An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena.

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Synthesis of an unusual alpha-zein protein is correlated with the phenotypic effects of the floury2 mutation in maize.

The soft, starchy endosperm of the maize (Zea mays L.) floury 2 mutant is associated with a reduction in zein mRNA and protein synthesis, unique protein body morphology, and enhanced levels of a 70 kDa protein, that has been shown to be the maize homolog of a chaperonin found in the endoplasmic reticulum. We found an unusual alpha-zein protein of 24 kDa to be consistently associated with the zein fraction from floury 2 mutants. Three additional alpha-zein proteins with molecular weights ranging from ca. 25 to 27 kDa are detected in the storage protein fraction of a high percentage of floury 2 kernels and a low percentage of normal kernels in a genetically segregating population. The four proteins in a genetically segregating population. The four proteins can be distinguished from one another by immunostaining on Western blots. Synthesis of the 24 kDa protein is regulated by Opaque2, since the 24 kDa protein is lacking in the storage protein fraction of opaque2/floury2 double mutants. The synthesis of an abnormal alpha-zein protein in floury2 could explain many features of the mutant, such as the abnormal protein body morphology, induction of the 70 kDa chaperonin, and hypostasis to opaque2 (o2). Although we cannot prove that the accumulation of this protein is responsible for the floury2 phenotype, we were able to detect a restriction fragment length polymorphism (RFLP) linked to the floury2 locus with a 22 kDa alpha-zein probe. We hypothesize that the unique characteristics of the floury2 mutant could be a response to the accumulation of a defective alpha-zein protein which impairs secretory protein synthesis.

Modification of maize-seed-protein quality.

The storage proteins of maize are a group of alcohol-soluble polypeptides called zeins. These proteins are synthesized in the developing endosperm, where they form protein bodies within the rough endoplasmic reticulum. Because they account for more than half of the total seed protein, zeins are the primary determinants of the amino acid composition of the seed. All of the zeins are devoid of lysine an essential amino acid for monogastric animals. We have modified the genes encoding zeins so that they encode proteins that contain lysine and tryptophan. Analysis of the synthesis and processing of these modified zein proteins indicates that the addition of lysine and tryptophan does not interfere with their association into protein bodies.

opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

A Drosophila phospholipase C gene that is expressed in the central nervous system.

A Drosophila phospholipase C (PLC) gene, designated as plc-21, was isolated by screening a genomic DNA library using a cDNA for a previously isolated Drosophila PLC gene, norpA, as probe under reduced stringency hybridization conditions. The gene maps to 21C on the left arm of the second chromosome. Two proteins of 1305 and 1312 amino acids, respectively, were deduced from two classes of cDNA which were isolated. The two putative plc-21 proteins are similar in sequence and overall structure to the beta-class of PLCs found in mammals and differ from each other only by 7 amino acid residues that are present near the C terminus of one of the proteins but not the other. Hybridization of plc-21 cDNA probes to blots of poly(A)+ RNA revealed that the gene encodes a 7.0-kilobase transcript that could be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that could be detected throughout development and in both heads and bodies of adults. In situ hybridization of cDNA sequences to tissue sections showed that the gene is expressed in the neuronal cell bodies of the optic lobe, central brain, and thoracic ganglia of adults and the brain of larvae. This tissue distribution of plc-21 transcripts is identical to the distribution of transcripts from a Drosophila Go alpha-subunit gene that we reported previously.

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase.

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.