D.E.S. Truman

A re-examination of the organ specificity of lens antigens

The cross-reacting antigenic determinants between lens proteins and other tissues of Xenopus laevis and the chick have been studied by immunodiffusion, immunoelectrophoresis and the Osserman technique, using antisera detceting minor as well as major antigenic determinants of the lens proteins. All tissues tested contained some antigenic determinants similar to those found in lens proteins and it was demonstrated that all of the major classes of lens proteins contained such cross-reacting groups. The pattern of cross-reactivity varied both qualitatively and quantitatively from tissue to tissue and there were no lens antigens which were found in all of the other tissues tested. The cross-reacting material from extralenticular tissue is not always identical in molecular form to that obtained from the lens. The results of the Osserman tests have been interpreted as indicating heterogeneity in a population of protein molecules which may be based either on a heteropolymer structure or on the selective binding of cross-reacting antigenic moieties. The results described are taken to mean that much tissue specificity is the result of a unique selection of antigens genetically available, any one of which may be found in other tissues, rather than being due exclusively to the possession of some specific protein restricted to that tissue.

Characterization of chick lens soluble proteins and the control of their synthesis.

Abstract A single step column procedure for the separation of chick lens soluble proteins is described. α-, β- and δ-crystallin, which account for 80–90% of the soluble protein are separated using this technique, with little or no cross-contamination between classes, as judged by immuoelectrophoresis. Analysis of the fractions in sodium dodecylsulphate-polyacrylamide (SDS) and isoelectric focusing (IEF) gels in dissociating conditions identifies and characterizes the major α-, β- and δ-crystallin subunits. Fresh lenses from day-old chicks were labelled by culturing in medium containing radioactive amino acids and the synthesis of individual crystallin subunits analysed. In both pulse and pulse-chase labelled lenses at least 70% of the incorporated radioactivity could be assigned to characterized crystallin subunits. Differences were observed in the radioactivity profiles from pulse and pulse-chase labelled lenses when analysed on SDS and IEF gels and some polypeptides showed labelling characteristics typical of post-translational modifications of existing protein chains.

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina.

Abstract Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)+ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.

Antigens of the lens of Xenopus laevis.

The antigens of the lens of Xenopus laevis were investigated by elect rophoresis, immunological double-diffusion methods and immuno-electrophoresis. Twenty-two lens antigens were detected. A new nomenclature for lens proteins is suggested.

Sequence complexity and tissue distribution of chick lens crystallin mRNAs.

Abstract Using hybridization reactions with a cDNA copy, the complexity of polysomal polyadenylated mRNA from the day-old chick lens was found to correspond to 5800–7200 sequences of average size, arranged in three abundance classes. Experiments with heterologous cDNAs suggest on a qualitative basis that many of the sequences expressed in 8-day embryonic neural retina and pigmented epithelium mRNAs are also present in lens mRNA. A cDNA fraction complementary to the most abundant lens mRNAs, representing an approximate minimum of four sequences, was used to assay the dosage of putative crystallin sequences in these and other embryonic tissues. Neural retina and pigmented epithelium cytoplasmic mRNAs have low concentrations of these sequences, which appear to be absent from mRNA prepared from headless bodies and muscle.

Isolation and identification of chick lens crystallin messenger RNA

Abstract After treatment of chick lens polysomes with EDTA, two ribonucleoprotein components sedimenting between the smaller ribosomal subunit and transfer RNA can be isolated on sucrose density gradients. The RNA from the larger ribonucleoprotein component sediments at approximately 15S and that from the smaller at approximately 9S, but in both cases the RNA is heterodisperse. When the pooled putative lens mRNA is added to a duck reticulocyte or mouse Landschutz ascites cell lysate system, the synthesis of chick lens crystallins is stimulated, as judged by specific immunological criteria and gel electrophoresis.

A method for direct assay of messenger RNA turnover for different crystallins in the chick lens

Abstract A method is described for an immunological fractionation of polysomes, based on the immunological specificity of their nascent chains, with cross contamination usually below 5%. The method was used to study turnover of RNA associated with specific polysome fractions in the chick lens and indicated that during embryonic development such RNA may become more stable. The extent of stabilisation varies between specific ribosome fractions.

Ultrastructural and biochemical abnormalities of lens cell membranes from two strains of chicks with epithelial hyperplasia.

Abstract A cell membrane fraction was isolated from lenses of three different genotypes of chickens, two of which (Hy-1 and Hy-2) are associated with epithelial hyperplasia of the lens. Ultrastructural analysis of the membrane fractions and of intact lens epithelial cells showed that Hy-1 and Hy-2 cells have a marked deficiency of gap junctions. Analysis of the detergent-solubilized membranes by isoelectric focusing and by SDS-polyacrylamide gel electrophoresis showed that the membrane protein polypeptides differ markedly from each other both by charge and size. Chemical analyses revealed that Hy-1 and Hy-2 membranes also have a high sialic acid content. These results indicated that Hy-1 and Hy-2 cells are characterized by abnormalities in the structure and composition of their cell membranes and it is suggested that these modifications may be related to their abnormal cellular behaviour in cell culture and the hyperproliferation of the lens epithelium in vivo.

Protein Synthesis and its Regulation in the Lenses of Normal Chicks and in two Strains of Chicks with Hyperplasia of the Lens Epithelium

Comparisons have been made of the regulation of the synthesis of crystallins and membrane proteins in the lenses of normal chicks (N) and two strains (Hy-1 and Hy-2) showing hyperplasia of the lens epithelium. Quantitative differences were found in the rate of crystallin synthesis and qualitative changes in membrane composition. Genetic differences between the strains were found in the response to preincubation, stability of synthesis to Actinomycin D and sensitivity to Daunomycin.

Comparative studies of normal and genetically hyperplastic lens epithelia II: Lectin-induced cell agglutination and 125I-lectin uptake by cells

The reactions of lectins with dissociated and cultured epithelial cells from hyperplastic lenses of two unrelated chick strains, Hy-1 and Hy-2, characterised by hyperplasia of the lens epithelium, and lenses of a normal genotype (N) were investigated using four different lectins. Three methods of monitoring lectin-binding to cell surfaces were employed. Each of the four lectins used showed an individual pattern of reactivity to separated membrane components. Data obtained with the three labelling methods showed the same trend viz: increased agglutinability and high affinity for binding of all four lectins by Hy-1 and Hy-2 cells. These results suggest that Hy-1 and Hy-2 lens epithelial cells are characterised by alteration in their cell surfaces.