Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Aggregates of partially purified mRNA coding for immunoglobulin light-chain

The 12S and 22S RNA fractions were isolated from phenol extract of the total polysome population of MOPC-321 mouse myeloma (a light-chain producer). Both RNA fractions exhibited mRNA activity when tested in the Krebs II ascites cell-free system. Fingerprint analyses of tryptic digests of the cell-free products showed that whereas the 12S RNA fraction contained L-chain mRNA together with a large amount of non-L-chain mRNAs, the 22S RNA fraction showed almost exclusively L-chain mRNA activity. The observed purification that was achieved by sucrose gradient centrifugation might be attributed to the tendency of L-chain to form aggregates upon exposure to phenol.     

Partial sequence of immunoglobulin light-chain isolated from purified plasma membranes of mouse myeloma cells.

Plasma membranes were prepared from MOPC-321 mouse myeloma cells incubated with [³H]leucine. The L-chain from the purified plasma membranes was isolated, it was subjected to radioactive sequence analysis, and leucine was found at positions 4, 11 and 15. This sequence matches with that of the mature L-chain (Leu at positions 4, 11, 15, etc.) and differs from that of the L-chain precursor that contains a hydrophobic N-terminal extra piece (Leu at positions 6, 7, 8, 11, 12, 13, 24, 31, 35, etc.). This result establishes mature L-chain in the surface membrane of plasmacytoma cells.     

The course of photooxidation of menadione.

The highly purified (> 95%) mRNA coding for immunoglobulin light (L)-chain yields on acrylamide gels a discrete 15.5S band and a “shoulder” ranging in size from 15.5 to 9.5S. The “shoulder” was isolated and found to be fragmented mRNA as judged from: 1. hybridization kinetic analysis, using the complementary-DNA to the L-chain mRNA; 2. capacity to form aggregates, similarly to the intact 15.5S mRNA. Partial cleavage of the mRNA probably occurs during mechanical disruption of the myeloma cells. Fragments with intact 3′ end are selected due to binding via the poly(A) moiety to oligo(dT)-cellulose, i.e., the fragments should be deficient at the 5′ end where mRNA translation is initiated. In agreement, the fragmented mRNA is essentially untranslatable in a cell-free system. The size of the L-chain mRNA is rather uncertain. A value of 15.5S is obtained from migration in acrylamide gels made in water or formamide, a value of 12S is obtained from sucrose gradient centrifugation.     

Evidence of new cadmium binding sites in recombinant horse L-chain ferritin by anomalous Fourier difference map calculation

We refined the structure of the tetragonal form of recombinant horse L-chain apoferritin to 2.0 Å and we compared it with that of the cubic form previously refined to the same resolution. The major differences between the two structures concern the cadmium ions bound to the residues E130 at the threefold axes of the molecule. Taking advantage of the significant anomalous signal (f′′ = 3.6 e⁻) of cadmium at 1.375 Å, the wavelength used here, we performed anomalous Fourier difference maps with the refined model phases. These maps reveal the positions of anomalous scatterers at different locations in the structure. Among these, some are found near residues that were known previously to bind metal ions, C48, E57, C126, D127, E130, and H132. But new cadmium binding sites are evidenced near residues E53, E56, E57, E60, and H114, which were suggested to be involved in the iron loading process. The quality of the anomalous Fourier difference map increases significantly with noncrystallographic symmetry map averaging. Such maps reveal density peaks that fit the positions of Met and Cys sulfur atoms, which are weak anomalous scatterers (f′′ = 0.44 e⁻). Proteins 31:477–485, 1998. © 1998 Wiley-Liss, Inc.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.