@misc{ivey_massel_phister_2013, title={Microbial interactions in food fermentations}, volume={4}, journal={Annual review of food science and technology, vol 4}, author={Ivey, M. and Massel, M. and Phister, T. G.}, year={2013}, pages={141–162} } @misc{mendu_massel_spiker_2001, title={Increasing loop domain size does not diminish effects of matrix attachment regions on transgene expression in tobacco cells in culture}, volume={496}, ISSN={["0014-5793"]}, DOI={10.1016/s0014-5793(01)02406-1}, abstractNote={It is now widely held that the chromatin of eukaryotic organisms is organized into loop domains in which chromatin fibers are attached to the nuclear matrix by specific interactions of nuclear matrix proteins with DNA sequences called matrix attachment regions (MARs) [1, 2]. Several MARs have been isolated and incorporated into constructs used for transformation. Work with animal cell culture systems has consistently shown much higher average levels of reporter gene expression in cell lines in which reporter genes are flanked with cloned MARs than in control lines without flanking MARs [1]. MARs have similar effects in plant cell systems. For example, in a tobacco cell culture system, we have shown that a heterologous MAR (a yeast MAR that binds weakly to the tobacco nuclear matrix) increases reporter gene expression by 12-fold, and a strong matrix-binding tobacco MAR increases reporter gene expression by 60-fold [3]. The mechanisms by which MARs increase reporter gene expression are unknown. MARs do not appear to act as typical enhancers, as they have little effect in transient expression assays [1, 2]. Most proposed mechanisms involve chromatin structure [1, 2]. According to one model, MARs affect transgene expression by creating independent, topologically isolated domains. The transgenes in these independent domains are insulated from chromatin structure of the native domains (either transcriptionally active or repressed) into which they become incorporated. If transgenic MARs do act by creating independent domains containing the transgene, the question remains of why the independent domains have a transcriptionally active chromatin structure. We have previously speculated that independent domains created by cloned MARs may have transcriptionally active chromatin structures because they are too small to form stable, transcriptionally repressed, condensed chromatin fibers in vivo [2, 3]. In our previous experiments, the putative domain formed by cloned MARs contains 3 kb of DNA, enough to form 16 plant nucleosomes. Even fewer nucleosomes may form on the transgenes in vivo, as close proximity to the nuclear matrix may sterically inhibit nucleosome formation. As the structure of the 30-nm chromatin fiber is not well understood, the number of nucleosomes necessary to form a stable, transcriptionally repressed structure is unknown. Formation of folded chromatin fibers presumably is dependent upon nucleosome–nucleosome interactions. If we consider the solenoid model of the 30-nm fiber (six nucleosomes per turn of the solenoid), it is logical to assume that a minimum of 12 nucleosomes would be required to form a stable, folded structure. This would allow each nucleosome to interact with at least one nucleosome other than its linear neighbors. Further turns of the solenoid would be expected to further stabilize the structure. For example, in a structure containing 18 nucleosomes (three turns of the solenoid), the nucleosomes of the middle turn could interact with nucleosomes of the outside two turns of the solenoid. Carruthers et al. [4] have provided evidence that a reconstituted linear DNA fragment containing 12 nucleosomes can form a stable, folded structure, but the relationship of this structure to the 30-nm fiber is unclear. The work of Butler and Thomas [5] indicates that more nucleosomes may be required. These workers observed a change in the hydrodynamic properties of native, rat liver nucleosome oligomers above the size of 50 nucleosomes. They attributed the change to higher-order folding. The considerations mentioned above suggest that in our previous work [2, 3] the 16 nucleosomes that would be expected to form on 3 kb of DNA bounded by MARs may be below or near the minimum required to form stable, folded chromatin fibers in vivo. Based on these ideas concerning the stability of higher-order structure in chromatin, we have asked if the enhancement of transgene expression by MARs can be counteracted by increasing the amount of DNA in the putative loop domain to a point at which a stable condensed chromatin fiber could be formed. In order to answer this question we have transformed plant cells in culture by microprojectile bombardment as we have previously described [3]. A co-transformation procedure is used in which a selectable marker (NPTII conferring kanamycin resistance) is carried on a separate plasmid from the reporter gene. As in our previous experiments we have used the reporter plasmids, pGHNC11 and pGHNC12 (Fig. 1A ). These plasmids contain the GUS reporter gene cassette flanked by the RB7-6 tobacco MAR and a control of the GUS cassette without flanking MARs. We also used a plasmid (pNMCS1 in Fig. 1A) in which λ DNA has been inserted between the GUS reporter cassette and the MAR. This ‘spacer’ DNA would increase the size of the putative MAR-bounded loop domain to 52 nucleosomes. The λ DNA (a 6.6-kb HindIII fragment, nucleotides 37586–44141) has an AT content of 51% and does not bind to the tobacco nuclear matrix (data not shown). A control plasmid (pNMCS2) with the λ DNA and the GUS reporter cassette but no MARs was also used. Kanamycin-resistant transformed cells were grown in liquid culture for 2 months with transfers every 7 days. At this time, cells were harvested and protein extracts were made in order to measure GUS specific activity [3]. Fig. 1B shows the results of the GUS specific activity measurements. Increasing the amount of DNA in the putative independent domain to a size that would support 8.6 turns of a nucleosome solenoid does not diminish the effect of MARs on enhancing reporter gene expression. The specific activity of the MAR-SPACER-GUS-MAR transformed cell lines is slightly higher than that in the MAR-GUS-MAR lines, but the difference is not statistically significant. In the lines transformed with constructs lacking MARs, the GUS activity was slightly lower in the SPACER-GUS lines than in the GUS-only lines, but again, this difference was not statistically significant. Because most DNA constructs used to test the effects of MARs on transgene expression contain amounts of DNA between the MARs that would be sufficient to form only a few nucleosomes [1-3], we hypothesized that the mechanism of MAR activity is the formation of loop domains that are too small to form a stable, transcriptionally repressed chromatin fiber. Increasing the amount of DNA in the putative MAR-bounded loop domains to a size that would accommodate 52 nucleosomes (nearly nine turns of a nucleosome solenoid) does not diminish the MAR-mediated enhancement of transgene expression in tobacco cells in culture. In vitro data indicate that 52 nucleosomes would be more than enough to support a stable, condensed chromatin fiber [5]. Thus, the explanation for MAR enhancement of transgene expression must lie elsewhere. We also note that the 5′ MAR is able to influence transgene expression at a fairly great distance (at least 6.6 kb from the promoter). It could be argued that the MAR located 3′ to the transgene is by itself causing the enhancement of transgene expression. But we have previously shown [2, 3] that both 5′ and 3′ flanking MARs are necessary to get the full MAR effect on increasing transgene expression. This work was supported by grant 9418491 from the USA National Science Foundation and by the North Carolina State University Agricultural Research Service.}, number={1}, journal={FEBS LETTERS}, author={Mendu, N and Massel, M and Spiker, S}, year={2001}, month={May}, pages={66–67} } @article{ascenzi_ingram_massel_thompson_spiker_weissinger_2001, title={The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions}, volume={10}, ISSN={["0962-8819"]}, DOI={10.1023/A:1012082602587}, abstractNote={The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.}, number={5}, journal={TRANSGENIC RESEARCH}, author={Ascenzi, R and Ingram, JL and Massel, M and Thompson, WF and Spiker, S and Weissinger, AK}, year={2001}, pages={465–470} } @inbook{pharr_stoop_studer feusi_williamson_massel_conkling_1995, title={Mannitol catabolism in plant sink tissues}, booktitle={Carbon Partitioning and Source-Sink Interactions in Plants, Current Topics in Plant Physiology, Vol. 13 (Madore, MA and Lucas, WJ, eds.)}, publisher={American Society of Plant Physiologists, Rockville, MD}, author={Pharr, D.M. and Stoop, J.M.H. and Studer Feusi, M.E. and Williamson, J.D. and Massel, M.O. and Conkling, M.A.}, editor={Madore, MA and Lucas, WJEditors}, year={1995}, pages={180–194} } @article{williamson_stoop_massel_conkling_pharr_1995, title={SEQUENCE-ANALYSIS OF A MANNITOL DEHYDROGENASE CDNA FROM PLANTS REVEALS A FUNCTION FOR THE PATHOGENESIS-RELATED PROTEIN ELI3}, volume={92}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.92.16.7148}, abstractNote={Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.}, number={16}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={WILLIAMSON, JD and STOOP, JMH and MASSEL, MO and CONKLING, MA and PHARR, DM}, year={1995}, month={Aug}, pages={7148–7152} } @article{pharr_stoop_williamson_feusi_massel_conkling_1995, title={The dual role of mannitol as osmoprotectant and photoassimilate in celery}, volume={30}, number={6}, journal={HortScience}, author={Pharr, D. M. and Stoop, J. M. H. and Williamson, J. D. and Feusi, M. E. S. and Massel, M. O. and Conkling, M. A.}, year={1995}, pages={1182–1188} }