The results of gel electrophoresis for both p2746 and p…. were inconclusive. Both bands
for p4726 were two faint to see, or possibly not present. One band showed up for p…., at
4000 bp. This band indicates the vector. There was no visible second band, which would
have indicated an insert in the vector. It is possible that the plasmid had no insert, or that
the band that would indicate the insert was too faint to show up. The faintness of the
bands may have resulted from the plasmid miniprep yielding less plasmid DNA than
anticipated. Because we could not say decisively whether or not our plasmids contained
cDNA inserts, we decided to proceed with a completely different cDNA, which had been
previously sequenced, but had not been analyzed.
In order to ensure the integrity of the sequencing data, we modified the raw data. The first
30 base pairs were eliminated due to inconclusive results. as were all the base pairs
following base pair number …. This modification allowed us to ensure that we were
working with the most accurate sequence possible. After we had ensured that the
remaining sequencing data was useable, we removed the remainder of the vector
sequence by cutting out all the nucleotides up to and including the first SalI restriction
enzyme site, which was the enzyme that was used to ligate the cDNA into the vector.
The results of our Blast search using the edited sequence indicated that our cDNA comes
from chromosome three of the Arabidopsis thaliana genome, part of which is stored on
BAC F25C20. The match for BAC F25C20 had a very low e-value, indicating that the
probability of the match resulting from random chance alone was very slim. The search
also revealed that the cDNA sequence matched that of an Arabidopsis protein called
RBP45, which is an RNA binding factor. There was, again, a very strong correspondence.
RBP45 is located at approximately 73294-76608 bp on the Crick strand of BAC F25C20.
Our segment of the cDNA matched up with the final two exons of the RBP45 gene,
located at …….-…… and ….-….. Neither of the matches covered an entire exon;
however, this was expected because the cDNA used for sequencing did not contain the
full sequence from the mRNA, due to the nature of the way cDNAs are created. The
DNA folds over at the 3’ end to act as a primer to itself. This causes a portion of the 3’
end to be lost. However, since the matches were very close, we can infer from this data
that the locations of these exons are accurately positioned in the RBP45 gene.
Since RBP45 has a role in RNA processing that has not yet been fully elucidated (1), one
direction we could take from here is attempting to locate similar genes in other
organisms, by locating similar sequences of DNA. This could help clarify the function of
RBP45 by determining if the gene is specific to one type of organism, or if it is more
universal. Also, along these lines, we could use the sequence of this gene to attempt to
find similar genes in the Arabidopsis genome, or genes with similar structures, which
may give clues as to the function of RBP45.
(1) RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with
poly(A)+ RNA in nuclei of plant cells.
Lorkovic ZJ, Wieczorek Kirk DA, Klahre U, Hemmings-Mieszczak M, Filipowicz W.