Western Transfer Analysis Research Articles

A biomolecular approach to the study of the expression of specific genes in the retina

The central nervous system contains a number of populations of neurons that are morphologically and functionally distinct. To study the genes responsible for the development and maintenance of proteins with unique structural and biochemical properties, polyspecific antisera were produced against normal mouse retinal proteins and used in combination with the rd (retinal degeneration) neurological mutant of the mouse to immunologically identify specific retinal proteins. Western transfer analysis of the proteins present in normal and in mutant retinas identified three classes of neural proteins: those found only in normal retina; those found in normal and in photoreceptorless retinas but not in other tissues; and those found in both normal and mutant retinas, as well as in brain, but not elsewhere. Some of these class 1 proteins were shown to be present in the retinas of other species, including humans, suggesting their importance in the process of vision. The poly A+ RNA was isolated from the retinas of normal mice and used to generate a cDNA expression library in lambda gt-11. This library was screened with polyspecific antisera absorbed with the proteins present in mutant retina, and a number of immunologically positive plaques were cloned. Four of these were shown to code for rhodopsin, the major visual protein in mammalian retinas. The approach described is applicable to other systems in order to generate specific immunological and recombinant DNA probes for examining the expression of specific genes during development and differentiation.

Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization.

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.

Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.

A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.

Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae.

Bacteroides nodosus is the primary causative agent of ovine foot rot. Virulent isolates of this bacterium contain fimbriae which appear to play a major role in both infectivity and protective immunity. This paper presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the fimbriae of B. nodosus. Total DNA was isolated from B. nodosus VCS 1001 (serogroup A), digested with HindIII, and inserted into the positive-selection vector pTR262. Recombinant E. coli clones were screened directly with anti-fimbrial antiserum by using a colony immunoassay. Several positive colonies were identified, each of which contained the same 5.5-kilobase HindIII insert. The prototype has been designated pBA101. Some clones also contained additional flanking sequences from the B. nodosus genome. Western transfer analyses verified that the positive clones were producing the B. nodosus fimbrial structural subunit, molecular weight ca. 17,500. The level of expression of the antigen in E. coli was comparable to that in B. nodosus itself and was unaffected by the insertion site or orientation of the cloned fragment, indicating that synthesis was being directed from an internal promoter. Restriction mapping and deletion analyses localized the fimbrial subunit gene to the vicinity of a PvuII site near the central region of the original HindIII insert. The expressed antigen was located in the membrane-cell wall fraction and may be exposed on the surface of the recombinant E. coli cells.

Pp60 c-src kinase is in chick and human embryonic tissues

The normal cellular protein pp60 c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60 c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60 c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60 c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60 c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60 c-src distributed throughout the neural tube and heart. The distribution of pp60 c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60 c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60 c-src kinase. The results of our studies suggest that pp60 c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.

Pp60c-src is developmentally regulated in the neural retina.

We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.

Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on the surface of Plasmodium falciparum merozoites.

A technique was developed for obtaining high yields of naturally released Plasmodium falciparum merozoites from synchronous cultures of parasitized erythrocytes. The cultured erythrocytes were treated with trypsin to prevent reinvasion (6), and the released merozoites that accumulated extracellularly were harvested by differential centrifugation. The total biosynthetically labeled proteins of schizonts and merozoites, and those immunoprecipitated by human immune serum were analyzed and compared. The surface antigens of free merozoites, labeled by lactoperoxidase-catalyzed iodination, were also described. A monoclonal antibody, specific for a 195,000 mol wt schizont protein, and processing fragments derived from it (3) were used in immunoprecipitation and Western transfer analyses to determine which of the processing fragments are associated with merozoites and which of them are located on the merozoite surface. It was found that processing of the 195,000 mol wt precursor down to an 83,000 mol wt fragment is complete in free merozoites, and that this fragment is expressed as one of the major surface antigens of P. falciparum merozoites.

The characterisation of an esterase derived from Babesia bovis and its use as a vaccine.

An esterase was isolated from a crude extract of Babesia bovis by affinity chromatography, using soy bean trypsin inhibitor as a ligand. In native form this enzyme had a molecular weight greater than 200 000, but on denaturing gels major bands were observed with molecular weights of 20 000, 10 000 and 7 000. Western transfer analysis revealed a major band with a molecular weight of 19 000-20 000. Both bovine and rabbit antisera avidly stained infected red cells, using indirect immunofluorescence. Weak parasite staining was also observed using this test. Two groups of five animals were vaccinated twice 4 weeks apart with esterase derived from 5 X 10(9) parasites as water-in-oil emulsions with Freund's complete adjuvant. Two control groups, each of five animals were also included. One group of vaccinates and a control group were challenged with virulent homologous B. bovis, whilst the other vaccinated and the remaining control group were challenged with virulent heterologous organisms. In the homologous groups two controls but no vaccinates died, whereas in the heterologous groups four animals in each group died. Significant differences in parasitaemia, temperature rise and total haemolytic complement were observed in the homologous vaccinated group compared to their controls but no differences were observed between heterologous groups.