Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

Theory of s-d exchange scattering in dilute magnetic alloys

The problem of s-d exchange scattering of conduction electrons off localized magnetic moments in dilute magnetic alloys is considered employing formal methods of quantum field theoretical scattering. It is shown that such a treatment not only allows for the first time, the inclusion of multiparticle intermediate states in single particle scattering equations but also results in extremely simple and straight forward mathematical analysis. These equations are proved to be exact in the thermodynamic limit. A self-consistent integral equation for electron self energy is derived and approximately solved. The ground state and physical parameters of dilute magnetic alloys are discussed in terms of the theoretical results. Within the approximation of single particle intermediate states our results reduce to earlier versions. The following additional features are found as a consequence of the inclusion of multiparticle intermediate states;

(i) A non analytic binding energy is pre sent for both, antiferromagnetic (J < o) and ferromagnetic (J > o) couplings of the electron plus impurity system.

(ii) The correct behavior of the energy difference of the conduction electron plus impurity system and the free electron system is found which is free of unphysical singularities present in earlier versions of the theories.

(iii) The ground state of the conduction electron plus impurity system is shown to be a many-body condensate state for J < o and J > o, both. However, a distinction is made between the usual terminology of "Singlet" and "Triplet" ground states and nature of our ground state.

(iv) It is shown that a long range ordering, leading to an ordering of the magnetic moments can result from a contact interaction such as the s-d exchange interaction.

(v) The explicit dependence of the excess specific heat of the Kondo systems is obtained and found to be linear in temperatures as T→ o and T ℓnT for 0.3 T_K ≤ T ≤ 0.6 T_K. A rise in (ΔC/T) for temperatures in the region 0 < T ≤ 0.1 T_K is predicted. These results are found to be in excellent agreement with experiments.

(vi) The existence of a critical temperature for Ferromagnetic coupling (J > o) is shown. On the basis of this the apparent contradiction of the simultaneous existence of giant moments and Kondo effect is resolved.

A database for the industrial trawl fishery of Cote d'Ivoire

Fishery statistics for the industrial trawl fishery of Cote d'Ivoire have been well documented since 1968. However, data processing has changed significantly with time and some of the data files have been lost. In 1997, the Centre de Recherches Oceanologiques d'Abidjan decided to retrieve and process all trawl data available from different sources. This paper gives an overview of the database covering the period 1968 to 1997 and describes its coverage, format, structure and use. The database was developed using MS ACCESS and is a powerful tool for storing information about this fishery, and for analysis of its dynamics over a period of 30 years.

Effects of physiological doses of vitamin D sub(3) (Cholecalciferol) on induced molting and growth in giant freshwater prawn, Macrobrachium rosenbergii (de Man)

Effects of three different doses of vitamin D sub(3) on molting, growth, and calcium and phosphate composition of tissue and molt during the grow-out of the giant freshwater prawn Macrobrachium rosenbergii (average weight 10.56 ± 0.20 g), obtained from a grow-out pond, were studied. Intramuscular doses of vitamin D sub(3) (100, 500 and 2000 IU/kg body weight) were given on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 20th, 25th and 30th days. All the experimental animals were fed with a basal diet containing fish meal, shrimp meal, wheat flour, groundnut de-oiled cake, soybean meal and wheat bran at 3% of the body weight. The numbers of molts were recorded as 20±0.50, 29±1.16, 51±1.87, and 30±1.60 in control, 100, 500 and 2000 IU/kg body weight physiological doses, respectively. Maximum growth was recorded in prawns given 500 IU/ kg dose. Survival was between 58.33 ± 9.13 and 77.77 ± 8.61%. The ash content and calcium level increased significantly (p<0.05) and recorded the highest values in 500 IU/kg physiological dose. However, the inorganic phosphate (P sub(i)) content recorded the highest values in tissue in 2000 IU/kg dose (p<0.05, F = 50.60613). There is no significant difference in calcium contents (p>0.05) in both tissue and molt at 500 and 2000 IU/kg doses. It was found that a higher physiological dose (2000 IU/kg) of vitamin D sub(3) increased the rate of mortality. Results have shown that vitamin D sub(3) has a positive impact on the growth and survival of M. rosenbergii and it interferes with the metabolism of Ca and P sub(i), in tissue, and alters molting frequency. Results on physiological dose suggest an alternative and effective dietary supplementation method of vitamin D sub(3) in the grow-out phase of M. rosenbergii.