Elucidation of the Cysteine Dioxygenase Gene (CDO1)
of the Pathogenic Dimorphic Fungus
Histoplasma capsulatum:
Isolation and Sequencing of a Gene
Thought to Play a Role in the
Pathogenicity of a Disease-Causing Fungus

Sarah Hasler



Preface Most may fail to find the importance of research on fungus. Histoplasma capsulatum is a fungus that exists in contaminated soil in temperate regions around the world, most notably for Mississippi, in the Mississippi River Valley. Furthermore, it can be found in diffuse areas where birds or bats are present. H. capsulatum causes a respiratory disease known as histoplasmosis in humans. It is estimated that 500,000 people are infected per year. One is infected by inhaling spores or fragments of the mold (mycelium) form. Once the fungus enters the lungs in the mycelium form, it transforms into yeast. As yeast, it lives within macrophages, phagocytic immune cells also known as histeocytes, where it kills lung tissue and creates pockets of necrosis. In immunocompromised individuals, it can spread to neurological, hepatic, and related tissues. In healthy adults, within two weeks of exposure, the intracellular antifungal immune response will activate to eliminate the pathogen. Research on H. capsulatum usually focuses on the shift from mold to yeast because this shift is where the pathogenesis lies. If one could prevent this shift or reverse this shift, histoplasmosis can be prevented or treated. This research focuses on cysteine dioxygenase, an enzyme that participates in creating the conditions necessary for the phase shift.

Abstract Histoplasma capsulatum is a dimorphic fungus that exists as a parasitic yeast at 37C and as a benign mycelium at 25C. This conversion and the cellular physiological components that transpire during the conversion are of considerable interest. Once these pathways are elucidated, the disease caused by this organism, histoplasmosis, can be treated more effectively or prevented entirely. One aspect of this pathway is the production of cysteine dioxygenase (CDO). The focus of this research was to identify and isolate the gene for cysteine dioxygenase.

Introduction
Histoplasma capsulatum H. capsulatum is a dimorphic fungus that intercoverts between February 9, 2007 2:56 PM>9, 2007 2:35 PMuces via budding. The yeast cells reside in macrophages and reticuloendothelial cells, where therein lies the source of their virulence. The mycelial phase thrives in 25C and reproduces via microconidia. The microconidia can develop to form yeast at 37C (Maresca and Kobayashi 1989).

The different strains of Histoplasma capsulatum vary in their level of virulence. Stains are classified into one of three classes based on mitochondrial DNA (mitDNA) and ribosomal DNA (rDNA) molecular weights, deciphered using Restriction Length Polymorphism (RFLP). Class One strains such as Downs, an avirulent strain. Class Two includes stains such as G217B. Finally, Class Three strains, such as the G184A and G186A, were used in this experiment. Of the A and B types, B is more virulent, but A grows more rapidly and can predominate a mixed A/B culture (Maresca and Kobayashi 1989).

Histoplasmosis The transition from mold to yeast is necessary for the pathogenesis of H. capsulatum. Once a mammalian host inhales the mycelia, they convert in the lungs into yeast. The yeast then takes residence in the aforementioned defense cells. This parasitic infection is referred to as histoplasmosis. Histoplasmosis occurs worldwide and is the most common fugal respiratory infection among humans and other animals. The highest occurrence of this infection exists in subtropical and tropical zones. In the United States, the Mississippi and Ohio River Valley areas provide favorable environmental conditions for H. capsulatum, resulting in 500,000 new cases of histoplasmosis per year. South America, Australia, Asia, Africa, and the Mediterranean foster virulent strains of H. capsulatum as well. Histoplasmosis is a major cause of death among immuno-compromised patients.

Description of Yeast Yeast cells have thin cell walls and reproduce via budding. With budding, a parent cell produces a daughter cell with the same cellular components and releases it. Yeast may be multinucleate or uninucleate. H. capsulatum, for instance, is uninucleate with lysosome-like functions. The cells lack microbodies.

Description of Hyphae Hyphae is the multicelluar form of H. capsulatum that characterizes the mycelial phase. Hyphae contain organelle components similar to yeast; however, hyphae are significantly morphologically different from yeast. Hyphae have a bilaminar cell wall that is approximately 20 nm thick. The cells may contain central pores in the septa that can be sealed with a Woronin body, a specialized organelle that regulates cytoplasmic flow. In addition to the yeast complement of organelles, hyphae contain Golgi-like cisternae.

Transition from Hyphae to Yeast The transition of interest is from the benign mycelial phase to the parasitic yeast phase. Superficially, it takes 18-24 hours for the earliest transformation changes to be evident. Most mycelial cells in a culture proceed at 37C through the change by the collapse of the hyphae and the development of oidial yeast cell. If the mycelium were already enlarged, then only oidial yeast cell formation occurs. The cell begins to assume a rounded appearance as the oidial yeast cell is liberated from the hyphal outer wall. After 48-72 hours, the oidial cells burst, releasing single yeast cells that reproduce by budding.

Environmental conditions such as temperature and the presence of particular vitamins and amino acids are required for the phase shift. The conditions, however, differ for the yeast-to-mycelia shift versus the mycelia-to-yeast shift. In the absence of the required nutrients, neither shift will take place, despite the temperature change. This suggests that there are specific mycelium genes and transitional yeast genes that are regulated by temperature as well as by the presence of particular nutrients. One necessary nutrient for the mycelia-to-yeast transition is sulfhydryl groups. The sulfhydryl groups, as part of the amino acid cysteine, must be in the media to see a shift. It has been suggested that the sulfhydryl groups regulate the reduction/oxidation (redox) potential of the media to allow for yeast formation. In fact, mycelia can convert to yeast regardless of the temperature as long as the redox potential of the media is lowered to +46mV. The mycelia can be maintained if the redox potential is +380mV (Rippon 1968).

Sulfur metabolism obviously plays a critical role in the conversion of phases. In the reaction of interest, cysteine stimulates oxygen uptake in the yeast phase, which is due to cysteine dioxygenase (CDO) activity (Maresca et al. 1981). CDO is not seen in the mycelial phase. CDO, an iron containing dioxygenase, produces cysteine sufonic acid, which is a key intermediate in cysteine metabolism (Figure 1); hence, it is believed to eiFebruary 9, 2007 2:56 PMuct necessary for transition to the yeast phase (Kumar et al. 1983). Cysteine is needed during the dormant phase to complete transition to the yeast form and to maintain the yeast form. Furthermore, experimental data suggests that cysteine is required to activate mitochondrial respiration (Maresca et al. 1981). Conclusively, it is known that cysteine metabolism plays a role in differentiation; however, the exact pathway and method are unclear. In addition, although CDO is expressed only in the yeast phase, it is unknown if CDO is inactive in the mycelial phase and if it is necessary for hyphae-to-yeast transition.


Figure 1 Cysteine reaction creating cystine (cysteine disulfide) or cysteine sulfonic acid (Hamsakutty, 2003).

Materials and Methods
Part One: Identify the CDO gene Previously the CDO gene has not been identified for H. capsulatum; however, the putative sequence for Neurospora, also a member of fungal kingdom, is available on the The National Center for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov/). This sequence was compared to Histoplasma 214 and 186 sequences via the Washington University Web site (http://www.genome.wustl.edu/projects/hcapsulatum/). All matches were rendered with a value that indicates the probability that the match is due to chance. The smaller the value, the less likely it is due to chance. The most promising match was chosen by this value, along with the size of continuity of the sequence. This sequence was evaluated for assumed exons and introns to create a theoretical map of the gene of interest. This is achieved by looking for AT GC donor/ acceptor signal sequence. These are traditionally indicative of intron splicing points.

Part Two: Amplify the CDO gene Once a map was created, primers were developed using the Mac Vector computer program (Figure 2). These primers (F1, F2, R1, and R2) were used in polymerase chain reactions (PCR) to amplify the region of interest in the 186ASY strain of the dimorphic fungus, Y signifing that the DNA was extracted from the yeast form.


Figure 2 F1, F2, R1, R2 Primer Amplification Map (217 Histoplasma capsulatum genome).

Gel electrophoresis was used to evaluate the product’s size, which can be used to test whether the gDNA sequence is as large as assumed by the map. This process is repeated with cDNA to experimentally test the length of the gene without the introns.

To determine the sequence of the gene, a cDNA RACE (Rapid Amplification of cDNA Ends) library of DNA from 186ASY was used as the DNA template for PCR reactions, using one of the CDO primers previously developed and a primer that will anneal to the end or beginning of the library. AP1 (anchor primer) is the primer that will anneal at the beginning or end of the library. In one reaction, AP1 and the CDO forward primer were used to amplify the region from the forward primer to the end of the library. Then, simultaneously, but in a different reaction tube, the AP1 and CDO reverse primer were used to amplify the beginning of the library to the region where the reverse primer anneals (Figure 3). In order to achieve visible bands, nested PCR techniques were used. Nested PCR employs AP2 primer, which will anneal within the AP1 primer site, thus only amplifying the DNA that contains this site from previous PCR reactions. It must be noted that AP1 PCR product bands were produced in 37 cycle runs where extra dNTPs and polymerase were added after 30 cycles. These products are separated on a gel and tested for the desired product by performing a southern blot. This membrane was hybrized at 65C with a radioactive probe of CDO DNA. The desired bands illuminated on the film, permitting the applications of cloning and transforming.


Figure 3 RACE Library Diagram In this figure, “Fwd” refers to the forward primer, “Rev” refers to the reverse primer, and “AP1” refers to the AP1 primer. The span of A’s denotes the poly-A tail in the RACE cDNA sequence.

Cloning of the DNA was achieved by using the InVitrogen Life Technologies TOPO TA Cloning kit. This kit places the target DNA in a TOPO vector that is then used to transfer the DNA into competent E. coli cells as plasmids. The E. coli were plated on LB + ampicillin plates, where they developed colonies. A sample of these colonies were removed and grown in liquid media. The target DNA from the chosen samples was extracted using a Zippy Zymoclean kit. This DNA was then separated on a gel and a southern blot was performed to determine if the CDO DNA was transformed and extracted. The CDO DNA was present, thus the DNA was sent for sequencing.

Unfortunately, only the 3’RACE was viable for the first sequencing. The cDNA library employed was deficient on the 5’end; hence another library was used to PCR the 5’RACE for sequencing. UPM primers with the standard CDO primers were used to extract the 5’RACE.

Part Three: Sequencing The cleaned CDO DNA sample was sent to the University of Maine. The results of the samples’ sequencing showed overlap to allow for the elucidation of the CDO gene sequence.

Results The gDNA and cDNA of 186ASY amplified with the possible primers pairs (F1/R1, F1/R2, F2/R1, F2/R2) were run on the gel shown in Figure 4. This gel clearly shows the variation in molecular weight between gDNA and cDNA. Using graphing methods, the gDNA PCR product sizes were determined: F1/R1, 311bp; F1/R2, 311bp; F2/R1, 296bp; and F2/R2, 296bp. The gDNA expected sizes were F1/R1, 379bp; F1/R2, 380bp; F2/R1, 373bp; and F2/R2, 374bp. The variation from expected values lies within the error range (5-10%) of the gel. The cDNA calculated sizes were determined as F1/R1, 182bp; F1/R2, 191bp; F2/R1, 182bp; and F2/R2, 173bp.


Figure 4 gDNA vs. cDNA for each primer pair (F1/R1, F1/R2, F2/R1, F2/R2) with LambdaHindIII and Amplisize molecular weight markers.

The RACE PCR performed with AP1 and F1, F2, R1, or R2 produced the gel shown in Figure 5. The southern blot associated with this gel, also in Figure 5, shows strong illumination after a 10-minute exposure to X-ray film (XAR film). Illumination indicates hybridization between the CDO probe, created with P32 labeled F1/R1 186ASY DNA, thus supporting the CDO identity of these bands.


Figure 5 RACE PCR blot and gel.

The gel and Southern blot shown in Figure 6 were derived from a nested PCR that used AP1/F1, AP1/F2, AP1/R1, and AP1/R2 PCR DNA as template DNA for AP2/F1, AP2/F2, AP2/R1, and AP2/R2 reactions, respectively. Again, the illumination denotes hybridization between the PCR products and the CDO probe, which indicates the CDO identity of the DNA. Constituents from the AP1 PCR and the nested PCR were separated on a singular gel (Figure 7). Bands from AP2/F1 and AP1/R1 were chosen to clone and transform.


Figure 6 Nested PCR gel and Southern blot.


Figure 7 AP1 and AP2 product gel.

The bands cloned into the TOPO vector and transformed into TOP10 E. coli cells were cut with EcoRI restriction enzyme to confirm the presence and size of the cloned product. Only AP2/F1 cells, shown as samples 1a-d in Figure 8, were of the proper size. After subsequent testing, this product from the 3’RACE was sent to the University of Maine for sequencing. The 3’RACE sequence is shown in Figure 9.


Figure 8EcoRI cut of AP2/F1 colonies (1a-d), AP1/R1 high band (2a-d), and AP1/R1 low band (3a-d).

AATTCGCCCTTACGAGAGCGAATGGGAACGGTATGCCT
TTGGTGACGCTGGCAGAGCGTATACGAGGAACCTGGTTGATGAGG
GCAATGGCAAATGTAATCTGCTTATCCTGGTCTGGAGCCCTGGAA
AGGGAAGCGCTATTCATGACCACGCCAACGCCCACTGTGTTATGA
AGGTGCTGAAAGGTTCTCTCCGAGAGACGTTATATGGGTGGCCAG
AGTCGGACAAGGTACAGAAGGGGGAGCCATCGCCCTTGACTGTCA
CCAGGGACAAGGTGTATGAAGAAGGCCAAGTCACATACATGTCAG
ACAAGCTGGGCTTGCATAAAATCTCCAATCCCGATCCGACAAATT
TTGCCATTTCTCTGCATCTCTACACGCCACCAAACGCTGCTCATT
ACGGGTTTTCCCTCTTTGACGAGAAGACGGGCAAGTCGCGCCACG
TCAAGCAATCCGTGCTCTTCTCCAGGAAAGGGCACAAACTATGAT
TTTAGCATTTGTGAAAAGAATCGGATGCTCCTTTCCGTCCTAAAT
ATCCGTTCTAAAGGGATAAAATGGAGGACCATATGCTGGTGAATC
TCTGGAGCACCTGTTTGAAGAAACCGTTCTTGCAGGGTGGAGTCT
GGAGTCTGGGGTTTTGGATTTAGGGTTTCGGGTTGGGTTAGCGCC
CTCATTTTTATTCCTCGTTCTTCAACAGACAGCGCTTTTCCACAC
ATGCGTCGAGCTAGTCGAGCTACATGGTCTCGCAACCCTCCAAGT
TTAGATGCATTCCTAACACGTGAACATTCATTATTGTTATCATTA
TTGGAGCCGTTGTAATTCCTCCTCTCCCTTTTTGTTAGTGTTTAA
GTGGTCCGCCATAAAGTTGTATAGAGTCAGAGCCAACTCCCCTCA
TACAGTTTGAATATGAGACATTAATAATACATGTATTCAATCAGC
GAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGGCCGTT
CTAGAATTCG
Figure 9 3’RACE of CDO gene derived from cDNA RACE library. The polyA tail is shown in italics, and the TOPO vector components that flank the sequence from the EcoRI cut are shown in italics.

The 5’RACE PCR product gel and Southern blot are shown in Figure 10. The illumination of the 5’RACE PCR product is a product of hybridization between the CDO probe and the PCR product, which suggests the CDO identity of the band.


Figure 105’RACE gel and blot

Following the confirmation of the 5’RACE and its sequencing at the University of Maine (Figure 10), the entire CDO gene was elucidated through the reconciliation of the 3’RACE and 5’ RACE. The gDNA sequence is shown in Figure 11. The gDNA sequence for CDO1 included 2282bp with five exons. The cDNA sequence spans 1200bp. The sequence is shown in Figure 12. The 213 amino acid CDO protein produced from the cDNA sequence is shown in Figure 13.

GTCCCCACATCATCACCAGATCATCCCCACAAAATCCCCCCGGA
ATCCCCTTTTCCTCGTCCCTGCATTCAGCCCCCGGCTCAACAAGTACGA
ACATTCCAACACAATGCCATATCTCGAGAACAGCGAGTCCTCTCCGGAC
CCCACCCCGCTCGATGCCTTCCACTGCTTGGTACAAGATATCAATAAGG
TCCTTGGTCCCAGTTCAGGCCTAGACTCGGACGACGTCGATCCGATGGA
TATCCAGAAGCTTATGGAGGACTATACTTCTAACGAGAGCGAATGGGAA
CGGTATGCCTTTGGTGACGCTGGCAGAGCGTATACGAGGAACCTGGTTG
ATGAGGGCAATGGCAAATGTAATCTGGTGAGTCTCCATGGTGGGAGTCG
ACCATCGGTTAGGATGGATTGATGATTTGAGATGAATTGATGTTCACGG
GGTCTCTGTGTTATGAGCGTGCTGACTGGTGTGAATAGCTTATCCTGGT
CTGGAGCCCTGGAAAGGGAAGCGCTATTCATGACCACGCCAACGCCCAC
TGTGTTATGAAGGTTAGTATCAGCAGAGGATCCCCGTTTCCTTTTGATT
TCGATAGTTACAGTGGGAATTTGTCTACTAATATTACTTACGTCACAGG
TGCTGAAAGGTTCTCTCCGAGAGACGTTATATGGGTGGCCAGAGTCGGA
CAAGGTACAGAAGGGGGAGCCATCGCCCTTGACTGTCACCAGGGACAAG
GTGTATAAAGAAGGCCAAGTCACATACATGTCAGACAAGGTTAGGGGAA
AAAAAAAAAAGAAAACAAATAAAAGCTCCTTGATCCTATTTTTAGAGCT
TTCTGGAAGAATGTTAACTGACAAACTAAAAAGCTGGGCTTGCATAAAA
TCTCCAATCCCGATCCGACAAATTTTGCCATTTCTCTGCATCGTAAGTG
CCTGCCGAACGGGGCAATATCCTTGTTCGTGCATTTAAATTTACTGATT
CAATTATTTTGGACCAATCCTAACCGGGGAACAGTCTACACGCCACCAA
ACGCTGCTCATTACGGGTTTTCCCTCTTTGACGAGAAGACGGGCAAGTC
GCGCCACGTCAAGCAATCCGTGCTCTTCTCCAGGAAAGGGCACAAACTA
TGATTTTAGCATTTGTGAAAAGAATCGGATGCTCCTTTCCGTCCTAAAT
ATCCGTTCTAAAGGGATAAAATGGAGGACCATATGCTGGTGAATCTCTG
GAGCACCTGTTTGAAGAAACCGTTCTTGCAGGGTGGAGTCTGGAGTCTG
GGGTTTTGGATTTAGGGTTTCGGGTTGGGTTAGCGCCCTCATTTTTATT
CCTCGTTCTTCAACAGACAGCGCTTTTCCACACATGCGTCGAGCTAGTC
GAGCTACATGGTCTCGCAACCCTCCAAGTTTAGATGCATTCCTAACACG
TGAACATTCATTATTGTTATCATTATTGGAGCCGTTGTAATTCCTCCTC
TCCCTTTTTGTTAGTGTTTAAGTGGTCCGCCATAAAGTTGTATAGAGTC
AGAGCCAACTCCCCTCATACAGTTTGAATATGAGACATTAATAATACAT
GTATTCAATCAGCATTGCAGACT
Figure 11 CDO gene sequence.

GTCCCCACATCATCACCAGATCATCCCCACAAAATCCCCCCGGAAT
CCCCTTTTCCTCGTCCCTGCATTCAGCCCCCGGCTCAACAAGTAC
GAACATTCCAACACAATGCCATATCTCGAGAACAGCGAGTCCTCTCCGG
ACCCCACCCCGCTCGATGCCTTCCACTGCTTGGTACAAGATATCAATAA
GGTCCTTGGTCCCAGTTCAGGCCTAGACTCGGACGACGTCGATCCGATG
GATATCCAGAAGCTTATGGAGGACTATACTTCTAACGAGAGCGAATGGG
AACGGTATGCCTTTGGTGACGCTGGCAGAGCGTATACGAGGAACCTGGT
TGATGAGGGCAATGGCAAATGTAATCTGCTTATCCTGGTCTGGAGCCCT
GGAAAGGGAAGCGCTATTCATGACCACGCCAACGCCCACTGTGTTATGA
AGGTGCTGAAAGGTTCTCTCCGAGAGACGTTATATGGGTGGCCAGAGTC
GGACAAGGTACAGAAGGGGGAGCCATCGCCCTTGACTGTCACCAGGGAC
AAGGTGTATGAAGAAGGCCAAGTCACATACATGTCAGACAAGCTGGGCT
TGCATAAAATCTCCAATCCCGATCCGACAAATTTTGCCATTTCTCTGCA
TCTCTACACGCCACCAAACGCTGCTCATTACGGGTTTTCCCTCTTTGAC
GAGAAGACGGGCAAGTCGCGCCACGTCAAGCAATCCGTGCTCTTCTCCA
GGAAAGGGCACAAACTATGATTTTAGCATTTGTGAAAAGAATCGGATGC
TCCTTTCCGTCCTAAATATCCGTTCTAAAGGGATAAAATGGAGGACCAT
ATGCTGGTGAATCTCTGGAGCACCTGTTTGAAGAAACCGTTCTTGCAGG
GTGGAGTCTGGAGTCTGGGGTTTTGGATTTAGGGTTTCGGGTTGGGTTA
GCGCCCTCATTTTTATTCCTCGTTCTTCAACAGACAGCGCTTTTCCACA
CATGCGTCGAGCTAGTCGAGCTACATGGTCTCGCAACCCTCCAAGTTTA
GATGCATTCCTAACACGTGAACATTCATTATTGTTATCATTATTGGAGC
CGTTGTAATTCCTCCTCTCCCTTTTTGTTAGTGTTTAAGTGGTCCGCCA
TAAAGTTGTATAGAGTCAGAGCCAACTCCCCTCATACAGTTTGAATATG
AGACATTAATAATACATGTATTCAATCAGCGAAAAAAAAAAAAAAAAAA
AAAAAAAAAA
Figure 12 CDO cDNA sequence

MPYLENSESSPDPTPLDAFHCLVQDINKVLGPSSGLDSDDVDPMDI
QKLMEDYTSNESEWERYAFGDAGRAYTRNLVDEGNGKCNLLILVWSPGK
GSAIHDHANAHCVMKVLKGSLRETLYGWPESDKVQKGEPSPLTVTRDKV
YEEGQVTYMSDKLGLHKISNPDPTNFAISLHLYTPPNAAHYGFSLFDEK
TGKSRHVKQSVLFSRKGHKL 
Figure 13 CDO protein sequence

Discussion The elucidation of the Cysteine Dioxtgenase Gene (CDO1) was achieved through extracting the desired sequence based upon a putative CDO sequence in Neurospora from 186 ASY cDNA via PCR techniques. The identities of the PCR products were confirmed with Southern Blots that differentiated the bands containing the CDO sequence. The cloned and transformed CDO DNA was checked again for its identity before the sample was sequenced. Due to this battery of conformational steps, one can have considerable confidence in authenticity of the determined CDO sequence.

The gDNA sequence for CDO1 is 2.282Kb with five exons. Exon one spans base pairs 802 to1059; exon two spans base pairs 1170 to 1241; exon three spans base pairs 1327 to 1464; exon four spans base pairs 1557 to 1614; and exon five spans base pairs 1705 to 1820. The cDNA sequence spans 1.2Kb. The 213 amino acids of the CDO protein are produced from the proposed cDNA.

According to Kumar (1983), enzyme assays of the CDO protein indicated that it has a molecular weight of 10,500. The average molecular weight of an amino acid is 110, thus his estimate would include 95 amino acids; however, the presented CDO protein is over twice this size. This variation warrants more investigation. An enzyme assay must be performed to ascertain the source of error and to rectify the conflicting data.

Although the sequence has been elucidated, the activity of this gene in both morphotypes must be determined. It has been suggested by Maresca et al. (1981) that this gene is only active in yeast; however, this has not been confirmed by subsequent research. The activity and the role the gene plays in morphology can be achieved by creating and introducing a knockout to the H. capsulatum genome and observing the consequences.

**Sarah is grateful to Dr. Glen Shearer for his direction and resources applied to this project.

References

Gilbert and Howard (1970). Uptake of Cysteine by the Yeast Phase of Histoplasma capsulatum. Infect. Immun. 2:139-144.

Hamsakutty (2003). Cysteine Free Radical and Radiation Biology. The University of Iowa. 5

Kumar, Maresca, Sacco, Goewert, Kobayashi, and Medoff (1983). Purification and Properties of Yeast Specific Cysteine Oxygenase from Histoplasma capsulatum. Biochemistry 22:762-768.

Maresca, Jacobson, Medoff, and Kobayashi (1978). Cysteine Reductase in the Dimorphic Fungus Histoplasma capsulatum. J. Bacteriol. 135:987- 992.

Maresca and Kobayashi (1989). Dimorphism in Histoplasma capsulatum: a Model for the Study of Cell Differentiation in Pathogenic Fungi. Microbiological Reviews 53:186-209.

Maresca, Lambowitz, Kumar, Grant, Kobayashi, and Medoff (1981). Role of Cysteine in Regulating Morphogenesis in the Dimorphic Fungus Histoplasma capsulatum. Proc. Natl. Acad. Sci. USA 78:4596-4600.

Rippon, J.W. (1968) Monitored Environment System to Control Cell Growth, Morphology, and Metabolic Rate in Fungi by Oxidation-Reduction Potentials. Appl. Microbio. 12:114-121.

Sarah Hasler is a senior biological sciences major and chemistry minor. She has been conducting research on Histoplasma capsulatum for 10 months under Dr. Glen Shearer. She has had the privilege of presenting her work at the Mississippi Academy of Sciences and at the Southern Miss Graduate Student Symposium. She has also worked with Dr. Jennifer Regan regarding the speciation of Gambusia affinis and Gambusia holbrooki. She also teaches two anatomy and physiology labs. In addition, she is a member of Kappa Alpha Theta fraternity, the honor society of Phi Kappa Phi, the Catholic Student Association, and Golden Key Honor Society. In four years of membership in Kappa Alpha Theta, she has served as vice president of finance for two terms, recruitment chair, and facility manager. Following her proposed graduation in May, she will relocate to Brandon, Miss., and begin medical school at the University of Mississippi School of Medicine in Jackson.