We describe accommodations that we have made to our applied computational–theoretical chemistry laboratory to provide access for blind and visually impaired students interested in independent investigation of structure–function relationships. Our approach utilizes tactile drawings, molecular model kits, existing software, Bash and Perl scripts written in-house, and three-dimensional printing in a process that allows a blind or visually impaired student to satisfy her or his curiosity about structure–function relationships with minimal assistance from sighted co-workers.
The discriminator base U73 at the acceptor terminus of Escherichia coli tRNA(Cys) is a determinant for the specific aminoacylation of this tRNA by the cognate cysteine tRNA synthetase. Substitution of U73 has a major deleterious effect on the catalytic efficiency of aminoacylation. Here, we show that an RNA hairpin minihelix and an RNA hairpin microhelix that recreate, respectively, the 12-base pair acceptor-T psi C stem and the 7-base pair acceptor helix of E. coli tRNA(Cys) were aminoacylated with cysteine. As in tRNA(Cys), alteration of U73 to A73, C73, or G73 in the cysteine mini- and microhelices eliminated aminoacylation. This established that the strong influence of U73 on aminoacylation is fully retained from the full-length tRNA(Cys) to the mini- and microhelixCys. Transfer of U73 to the noncognate minihelixAla conferred cysteine acceptance to the latter, despite the presence of the major determinant for alanine tRNA synthetase. Even minihelixGly, which shares U73 with minihelixCys, was an efficient substrate for aminoacylation with cysteine. Conversely, as long as U73 was present in minihelixCys, introduction of the glycine or alanine determinant could not block charging by cysteine tRNA synthetase. Although the catalytic efficiency of aminoacylation of these small RNA helices with cysteine was reduced by orders of magnitude from that of tRNA(Cys), the single nucleotide U73 determines the ability of these RNA helices to be aminoacylated with cysteine. These results demonstrated a dominant role of U73 for aminoacylation of small RNA helices by cysteine tRNA synthetase.
Curricula for three chemistry camp experiences for blind and visually impaired (BVI) individuals that incorporated single-and multiday activities and experiments accessible to BVI students are described. Feedback on the camps from students, mentors, and instructors indicates that these events allowed BVI students, who in many cases have been discouraged from doing science, to understand that chemistry can be made accessible and that they can think about chemistry on a level comparable to their sighted peers.
Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfRl) and small (PfR2) subunits of Plasmodium falkiparum ribonucleotide reductase. The small subunit (PfR2) contains the majior catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations "10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfRl) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfRl and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle.The spread of chloroquine resistance and the emergence of resistance to newer antimalarial agents is a serious problem in the therapy of malaria caused by Plasmodiumfalciparum, and clearly, additional agents are needed. Inhibition of ribonucleotide reductase (RR) encoded by pathogenic organisms is now recognized as a target for the design of antimicrobial agents. This approach has been most extensively investigated in the design of therapies for the herpes viruses (1-4 complex between the large subunit (Rl) dimer and the small subunit (R2) dimer. The association constant for this interaction is 0.5-1 x 107 M-1 (6). Kinetic studies of the association of E. coli Rl with full-length E. coli R2 compared to the association of Rl with a C-terminal truncated R2 strongly indicate that the interaction between Rl and R2 is entirely accounted for by C-terminal sequences of R2 (7). Synthetic peptides identical to the C terminus of R2 effectively inhibit RR activity by competing with R2 for association with Rl. This phenomenon was first demonstrated for herpes simplex virus RR where the acetylated nonapeptide, AcYAGAV-VNDL, had an IC50 value of 10-20 uM (2, 3, 8, 9). Further work showed that even modest changes in the sequence of the parent peptide can lead to large decreases in inhibitory potency (10, 11). We demonstrated that AcFTLDADF, corresponding to the C terminus of mouse R2, inhibits mammalian RR with an IC50 value of 20 uM (12). It was later shown that C-terminal peptides derived from E. coli, viral, or mammalian R2 sequences inhibited the RRs from which they were derived but did not cross-inhibit RRs from other species (13). In principle, therefore, a ...
Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP alpha) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group of seven naphthalene-degrading Pseudomonas strains showed strong hybridization with the ndoB probe, and five Gordona, Mycobacterium, Rhodococcus and Pseudomonas strains able to degrade higher molecular weight PAH showed weaker hybridization signals. Using a polymerase chain reaction (PCR) approach, seven naphthalene-degrading Pseudomonas strains showed a PCR fragment of the expected size with ndoB-specific primers and additionally ten strains of Gordona, Mycobacterium, Pseudomonas, Sphingomonas and Xanthomonas able to degrade higher molecular weight PAH were detected with degenerate primer-pools specific for the ISP alpha [2Fe-2S]-Rieske center of diverse aromatic hydrocarbon dioxygenases. This suggests a molecular relationship between genes coding for PAH catabolism in various PAH-degrading bacterial taxa, which could be used to evaluate the PAH-degradation potential of mixed populations.
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