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The molecular organization of the yeast transcriptional coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA) was analyzed by single-particle electron microscopy. Complete or partial deletion of the Sgf73 subunit disconnects the deubiquitination (DUB) module from SAGA and favors in our conditions the cleavage of the C-terminal ends of the Spt7 subunit and the loss of the Spt8 subunit. The structural comparison of the wild-type SAGA with two deletion mutants positioned the DUB module and enabled the fitting of the available atomic models. The localization of the DUB module close to Gcn5 defines a chromatin-binding interface within SAGA, which could be demonstrated by the binding of nucleosome core particles. The TATA-box binding protein (TBP)-interacting subunit Spt8 was found to be located close to the DUB but in a different domain than Spt3, also known to contact TBP. A flexible protein arm brings both subunits close enough to interact simultaneously with TBP.

Intracellular transport of karyophilic cargos comprises translocation to the nuclear envelope and subsequent nuclear import. Small cargos such as isolated proteins can reach the nuclear envelope by diffusion but movement of larger structures depends on active translocation, typically using microtubules. Centripetal transport ends at the perinuclear microtubule organizing centre called the spindle pole body (SPB) in yeast. Previously, we found by two hybrids that the karyophilic lentiviral-encoded integrase (IN) interacts with two yeast microtubule-associated proteins, Dyn2p (dynein light chain protein) and Stu2p, a centrosomal protein (de Soultrait et al., 2002). Thus, to investigate the hinge between cytoplasmic retrograde transport and nuclear import, we decided to analyse HIV-1 IN trafficking in yeast as the model, since each of these biological mechanisms is evolutionarily conserved in eukaryotic cells. Here, we found an accumulation of IN at the SPB in yeast via Stu2p colocalization. Disruption of the microtubule network by nocodazole or IN expression in a dynein 2-deficient yeast strain prevented IN accumulation in the nuclear periphery and additionally inhibited IN transport into the nucleus. By mutagenesis, we showed that trafficking of IN towards the SPB requires the C-terminus of the molecule. Taking our findings together, we proposed a model in which IN nuclear import seems to depend on an essential intermediate step in the SPB. We found that Dyn2p and Stu2p play an important role in driving IN toward MTOC and could optimize nuclear entry of the retroviral enzyme. Our results suggest a new hypothesis in keeping with the current HIV-1 intracellular trafficking model.

Chlorpromazine (CPZ) is a small permeable cationic amphiphilic molecule that inserts into membrane bilayers and binds to anionic lipids such as poly-phosphoinositides (PIs). Since PIs play important roles in many cellular processes, including signaling and membrane trafficking pathways, it has been proposed that CPZ affects cellular growth functions by preventing the recruitment of proteins with specific PI-binding domains. In this study, we have investigated the biological effects of CPZ in the yeast Saccharomyces cerevisiae. We screened a collection of approximately 4,800 gene knockout mutants, and found that mutants defective in membrane trafficking between the late-Golgi and endosomal compartments are highly sensitive to CPZ. Microscopy and transport analyses revealed that CPZ affects membrane structure of organelles, blocks membrane transport and activates the unfolded protein response (UPR). In addition, CPZ-treatment induces phosphorylation of the translation initiation factor (eIF2alpha), which reduces the general rate of protein synthesis and stimulates the production of Gcn4p, a major transcription factor that is activated in response to environmental stresses. Altogether, our results reveal that membrane stress within the cells rapidly activates an important gene expression program, which is followed by a general inhibition of protein synthesis. Remarkably, the increase of phosphorylated eIF2alpha and protein synthesis inhibition were also detected in CPZ-treated NIH-3T3 fibroblasts, suggesting the existence of a conserved mechanism of translational regulation that operates during a membrane stress.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the insertion of the viral genome into the host cell DNA, an essential reaction during the retroviral cycle. We described previously that expression of HIV-1 IN in some yeast strains may lead to the emergence of a lethal phenotype which was not observed when the catalytically crucial residues D, D, (35)E were mutated. The lethal effect in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonucleolytic activity carried by this enzyme. This non-sequence-specific endonuclease activity was further characterized. Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences, the presence of LTR regions stimulated significantly this activity. Genetic experiments were designed to show that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A has no significant effect. A close interaction was demonstrated between integrase activity and in vivo/in vitro recombination process, suggesting that retroviral integration and recombination mechanism are linked in the infected cell. Our results show that the yeast system is a powerful cellular model to study the non-sequence-specific endonucleolytic activity of IN. Its characterization is essential since this activity might represent a very important step in the retroviral infectious cycle and would provide further insights into the function of IN. Indeed, effectors of this activity should be sought as potential antiviral agents since stimulation of this enzymatic activity would induce the destruction of early synthesized proviral DNA.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.

Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects of ribosome biogenesis in eukaryotic cells. Scores of snoRNAs have been discovered in recent years from various eukaryotes, and the total number is predicted to be up to 200 different snoRNA species per individual organism. We have created a comprehensive database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured). The database consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual snoRNAs. The utilities section provides an introduction to the database. The master table lists all known S. cerevisiae snoRNAs and their major properties. Information in the individual tables includes: alternate names, size, family classification, genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene disruption phenotypes, functional properties and associated RNAs and proteins. All information is accompanied with appropriate literature references. The database is available on the World Wide Web (http://www.bio.umass. edu/biochem/rna-sequence/Yeast_snoRNA_Database/snoRNA_ DataBase.html), and should be useful for a wide range of snoRNA studies.

The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides. Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes. The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae. Intronic snoRNA production was determined to depend on Dbr1p. Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats. Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs. Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39. This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs. These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs. Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA.

Most small nucleolar RNAs (snoRNAs) fall into two families, known as the box C/D and box H/ACA snoRNAs. The various box elements are essential for snoRNA production and for snoRNA-directed modification of rRNA nucleotides. In the case of the box C/D snoRNAs, boxes C and D and an adjoining stem form a vital structure, known as the box C/D motif. Here, we examined expression of natural and artificial box C/D snoRNAs in yeast and mammalian cells, to assess the role of the box C/D motif in snoRNA localization. The results demonstrate that the motif is necessary and sufficient for nucleolar targeting, both in yeast and mammals. Moreover, in mammalian cells, RNA is targeted to coiled bodies as well. Thus, the box C/D motif is the first intranuclear RNA trafficking signal identified for an RNA family. Remarkably, it also couples snoRNA localization with synthesis and, most likely, function. The distribution of snoRNA precursors in mammalian cells suggests that this coupling is provided by a specific protein(s) which binds the box C/D motif during or rapidly after snoRNA transcription. The conserved nature of the box C/D motif indicates that its role in coupling production and localization of snoRNAs is of ancient evolutionary origin.

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C' and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5' portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic precursor rRNA. The U14 RNA from Saccharomyces cerevisiae is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function. This element, known as the Y-domain, is located in the U14 sequence between two universal sequences that base pair with 18S rRNA. Sequence data obtained for the U14 homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to S.cerevisiae. Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight invariant nucleotides. Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction. Several plant U14 RNAs were found to contain similar structures, though with an unrelated consensus sequence in the loop portion. The U14 gene from the most distantly related yeast, Schizosaccharomyces pombe, was found to be active in S.cerevisiae, showing that Y-domain function is conserved and that U14 function can be provided by variants in which the essential elements are embedded in dissimilar flanking sequences. This last result suggests that U14 function may be determined solely by the essential elements.

U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.

Yeast U14 small nuclear (sn) RNA is required for normal processing of rRNA. The sequence and folding properties of U14 were analyzed in the present study, with the aim of defining the structures of natural U14 subspecies and characterizing the folding properties of free U14 RNA. Natural U14 was determined to consist of four subspecies of 125-128 nucleotides, none containing a 5'-cap structure. Length heterogeneity occurs at both ends and is presumed to reflect post-transcriptional processing of U14 precursors. Results from nuclease and chemical probing revealed that U14 has surprisingly little secondary structure overall. Three essential sequence elements conserved among all U14 RNAs occur in regions that are largely single-stranded, i.e. box C, box D, and a 13-nucleotide segment complementary to 18 S rRNA; a non-essential 14-nucleotide sequence complementary to 18 S rRNA is also unpaired. Two non-conserved segments required for activity are part of a stably folded 32-base domain that is unique to yeast U14. Finally, a 5'-, 3'-stem shown earlier to be required for U14 accumulation appears to exist only in precursors to U14 and not in protein-free mature RNA. The implications of these results are discussed in terms of U14 synthesis and function.

Genes for three novel yeast snRNAs have been identified and tested for essentiality. Partial sequence information was developed for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database. The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively. Each RNA is derived from a single copy gene. The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein eIF-4E. The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two open reading frames with no known function. Genetic disruption analyses showed that none of the three snRNAs is required for growth. The new RNAs bring the number of non-spliceosomal snRNAs characterized thus far in S. cerevisiae to 14, of which only three are essential.

Despite their early discovery, research into the small RNAs associated with the eukaryotic nucleolus (snoRNAs) has lagged behind that of their cousins, the small nuclear RNAs which are known to function in mRNA splicing (spliceosomal snRNAs). Recent progress has now shown that the snoRNAs also occupy a vital niche in the RNA world, participating in the processing of ribosomal RNA. Like the spliceosomal snRNAs, the snoRNAs exist as ribonucleoprotein (RNP) particles which appear to assemble into a large multi-RNA RNP complex for pre-rRNA maturation.

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

The functional equivalency of yeast and mouse U14 RNAs was examined in Saccharomyces cerevisiae. The test RNAs included mouse U14 and several yeast-mouse bi- and tri-partite hybrid RNAs, all transcribed from yeast U14 gene signals. The ability of the heterologous RNAs to provide essential U14 function was assessed in a test strain containing a single glucose-repressible wild-type U14 gene. Mouse U14 was not functional in yeast. However, wild-type growth was supported by hybrid RNAs that included universal sequence elements from either source, two yeast-specific segments and a 5',3' terminal stem domain. The universal sequences include box C, box D and a sequence complementary to 18S rRNA, all shown previously to be required for function of yeast U14. Deletion and substitution mapping defined the yeast-specific elements and showed that a major portion of neighboring non-conserved RNA is dispensible. The results are discussed with a view to defining a minimal consensus U14 molecule.

The U14 RNA of Saccharomyces cerevisiae is a small nucleolar RNA (snoRNA) required for normal production of 18S rRNA. Depletion of U14 results in impaired processing of pre-rRNA, deficiency in 18S-containing intermediates and marked under-accumulation of mature 18S RNA. The present report describes results of functional mapping of U14, by a variety of mutagenic approaches. Special attention was directed at assessing the importance of sequence elements conserved between yeast and mouse U14 as well as other snoRNA species. Functionality was assessed in a test strain containing a galactose dependent U14 gene. The results show portions of three U14 conserved regions to be required for U14 accumulation or function. These regions include bases in: (i) the 5'-proximal box C region, (ii) the 3'-distal box D region, and (iii) a 13 base domain complementary to 18S rRNA. Point and multi-base substitution mutations in the snoRNA conserved box C and box D regions prevent U14 accumulation. Mutations in the essential 18S related domain do not effect U14 levels, but do disrupt synthesis of 18S RNA, indicating that this region is required for function. Taken together, the results suggest that the box C and box D regions influence U14 expression or stability and that U14 function might involve direct interaction with 18S RNA.

The structure of a Saccharomyces cerevisiae gene that encodes a small nuclear RNA (snRNA) of 189 nucleotides is described. This gene, designated SNR189, is located 400 base pairs upstream of the CRY1 gene on yeast chromosome III. Gene replacement analysis revealed the SNR189 gene to be dispensable for growth under a variety of culture conditions. The snR189 sequence lacks homology with other sequenced yeast or metazoan snRNAs.

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.

A genetic locus is described that specifies two Saccharomyces cerevisiae small nuclear RNAs (snRNAs). The genes specifying the two snRNAs are separated by only 67 base pairs and are transcribed in the same direction. The product RNAs contain 128 and 190 nucleotides and are designated snR128 and snR190, respectively. These RNAs resemble snRNAs of other eucaryotes in nuclear localization and possession of a 5' trimethylguanosine cap. Neither snRNA is related in sequence to previously described vertebrate or yeast snRNAs. Both RNAs exhibit properties consistent with nucleolar organization and hydrogen bonding to pre-rRNA species, suggesting possible roles in ribosome biogenesis. The snR128 species cosediments with deproteinized 27S pre-rRNA, whereas snR190 is associated with a 20S intermediate. Gene disruption in vitro followed by replacement of the chromosomal alleles reveals that SNR128 is essential, whereas SNR190 is not.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.