Oogenesis of amphibians is an atypical situation in which histone mRNA is polyadenylated. The poly(A) tract on H4 mRNA has been examined by Sl nuclease analysis. Throughout oogenesis the poly(A) tract is very short, and nonexistent on some mRNA molecules. The poly(A) tract is completely removed during maturation of the oocyte, and is absent in embryos and cultured cells. Xenopus development H4 Histone mRNA Polyadenylation Depolyadenylation
We have isolated several clones containing Xenopus histone genes from genomic libraries of X. laevis and X. borealis DNA. Each genomic clone has been mapped and the positions of 26 histone genes in seven laevis clones and 5 histone genes in one borealis clone have been determined. In laevis, the histone gene clusters show considerable variation in gene order within a single individual. When the cloned DNAs were microinjected into the nucleus of Xenopus oocytes, expression of cloned genes at the transcriptional and translational level was readily detectable. Previously unknown histone variants were revealed by the microinjection experiments.
Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34+ cells. We transduced HeLa and CD34+ cells with self-inactivating HIV-1 vector at low and 10-fold higher MOIs, and evaluated vector amounts at various timepoints based upon the rationale that if a given step was not limiting, 10-fold greater vector amounts would be obtained at the 10-fold higher MOI. We observed slower internalization (>60 minutes), a peak of reverse transcription at 24 hours, and completion of integration at 3 days in CD34+ cells. In HeLa cells, vector amounts at high MOI achieved ~10-fold greater values over all timepoints. When compared to HeLa cells, CD34+ cells had a larger difference of vector amounts between high and low MOIs at 2–6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34+ cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts didn’t decrease once detected among CD34+ cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34+ cells in vitro and in xenografted mice. These data suggest that initiation of reverse transcription mainly limits lentiviral transduction for human CD34+ cells. Our findings provide an avenue for optimizing human CD34+ cell transduction.
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.