Agrobacterium tumefaciens is a bacterium that infects a wide range of broad-leaved plants. During infection, a plasmid called Ti is transferred into the plant cells, where that plasmid DNA is integrated into the plant’s genome. Some of the genes encoded by this plasmid are expressed by host cell machinery in the plant cell. These gene products produce plant hormones which stimulate cell division in the infected cells, causing the development of a plant tumor which provides an environment for that bacterium to grow.
You are studying a novel isolate of A. tumefaciens and want to determine which of the genes on the Ti plasmid are expressed in the bacterium, and which are expressed in the plant. You start by sequencing the Ti plasmid.
a) Considering the DNA sequence of Ti, describe two specific genetic features that would help you to differentiate whether a given gene is likely expressed in the plant or bacterium.
b) You collect plant tumor tissue, containing both bacterial and plant cells, and sequence the RNA collected from this mixed population of cells. Will this RNA sequence provide you with any additional information to influence your conclusions about which Ti-encoded genes are expressed in the plant vs. bacterium beyond what you saw in the DNA sequence? Why or why not (provide specific examples)?
c) You want to make a new transgenic plant where the gene yfgA is integrated into a specific location in the plant chromosome, completely replacing an existing gene yfgZ. Your Ti plasmid contains yfgA and you confirm that the plasmid is entering plant cells, but the integration of yfgA turns out to be random and yfgZ is still present in all plants examined. Describe how you might change the Ti plasmid sequence to improve the ability of this plasmid to completely replace yfgZ with yfgA. (e.g. describe the type/source of sequences you might add/remove/change on the plasmid, and where they will be located, to enable this outcome; no specific sequences are of course needed).
Please answer parts a, b, and c as listed above
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