Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s10577-014-9404-1) contains supplementary material, which is available to authorized users.
Hori et al. show that centromere position can be drifted during cell proliferation in chicken DT40 cells. However, the centromere drift is suppressed in short-term cultures, and a complete constitutive centromere-associated network organization contributes to the suppression of the centromere drift.
CENP‐R is a component of the CENP‐O complex, including CENP‐O, CENP‐P, CENP‐Q, CENP‐R, and CENP‐U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP‐R‐deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP‐R in vivo, we generated CENP‐R‐deficient mice (Cenp‐r −/−). Mice heterozygous or homozygous for Cenp‐r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp‐r is not essential for normal development. Accordingly, to investigate the role of the Cenp‐r gene in skin carcinogenesis, we subjected Cenp‐r −/− mice to the 7,12‐dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp‐r −/− mice initially developed significantly more papillomas than control wild‐type mice. However, papillomas in Cenp‐r −/− mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp‐r −/− mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp‐r functions bilaterally in cancer development: during early developmental stages, Cenp‐r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor‐promoting factor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.