Craig Bierle
|
 | Research Advisor & Department:
Stephen Zinder - Microbiology
Name of Project:
Isolation and Characterization of Cellulose Utilizing Microbes From Bogs
Abstract:
Peatlands are vast reserves of organic matter that are formed when dead plant matter accumulates faster than it is degraded. One type of peatland, the ombrotrophic Sphagnum bog, is fed only by rainwater and becomes an acidic and mineral poor environment. The complete degradation of cellulose, the structural sugar of plant matter, to methane or carbon dioxide is a complex process that involves entire communities of microorganisms. The hydrolysis of cellulose into its glucose subunits is a difficult process due to the b-1,4- bonds that connect the monomers. Only fungi and several groups of bacteria can hydrolyze cellulose under aerobic conditions and only a few groups of anaerobic bacteria have been shown to truly degrade crystalline cellulose. In ombrotrophic bogs, cellulose degradation occurs both aerobically near the surface and anaerobically in the deeper regions of the peat. The harsh environment of the bog makes it inhospitable to the most well understood cellulose-utilizing microbes. In order to obtain a greater knowledge of the microbial community of the bog and its role in the global carbon cycle, it is important to identify and characterize, if not isolate, the key cellulose degraders, both aerobic and anaerobic, of the bogs. Our preliminary results have shown that supplementing samples of peat with microcrystalline cellulose can lead to substantial increases in methane production in a low pH anaerobic environment. New enrichment cultures will be produced from peat material supplemented with cellulose under both aerobic and anaerobic conditions. These enrichments will then be used as a source of inoculum for isolation, as will direct MPNs followed by serial dilution. Two forms of cellulose will be used: microcrystalline cellulose and filter paper. Because cellulolytic organisms tend to directly colonize cellulose, partially digested filter paper could be used as a source of DNA, which can be used to identify present bacteria and provide information as to which bacterial or fungal groups are involved. These methods should yield a greater understanding of the carbon cycle in the bogs, especially in relation to the hydrolyzing bacteria.
|
Sabina Bis
|
 | Research Advisor & Department:
Eric Alani - Molecular Biology and Genetics
Name of Project:
A Search for Downstream Mismatch Repair Factors in Saccharomyces cervisiae
Abstract:
DNA mismatch repair (MMR) plays a key role in maintaining the integrity of DNA by correcting DNA replication errors such as DNA polymerase slippage or misincorporation events. The MMR system has been well studied in E. coli, which have a MutHLS system. A similar system can be found in yeast and other eukaryotes. Homologs of both MutS (MSH) and MutL (MLH) have been identified. MSH2-MSH3 and MSH2-MSH6 are heterodimers that recognize different types of mismatches such as insertion/deletion loops or single nucleotide substitutions. This MSH-mispair complex then recruits an MLH heterodimer. The primary MLH heterodimer in MMR is MLH1-PMS1. Like MutL, this heterodimer is a key player in MMR system, coordinating mismatch recognition with downstream repair factors. However, these downstream factors have yet to be found - no MutH homologue has been identified. We are interested in identifying new MMR factors using a genetic suppression approach. We have identified a set of conditional mlh1 alleles and are performing a screen to identify genes expressed in high copy that suppressed the mutator phenotype observed in mlh1-7 strains grown at the restrictive temperature (35°C). A "blue-white" assay was performed, using a reporter plasmid with a LACZ gene out of frame, to screen the yeast library for candidate suppressors of the mlh1-7 defect. After growth at 35°C, colonies containing mlh1-7 and the LACZ reporter turn blue on X-gal plates due to a high rate of mutations that restore the LACZ gene reading frame. However, any clone that can suppress the mlh1-7 defect lowers the mutation rate, the LACZ gene remains out of frame, and the resulting colonies are white. Testing suppression using other types of mutator assays, such as the lys2-14A assay and canavanine resistance, is crucial to see if these are indeed true suppressors.
|
Jocelyn Bosco
|
 | Research Advisor & Department:
Stephen Zinder - Microbiology
Name of Project:
Knock-out in the Operons Involved with Nitrogenase Fixation in the Anaerobe, Methanosarcina acetiverans CZA
Abstract:
The anaerobic archaeon Methanosarcina acetivorans C2A contains the nif operon, anf operon, and the vnf operon, each of which may play a role in the ability of this anaerobic archaeon to perform nitrogen fixation. The nif operon contains the structural genes that code for the molybdenum nitrogenase, the principle nitrogen fixation enzyme in the organism. There is strong evidence that the regulation of the genes in the nif operon is controlled at the level of transcription. A genetic system of M. acetivorans C2A recently developed by William Metcalf in 1997 will be used to determine the regions of the nif promoter that are important for regulation of the nitrogenase structural genes within the nif operon.
Deletion mutagenesis is a technique that will be used in order to develop different truncations of the nitrogenase promoter region in M. acetivorans C2A. Once these truncations are made, each new mutated promoter region will be fused to a reporter gene known as uidA (a.k.a GUS) at the promoter’s translational start site. These constructs will then be cloned into an E. coli shuttle vector and E. coli suicide vector. E. coli will be used to produce large amounts of the vectors that contain the insert of the reporter gene and truncated promoter region. Then these vectors will be used to transform M. acetivorans C2A. The effect of each promoter truncation on the activity of the reporter will hopefully indicate the areas within the nif promoter that are crucial to the transcription of the structural genes that code for the nitrogenase enzyme.
|
Karim Boudadi
|
 | Research Advisor & Department:
Thomas Cleland - Neurobiology and Behavior
Name of Project:
Effects of Reward Quality on Olfactory Acuity in Mice
Abstract:
Olfactory acuity represents the degree to which odors of differing quality are behaviorally distinguished in the context of specific training paradigms. Using a rewarded forced-choice odor generalization task, Cleland and colleagues have shown that both the amount of training and the intensity of the conditioned stimulus (odor concentration) narrow olfactory generalization acuity. These results suggest that the regulation of olfactory acuity is a learning process in the classical sense, in that it follows certain Rescorla-Wagner associative learning rules. We will test this hypothesis further by testing a third variable in the Rescorla-Wagner relationship: the strength of the unconditioned stimulus (reward). Specifically, we will examine the effect of reward quality on olfactory acuity using the same generalization task performed in previous studies. We predict, all else being equal, that mice will learn more about odors and demonstrate sharper olfactory acuity when trained with a high-quality reward compared to a low-quality reward. The long-term goal of this project is to determine whether olfactory acuity, and its neural substrate in the olfactory bulb, can be used as a reduced model to study the neural mechanisms of cortical learning and dementia.
|
Timothy Brown
|
|
Research Advisor & Department:
Jeffrey Doyle - Plant Biology
Name of Project:
Evolution of Polyploidy in Perennial
Abstract:
Allopolyploidy, the process by which two genomes from different species come together to form a doubled genome, allows for interactions between homoeologous gene copies. Plant disease resistance (R) genes confer resistance to many pathogens and are often clustered in plant genomes. One particular R gene, Rpg1-b, is a gene that confers resistance to the bacterium Pseudomonas syringae, which causes soybean leaf spots. Only a single locus is known in diploid soybean, although six phylogenetically closely related loci lie within a 220 kilo-base region, which may be the byproducts of tandem duplications. There may be additional loci produced by polyploidy that have yet to be identified. Recent studies have clarified several aspects of the phylogenetic history of soybean (Glycine max, 2n = 40) and its close relatives. This work verified the ancient polyploid nature of the genus, as well as the allopolyploid origins of the recent (2n = 78, 80) polyploids. Glycine tomentella is the name applied to what has been discovered to be group of more than ten species containing diploids and most of the allotetraploids in the genus. Two of the G. tomentella diploid species (D3 and D4) combined to form the allotetraploid T2. We wish to investigate the molecular evolution of the Rpg1-b locus (or loci) in this tetraploid. Have the sequences evolved independently or interacted in some way since the allopolyploid event? Are there multiple loci derived from the ancient polyploid event shared by the whole genus? Can the hypothesis tandem duplicated nature of Rpg1-b be confirmed? Also, there may be levels of interaction which promote sequence and allelic diversity between the loci.
|
Adam Buck
|
 | Research Advisor & Department:
Stephen Ellner - Ecology and Evolutionary Biology
Name of Project:
The Migration of the Rapid Evolution Project with Chlamydomonas as Prey
Abstract:
For a decade the Hairston lab has investigated the predator prey dynamics of rotifers and green algae using a simple nutrient flow-through system called a chemostat. The original goal was to induce chaos into a biological system, but we were sidetracked by the long, nearly in-phase population cycles which were not predicted by traditional predator-prey models. Adding into our computer simulations rapid evolution of defense on the part of the algae produced the same cycles we saw in the lab. This summer I will migrate the system prey species from Chlorella vulgaris to Chlamydomonas reinhardtii. Chlamydomonas has its entire genome sequenced and has many genetically marked clones available for use, so it will be easier to quantitatively track the evolution in the system and perhaps identify the gene or genes that are changing.
More specifically, it is my task to completely get the updated system running, starting from the basics. I will construct chemostats to run experiments. I will decide what type of medium to feed the algae with. The medium used for Chlorella used nitrogen in the form of nitrate, but some of the Chalmydomonas clones we have do not have the ability to reduce nitrate. Currently we are looking at substituting the nitrate with either nitrite or ammonium. After the choice of medium is finalized, I will inoculate chemostats with Chlamydomonas and Brachionus (a rotifer), determine at which dilution rates the populations cycle, and calculate the consumption and recruitment rates of the two species. These parameters are needed so that we can match our computer simulations with the system. With the updated model we can match the predicted bifurcation points with results from the actual system. Lastly, using various marked clones of Chlamydomonas, I hope to track clonal variation in the chemostat and prove that rapid evolution is occurring in the chemostat.
|
Frank Castelli
|
 | Research Advisor & Department:
Paul Sherman - Neurobiology and Behavior
Name of Project:
An investigation of odor use as the mechanism underlying intra-colonial discriminatory behaviors of the naked mole-rat.
Abstract:
Naked mole-rats (Heterocephalus glaber) are subterranean rodents that inhabit dry regions of Somalia, Ethiopia, & Kenya. They are eusocial1, meaning that they live in colonies with overlapping generations, cooperate to raise offspring, and exhibit a reproductive division of labor. The discovery of eusociality in naked mole-rats was a milestone in the investigation of the evolution of social behavior; prior to their discovery, examples of eusociality were restricted to insects (ants, bees, wasps, and termites).
Naked mole-rats live entirely underground and so their vision is greatly reduced from that of their surface-dwelling ancestors. They rely primarily on their tactile, auditory, and olfactory senses. Researchers have shown that naked mole-rats leave behind odor trails that other colony members follow to newly discovered food sources2. Naked mole-rats also use odor cues to distinguish fellow colony members from those of foreign colonies and will even behave aggressively toward fellow colony members that have odors of foreign colony members smeared on them3.
Naked mole-rats exhibit discriminatory behaviors among members of their own colony as well. Breeding females have been shown to act more aggressively towards fellow colony members that are less genetically related4. Also, breeding females have complete control over mate choice and discriminate among all male members of their colony when selecting sexual partners5. Additionally, naked mole-rats discriminate between individuals to coordinate their efforts to efficiently forage for food. Unpublished data indicate that odor trails laid by individual scouts are followed preferentially, depending on the quality of the food brought back by each individual scout6. The exact mechanism for these intra-colonial discriminatory behaviors has not been determined.
For my project, I will be testing to see if naked mole-rats behaviorally discriminate between the body odors of other individual naked mole-rats from the same colony, as well as those differing in category, including breeding status, sex, age, and size. I have designed and built a special glass apparatus to present naked mole-rats with the body odors of other naked mole-rats. The apparatus is a glass ring that acts as a "room" in which naked mole-rats are isolated so that the walls absorb their odor. After this preparation step is complete for two rings from individuals of different categories, both rings will be positioned at either end of a T-maze connected to the colony. Subjects will be allowed to enter the T-maze one at a time and their behavior and time spent in each glass ring will be compared. Trials will be repeated for three separate colonies.
Understanding the mechanism of colony member discrimination will provide insight into many components of the social behavior of naked mole-rats which can then be compared to the social insects species and add to our understanding of communication in eusocial colonies.
|
| |
>>next previous<< |