Use of epitope tags for routine analysis of transgene expression.

Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.

Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast.

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Leaky transcription of variant surface glycoprotein gene expression sites in bloodstream african trypanosomes.

Trypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20-40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45-60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and the vsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versus bloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere.

FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells.

The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.

Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.

In complex with the prolyl isomerase FKBP12, the natural product rapamycin blocks signal transduction in organisms as diverse as yeast and man. The yeast targets of FKBP12-rapamycin, TOR1 and TOR2, are large proteins with homology to lipid and protein kinases. A mammalian FKBP12-rapamycin binding protein, RAFT1, shares 39% and 43% identity with TOR1 and TOR2 proteins, respectively but has not been linked to rapamycin action in vivo. We find that when expressed in yeast, neither wild-type nor mutant RAFT1 complemented tor mutations or conferred rapamycin resistance. In contrast, TOR1-RAFT1 and TOR1-RAFT1 hybrid proteins containing the carboxy-terminal RAFT1 kinase domain complemented tor2 and tor1 mutant strains, respectively. Moreover, TOR2-RAFT1 and TOR1-RAFT1 hybrid proteins mutated at the position corresponding to rapamycin-resistant TOR mutants (S20351) conferred rapamycin resistance. Like the TOR2 protein, the TOR2-RAFT1 proteins were stably expressed, localized to the vacuolar surface, and associated with a phosphatidylinositol-4 kinase activity. These findings directly link the mammalian TOR homolog RAFT1 to rapamycin action in vivo and indicate that the TOR/RAFT1 kinase domain has been functionally conserved from yeast to man.

cDNA expressed sequence tags of Trypanosoma brucei rhodesiense provide new insights into the biology of the parasite.

A total of 518 expressed sequence tags (ESTs) have been generated from clones randomly selected from a cDNA library and a spliced leader sub-library of a Trypanosoma brucei rhodesiense bloodstream clone. 205 (39%) of the clones were identified based on matches to 113 unique genes in the public databases. Of these, 71 cDNAs display significant similarities to genes in unrelated organisms encoding metabolic enzymes, signal transduction proteins, transcription factors, ribosomal proteins, histones, a proliferation-associated protein and thimet oligopeptidase, among others. 313 of the cDNAs are not related to any other sequences in the databases. These cDNA ESTs provide new avenues of research for exploring both the novel trypanosome-specific genes and the genome organization of this parasite, as well as a resource for identifying trypanosome homologs to genes expressed in other organisms.

The putative promoter for a metacyclic VSG gene in African trypanosomes.

During their metacyclic developmental stage, African trypanosomes are coated with one of 12-15 variant surface glycoproteins (VSGs) that define different metacyclic variant antigen types (MVATs). The MVAT VSG genes are located near telomeres of large chromosomes and are expressed without rearrangement in the metacyclic stage. We have cloned and examined the telomere-linked MVAT5 VSG gene and its upstream expression site associated gene (ESAG I) which are separated by 4.5 kb. Within this 4.5-kb intergenic region is an 87-bp sequence that serves as a strong promoter for a luciferase reporter gene in transient transfection assays. This 87-bp sequence is similar, but not identical, to the promoter for another MVAT VSG gene. UV irradiation experiments were used to detect RNA synthesis from this MVAT5 promoter in bloodstream trypanosomes expressing an unrelated VSG. We propose that this sequence is a specific promoter for the MVAT5 VSG mRNA that occurs in about 10% of the trypanosome population during the metacyclic stage of the parasites' life cycle.

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

Many transcribed regions of the Onchocerca volvulus genome contain the spliced leader sequence of Caenorhabditis elegans.

Genomic DNAs of the related parasitic nematodes Onchocerca volvulus and Dirofilariae immitis, and a cDNA library of O. volvulus, were examined for the presence of the 22-nucleotide spliced leader (SL) found at the 5' ends of 10 to 15% of the mRNAs in the free-living nematode Caenorhabditis elegans. As in C. elegans, genes for the SL RNA are linked to the repetitive 5S rRNA genes of O. volvulus and D. immitis, but unlike C. elegans, they are in the same orientation as the 5S rRNA genes within the repeat unit. In O. volvulus the SL sequence is also encoded at more than 30 additional genomic locations and occurs at interior sites within many transcripts. Sequence determinations of four different cDNAs of O. volvulus, each containing an internal copy of the SL within a conserved 25mer, and one corresponding genomic DNA clone indicate that this sequence is not trans spliced onto these RNAs, but is encoded within the genes. The RNAs of two of these cDNAs appear to be developmentally regulated, since they occur in adult O. volvulus but were not detected in the infective L3 stage larvae. In contrast, actin mRNAs are present at all developmental stages, and at least one actin mRNA species contains a trans-spliced 5' SL. The internal locations of the SL in various transcripts and its perfect sequence conservation among parasitic and free-living nematodes argues that it serves specific, and perhaps multiple, functions for these organisms.