The methylation of tRNA in KB cells after treatment with actinomycin D

Extensive shifts in the distribution of labeled methylated constituents of tRNA were observed in KB cells treated with actinomycin D for 30 min prior to a 90-min pulse with ³H-CH₃-methionine. Although this treatment completely blocked the synthesis of tRNA, methylation continued to the extent of 12–15% of controls (pulsed without antibiotic). Under this condition the relative proportion of radioactivity incorporated into 3-methylcytosine, N²-methylguanine and 2′-O-methylribose was markedly increased (170–235% of control values), it was moderately reduced in 1-methyladenine, 5-methyl-cytosine and 5-methyluracil (35–70% of controls) and markedly reduced in 1-, 7- and N²N²-methylguanines (15–30% of controls). These data suggest that specific types of methylations occur at particular times during the processing of pre-tRNAs.     

Spontaneous Autoimmunity in the Absence of IL-2 Is Driven by Uncontrolled Dendritic Cells

BALB/c IL-2-deficient (IL-2-KO) mice develop systemic autoimmunity, dying within 3 to 5 wk from complications of autoimmune hemolytic anemia. Disease in these mice is Th1 mediated, and IFN-γ production is required for early autoimmunity. In this study, we show that dendritic cells (DCs) are required for optimal IFN-γ production by T cells in the IL-2-KO mouse. Disease is marked by DC accumulation, activation, and elevated production of Th1-inducing cytokines. IL-2-KO DCs induce heightened proliferation and cytokine production by naive T cells compared with wild-type DCs. The depletion of either conventional or plasmacytoid DCs significantly prolongs the survival of IL-2-KO mice, demonstrating that DCs contribute to the progression of autoimmunity. Elimination of Th1-inducing cytokine signals (type 1 IFN and IL-12) reduces RBC-specific Ab production and augments survival, indicating that cytokines derived from both plasmacytoid DCs and conventional DCs contribute to disease severity. DC activation likely precedes T cell activation because DCs are functionally activated even in an environment lacking overt T cell activation. These data indicate that both conventional and plasmacytoid DCs are critical regulators in the development of this systemic Ab-mediated autoimmune disease, in large part through the production of IL-12 and type 1 IFNs.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Estrogen induction of 20alpha-hydroxysteroid dehydrogenase in mouse vaginal tissue

The conversion of progesterone to 20α-hydroxy-4-pregnen-3-one by 20α-hydroxysteroid dehydrogenase was measured in mouse vaginal tissue. The enzyme was confined to the 105,000 × g supernatant of tissue homogenates and the requirement for reduced NADP demonstrated. The Initial rates of 20α-hydroxysteroid dehydrogenase were determined in the cytosol of tissues from four-day estrogen-treated and untreated animals. The rate of 20α-hydroxy-4-pregnen-3-one formation per vagina was increased 15-fold by estrogen stimulation. This increase could not be accounted for on the basis of increased organ weight or increased availability of cofactor. These findings indicate that 20α-hydroxy steroid dehydrogenase induction in the mouse vaginae is under estrogen control.     

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.     

A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5’ splice site identity

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.     

Enzyme kinetics: Inhibition equations derived from the inelastic collision hypothesis

The inelastic collision hypothesis of enzyme action has been proposed by G. Medwedew. This theory has been here extended to the cases of competitive and non-competitive inhibitors, which form enzyme-inhibitor complexes, and to the cases of competitive and non-competitive substrates. The resulting equations are discussed and contrasted to those derived from the classical enzyme-substrate hypothesis. The original formulation of Medwedew neglects the presence of the enzyme-substrate complex although the general theory admits the formation of this complex. The formulation has been revised and extended to include complex formation. The resulting equation is discussed in terms of the usual criteria used to evaluate the Michaelis-Menten-Briggs-Haldane equation. Taken in part from a thesis submitted by Robert Katzman to the faculty of the Department of Physiology, University of Chicago, in partial fulfillment of the requirements for the Degree of Master of Science.