Nucleic acid hydrolysing enzymes of Aspergillus candidus M16a

Pectin-degrading strains of fungi

Fungi elaborating proteolytic enzymes

Gelling polysaccharide of bacteria

Antimicrobial activity of some plant extracts

Bacterial strains

In an effort to understand the molecular mechanisms of DNA alkylation repair, I initiated molecular cloning of a novel gene, ' NGS1'. The <i>Saccharomyces cerevisiae ngs1-1</i> mutant was previously identified by its enhanced sensitivity to simple DNA alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of 'NGS1' as a putative DNA alkylation repair gene revealed that it is identical to 'MRE11'/'RAD58', a gene that is involved in meiotic recombination and DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, I determined the nucleotide sequence alterations of a number of 'mre11' mutant alleles, including 'ngs1-1', 'mre11-1' ('ts'), 'mre11-2', 'mre11-3' and 'mre11-58'. The location of various 'ngs1'/' mre11'/'rad58' mutations combined with the deletion analysis indicates that the functional domain(s) resides in the highly conserved N-terminus of Mre11. I also investigated various roles of Mre11 in spontaneous and DNA damage-inducedmitotic recombination. The assays used in this study show that the <math> <f> mre<it>11<g>D</g></it></f> </math> mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, 'MRE11' appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination. Physical interactions between members of the 'RAD52' epistasis group have been detected genetically and biochemically. These protein interactions also appear to be important at the early stage of meiotic recombination. Mre11 has been shown to interact with itself, Rad50 and Xrs2 in a yeast two-hybrid system. Preliminary studies employing deletion analysis predicted that the self-interaction domain of Mre11 resided at the N-terminus of the protein. To determine domain(s) required for Mre11 complex formation, and to elucidate the relationship between this complex formation and 'MRE11' DNA repair function, I employed a combined yeast two-hybrid and functional analyses. My results indicate that both Mre11 dimerization and interaction with Rad50 are essential for recombinational repair. I found that the N-terminus of the Mre11 protein constitutes the core homodimerization and heterodimerization domain and is sufficient for Mre11 DNA repair activity. Collectively, these studies support the hypothesis that Mre11 self-association as well as its assembly into a multi-protein complex consisting of Mre11 and Rad50 are essential for effective DNA recombinational repair. Using the sequence of yeast 'MRE11', isolated the full-length ' hMRE11B' cDNA from a human HeLa cell cDNA library. Compared to the previously identified 'hMRE11', 'hMRE11B' contains an additional 84 base pair sequence that results in a 28 amino acid insertion close to the C-terminus. Overexpression of 'hMRE11B' does not complement the alkylation sensitivity of the 'mre11' null and temperature sensitive mutant strains. My results suggest that species-specific protein interaction determines the functional specificity of 'MRE11 ' and that the participation of the C-terminus of Mre11 protein plays an important role in this regard. (Abstract shortened by UMI.)

The 'mms2-1' mutant was initially isolated by its sensitivity to the alkylating agent methyl methanesulfonate (MMS). I screened a yeast genomic library and identified the 'MMS2' gene. Disruption of 'MMS2' results in enhanced sensitivity to both MMS and UV and 'mms2-1' was found to be a missense mutation with partial loss of functions. Mms2 shares homology with ubiquitin-conjugating enzymes (Ubcs), but does not function as a typical Ubc in ubiquitination. The only other 'ubc' mutant that displays a UV and MMS sensitivity is 'ubc2' ('rad6') which is involved in postreplication repair (PRR) and mutagenesis. Epistatic analysis placed 'MMS2' within the 'RAD6' pathway but is parallel to the error-prone PRR subpathway consisting of 'REV1, REV3' and 'REV7 '. Mms2 is defined as a member of error-free PRR, since 'mms2 '[Delta] cells display an increase in the spontaneous and UV-induced mutations, and these phenotypes depend on 'REV3. mms2'[Delta] and 'rev3'[Delta] are synergistic with respect to killing by DNA damaging agents, indicating that they function in alternative pathways. Study of 'rad6'[Delta]'1-9, po130-46, po13-13, rad5'[Delta] and 'rad30'[Delta] alleles has placed these genes in the error-free PRR pathway. I found that 'rad6'[Delta]' 1-9' is epistatic to 'mms2'[Delta], while ' pol30-46, rad5'[Delta] and 'rad30'[Delta] all display an additive effect with 'mms2'. A 'srs2'[Delta] mutation is able to rescue the UV sensitivities of the 'rad6'[Delta] and 'rad18'[Delta] mutants, and is epistatic to 'mms2 '[Delta] and 'rev3'[Delta]. Furthermore, 'srs2 '[Delta] suppresses the MMS sensitivity of 'pol30-46' and 'rad5'[Delta], indicating that Srs2 controls the entire PRR pathway. In addition to PRR, the Ubc activity of Rad6 is involved in other cellular functions including sporulation, telomere silencing and protein degradation. The 'mms2'[Delta] mutation results in moderate defects in sporulation and protein degradation but has no effect on telomere silencing. This indicates that Mms2 may act as an accessory protein for Rad6, or alternatively, may provide an similar activity via a pathway distinct from Rad6. Two human Mms2 homologues, 'hMMS2' and 'CROC1', have been isolated. 'hMMS2' can suppress the yeast ' mms2'[Delta] UV and MMS sensitivity and elevated mutation rate. Further analysis is required to assess whether 'hMMS2' and ' CROC1' possess analogous activities in mammalian cells.

The current study involved students enrolled in the Fall 2007 Biosc 0150 and Spring 2008 Biosc 0160 lecture sections of a single Faculty member. Most students were incoming freshmen, and their prior exposure to the natural sciences varied. A single recitation section was used as the experimental section, taken from each lecture section. In addition to regular instruction, the students enrolled in the experimental section were also assigned readings from peer-reviewed journals. The performance of these students was then tracked, as it was expected to vary, and compared to that of the students enrolled in the control recitation sections. There was found to be a significant difference between the achieved mean course grade of the experimental section and the control section, for the Biosc 0150 lecture section, but not in the Biosc 0160 lecture section. Important trends, though, were noted in both semesters and are examined herein.

Although conventional therapies for metastatic cancers have made significant progress in recent years, they are relatively nonspecific and have many deleterious side-effects. Recently, novel therapies, including adoptive cellular immune therapies have had sporadic, but spectacular success in cancers such as malignant melanoma and renal cell carcinoma: tumors in which an immune response has been demonstrated. However, other physiological mechanisms, such as blood coagulation inhibit the immune response against cancers. Our previous work has shown that one of these coagulation proteins, soluble fibrin (sFn), inhibits unstimulated and activated lymphocyte adherence to tumor cells by blocking leukocyte integrin (CD11a/CD18) binding to tumor cell CD54, suggesting that sFn is an immunosuppressive agent in cancer. Since these receptors are also involved in lymphocyte/endothelial cell adherence and diapedesis (a necessary step in the immune response to cancer), it was hypothesized that sFn inhibits these functions, and that blockade of this inhibition using specific peptides would restore these immune responses. Fluorescently labeled lymphocytes and Interleukin-2 activated lymphocytes (LAK cells) were incubated with sFn (or its components; fibrinogen, Gly-Pro-Arg-Pro, or thrombin) in the presence or absence of specific blocking peptides. Lymphocyte and LAK cell adherence to endothelial cell monolayers was measured by perfusion at physiological shear rates in a microscope-mounted closed perfusion chamber, followed by image analysis using Image Pro Plus software. Diapedesis was measured by detection of fluorescence in 24-well microplates following immune cell incubation (18 h) with endothelial cell monolayers grown in transwells. SFn inhibited lymphocyte (54.1 + 11.3 %) and LAK cell (43.9 + 4.4 %) adherence to sFn pretreated endothelial cells, and intermediate values were obtained from sFn pre-treatment of only one cell type. Adherence was restored by peptide mediated blockade of sFn/CD54 binding, but not by CD11b blocking peptides. Diapedesis was also inhibited by sFn (lymphocyte 29.6 + 7.7 %; LAK 12.2 + 4.9 %) and restoration was observed using blocking peptides. These results confirm the stated hypotheses, and if physiologically relevant, suggest that sFn is an etiological agent in tumor growth and metastasis, and that blockade using fibrin specific peptides may enhance the effectiveness of adoptive immunotherapies.