Chemical Biodynamics Division: Annual report, October 1, 1986-September 30, 1987 Page: 27 of 69
This report is part of the collection entitled: Office of Scientific & Technical Information Technical Reports and was provided to UNT Digital Library by the UNT Libraries Government Documents Department.
Extracted Text
The following text was automatically extracted from the image on this page using optical character recognition software:
Accomplishments
We are working on several novel techniques to assay and visualize DNA-protein complex
formation between leaf or root cellular extracts and portions of the pRi T-DNA. These are
filter-binding assays and gel retardation technique s
We have made several DNA constructs, where the acetyl-chloramphenicol transferase gene from
Tn 9 has been fused to the Ri root or leaf specific promoters. The constructs have all been
sequenced to test for the orientation of the CAT gene in regard to the Ri promoters. The aim
is to test for the transient CAT activity after transformation of Nicotiana tabacum leaf or root
protoplasts. We are expecting the leaf protoplasts to contain the necessary factors to activate
the leaf promoter, but not the root promoter. We are hence expecting to observe CAT activity
after transformation of leaf protoplasts by the pRi T-DNA leaf-promoter/CAT construct, but not
by the root-promoter/CAT construct, and reciprocally after transformation of root protoplasts.
We are testing the length of 5’ upstream sequences necessary for activity of these two pRi
promoters. We have established the optimal conditions for protoplast isolation, cell suspension
cultures, electroporation and PEG transformation. We are now testing CAT aevtivity from the
CAT plasmids constructed.
We have tested conditions that will allow plant protein-DNA complex formation. Tobacco root-
and leaf-protein extracts have been tested for their DNA-binding capacities after separation of
the proteins by electrophoresis and transfer to a nitrocellulose membrane. A number of proteins
which show DNA-binding capacities have been observed, and large differences in the DNA-
binding pattern of extracts from the two tissues were noted.
We are hoping to isolate, if they exist, proteins which bind specifically with the regulatory
sequences of the root-specific and the leaf-specific T-DNA genes.
To visualize DNA-binding proteins we transfer proteins to nitrocellulose filters and probe with
nick-translated labelled DNA. The DNA sequences we are using for these experiments are
subclones containing the leaf-specific and the root-specific promoters of the A. rhizogenes T-
DNA. The goal is to show that there is indeed a specificity in the recognition of each of these
sequences by the two types of extracts and to identify what proteins are involved.
We are expecting difficulties in this part of the project in defining conditions that will enhance
the sequence specificity of the DNA-binding proteins. This has been a major difficulty in the
identification of sequence-specific recognition factors. Retarded electrophoretic migration of
DNA complexed to proteins has proven to be a useful method in detecting specific over non-
specific binding of proteins to isolated sequences. It has recently been used to identify specific
transcription factors in SV40 infected cells, and in 02 induced cytochrome expression in yeast.
We are now testing several approaches in order to determine the general and sequence-specific
DNA-binding properties of the Tobacco root and leaf proteins. These include investigating
factors influencing the formation of DNA-protein complexes in solution, such as salt conditions,
length of reaction and subsequent washes, and competition experiments with random sequence
DNA such as calf-thymus DNA.
21
Upcoming Pages
Here’s what’s next.
Search Inside
This report can be searched. Note: Results may vary based on the legibility of text within the document.
Tools / Downloads
Get a copy of this page or view the extracted text.
Citing and Sharing
Basic information for referencing this web page. We also provide extended guidance on usage rights, references, copying or embedding.
Reference the current page of this Report.
Chemical Biodynamics Division: Annual report, October 1, 1986-September 30, 1987, report, September 1, 1987; [Berkeley,] California. (https://digital.library.unt.edu/ark:/67531/metadc1093053/m1/27/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.