Beta Complex Research Articles

Structural analysis of a new B-cell-differentiation antigen associated with products of the I-A subregion of the H-2 complex.

Ia.W39 is a private specificity of the I-Ab subregion of the H-2 complex. It is selectively expressed on a subset of B lymphocytes that is absent in newborn normal and adult mutant mice carrying the xid gene. Immunoprecipitation and one-dimensional NaDodSO4/polyacrylamide gel electrophoresis showed that the molecule bearing Ia.W39 consists of two noncovalently linked glycoproteins of apparent Mr 33,000 and 28,000. Anti-Ia.W39 serum did not preclear the Iab molecule; however, the conventional allo-anti-I-Ab serum cleared Ia.W39 completely. In view of the identical two-dimensional gel pattern generated by the Ia.W39 and the conventional Iab immunoprecipitates, we believe that all Ia molecules bear the conventional specificities and only a subset would in addition express Ia.W39. Ia.W39 is probably not a carbohydrate antigen, because the antibiotic tunicamycin had no influence on its expression. It may be a conformational determinant on the A alpha and A beta complex induced by the association of an unknown molecule with these chains.

Detection of CML determinants associated with H–2 controlled Eβ and Eαchains

The serologically detectable molecules encoded by the H-2 complex, the major histocompatibility complex (MHC) of the mouse, fall into two classes-class I and class II (ref. 1). The class I molecules encoded by the K and D loci have a molecular weight of 44,000 and are noncovalently associated with beta 2 microglobulin which is not controlled by the MHC. The class II molecules are of two kinds, A and E, each consisting of two noncovalently associated polypeptide chains, alpha (Mr approximately 34,000) and beta (Mr approximately 28,000). Three of the four chains, A alpha, A beta and E beta, are controlled by loci in the I-A subregion, whereas the locus controlling the E alpha chain is located in the I-E subregion of the H-2 complex. Thus the loci coding for the E alpha and E beta chains are separated by at least one (J) and perhaps more loci. It has been shown that the E alpha and E beta chains are synthesized independently, and that the E alpha chain is required for the insertion of the E beta chain in the plasma membrane. We demonstrate here that the E alpha E beta complex (the E molecule) can evoke in vitro cell-mediated lymphocytotoxicity (CML) without previous sensitization in vivo, and that the strong CML reactivity is directed against allele-specific determinants on the highly polymorphic E beta chain.

Studies on the High Molecular Weight Form of Polypeptide Chain Elongation Factor-1 from Pig Liver

The high molecular weight form of the polypeptide chain elongation factor-1 (EF-1H) from pig liver was purified to an apparently homogeneous state. The procedure included aqueous two-phase separation, ammonium sulfate fractionation, four successive column chromatographies on Sephadex G-200, CM-Sephadex C-50, hydroxylapatite and octyl-Sepharose CL-4B, and glycerol density gradient centrifugation. By this procedure, the specific activity of EF-1H increased about 60-fold with a recovery of the total activity of 6%, as compared to the material obtained by aqueous two-phase separation followed by ammonium sulfate fractionation. Purified EF-1H was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions and glycerol gradient centrifugation, revealing that it is apparently homogeneous and has both EF-1 alpha and EF-1 beta gamma activities. The subunit structure of EF-1H was investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as well as two-dimensional gel electrophoresis, which indicated that EF-1H consists of three different subunits, i.e., EF-1 alpha, EF-1 beta, and EF-1 gamma, in an equimolar ratio. The Stokes radius and the sedimentation coefficient of EF-1H were estimated to be 5.9 nm and 6.2S, respectively, from which the molecular weight of EF-1H was calculated to be about 157,000. This value is close to the sum of the molecular weights of EF-1 alpha (53,000), EF-1 beta (30,000), and EF-1 gamma (53,000). Thus, we concluded that EF-1H is a monomer of a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, or EF-1 alpha beta gamma and its molecular weight is 136,000. This conclusion was further confirmed by measuring the amounts of EF-1 alpha and EF-1 beta gamma in EF-1H by enzymatic assays. The amino acid composition of EF-1H was determined, and the results revealed that there was a close resemblance between the values of EF-1H and those of EF-1 alpha beta gamma, which were calculated from the reported values for EF-1 alpha, EF-1 beta, and EF-1 gamma.

Autogenous and post-transcriptional regulation of RNA polymerase synthesis

The regulation of gene expression was studied, for the Escherichia coli rpoBC operon, which includes the genes, rpoB and rpoC, for the beta and beta subunits of RNA polymerase, and rplJ and rplL, for the two proteins, L10 and L7/12, of the 50S ribosome. The gene organization agrees well with the accumulated observations indicating the coordinate synthesis of RNA polymerase and ribosomes under various growth conditions for wild-type E. coli cells. On the other hand, the differential regulation of the two essential components observed under restrictive growth conditions, after addition of various drugs or with certain mutants, in particular those carrying mutations in the RNA polymerase genes, was found to take place through two novel regulation systems: The transcriptional termination at an internal attenuation site and the two autogenous and posttranscriptional controls, being specific for the two ribosomal protein genes and the two RNA polymerase subunit genes, respectively. The majority of the transcription initiated from the promoter rpoP beta terminates at an attenuator site between the promoter-proximal rplJL and the promoter-distal rpoBC genes. The frequency of the attenuation seems to control the relative level of RNA polymerase synthesis to that of ribosomes. The expression of rpoBC genes is subject to an autogenous regulation, in which both RNA polymerase holoenzyme and alpha 2 beta complex function as regulatory molecules with repressor activity. The autogenous regulation was found to operate at post-transcriptional step(s), probably at the level of translation. During the study on the regulation of RNA polymerase synthesis, we noticed that the rpoBC operon contained another autogenous regulation circuit, in which the synthesis of L10 and L7/12 was specifically repressed by the L10-L7/12 complex. Molecular mechanisms and physiological meanings of the novel regulations are discussed.

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.

Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.

Allozyme genetics in permanent translocation heterozygotes of the Oenothera biennis complex.

Allozyme inheritance and transmission genetics of 11 enzyme systems were determined in the permanent translocation heterozygotes Oenothera biennis, Oe. strigosa, and Oe. parviflora. Electrophoretic variation was examined first among 164 strains of structural heterozygotes. Allelic configurations were then judged from inheritance patterns in reciprocal F1 hybrids between each of 22 ring-forming strains and tester strains of the related bivalent-formers, Oe. hookeri and Oe. grandiflora. Allozymes are inherited as codominant markers, and, as dictated by the genetic system, within a strain individual allelic variants are generally transmitted through only one germ line. Of the 20 loci resolved, only eight are polymorphic in any species, and, within species, generally only two alleles are present at each polymorphic locus. Despite the relatively meager allelic array, each of the 22 strains whose chromosome complexes were characterized is genotypically unique. Generally, within taxa, alpha (egg) and beta (sperm) complexes differ in allele frequency at several polymorphic loci. Such variability is correlated with differences in the phylogenetic origins of complexes and not with differences in segmental arrangement within a group of related complexes.

INCOMPATIBILITY STUDIES IN OENOTHERA : THE DISTRIBUTION OF S I ALLELES IN BIENNIS 1 POPULATIONS

The studies of the population structure of the North American Euoenotheras which have been carried out largely by Cleland and his associates have analyzed the segmental arrangement and phenotypes of the genomes in plants collected from a wide range of geographical locations (see Cleland, 1958 for a summary of this work). Although some collections have included three or four different plants from the same population, other than the work of Winterheimer (unpublished), relatively little has been done to investigate in more detail a sizable sample from a single stand of plants. Because this type of study requires on the average 15 to 20 hybrids for each collection analyzed, the size of the population sample undertaken for investigation tends to be limited to modest numbers. The discovery of an incompatibility allele system in the complex heterozygotes of Oenothera (Steiner, 1956) suggested another approach to population analysis, which, although providing in part different data, allows an estimate of the diversity of single populations as well as enabling an appreciable number of plants to be included in the study. The group of oenotheras best suited for this type of analysis is known as the biennis group 1 (Cleland et al., 1950). Members of this group are typical complex heterozygotes, composed of two genomes or involving seven chromosomes each. These differ completely in the arrangement of their chromosome segments, so that metaphase I of meiosis is characterized by a ring of 14 chromosomes. The adjacent chromosomes of the ring regularly go to opposite poles at anaphase so that only two kinds of spores, and subsequently, gametes, are formed. Although these plants are normally self-pollinated, the homozygous complex combinations do not occur because of a balanced lethal system. Thus, the offspring are, like the parent, complete translocation heterozygotes which continue to breed true in succeeding generations. When different strains of complex heterozygotes are outcrossed, it becomes apparent that of the two complexes making up a plant, one is usually transmitted more readily through the egg, and the other more frequently through the pollen. On this basis, the two complexes are identified as alpha and beta, the alpha characteristically coming through the egg, the beta through the pollen. In addition, the alpha and beta complexes of a particular strain differ not only in the segmental arrangement of their chromosomes and their transmission behavior, but also produce distinctive phenotypes. Races differ in the extent of transmission of their complexes through egg and pollen. Some pass on only the alpha through the egg and the beta through the pollen; others may transmit the alpha through the pollen and the egg but only the beta through the pollen. Still others show offspring receiving both alpha and beta through pollen and egg; races also occur which transmit the alpha alone through the egg, but both complexes through the pollen. By intercrossing appropriate biennis group 1 races, alpha alpha combinations can readily be obtained. These are invariably self-incompatible because of the failure of pollen tubes to develop; when these plants are outcrossed either as male or female parents, they are perfectly compatible. An analysis of the compatibility behavior of biennis 1 alpha alpha showed that the alpha complexes carry an incompatibility allele system of the gametophytic type 1 Supported by Grant G-23596 from the National Science Foundation.