RNA interference in embryonic stem cells and the prospects for future therapies.

In 1998, two distinct and exciting scientific fields emerged which have profoundly shaped the current direction of biomedical research. The discovery of RNA interference (RNAi) and the derivation of human embryonic stem (ES) cells have yielded exciting new possibilities for researchers and clinicians alike. While fundamentally different, aspects from these two fields may be combined to yield extraordinary scientific and medical benefits. Here, we review the prospects of combining RNAi and ES cell manipulation for both basic research and future therapies, as well as current limitations and obstacles that need to be overcome.

Ultrastructural changes to the midgut of the black field cricket (Teleogryllus commodus) following ingestion of potato protease inhibitor II.

Ultrastructural changes to the midgut epithelium of nymphs of the black field cricket (Teleogryllus commodus) after ingestion of potato protease inhibitor II (PPI-II) (0.6% (w/v) in artificial diet) were determined by light and electron microscopy. Crickets fed diet containing PPI-II grew more slowly than those fed control diet and changes observed to the PPI-II-fed nymphs included reduction of midgut wall depth, vacuolisation of the epithelial cells, swelling of the microvilli, cellular protrusions into the midgut and eventual rupture of individual or small groups of epithelial cells. These changes were first seen 2 days after PPI-II ingestion. Complete disintegration of the midgut to the basement membrane was not seen during the 27-day observation period and repair and regeneration of pockets of epithelial cells was observed. Immunocytochemistry revealed that PPI-II was localised within the ectoperitrophic matrix space of the gut. The location of the peritrophic matrix was determined by labelling with wheat germ agglutinin (WGA), but no rupture of this structure was observed in PPI-II-fed nymphs.

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

Processing of polycistronic guide RNAs is associated with RNA editing complexes in Trypanosoma brucei.

In kinetoplastid mitochondrial mRNA editing, post-transcriptional insertion or deletion of uridines is templated by guide RNAs (gRNAs). Pre-mRNAs are encoded by maxicircles, while gRNAs are encoded by both maxicircles and minicircles. We have investigated minicircle transcription and the processing of gRNAs in Trypanosoma brucei. We find that minicircles are transcribed polycistronically and that transcripts are accurately processed by an approximately 19S complex. This gRNA processing activity co-purifies with RNA editing complexes, and both remain associated in 19S complexes. Furthermore, we show that RNA editing complexes associate preferentially with a polycistronic gRNA over non-processed RNAs. We propose that the approximately 19S complexes initially described as RNA editing complex I are gRNA processing complexes that cleave polycistronic gRNA transcripts into monocistrons.

A novel alliinase from onion roots. Biochemical characterization and cDNA cloning.

We have purified a novel alliinase (EC 4.4.1.4) from roots of onion (Allium cepa L.). Two isoforms with alliinase activity (I and II) were separated by concanavalin A-Sepharose and had molecular masses of 52.7 (I) and 50.5 (II) kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 51 (I) and 57.5 (II) kD by gel filtration fast-protein liquid chromatography. Isoform I had an isoelectric point of 9.3, while isoform II had isoelectric points of 7.6, 7.9, 8.1, and 8.3. The isoforms differed in their glycosylation. Both contained xylose/fucose containing complex-type N-linked glycans, and isoform II also contained terminal mannose structures. Both isoforms had activity with S-alk(en)yl-L-cysteine sulfoxides. Unlike other allium alliinases, A. cepa root isoforms had cystine lyase activity. We cloned a gene from A. cepa root cDNA and show that it codes for A. cepa root alliinase protein. Homology to other reported allium alliinase genes is 50%. The gene coded for a protein of mass 51.2 kD, with two regions of deduced amino acid sequence identical to a 25- and a 40-amino acid region, as determined experimentally. The A. cepa root alliinase cDNA was expressed mainly in A. cepa roots. The structure and function of the alliinase gene family is discussed.

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA.

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.

Expression of 1-aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white clover.

We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3'-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to 1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development.

A cysteine proteinase inhibitor purified from apple fruit.

A cysteine proteinase inhibitor has been purified from immature fruit of Malus domestica (var. Royal Gala). The M(r) of this apple cystatin is estimated to be 10,700 by MALDI-TOF mass spectrometry, 11 300 by SDS-PAGE and 11,000 by gel filtration. It is a relatively strong inhibitor of papain with a Ki value of 0.21 nM and also inhibits ficin and bromelain but not cathepsin B. An amino acid sequence was obtained from a peptide produced by trypsin digestion of the inhibitor. Comparison with other plant sequences shows a high degree of homology with other phytocystatins. As the single cysteine proteinase inhibitor detectable in immature apple fruit (5-8 mm diameter), levels of 83.3 pmol/g FW were determined. In larger fruit (up to 16 mm diameter) significantly less inhibitor was present (6.9 pmol/g FW). Given these low levels, it is postulated that this inhibitor has an endogenous role in apple fruit development rather than one of protection against pest or microbial attack.

Biochemical methods for analysis of kinetoplastid RNA editing.

RNA editing is a posttranscriptional process involving mRNAs [reviewed by K. Stuart et al. (1997) Microbiol. Mol. Biol. Rev. 61, 105-120; G. J. Arts and R. Benne (1996) Biochim. Biophys. Acta 1307, 39-54; and S. L. Hajduk and R. S. Sabatini (1996) in Molecular Biology of Parasitic Protozoa (Smith, D. S., and Parsons, M., Eds.), pp. 134-158, Oxford Univ. Press, Oxford] and tRNAs [K. M. Lonergan and M. Gray (1993) Science 259, 812-816] that has now been described in an increasing number of eukaryotic organisms. In this process sequences differ from their gene sequences by the addition, removal, or conversion of specific ribonucleotides. RNA editing was first described within the mitochondrion of kinetoplastid protozoa. Several of the mitochondrial mRNAs in these flagellates have uridine residues inserted and deleted at specific sites. In some cases, more than 50% of the mRNA is created by RNA editing. In this article, we describe some of the biochemical methods used in analyzing the process of RNA editing in kinetoplastid mitochondria.

Glycans of higher plant peroxidases: recent observations and future speculations.

Plant peroxidases are composed of a peptide and associated heme, calcium and glycans. The 3D structure of the major cationic peanut peroxidase has revealed the sites of the heme and calcium. But the diffraction of the glycans was not sufficient to show their structure. This review presents research that has been executed to obtain putative glycans and their binding sites, and to gain an indirect insight into these glycans. It also offers approaches that will be used to determine the function of the glycans on the peanut peroxidase. Some comparisons are made with other plant glycoproteins including peroxidases from plants other than peanut.

Wounding induces a series of closely related trypsin/chymotrypsin inhibitory peptides in leaves of tobacco.

Wounding of tobacco (Nicotiana tabacum) leaves induced the expression of acid-stable trypsin/chymotrypsin inhibitory activity. Analysis by gel filtration determined that the inhibitory activity was contained within a fraction with a native M(r) of ca 5-7 x 10(3). Using ion-exchange column chromatography, this was resolved further into two major fractions, each of which inhibited both trypsin and chymotrypsin. Reverse-phase HPLC identified a total of six peptides from both fractions and each was purified to homogeneity. Four of these peptides inhibited both trypsin and chymotrypsin, a fifth inhibited trypsin only, while the sixth inhibited chymotrypsin almost exclusively. Sequencing of the N-terminal revealed that each peptide had an identical amino acid sequence and that these proteins are similar to a series of trypsin/chymotrypsin inhibitory peptides that are expressed predominantly in the stigmas of Nicotiana alata flowers.

Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.

Identification of a monoclonal antibody to abscission tissue that recognises xylose/fucose-containing N-linked oligosaccharides from higher plants.

Monoclonal antibodies raised against extracts of the rachis abscission zone of Sambucus nigra L. were selected for high reactivity towards abscission-zone proteins. One antibody (YZ1/2.23) has been shown to cross-react, by both indirect and competition enzyme-linked immunosorbent assay and by Western blotting, with a number of plant enzymes including horseradish peroxidase, rice α-glucosidase, almond ß-glucosidase and the lectins from Phaseolus vulgaris and Erythrina cristagalli.The major N-linked oligosaccharide isolated from horseradish peroxidase has the sequence Manα 3(Manα6)(Xylß2)Manß4GlcNAcß4(Fucα3) GlcNAc. This oligosaccharide was found to be a potent inhibitor of the binding of YZ1/2.23 to the intact glycoprotein. The common determinant is therefore contained within this structure.