Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Cyst germination proteins of the potato pathogen Phytophthora infestans share homology with human mucins.

We have cloned genes of Phytophthora infestans, the causal agent of potato late blight, that are activated shortly before the onset of invasion of the host tissue. The three genes isolated appear to be arranged in a genomic cluster and belong to a small polymorphic gene family. A conspicuous feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAP TEE. Because of this structural motif, these novel P. infestans proteins were named Car (Cyst-germination-specific acidic repeat) proteins. One of the genes, car90, codes for 1,489 amino acids including 120 octapeptide tandem repeats. Car proteins are transiently expressed during germination of cysts and formation of appressoria and are localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides also occurs in a prominent class of proteins, the mucins, from mammals. The P. infestans Car proteins share 51% sequence homology with the tandem repeat region of a subfamily of human mucins. According to the physiological functions ascribed to mucins, we suggest that Car proteins may serve as a mucous cover protecting the germling from desiccation, physical damage, and adverse effects of the plant defense response and may assist in adhesion to the leaf surface.

An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus.

A cDNA clone of a defence response transcript was isolated from a library prepared from barley leaves expressing papilla resistance towards the powdery mildew fungus, Blumeria (syn. Erysiphe) graminis f.sp. hordei (Bgh). The 904 bp sequence encodes a 229 amino acid polypeptide with a putative signal peptide of 23 amino acids. After cleavage, the protein has a mass of 22.3 kDa and exhibits up to 60% amino acid identity to certain dicot proteins, and 46% amino acid identity to barley oxalate oxidase; therefore we designated it HvOxOLP (for Hordeum vulgare oxalate oxidase-like protein). Single-base substitutions among several cDNA and RACE clones demonstrate a gene of many copies. Both the transcript and protein accumulate from 3 h after inoculation with Bgh. The transcript level peaks at 18-24 h and subsequently decreases, whereas the protein level is stable from 24 h after inoculation. The accumulation patterns are independent of the outcome of the barley/powdery mildew interaction, unlike that of PR proteins, for example. The transcript accumulates specifically in the inoculated epidermal tissue. This temporal and spatial expression pattern suggests a very close relationship to papilla formation. Immunoblot analyses have facilitated a demonstration that HvOxOLP, like oxalate oxidase, is a water-soluble 100 kDa oligomeric protein. The oligomer is heat-stable and SDS-tolerant, and it can be denatured into a 25 kDa monomer. Attempts to demonstrate oxalate oxidase activity for this protein have failed. However, the relationships to oxalate oxidase suggests that HvOxOLP may be involved in H2O2 generation necessary for, for example, cross-linking of cell wall components during formation of papillae.

Primary structure and expression of acidic (class II) chitinase in potato.

Infection of potato (Solanum tuberosum) leaves by the late blight fungus Phytophthora infestans or treatment with fungal elicitor leads to a strong increase in chitinase activity. We isolated cDNAs encoding acidic (class II) chitinases (ChtA) from potato leaves and determined their structures and expression patterns in healthy and stressed plants. From the total number of cDNAs and the complexity of genomic DNA blots we conclude that acidic chitinase in potato is encoded by a gene family which is considerably smaller than that encoding basic (class I) chitinase (ChtB). The deduced amino acid sequences show 78 to 96% identity to class II chitinases from related plant species tomato, tobacco) whereas the identity to basic chitinases of potato is in the range of 60%. RNA blot analysis revealed that both acidic and basic chitinases were strongly induced by infection or elicitor treatment and that the induction occurred both locally at the site of infection and systemically in upper uninfected leaves. In contrast, a differential response to other types of stress was observed. Acidic chitinase mRNA was strongly induced by salicylic acid, whereas basic chitinase mRNA was induced by ethylene or wounding. In healthy, untreated plants, acidic chitinase mRNA accumulated also in an organ-, cell-type- and development-specific manner as revealed by RNA blot analysis and in situ RNA hybridization. Relatively high transcript levels were observed in old leaves and young internodes as well as in vascular tissue and cells constituting the stomatal complex in leaves and petioles. Lower, but appreciable mRNA levels were also detectable in roots and various flower organs, particularly in sepals and stamens. The possible implications of these findings in pathogen defense, development and growth processes are discussed.

Disease resistance results from foreign phytoalexin expression in a novel plant.

Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants.

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3-ß-glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and 'pathogenesis-related' proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.

Synthesis of rat-liver lactate dehydrogenase and characterization of its mRNA.

Rat liver lactate dehydrogenase (LDH) has been synthesized in hepatocytes and in a cell-free translation system. The subunit synthesized in both systems displayed the same electrophoretic mobility upon sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. Sequence analysis of the subunits translated in vitro and synthesized in vivo indicated N-acetyl-alanyl-alanine for both N termini. Thus, the newly synthesized subunit does not exhibit an amino-terminal extension. The mRNA for the lactate dehydrogenase subunit was exclusively found in free polysomes. A size of 2120 +/- 240 nucleotides was estimated for the mRNA. Only about 50% of these nucleotides are needed to code for the polypeptide chain of the enzyme.

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes.

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.

Proteolytic precursor processing in the biosynthesis of mitochondria.

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.

Characterization of 20-S and 40-S non-polysomal cytoplasmic messenger ribonucleoprotein particles from rat liver.

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system.

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.