996, I joined the laboratory of Dr. Judith Berman in the Department of Genetics, Cell Biology and Development at the University of Minnesota.  I studied a protein from the algae Chlamydomonas reinhardtii known as Gbp1p, which may bind the ends of chromosomes.  Isolated Gbp1p prefers to bind to single-stranded RNA molecules, whose sequence mimics that found at the end of the chromosome.  In contrast, dimerized Gbp1p (two molecules of the protein bound to each other) prefers to bind to single-stranded DNA.  In the Berman lab, I also began my studies on chromatin assembly and signal transduction in yeast.  This work is described on my research homepage and is now continuing at North Central College.

Research Publications:

Pratt, Zachary L.,  Bethany J. Drehman, Mary E. Miller, Stephen D. Johnston. 2007.  Mutual Interdependence of MSI1 (CAC3) and YAK1 in Saccharomyces cerevisiae Journal of Molecular Biology.  368:30-43.

The MSI1 (CAC3) gene of Saccharomyces cerevisiae has been implicated in diverse cellular functions including suppression of the RAS/cAMP/Protein Kinase A signaling pathway, chromatin assembly and transcriptional co-repression.  Seeking to identify the molecular mechanisms by which Msi1p carries out these distinct activities, a novel genetic interaction was uncovered with YAK1, which encodes a kinase that antagonizes the RAS/cAMP pathway.  MSI1 was capable of efficiently suppressing the heat shock sensitivity caused by deletion of yak1.  Surprisingly, the YAK1 gene is required for Msi1p to associate with Cac1p in the yeast two-hybrid system.  A new activity of Msi1p was identified: the ability to activate transcription of a reporter gene when tethered near the promoter, but only in the absence of fermentable carbon sources.  This transcriptional activation function was substantially diminished by the loss of YAK1.  Furthermore, MSI1 influences YAK1 function; overexpression of YAK1 decreased the growth rate, but only in the presence of a functional MSI1 gene.  Finally, it is shown that YAK1 antagonizes nuclear accumulation of Msi1p in nonfermenting cells.  Taken together, these data demonstrate a novel interaction between Msi1p and Yak1p in which each protein influences the activity of the other.  Click here to download a preprint of this paper.I 

Cac3p/Msi1p, the S. cerevisiae homolog of Retinoblastoma-associated protein 48 (RbAp48) is the smallest of three components of Chromatin Assembly Factor-I (CAF-I), a complex that assembles tetramers of histones H3 and H4 onto newly replicated DNA.  RbAp48 is also associated with histone deacetylase and histone acetyltransferase activities.  In addition, CAC3 overexpression suppresses the RAS/cAMP signal transduction pathway by an unknown mechanism.  We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of both CAC1 and CAC2, subunits required for CAF-I function.  CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function.  Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm.  In addition, we found that Npr1p, a kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway, interacts with Cac3p.  Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway.  Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, suggesting that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p.  Deletion of NPR1 did not affect the quantity, phosphorylation state or localization of Ras protein.  Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, extra copies of UBI4, the gene encoding ubiquitin, suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway.  Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.  Follow this link to download a reprint of this paper.

Asleson, Catherine M., John C. Asleson, Emily Malandra , Stephen D. Johnston, and Judith Berman. 2000. Filamentous Growth of Saccharomyces cerevisiae is Regulated by Manganese.  Fungal Genetics and Biology, 30 (2):155-162.

The Candida albicans INT1 gene is a virulence factor that contributes to both adhesion and filamentous growth of the fungus (Gale et al.,1996).   Expression of INT1 in the budding yeast Saccharomyces cerevisiae directs both adhesion and filamentous growth (Gale et al.,1998).  Because Int1p contains two predicted divalent cation-binding motifs, we asked if divalent cations are important for the role of Int1p in filament formation.  In this study, we found that INT1-induced filamentous growth (I-IFG) is sensitive to the divalent cation chelator EDTA and that this EDTA sensitivity can be ameliorated by the addition of Mn²⁺, but not Mg²⁺ or Ca²⁺ ions.  The addition of MnCl₂ restored both the proportion of cells forming filaments as well as the length of filaments formed.  Expression of INT1 in  S. cerevisiae mutants that reduce the intracellular concentration of Mn²⁺ did not affect I-IFG.  Interestingly, the Mn²⁺ dependence of I-IFG is not dependent upon the presence of the putative divalent cation binding domains found in INT1.  Rather, we found that polarized growth induced by mutations in CDC12, CLA4 or by expression of excess SWE1 was also sensitive to EDTA treatment and was restored by the addition of MnCl₂ but not by the addition of CaCl₂.  Thus, our results suggest that in S. cerevisiae polarized growth is dependent upon the presence of Mn²⁺ ions.

Using a screen for genes that affect telomere function, we isolated sir3^rlf3, an allele of SIR3 that reduces telomeric silencing yet is mating competent.  While sir3^rlf3 alleles are able to mate in quantitative assays, they exhibit synergistic mating defects in combination with mutations such as sir1, that affect the establishment of silencing.  In contrast, in combination with mutations such as cac1, which affect the maintenance of silencing, no synergistic mating defects are observed.  These results imply that wild-type Sir3p has an important role in the maintenance of silencing, that can be separated from its role in the nucleation or establishment of silencing.  In addition, we found that overexpression of the C-terminal fragment of Sir3^rlf3p cause a dominant non-mating phenotype:  HM silencing is completely lost at both HML and HMR.  Furthermore, HM silencing is most vulnerable to disruption by the Sir3^rlf3p C-terminus immediately after S-phase, the time when new silent chromatin is assembled onto newly replicated DNA.  Follow this link to download a reprint of this paper.

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1p can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.      Follow this link to download a reprint of this paper.

Qian, Zhijian, Hanhua Huang, Joo Yun Hong, Carol L. Burck, Stephen D. Johnston, Judith Berman, Andy Carol, and Susan W. Liebman. 1998. Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins. Molecular and Cellular Biology, 18:4783-4792.

A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutations of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.      Follow this link to download a reprint of this paper.

Johnston, Stephen D., Xuedong Liu, Fengrong Zuo, Theresa L. Eisenbraun, Steven R. Wiley, Richard J. Kraus, and Janet E. Mertz. 1997. Estrogen-related receptor a1 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements. Molecular Endocrinology, 11:342-352.

The human estrogen-related receptor a1 (hERRa1) is an orphan member of the steroid/thyroid hormone receptor superfamily. A cDNA encoding this protein was originally isolated on the basis of sequence similarity in its DNA-binding domain with estrogen receptor  (ER). Previously, we reported the purification of hERRa1 from HeLa cell nuclear extracts on the basis of its ability to bind two sites in the late promoter of simian virus 40 (SV40). We have now determined the primary structure and the DNA and protein binding specificities of hERRa1 and developed in vivo and in vitro assays for its functional activities. hERRa1 was found to bind as a monomer, with a high-affinity binding site containing the extended half-site sequence 5´-TCAAGGTCA-3´. Binding sites for hERRa1 were identified in many cellular promoters, including some that were previously shown to function as estrogen-response elements (EREs). hERRa1 was shown to function as a sequence-specific repressor of the SV40 late promoter in both cell culture and cell-free transcription systems. It was also shown to interact with both ER and the transcription factor TFIIB by direct protein-protein contacts. Thus, hERRa1 may play a role in the response of some genes to estrogen via heterodimerization with ERs or competition with ERs for binding to EREs.      Follow this link to download a reprint of this paper or see this page for more info on hERRa1 (now called ESRRA).

Johnston, Stephen D., Xian-Ming Yu, and Janet E. Mertz. 1996. The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex. Journal of Virology, 70:1191-1202.

Simian virus 40 (SV40) large T antigen (Tag) is a promiscuous transcriptional transactivator; however, its mechanism of transactivation remains unknown. Recent studies have suggested the possible involvement of protein-protein interactions with TBP, the TATA box-binding protein of TFIID, and TEF-1, an enhancer-binding factor. We show here that (i) the Tag domain containing amino acids 133 to 249 directly interacts with the general transcription factor TFIIB, the activator protein Sp1, and the 140-kDa subunit of RNA polymerase II, as well as with TBP and TEF-1; (ii) these interactions can also occur when these transcription factors are present in their functional states in cellular extracts; (iii) binding of Tag to TBP is eliminated by preincubation of TBP either at 48 degrees C or with the adenovirus 13S E1a protein; (iv) this domain of Tag cannot bind concurrently to more than one of these transcription factors; and (v) the substitution of Tag amino acid residues 173 and 174 inactivates the ability of this Tag domain both to associate with any of these transcription factors and to transactivate the SV40 late promoter. Thus, we conclude that SV40 Tag probably does not transactivate via the concurrent interaction with multiple components of the preinitiation complex. Rather, we hypothesize that transactivation by Tag may primarily occur by removing or preventing the binding of factors that inhibit the formation of preinitiation complexes.      Follow this link to download a reprint of this publiction.

Boettcher, Paul J., A.E. Freeman, Stephen D. Johnston, Renotta K. Smith, Donald C. Beitz, and Benjamin T. McDaniel. 1996. Relationships between polymorphism for mitochondrial deoxyribonucleic acid and yield traits of Holstein cows. Journal of Dairy Science, 79:647-654.

Two independent data files from the breeding herd of Iowa State University and six North Carolina herds were used to examine relationships between yield traits and mtDNA polymorphism. Maternal lineages were established by tracing ancestry of cows to founder females in the herd book of the Holstein Association. Data from Iowa State University were 1476 records from 602 cows from 29 maternal lineages. The nucleotides of mtDNA encoding rRNA were sequenced. Eleven sites of polymorphism were found. An animal model for gene substitution was used to examine the relationship between sequence differences and yield traits. Traits analyzed were mature equivalent yield of milk, fat, SNF, and milk energy as well as concentrations of fat, SNF, and milk energy. Effects of sequence differences were significant for most traits. Sequence information from the D-loop was available for 12 lineages from North Carolina. The effect of polymorphism at 4 sites was examined using 1472 records from 668 cows. Traits measured were the same, except that protein replaced SNF. No significant relationships existed between any of the traits and D-loop polymorphism, but results suggested that an association might exist between polymorphism and concentrations of milk yield, fat percentage, and energy. Whenever a significant relationship was detected, the effect of mutation (rare genotype) was detrimental.

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Last updated: March 26, 2007