No abstract

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class‐specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class‐specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg‐positive C4A 3a and Ch‐positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101‐1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion‐like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21‐hydroxylase genes (21‐OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28‐30 kb that included both the C4A and 21‐OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21‐OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one‐half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross‐over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross‐over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21‐OHA gene is discussed.

Human CD1 antigens have a similar tissue distribution and overall structure to (mouse) TL. However recent data from human CD1 suggest that the mouse homologue is not TL. Since no human TL has been conclusively demonstrated, we have analysed the murine CD1 genes. Two closely linked genes are found in a tail to tail orientation and the limited polymorphism found shows that, as in humans, the CD1 genes are not linked to the MHC. Both genes are found to be equally transcribed in the thymus, but differentially in other cell types. The expression in liver, especially, does not parallel CD1 in humans. This demonstrates conclusively that CD1 and TL are distinct and can co-exist in the same thymus. It is paradoxical that despite the structural similarity between mouse and human CD1, the tissue distribution of human CD1 is closer to TL. The possibility of a functional convergence between MHC molecules and CD1 is discussed.

Mitochondria are known for their role in ATP production and generation of reactive oxygen species, but little is known about the mechanism of their early involvement in plant stress signaling. The role of mitochondrial succinate dehydrogenase (SDH) in salicylic acid (SA) signaling was analyzed using two mutants: disrupted in stress response1 (dsr1), which is a point mutation in SDH1 identified in a loss of SA signaling screen, and a knockdown mutant (sdhaf2) for SDH assembly factor 2 that is required for FAD insertion into SDH1. Both mutants showed strongly decreased SA-inducible stress promoter responses and low SDH maximum capacity compared to wild type, while dsr1 also showed low succinate affinity, low catalytic efficiency, and increased resistance to SDH competitive inhibitors. The SA-induced promoter responses could be partially rescued in sdhaf2, but not in dsr1, by supplementing the plant growth media with succinate. Kinetic characterization showed that low concentrations of either SA or ubiquinone binding site inhibitors increased SDH activity and induced mitochondrial H 2 O 2 production. Both dsr1 and sdhaf2 showed lower rates of SA-dependent H 2 O 2 production in vitro in line with their low SA-dependent stress signaling responses in vivo. This provides quantitative and kinetic evidence that SA acts at or near the ubiquinone binding site of SDH to stimulate activity and contributes to plant stress signaling by increased rates of mitochondrial H 2 O 2 production, leading to part of the SA-dependent transcriptional response in plant cells.

Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in , resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in () and , a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins ( [] and ) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap is presented for promoting future complex I research in Arabidopsis thaliana.

Six a2-macroglobulin (a2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, pa2M1, carries a 4.6-kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature a2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor proa2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published a2M amino acid sequence for all except three residues. The a2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using a2M cDNA as a hybridization probe.a2-Macroglobulin (a2M) is a serum glycoprotein and a major plasma proteinase inhibitor with a wide specificity. a2M-related proteins are present in all vertebrate species (1-4). Human a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfidelinked monomers (5, 6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor. The bait region, composed of a series of target peptide bonds for plasma proteinases (7, 8) (13,14).This suggests that the conformational change, which accompanies the complex formation between a2M and proteinases and the hydrolysis of the thiol ester bond, exposes regions of the a2M molecule that are recognized by these receptors. Receptor-mediated endocytosis of proteinase-a2M complexes by macrophages and liver cells leads to clearance of the complexes from the circulation.Internal thiol ester bonds are also found in the complement proteins C3 and C4 (15), which are derived from precursor polypeptides of size similar to a2M (180-200 kDa). Their thiol ester sites are found in positions comparable to those in a2M, and the amino acid sequences of all three thiol ester sites are conserved. These observations led to the proposal that the C3, C4, and a2M genes are derived from a common ancestral gene (16). The sequences of murine and human C3 and human C4 and partial cDNA sequences of murine C4 have recently been determined (17-21). Comparison of human a2M with murine C3 revealed a 25% overall sequence homology (17,18,22