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Saline seeps are a known environmental hazard in eastern Montana. Identifying the salinity threshold value for fish using a 96-hour acute toxicity test will allow fish salinity tolerance to be taken into account when biotic integrity is used to determine the health of a waterbody. Acute toxicity tests were run 10 fish per 8 gallons of test solution in glass aquaria. The test solution was made using Na2SC>4 dissolved in carbon filtered city water and was renewed after 48 hours. Mortalities were counted and removed every 6 hours for the first 24 hours and every 12 hours there after. Results obtained from the 96-hour acute toxicity test determined the LC50 range to be 10-20 g/L using the binomial method. The geometric mean being 14.1 g/L. Results from a follow up experiment showing 100% mortality at 13 g/L lead to the conclusion that the actual LC50 lies below 14.1 g/L. The calculated LC50 value of 14.1 g/L leads to the conclusion that northern redbelly dace are a salt tolerant species well adapted to the saline waters of eastern Montana. Affected streams in eastern Montana that have salinity levels regularly approaching 9 g/L are approaching toxic levels.

The herpes simplex virus (HSV-1) encodes a trans-activator protein VP16. VP16 activates the immediate early genes (IE) of the viral genome during lytic infection. VP16 has been heavily studied by mutational analysis as a model for transcriptional activation. The goal of this project was to examine two specific aspects of VP16. First, to compare the properties of a previously constructed insertion mutant, in1814, to other mutant strains constructed in our laboratory; this insertion was moved from strain 17 of HSV-1 into the KOS strain, the strain used in our lab. The second aspect of this examination is a mutational analysis of a possible cryptic N-terminal activation domain. To assess whether this region contributes to transcriptional activation of the IE genes, deletion mutants were constructed in the N terminus of VP16, with or without the deletion of the Cterminal activation domain. The constructed viruses will be verified for the proper mutations, before the properties of these viruses are characterized.

The main objective ofthis study was to determine the presence and level of mosaicism for chromosomal aneuploidy in several cases of fetal demise that had previous indications of chromosome aberrations. The presence of aneuploidy was determined using fluorescence in situ hybridization (FISH) probes. Techniques that were utilized in attempts to prepare several tissue types for FISH are described. The analyses made with FISH provided useful data on the chromosomal makeup oftwo of the cases studied. In one case FISH results showed that the product of conception (POC) did not contain the trisomy 18 that is present at low levels in the mother’s blood cells. In the other case the miscarried fetus was previously identified as being mosaic for trisomy X. In this research project the presence ofvarying levels of trisomy X in both embryonic and placental tissue was confirmed

Breast cancer is the most prevalent endocrine-related malignancy in women. Both endogenous and exogenous estrogen exposure is associated with an increased breast cancer risk. The main goal of the present research project was to study the early morphological changes and expression of PCNA and ER-a during estrogen induced mammary carcinogenesis in the female ACI rats. The presence of ER-a was further studied by Western blot analysis. The ACI rat is an excellent model to study estrogen-induced mammary cancer, since treatment of estrogen alone induces a high incidence of mammary gland tumors after 5-7 months of treatment. In the present study, ACI female rats were treated with estrogen pellets (5 mg of 17-beta estradiol in a 20-mg pellet) for 4 to 8 months. After 4.5 months of treatment, significant morphological changes were observed when compared to age-matched, untreated controls. The mammary glands of the treated rats displayed dysplastic lesions, and the mammary gland ductules were filled with stratified squamous epithelial cells. Tumors were observed after 6.5 months of estrogen treatment. Immunohistochemical studies indicated the presence of numerous cells positive for proliferating cell nuclear antigen (PCNA) and for estrogen receptor-a (ER). Mammary glands of untreated age-matched rats revealed that most ductule epithelial cells had moderate cytoplasmic and nuclear ER staining, while the mammary glands ofestrogen-treated displayed increased intensity in nuclei ER staining.

The number of habitat types that bats can efficiently exploit may be limited by bat morphology (mass, wingspan, wing area, wing shape) and echolocation calls. Analysis of these characteristics can be used to predict which habitats bats are most likely to use. Slower, more maneuverable bats such as Myotis spp. should be able to exploit cluttered habitats in which prey densities are high, whereas larger, faster bats such as Eptesicus fuscus should be less able to use cluttered habitats due to an inability to maneuver efficiently in such habitats. I surveyed bats belonging to two genera, Myotis and Eptesicus, between 29 July and 30 August, 1997 at Spring Meadow Lake, a state recreation area on the western outskirts of Helena, Montana. Five habitat types were monitored for relative bat activity. The habitats were an open field (OP), a canopied area (CA) characterized by Salix spp., and three shoreline habitats representing low (SW), medium (ME), and high (TH) vegetation densities. Overall, bats were found to be most active at the shoreline sites of medium vegetation density, with relative abundance at other habitat types decreasing in the following order: SW, TH, CA/OP. Myotis spp. activity reflected this same trend. However, Eptesicus spp. activity was not significantly associated with any of the habitat types. Both field and canopy sites were virtually devoid of any bat activity. Greatest bat foraging activity (feeding buzz ratio) was associated with the ME habitat type, reflecting the overall relative abundance of bats. There was a significant difference in foraging activity between the ME and OP habitat types, but not between other habitat types for Myotis spp. Results are consistent with research showing that some species of Myotis favor aquatic habitats over terrestrial habitats. Greatest relative bat activity at shoreline sites of medium vegetation may reflect a cost-benefit “decision” to forage in an area in which the combination of prey density and extent of su

I studied the effects of gender and habitat patch size * on the home range of the Florida Scrub Lizard (Sceloporus woodi) for a period of three months during the summer of 1997. Scrub Lizards were trapped using 5-gallon bucket traps, noosing, and visual recapture. Location data collected for the lizards on eight 1-ha trapping grids was used to estimate the lizards' home range size. Significant differences between home ranges of males and females were found. Males had larger home ranges than females. There was no significant difference in home range size between lizards in small versus large patches. In addition, there was no significant interaction between sex of the lizard and patch size in their effect on home range size. Although patch size did not significantly influence home range size, there was a trend toward such an effect, especially for females. Males may have larger home ranges in order to have access to a greater number of females. Both sexes may have slightly smaller home ranges in large patches because lizard density is higher in large patches leading to increased competition. My results suggest that gender and possibly patch size can have significant effects on life history parameters such as home range.

Allozyme electrophoresis was used to discriminate between two types of freshwater sponges that previously could not be distinguished using morphological criteria. One of these types was a well-characterized species, Ephydatia muelleri, whereas the other could not be identified because of an absence of gemmules and gemmoscleres, structures on which classification of freshwater sponges largely depends. Homogenates prepared from five individuals of each sponge type were electrophoresed in cellulose acetate gels. The gels were then stained to localize bands of enzyme activity. Analyzing the stained gels revealed migration rates of hexokinase and sorbitol dehydrogenase that differed between the two sponge types. The absence of any electrophoretic bands common to the two sponge types argues that the sponges are different species.

The molecular cytogenetic techniques of fluorescence in situ hybridization (FISH) and karyotyping by means of GTG-banding were used to determine if a chromosomal abnormality could be detected in cell lines lacking expression of the Interferon Regulatory Factor -1 gene (IRF-1). It is believed that IRF-1 plays an important role in the biological immune response leading to the presence of Human Leukocyte Antigens (HLA) class II. These molecules are important indicators for some cancerous cells. Without HLA class II expression a cell may avoid an immune attack by T-cell receptors of CD4+ cells, thus the cell may continue on its cancerous course. Four malignant cell lines that lacked IRF-1 expression (WERI, Y-79, Hl734, and H322) were examined by focussing on the long (q) arm of chromosome 5 at band region q31, the IRF-1 gene locus. The results for each cell line did not indicate a deletion at 5q31. However, multiple but inconsistent abnormalities could be characterized, and future analysis is recommended.

The antibiotic activities of clarithromycin, clarithromycin plus 14-hydroxy clarithromycin and azithromycin against Haemophilus influenzae were compared. This comparison was performed because both clarithromycin and azithromycin have proven to be effective against infections due to H. influenzae. H. influenzae is partially responsible for the prevalence of community-acquired pneumonia (CAP) in the United States. Currently, many infectious and non-infectious conditions can mimic CAP, thereby hindering diagnoses of the causative agents and the administration of the most effective treatments. The initial treatment of CAP is crucial in reducing the mortality and morbidity due to CAP, yet the difficulties in diagnosis and administration of appropriate treatments prevent the initial treatment from being precise. Since H. influenzae is partially responsible for CAP, the use of an antibiotic that actively inhibits its growth would decrease the prevalence of CAP due to this bacteria. Minimum inhibitory tests, minimum bactericidal tests, and time kill assays were performed on five different strains of H. influenzae in order to determine if one antibiotic was more active over another. The results were not conclusive, but did show that all three antibiotics were bactericidal towards H. influenzae. Further studies will need to be performed to determine if one antibiotic over another or a combination of antibiotics should be initially administered to CAP patients in which H. influenzae is the responsible pathogen.

Freshwater sponges often contain symbiotic algae, genus Chlorella, within their cells, yet there is no quantitative data available on either the number of algae present in sponge host cells or the proportion of the host cell occupied by algae. The objectives of my study were (1) to determine the number of algae in archaeocytes, the sponge cell type containing most of the symbiotic algae, and the ratio of algal volume both to total host cell volume and to host cytoplasmic volume and (2) to determine the effect of light deprivation on the number of algae per host cell and the ratio of algal volume to host cell volume. Samples of the freshwater sponge, Ephydatia muelleri, were collected during the summer from Salmon Lake in Western Montana. The samples were dissociated and observed using brightfield and epifluorescence microscopy. There was an average of 20.5 + 16.5 algae per host cell, which occupy on average 3.8% + 6.5% of the host cell volume and 4.0% + 7.0% of the total cytoplasmic volume. The effect of light deprivation on the algae was a continuous decrease in algae numbers per host cell over time (numbers remain almost constant over time when sponges are kept in alternating light and dark). Unexpectedly, the ratio of algal volume to host cell volume in light deprived sponges remained almost constant over time. Further study is needed to reconcile the decrease in the number of algae with the constancy of the volume ratio.

Freshwater sponges containing symbiotic algae can serve as a model for the study of relationships in which a host and an intracellular symbiont benefit. In freshwater sponges, symbiotic algae belonging to the genus, Chlorella, are housed in large cytoplasmic vacuoles, called perialgal vacuoles. I used microscopic techniques in an effort to determine whether the perialgal vacuoles ofthe freshwater sponge, Ephydatia muelleri, (1) contain active acid phosphatase, a lysosomal enzyme and (2) are acidic. Whereas the postcoupling method for localizing active acid phosphatase gave equivocal results, the Gomori method for localizing the enzyme revealed that few of the perialgal vacuoles possesed active enzyme. The fluorescence of LysoSensor pH-sensitive probes suggested that the perialgal vacuoles are not strongly acidic and may have a pH of between -5.5 and —8.0. The latter conclusion is considered tentative, however, since the precise location of the fluorescing LysoSensor probe within the perialgal vacuoles could not be ascertained.

Earlier studies have indicated particle selection as a means of controlling interspecific competition between freshwater sponges. Thus, an attempt was made to observe differences in two sponge types, Ephydatia muelleri and a possible ecotype, from two different lakes, Salmon Lake and Coopers Lake, in the same drainage system. The results support particle selection but the means of selection remains unclear. A comparison of the algae found in the two lakes is also made showing significant differences.

A novel tissue digestion and culture technique was used to enrich for macrophages and isolate cells from reduction mammoplasty and non-cancerous mastectomy tissue obtained from pre- and postmenopausal women. The effectiveness of the isolation procedure was tested with immunohistochemical techniques which characterized cells for the following proteins: CD 68 and Ham 56 (known markers of macrophage cells); keratin; and smooth muscle actin. The results of the immunohistochemistry verified the presence of macrophages in the cultures. Cell cultures were also tested for macrophage biological activity which was measured by the release of oxygen free radicals. Also studied was whether this response could be modified after cells were cultured with a reproductive hormone believed to affect the development of breast cancer. Estradiol (estrogen) showed high free radical production in this preliminary study and supports the immunohistochemical findings. This also suggests that macrophage response to stimulus is modified following the treatment with estradiol.

Uniparental disomy is a condition that results in many genetic anomalies. The condition involves an individual who has inherited both members of a homologous pair of chromosomes from one parent, while all other chromosome pairs come from biparental inheritance. That is, each parent contributes one chromosome to each homologous pair. The intent of the study was to find individuals with this condition. To test for uniparental disomy PCR technology was used to amplify regions of polymorphic microsatellite repeats. This PCR technique allows the experimenter to determine the origin of the child's chromosomes. Two individuals suspected of uniparental disomy were tested. The result was negative for uniparental disomy in both cases.

Carbohydrates have been implicated in cell adhesion and in developmental processes. Lectin histochemistry using ConcanavalinA and Lotus lectin was used to localize lectin-accessible mannosyl and fucosyl residues in a maturing sponge developing from histoblasts generated during germination of a gemmule. With the exception of cystencytes found in the sponges stained with Lotus lectin, a similar staining pattern was seen with the two lectins, but there was a rather significant difference in the intensity of staining. This difference in staining intensity was thought to be a result of either more mannosyl residues available to the lectin or that the concentration of mannosyl residues in Ephydatia mulleri is greater than the concentration of fucosyl residues.

In a continuing attempt to purify mitochondrial DNA from the thatching ant, modifications were made to a previously developed extraction procedure. Although mitochondria were highly enriched in a subcellular fraction derived from the heads of the ants, when DNA purified from the fraction by an alkaline lysis procedure was electrophoresed, inconsistent results were obtained. Bands in the gel show a molecule larger than 22 kilobase pairs, which is not expected of mtDNA.

A retroviral vector (pSFF) derived from murine Friend spleen focus forming virus was used to transduce murine hematopoietic stem cells and express a cell surface marker protein, mutated murine prion protein, in vivo after transplantation. To enhance retroviral vector integration in bone marrow cells, mice were treated with 5 - fluorouracil (5- FU) to increase stem cell mitotic activity. The infectivity titer of the vector, pSFF-mPrP-3F4, was determined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cultures of mixed packaging cell lines. Infection of Sca- 1 + /Lineagene9''low cells with pSFF-mPrP-3F4 resulted in marker protein expression in 40% of the progeny cells after 7 days of culture. Transplantation of marrow cells or sorted Sca- 1 + /Lineagene9'low cells transduced with vector resulted in 3F4- positive mPrP expression in 11-37% of donor-derived peripheral blood leukocytes at two weeks. Although the percentage of 3F4-positive blood cells gradually declined, at 28 weeks 23% of recipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid and erythroid lineages and was detected in Scal + /Lineagene9_low marrow cells. The multi-lineage, high frequency expression observed suggests that pSFF may have utility in gene therapy directed to hematopoietic stem cells and their differentiated progeny.

An unknown sequence of DNA discovered in Bartonella henselae was studied in order to determine rts conformation, size, and origin. The DNA was isolated from B. henselae cells using a standard miniprep method, commonly used to isolate small extrachromosomal DNA from cells. This DNA was subjected to a series of experiments including digestion with restriction endonucleases, SI, and Mung Bean nucleases, and hybridization to B. henselae genomic DNA. in order to determine whether there are any sequences in common. DNA fragments produced by digestion were separated using standard gel as well as Pulse-Field Gel Electrophoresis, and viewed after being stained with ethidium bromide. Fragments used for hybridization experiments were labeled with 32P. The results indicated that the unknown DNA contained many restriction sites for a majority of the restriction enzymes used in the experiment. Pulse Field Gel Electrophoresis of the unknown DNA digested with the SI and Mung Bean nucleases suggested that the DNA may be linear since both enzymes, which cut strictly circular DNA, did not seem to attack the unknown DNA. The results of the hybridization experiment indicated that the possibility of the unknown DNA sharing sequences with genomic DNA is unlikely. Finally, Pulse Field Electrophoresis of undigested B. henselae DNA suggested that the undigested DNA itself consists of a number of fragments of various sizes which were most likely compressed into a smgle band on the standard electrophoresis gel. This result ultimately disproves the t hypothesis that the unknown DNA was produced by a plasmid, bacteriophage, or a transposon, since such DNA would be expected to consist of fragments of uniform size.

This project was intended to be a pair of preliminary experiments from which more detailed and specific experiments could be expanded. There were two procedures used in carrying out this study involving the isolation of DNA from a freshwater sponge. First, genomic DNA was isolated and purified. This genomic DNA was digested with the restriction endonuclease EcoR1 and then analyzed using agarose gel electrophoresis. The results showed an accumulation of the sponge DNA in one major band roughly slightly larger than the 21226 bp band generated by EcoR1 digestion of X-phage DNA. The second part of this experiment was an attempt to isolate mitochondrial DNA (mtDNA) from the sponge. Prior to isolation a succinate-INT reductase (SIR) assay was done on fractionated sponge tissue. SIR activity is due to succinate dehydrogenase, an enzyme that is specific to mitochondria. The results of this assay confirmed that the fractionation procedure used in the purification procedure yielded a fraction rich in mtDNA. A simple and relatively quick procedure was then used in an attempt to isolate mtDNA from the sponge tissue, but upon analysis by agarose gel electrophoresis no mtDNA was detectable.

Populations of many amphibian species appear to be declining in locations all over the world, including the Pacific Northwest (Bartelt and Peterson 1994). As an example, the populations of Bufo boreas have undergone drastic declines throughout much of their range (Com and Fogelman 1984, Peterson et al. 1992, Carey 1993, and Blaustein et al. 1994). If we want to answer questions about amphibian decline and plan to conserve species such as Bufo boreas, we must first have knowledge of the natural history and normal behavior of healthy animals, preferably in their natural environments. With the use of radiotelemetry techniques I conducted a focal study and gathered information on the daily activity, behavior, requirements, and location during different times of the day of Bufo boreas. I found that the surface activity rate increased during the night, and increased moderately during the mid-day. Surface activity rate occurred over a broad range of temperatures and increased as relative humidity increased. Locomotion rate increased during the night as toads moved from a daily retreat site to water or warm substrate. Locomotion rate was low at temperatures above 16 °C, with most locomotion between 5 and 16°C. Locomotion rate increased as relative humidity increased. Feeding strike rate was highest during the mid-day and occurred over a broad range of temperatures and relative humidities. My study uncovered aspects of activity and behavior that were not previously known. This information provides the raw material from which further research questions and hypotheses can be formulated to determine the vulnerabilities of Bufo boreas.

Tomato plants respond to wounding caused by herbivorous insects with the induction of certain defensive genes. This wound response is mediated by systemin, an 18 amino acid polypeptide derived from a larger precursor called prosystemin. Transgenic plants that overexpress the systemin precursor exhibit a constitutive wound response allowing the accumulation of high levels of proteins produced by these defensive genes. Reported here is the purification of one of these defense related proteins from transgenic plant extracts by polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was obtained after blotting to PVDF-membranes. An oligonucleotide corresponding to the amino acid sequence was then designed and used as a primer in the polymerase chain reaction (PCR). Three specific PCR products were obtained. The largest one (900 bp) was used to screen a tomato leaf cDNA library. A cDNA clone of 2.1 kb was retrieved. Preliminary sequence data has identified the clone as a ketol-acid reductoisomerase. The second largest PCR product (500 bp) was cloned via a plasmid vector and preliminary sequence data has identified it as an acyl-CoA binding protein. The role that these proteins play in the systemic wound-response remains to be found.

Calcitonin gene-related peptide (CGRP) has been shown to interact specifically with at least two types of receptors. Cloning of either receptor would be a major breakthrough since this knowledge could be applied to the development of drugs to augment or antagonize CGRP's actions in the body. With this goal in mind, Fisher and Chatterjee used an approach applying cDNA library screening combined with polymerase chain reaction (PCR) to obtain a partial cDNA sequence corresponding to a gene that could code for the CGRP receptor. A new project to find the entire sequence involved screening a human liver cDNA library using a putative CGRP receptor gene-specific probe. PCR was used as a means of verifying the presence of the correct cDNA. The cDNA was prepared for amplification and subsequent sequencing by in vivo excision. The DNA sequence that was finally obtained apparently was not the same sequence as the putative CGRP receptor DNA and contained no homology to any known gene.

Thirty-seven freshwater sponge populations were found in 25 lakes in Western Montana. Three species were identified: Eunapiusfragilis (E.f.), Spongilla lacustris (SI), and Ephydatia muelleri (E.m.). In an effort to describe factors correlating with sponge distribution, limnological data were obtained from 16 ofthe lakes which contained sponges and an additional 9 lakes where no sponges were found. New limits oftolerance with respect to hardness (E.m.,E.f), magnesium (E.m., S.I.), silica (E.m., S.I.), calcium (E.m ), and conductivity (E.m.) were established . Reasons for the lack offindings in regard to differences between species habitats are discussed and attributed to the similar habitat requirements for the three species found in Western Montana. These species are essentially cosmopolitan and are commonly found in association with one another. Spicule measurements greatly expanded previously recorded size ranges. This great size variation plus spicule malformation are discussed as points which lead to the difficulty of using spicule morphology as the major indicator for species identification. The need to look more closely at factors such as food preference and interspecific competition is discussed as a means for more specifically defining a species' ecological niche, especially in species which commonly associate with one another.

A simple and relatively quick procedure was developed for the extraction of mitochondrial DNA from the thatching ant Formica obscuripes . DNA obtained using this procedure was analyzed both after digestion with restriction enzymes (Eco R1 and Bam H1) and without digestion by electrophoresis on agarose gels. Although the DNA isolated appeared to be larger than the usual upper size limit (approximately 20 kb) for mitochondrial DNA, the undigested and digested molecules behaved as would be expected of a circular molecule with one restriction enzyme recognition site (i.e., the undigested form migrated further into the gel than the digested form).

The purpose of this study was to further the understanding of the causes of triploidy in spontaneous abortions in humans. Triploidy is a chromosomal aberration that can be caused by several erroneous occurrences during gamete formation or fertilization. In the present study, reports that involve cases of triploidy from prenatal genetics laboratories in the United States were collected. The emphasis was put on recurrent triploidy. The percentage of triploidy cases out of all triploid spontaneous abortions, as well as the percentages of XXX, XXY, and XYY cases, respectively, have been determined. Based on literature review, possible etiology of triploidy was established. Case reports of patients from Shodair Hospital in Helena, MT, who had recurrent spontaneous abortions caused by triploidy, were included.

Borrelia spirochetes, the etiologic agents of Lyme disease, are unique among bacteria because they contain linear, rather than circular, molecules of DNA. In addition, a significant amount, sometimes up to 25%, of the bacteria’s genome, is in the form of plasmids, small pieces of DNA which can replicate autonomously and can contain functional genes. This study was undertaken to investigate the structure and origin of several unusually large plasmids in four isolates of Borrelia, HO14, BO23, VS116, and R100. Isolating the desired plasmids from the bacterial cells was accomplished through gel electrophoresis, making it possible to isolate the desired plasmid by extracting it from the gel. After the plasmid-containing band was cut out of the gel, the DNA was purified and cut with restriction endonucleases. The DNA was then labeled with radioactive phosphorus and used to probe fractionated DNA previously transferred to a membrane. After allowing the radioactive probe to hybridize to the membrane-bound DNA, the membrane was covered with Hyperfilm. The radioactive molecules caused the film to be exposed, making it possible to determine which bands hybridized with the probe. In this project, I tried to determine the origin of the large plasmids in question. Were they part of the bacterial chromosome that had somehow become excised? Or were they created from a smaller plasmid that had made a mistake during replication and formed a dimer?

Ephydatia muelleri is a freshwater sponge abundantly found in the lakes and streams of Western Montana. In this study, the microscopic anatomy of E. muelleri in Salmon Lake, a lake located ca. 35 miles east of Missoula, MT, is described for specimens taken at ca. two week intervals between the months of May and August, 1994. The major cells (pinacocytes, choanocytes, and archaeocytes) are described. Although no sperm forming structures were noted, an egg and embryo indicate that sexual reproduction occurred.

This preliminary study investigated the distribution and role of cell surface carbohydrates which were suspected to affect proximal cell interactions such as recognition and adhesion. Recognition and adhesion play roles in the development of many organisms but are easily followed in freshwater sponges. The distribution of glycoconjugates in a freshwater sponge Ephydatia mulleri was investigated with the Periodic acid-Schiff (PAS) reaction and lectin histochemistry. PAS revealed the distribution of general carbohydrate in the sponge. Lectin histochemistry used Concanavalin A (ConA), a mannosebinding lectin, coupled through avidin and biotin to horseradish peroxidase enzyme (HRP) to reveal the location of mannose-containing glycoconjugates. PAS disclosed an abundance of carbohydrates in the extracellular matrix. Localization of ConA-binding glycoconjugates revealed their presence in the extracellular matrix, inside the cells, and on cell surface. The role of these glycoconjugates in sponge development from gemmules was assessed by ConA blockage of cell surface glycoproteins. Blocking the cell surface recognition glycoproteins with Concanavalin A showed no clear cut effect on cell adhesion and sponge fusion.

Spongilla lacustris is a freshwater sponge (Phylum Porifera, Family Spongillidae) that is present in the lakes of Western Montana. In an effort to understand more about its sexual reproductive cycle and about its microscopic anatomy, sections prepared from samples obtained from Salmon Lake (T15N/R14/S8) between May-August 1994 were examined using light microscopy Spermatogenic cysts, eggs, and larvae were found in samples obtained. The structures of pinacocytes, choanocytes, and archaeocytes are described.

Assays based on histocompatibility were used to test whether sponges belonging to the species Ephydatia muelleri and Spongilla lacustris occupy single clonal populations. The histocompatibility assay consists of two individual sponges of the same species being bound together to form a sample pair. After seven days the sample pairs were observed as exhibiting fusion or non-fusion. Two individuals that did not fuse with each other were considered to be of different strains. The results show that neither species of sponge, E. muelleri or S. lacustris. grow as a single clonal population in Salmon Lake. Three strains of E. muelleri and two strains of S. lacustris were identified.

In this study, a simplified procedure for the isolation of mitochondrial DNA from the giant stonefiy, Pteronarcys californica, was developed. The mitochondrial DNA was purified, cut with restriction endonucleases, electrophoresed on agarose gels, and stained. The isolation of mitochondrial DNA has been used in the past to analyze variations within populations of organisms. This application has proven useful in better understanding how populations have developed and evolved through time. However, until recently the isolation of mitochondrial DNA and its analysis has been a tedious process requiring extensive laboratory facilities. The generation of a simplified procedure should make these applications possible for the giant stonefiy. Although the procedure I developed did not yield mitochondrial DNA free of contaminants, the modifications introduced here do point the way for further work.

Two types of histocompatibility bioassays were used to assess the genetic variability within a population of Ephydatia muelleri, a freshwater sponge found at the north and south ends of Salmon Lake. One type of assay, relying on outgrowth from gemmules, did not work because of poor gemmule viability. The second type of assay, involving grafts of sponge tissue, indicated that the lake contains a single strain of E. muelleri.

Sandwich cultures of sponges provide a convenient means to study a sponge's internal organization and the physiological activities of sponge cells. In this study, the feasibility of using sponge explants as a source for sponge tissue in sandwich cultures was evaluated and the organization of the sponge's canal system was observed. Explants from Eohydatia muelleri. obtained from Salmon Lake, were attached to sandwich culture assemblies. Cells were allowed to grow out from the explants between closely spaced glass slides. Culturing was carried out either in Salmon Lake or in the laboratory for two to three weeks. Although there was little outgrowth in the laboratory, in the lake, tissue spread approximately 1.5cm from the sponge explant. Flagellated chambers were visible in the sponge tissue between the glass slides; however, connections between them were not. A model for the development of flagellated chambers is proposed.

A population of archeocytes was isolated from the freshwater sponge, Ephydatia muelleri, using a Ficoll gradient. From these archeocytes an endozoic Chlorella sp. was removed and cultured. The cultured Chlorella sp. were then used to investigate the uptake of symbiotes by E. muelleri. The cultured algae were added to germinated gemmules. The outgrowth from the gemmules was then examined for algae uptake by the sponge cells using brightfield and phase contrast photomicroscopy. The results were inconclusive because algal symbiotes inhabited sponge cells of both the experimental group (the sponges exposed to the algae culture) and the control group (the sponges not exposed to algae culture).

Prions are infectious particles composed exclusively of protein and can cause a number of neurodegenerative diseases. Prions are coded for by a normal chromosomal gene which is found in the genomes of all mammals. A posttranslational event, as yet unidentified, converts the normal prion protein into a pathogenic isoform. Incubation time in prion diseases is controlled by either the prion gene itself or a scrapie incubation time gene closely linked to the prion gene. Gene cloning techniques were used to attempt to demonstrate the existence of the incubation time gene. DNA of two control mice, B6.I and I/lnJ, was compared to DNA from congenics with B6.I and I/lnJ parents. B6.I mice are known to have a shorter incubation time than the I/lnJ mice. Both mice have different alleles of the prion gene. Analysis of the DNA surrounding the prion gene was accomplished by using the polymerase chain reaction. Two markers showed polymorphisms between DNA of a control mouse and the DNA of a congenic. A polymorphism in these markers would allow the PCR to show the parental origin, either B6.I or I/lnJ, of the flanking DNA. A difference in incubation time of these mice from their parents shows that an incubation time gene, rather than the prion gene, is responsible for incubation time.

Acquired pure red cell aplasia (PRCA) is an autoimmune disease characterized by poor red blood cell formation. In no instance has the molecular target of the autoimmunity been identified. In one case, a unique antibody, binding to a membrane protein of 130 kDa (P130) has been shown on immunoblots of mouse erythroleukemia (MEL) cells and other erythroid progenitor cells. Because P130 may be important in red cell development, the cloning and sequencing of its cDNA is of interest. Initially, I reproduced the detection of this protein with patient antisera using an indirect immunoblotting technique and enhanced chemiluminescence (ECL) detection. Experimenting with a variety of conditions, I found that pl30 could best be visualized on autoradiograms using 1:50 dilution of the patient’s plasma to blocking buffer and by performing three ten-minute washings at 30°C between the additions of each subsequent antibody. I began the molecular cloning of the pl30 cDNA by plating on a Clontech MEL cDNA expression library (using the phage lambda gtll expression vector) on E. coli strain Y1090. Nitrocellulose lifts of the plates were made, and these were screened with the patient’s antibody using the same conditions described above. Several positive plaques were identified, potentially indicating the desired fusion protein. These plaques remain to be screened repeatedly to obtain the purified protein.Acquired pure red cell aplasia (PRCA) is an autoimmune disease characterized by poor red blood cell formation. In no instance has the molecular target of the autoimmunity been identified. In one case, a unique antibody, binding to a membrane protein of 130 kDa (P130) has been shown on immunoblots of mouse erythroleukemia (MEL) cells and other erythroid progenitor cells. Because P130 may be important in red cell development, the cloning and sequencing of its cDNA is of interest. Initially, I reproduced the detection of this protein with patient antisera using an indirect immunoblotting te

Measles Virus (MV) Enders and MV Edmonston-Zagreb (EZ) were grown in both U937 cells and PBLs to study their dissemination. The > presence of aggregation and formation of giant cells was observed in those cultures infected with MV EZ, while it was notably absent from cultures infected with the MV Enders. Believing this difference to be a possible link in the understanding of measles virus dissemination, further studies were conducted to test the idea that MV EZ may disseminate intracellularly, while MV Enders disseminates extracellularly. Gamma Gard was added to cultures to study its effects on the dissemination of the two viral strains. Results of this experiment indicated the spread of MV Enders can be completely inhibited by antibody to measles virus, while the spread of MV EZ cannot be. Also, the two vaccine strains were grown in PBLs to ensure that results of studies in U937s could be applied to the animal model. Differences (as compared to growth in U937s) resulted for MV Enders, but not MV EZ. Apparently, the MV Enders could not infect all cells in the cultures and was dying off. So, experiments were conducted whereby aggregation was artificially induced in PBLs infected with MV Enders to see if it could facilitate the dissemination of the virus to normal lymphocytes previously unavailable to infection. While the viral quantification plates were contaminated, the aggregation and giant cell assays showed that MV Enders may disseminated intracellularly if given the opportunity.

Because of the similar properties of mitochondrial DNA and plasmid DNA, methods for the isolation of plasmid DNA can be adapted for the isolation of mitochondrial DNA. In the present study, a method for mitochondrial DNA isolation and analysis was used in an attempt to gain information about variability in a local population of snails (Helisoma sp.). DNA obtained from the foot of the snail was cleaved with the restriction endonuclease EcoR1, electrophoresed on agarose gels and stained. Although problems with the method prevented the principal goal of the project from being reached, a method was developed which should permit future analyses of genetic variability.

The object of this study was to identify types of freshwater sponges present in western Montana and to determine destribution of the different species. An attempt was also made to estimate growth rate. In Salmon Lake, Ephydatia muelleri. and Spongilla lacustris were found. In Blanchard Lake, Ephvdataia muelleri. Sponailla lacustris. and Eunapius fragilis were identified. Within Rock Island Lake, Eunapius fragilis and Ephydatia fluviatilis (tentative) were identified.

Taxonomically, sponges belong to the Phylum Porifera. The name Porifera (from the Latin porus, pore) was coined in the mid-nineteenth century by R.E. Grant to designate the phylum of these simple animals. The Phylum Porifera is divided into three classes. Freshwater sponges belong to the Class Demospongiae, comprising 95% of total sponge species (Pearse and Buchsbaum 1987). This class is defined by its skeletal structure which may consist of horny fibers (spongin), siliceous spicules (not triaxon), or both. Of the eight orders, freshwater sponges belong to the Order Haplosclerina, consisting of monaxon spicules and spongin. Most freshwater sponges belong to the Family Spongillidae while a few belong to the recently described family, the Metaniidae (Frost 1991). The Spongillidae consists of 125 species, 25 of which have been identified in the U.S.. While much work and study has been carried out on marine sponges, there has been less study of freshwater sponges, with little or no study of sponges indigenous to Montana. The present study will attempt to provide a preliminary focus on three aspects of freshwater sponges in this area. My main objectives focus on laying a foundation for future studies in morphology, cell dissociation and culture, histocompatibility, and to work out basic procedures in each section. Since this thesis encompasses three distinct sections, each section will be considered separately.

Means of localizing molecules are essential in contemporary biological research attempting to unlock the mysteries of how cells function, interact, and go wrong. Enzyme cytochemistry and immunohistochemistry are two methods which utilize different characteristics of given molecules to localize them in tissue. The objective of this study was to localize two proteins, Na,K-ATPase and vinculin, in frog and mouse liver sections using these methods. Na,KATPase activity contributes to overall p-nitrophenylphosphatase (jpNPPase) activity, and sites of p-NPPase activity were determined using an enzyme cytochemical method. Sites of p-NPPase activity were thus extrapolated to indicate regions of probable Na,K-ATPase activity. Vinculin was localized immunohistochemically using an antibody prepared specifically against the vinculin molecule. Liver sections mounted on glass slides were subjected to appropriate reaction conditions to carry out either the enzyme cytochemical method for p-NPPase, or the immunohistochemical method for vinculin. Results of the vinculin localization were inconclusive, but results of the p-NPPase localization enabled probable identification of Na,K-ATPase at lateral hepatocyte membranes, along the lining of the sinusoids, and associated with the bile canalicular membranes.

The major objective of this study was to determine what effect, if any, rainfall had on the concentrations of certain ions and other chemical species present in soil pore water. A transect was established in the Upper Three Runs Creek watershed on the U.S. W Department of Energy's Savannah River Site in Aiken, South Carolina. Water was collected as rain, throughfall, soil pore water, and stream water. Over three months, collections were made during five storm periods and five nonstorm periods. Samples were analyzed for anions and cations including Ca+2, Na+, and SO4'2 and for dissolved organic carbon (DOC). The effect of storms on the composition of soil pore water was evaluated using soil pore water samples taken from sites in the bottomland regime (which included the floodplain and riparian zone) during storm and nonstorm periods. No significant differences were found between storm and nonstorm concentrations of Ca+2, Na+, SO4'2, or DOC. Sampling with zero tension lysimeters is suggested to intercept macropore flow so that the chemistry of water flowing quickly through the profile immediately after a rain can be determined.

The aim of this study was to examine the change in the dissociation curve of oxygen from human hemoglobin at the temperatures of 37°, 25°, 18°, 12°, 7°, and 3° C. The partial pressures of oxygen and the calculated hemoglobin saturations were determined for samples at the various temperatures. From this data the partial pressures at 75% saturation were calculated, and the differences in the partial pressures at 75% saturation were compared. The dissociation curve was shown to shift to the left as the temperature was decreased down to 12°. Below 12°, the shift was not further observed. At the lower temperatures, the oxygen did not dissociate as well as it did at the higher temperatures.

Aleutian Mink Disease Parvovirus (ADV) belongs to the parvovirus family. Although ADV can be grown in tissue culture and has been cloned in procaryotic systems, a eucaryotic expression system was desired. This would allow the opportunity to examine viral synthesis in absence of ADV replication. I attempted to develop a eucaryotic cloning system using vaccinia virus to express the ADV virion protein genes. My goal was to clone a segment of a cDNA clone (pIB21), that coded for the open reading frame containing both virion protein-1 (VP1) and virion protein-2 (VP2) of ADV, into pSCll, a modified vaccinia virus co-insertion vector. I attempted this cloning procedure with clones constructed from segments of two different strains of the ADV virus, ADV-G and ADV-Utah-1. The recombinant vaccinia virus coinsertion vectors generated from these clones were used by members of Dr. Marshall Bloom's research team at Rocky Mountain Laboratories. They homologously recombined them with live vaccinia virus by transfection into cells previously infected with vaccinia virus. The vaccinia system was successful in expressing the ADV genes and synthesizing the viral proteins. It was later demonstrated that these proteins self-assembled into viral particles.Aleutian Mink Disease Parvovirus (ADV) belongs to the parvovirus family. Although ADV can be grown in tissue culture and has been cloned in procaryotic systems, a eucaryotic expression system was desired. This would allow the opportunity to examine viral synthesis in absence of ADV replication. I attempted to develop a eucaryotic cloning system using vaccinia virus to express the ADV virion protein genes. My goal was to clone a segment of a cDNA clone (pIB21), that coded for the open reading frame containing both virion protein-1 (VP1) and virion protein-2 (VP2) of ADV, into pSCll, a modified vaccinia virus co-insertion vector. I attempted this cloning procedure with clones constructed from segments of two different strains of the ADV vi

Previous studies have shown that epithelial cells immortalized by human papillomaviruses (HPVs) at late passages were more resistant to the effects of retinoic acid than late passage normal keratinocytes. Some of these late passage HPV-immortalized cells, however, lose the ability to form a cornified envelope with continued culturing. For this reason, early passage normal keratinocytes and early passage HPV-immortalized cells were studied to see if they also showed this resistance to retinoic acid. Normal epithelial cells and cells immortalized by HPVs were grown in organotypic raft cultures and treated with varying amounts of retinoic acid, which normally blocks terminal differentiation in keratinocytes. The cultures were grown for two weeks, fixed, sectioned, and stained with either hematoxylin and eosin (H&E) or an antibody against the keratin K1. The HPV-immortalized cell lines were found to be more resistant to the action of retinoic acid than the normal cells. The same cell lines were also grown in culture plates in the same concentrations of retinoic acid. These cultures were counted daily for one week, and the results were used to construct growth curves and viability charts. It was found that the normal and HPV-immortalized cells show the same response to the growth inhibitory effects of retinoic acid. In addition, the viability of all cell lines was not significantly reduced by the higher concentrations of retinoic acid This showed that the differences in morphology seen in the raft cultures were probably due to a different mechanism than that which causes growth inhibition.

Tissue sections of chick embryos were immunohisto- chemically stained for the presence of A-CAM, a cell surface protein associated with epithelial tissues. This was to determine an effective method of immunohistochemical staining for A-CAM and to study the distribution of A-CAM in the embryonic chick. A method of immunohistochemical staining was developed.

Pili show remarkable variation in Neisserria gonorrhoeae. This variation is due to variable pilin, the primary protein component of pili. The variation in pilin is in turn due to events at the genetic level. Intragenic recombination (i.e., recombination from a homologous duplex located on the same chromosome) is one means by which variation could be brought about. The presence of the recBCD gene wpuld be consistent with such a mechanism since the presence of this gene is involved in recombinational events. This past summer at the Rocky Mountain Labs under the direction of Stuart Hill, I attempted to identify a recBCD gene in gonococcus.