Title page for ETD etd-1513132149731401


Type of Document Dissertation
Author Wen, Xiao
Author's Email Address xwen@vt.edu
URN etd-1513132149731401
Title Cloning and Characterization of Replication Protein A from Dictyostelium discoideum
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Larson, Timothy J.
Walker, Richard A.
Rutherford, Charles L. Committee Chair
Keywords
  • glycogen phosphorylase 2
  • DNA replication
  • Dictyostelium discoideum
  • Replication Protein A
  • transcription factor
Date of Defense 1997-08-05
Availability unrestricted
Abstract

The gene encoding the Dictyostelium replication protein A

large subunit (DdRPA1) has been cloned by screening of

an EcoR I partial genomic library and a Hind III genomic

sub-library. The complete nucleotide sequence, including

the promoter region of the gene has been obtained by

sequencing. Though the DdRPA1 protein has a size shift

during development, 62 kDa in undifferentiated cells and

81 kDa in differentiated cells; they are the products of the

same gene. Northern blot analysis revealed that the

expression level of the DdRPA1 was constant throughout

differentiation and the size of mRNA is the same at all

stages, corresponding to a 81 kDa protein. Thus, it seems

that the size change between the 62 kDa and 81 kDa is

probably due to posttranslational modification, most likely,

proteolytic cleavage. The transcription start site for both

sizes of DdRPA1 has been identified at 306 bp upstream

of the coding sequence by primer extension reaction.

A PCR fragment representing 27% of the gene encoding

the DdRPA middle size subunit (DdRPA2) has been

generated by using the degenerate primers. This PCR

fragment has been cloned and sequenced. The mRNA for

this subunit corresponds to a protein of about 35 kDa. A

decrease of the DdRPA2 mRNA expression level during

differentiation was found by comparison between

undifferentiated and differentiated cells.

In Dictyostelium, replication protein A is a heterotrimeric

protein that can bind with specific DNA sequences in a

stage-dependent pattern. These DNA sequences were

identified as the cis-acting regulatory sites in

differentiation-related genes, including the glycogen

phosphorylase 2 gene (gp2). Therefore, it is possible that

DdRPA is not only a single-stranded DNA binding protein

that is used in multiple essential DNA metabolic

processes, such as DNA replication, repair and

recombination in undifferentiated cells, but also involved in

the transcriptional regulation process during differentiation.

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