Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
Browse
4 results
Search Results
Item STRUCTURAL AND FUNCTIONAL ANALYSIS OF DNA REPLICATION INITIATION PROTEINS FROM THE ARCHAEON METHANOTHERMOBACTER THERMAUTOTROPHICUS(2005-12-01) Kasiviswanathan, Rajesh; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The faithful duplication of the chromosome requires the combined efforts of numerous proteins. Cdc6 and MCM are two such proteins involved in the initiation of DNA replication. The genome of the euryarchaeon Methanothermobacter thermautotrophicus contains one MCM and two Cdc6 homologues (Cdc6-1 and -2). While MCM is the replicative helicase that unwind the duplex DNA to provide single-stranded DNA substrate for the replicative polymerases, the Cdc6 proteins are presumed to function in origin recognition and helicase assembly at the origin. This thesis elucidates the structure, function and regulation of these archaeal initiation proteins. The M. thermautotrophicus MCM helicase is a dumb-bell shaped double hexamer. Each monomer can be divided into two portions. The C-terminal catalytic region contains the ATP binding and hydrolysis sites essential for helicase activity. This thesis concentrates its efforts to determine the functional role of the N-terminal region. Using a variety of biochemical approaches it was found that the N-terminal portion of MCM is involved in hexamer/dodecamer formation. The study also identified two structural features at the N-terminus, the zinc- and the beta-finger motifs, essential for DNA binding, which in turn is essential for helicase activity. In addition, the N-terminal portion of MCM interacts with both Cdc6 proteins. The role of the Cdc6-1 and -2 proteins in origin recognition and helicase loading was also elucidated. The results presented in this thesis show that Cdc6-1 has binding specificity to origin DNA sequences suggesting a role for the protein in origin recognition. While both Cdc6 proteins interact with the MCM helicase, Cdc6-2 exhibited tighter binding compared to Cdc6-1 suggesting a role for Cdc6-2 in helicase loading. Summarizing the observations of this study, a model for the replication initiation process in M. thermautotrophicus has been proposed, outlining separate role for the two Cdc6 proteins, Cdc6-1 in origin recognition and Cdc6-2 in MCM helicase assembly at the origin.Item COMPUTATIONAL ANALYSES OF MICROBIAL GENOMES - OPERONS, PROTEIN FAMILIES AND LATERAL GENE TRANSFER(2005-05-15) Yan, Yongpan; Moult, John; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)As a result of recent successes in genome scale studies, especially genome sequencing, large amounts of new biological data are now available. This naturally challenges the computational world to develop more powerful and precise analysis tools. In this work, three computational studies have been conducted, utilizing complete microbial genome sequences: the detection of operons, the composition of protein families, and the detection of the lateral gene transfer events. In the first study, two computational methods, termed the Gene Neighbor Method (GNM) and the Gene Gap Method (GGM), were developed for the detection of operons in microbial genomes. GNM utilizes the relatively high conservation of order of genes in operons, compared with genes in general. GGM makes use of the relatively short gap between genes in operons compared with that otherwise found between adjacent genes. The two methods were benchmarked using biological pathway data and documented operon data. Operons were predicted for 42 microbial genomes. The predictions are used to infer possible functions for some hypothetical genes in prokaryotic genomes and have proven a useful adjunct to structure information in deriving protein function in our structural genomics project. In the second study, we have developed an automated clustering procedure to classify protein sequences in a set of microbial genomes into protein families. Benchmarking shows the clustering method is sensitive at detecting remote family members, and has a low level of false positives. The aim of constructing this comprehensive protein family set is to address several questions key to structural genomics. First, our study indicates that approximately 20% of known families with three or more members currently have a representative structure. Second, the number of apparent protein families will be considerably larger than previously thought: We estimate that, by the criteria of this work, there will be about 250,000 protein families when 1000 microbial genomes are sequenced. However, the vast majority of these families will be small. Third, it will be possible to obtain structural templates for 70 - 80% of protein domains with an achievable number of representative structures, by systematically sampling the larger families. The third study is the detection of lateral gene transfer event in microbial genomes. Two new high throughput methods have been developed, and applied to a set of 66 fully sequenced genomes. Both make use of a protein family framework. In the High Apparent Gene Loss (HAGL) method, the number and nature of gene loss events implied by classical evolutionary descent is analyzed. The higher the number of apparent losses, and the smaller the evolutionary distance over which they must have occurred, the more likely that one or more genes have been transferred into the family. The Evolutionary Rate Anomaly (ERA) method associates transfer events with proteins that appear to have an anomalously low rate of sequence change compared with the rest of that protein family. The methods are complementary in that the HAGL method works best with small families and the ERA method best with larger ones. The methods have been parameterized against each other, such that they have high specificity (less than 10% false positives) and can detect about half of the test events. Application to the full set of genomes shows widely varying amounts of lateral gene transfer.Item Human Immunodeficiency Virus Nucleocapsid Protein: Analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity.(2004-06-15) Heath, Megan Joy; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human immunodeficiency virus genome is coated by the nucleocapsid protein (NC). NC is a 55 amino acid highly basic protein. It has two zinc fingers that differ by five amino acids. NC contains nucleic acid chaperone activity that aids in the formation of highly stable nucleic acid structures by destabilizing and preventing the formation of weaker structures. This activity is important for genome dimerization and maturation, tRNA:primer binding site annealing, and many steps in reverse transcription. Annealing experiments were performed with four different RNA structures and complementary DNAs. NC enhanced annealing of all structures showing that NC enhances both unwinding of nucleic acid structure and hybridization of unstructured sequences. NC mutant proteins were used in annealing assays. 1.1 NC had two copies of the first zinc finger, 2.2 NC had two copies of the second zinc finger, and 2.1 NC had both zinc fingers with their positions switched. Experiments showed that all mutants could enhance the annealing of weakly structured nucleic acids but only 1.1 NC and 2.1 NC enhanced annealing of strongly structured nucleic acids. Results suggest that finger one is important for nucleic acid unwinding while finger two plays an accessory role in annealing. The mechanism of strand exchange, another important aspect of NC chaperone activity, was also investigated. Experiments were performed using RNA:DNA hybrids with either the DNA or RNA radioactively labeled. Hybrids were incubated with different types of RNA acceptor molecules to which the DNA could transfer. The transfer of the DNA or the displacement of the original donor RNA was monitored. Experiments showed that optimal enhancement of strand exchange by NC occurred with acceptors that had more than 22 nucleotides that could anneal to the single stranded region of the DNA. Also, experiments with acceptors that had point mutations showed that the region of the acceptor that binds to the single stranded region of the DNA should be completely complementary for optimal NC stimulation. These results indicate the annealing of the acceptor and DNA outside the donor:DNA hybrid region can be an important initiation step for NC enhanced strand exchange.Item Production and Characterization of New Alleles of SCAR in Drosophila melanogaster(2003-12-04) Valentine, Summer Danyse; de Cuevas, Maragaret; Mount, Stephen; Baehrecke, Eric; Cell Biology & Molecular GeneticsGermline cysts are clusters of germ cells that arise by division from a single cell and are connected to one another by stable intercellular bridges. Cysts are a highly conserved stage of pre-meiotic germ cell development, but the molecular mechanisms that control their formation are highly unknown. Developing cysts in Drosophila contain a cytoplasmic organelle called a fusome, which plays a critical role in cyst formation. The SCAR gene, which encodes an actin regulatory protein, was identified in a screen for mutations that disrupt fusomes and cyst formation. It was not clear, however, if the mutation l(2)k03107 disrupts only SCAR. To address this question, new alleles were created by excising the P-element in l(2)k03107. These alleles were then characterized molecularly and phenotypically. The results show that two alleles have deletions in the SCAR gene. Further testing must be done to determine the role of SCAR in cyst formation.