From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas

Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.     

Initiation of translation at an upstream non-AUG codon accounting for N-terminally extended minor forms of recombinant proteins expressed in insect cells

When the 34 kDa kinase domain of human spleen tyrosine kinase (Syk-KD) was expressed as a C-terminally His-tagged protein in baculovirus-infected Sf-21 insect cells, the purified protein included two forms that migrated slightly differently in SDS-polyacrylamide gel electrophoresis. Intact mass analysis and LC-MS/MS peptide mapping showed that the major and faster-migrating product had the intended amino-acid sequence and 0-6 phosphorylations. This material accounted for about 95% of the purified protein. The minor product was Syk-KD with a 26 amino-acid N-terminal extension. The result suggested the existence of an upstream alternative site for the initiation of translation, and this proved to be an ACG codon derived from the pBacPAK9 vector used to express Syk-KD. The ACG codon was preceded and followed by Kozak-type sequence elements (a purine in the -3 position and a G in the +4 position) that would have enhanced the viability of initiation at ACG. The initiating amino-acid residue was Met for both minor and major products, and both forms of the protein were α-N-acetylated. For the minor product, protein intact mass analysis and peptide mapping both gave results in agreement with the sequence predicted from the DNA. A similar result with the same underlying cause was obtained with insect cell expression of full-length Syk. It appears that similar results are possible whenever this vector is used.     

Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.     

Ribosomal initiation from an ACG codon in the Sendai virus P/C mRNA.

The Sendai virus P/C mRNA expresses the P and C proteins from alternate reading frames. The C reading frame of this mRNA, however, is responsible for three proteins, C', C and Y, none of which appear to be precursors to each other in vivo. Using site-directed and deletion mutagenesis of the P/C gene cloned in SP6 and in vitro translation of the mRNAs, we show that the 5' most proximal initiation codon of the mRNA is an ACG at position 81, responsible for C' synthesis. The succeeding initiation codons, all ATGs, are responsible for the P protein (position 104), the C protein (position 114) and the Y protein(s) (either positions 183 or 201). Examination of the relative molar amounts of the C', P and C proteins found in vivo suggests that an ACG in an otherwise favorable context is almost as efficient for ribosome initiation as an ATG in a less favored context, but only 10-20% as efficient as an ATG in a more favored context. The judicious choice of increasingly more favorable initiation codons in the P/C gene allows multiple proteins to be made from a single mRNA.     

Redundancy of non-AUG initiators. A clever mechanism to enhance the efficiency of translation in yeast

It was recently shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, uses two successive ACG triplets as the translation initiators for its mitochondrial form. Evidence presented here argues that the second ACG triplet not only acts as a remedial initiation site for scanning ribosomes that skip the first ACG, but also enhances the activity of the preceding initiator by providing a preferable "A" at its relative position +4. Therefore, ALA1 constructs with redundant ACG initiators exhibit stronger complementing activity and express a higher level of protein than do those with a single ACG initiator. A similar scenario is seen when a single or redundant ACG triplets are placed in the positions of the first and second AUG initiators of VAS1, which serve as the start sites of the mitochondrial and cytoplasmic forms of valyl-tRNA synthetase, respectively. Cumulatively, the results suggest that this feature of redundancy of non-AUG initiators in a single mRNA per se may represent a novel paradigm for improving the efficiency of a poor or otherwise nonproductive initiation event.     

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

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.     

Steroid 5alpha-reductase 1 polymorphisms and testosterone/dihydrotestosterone ratio in male patients with hypospadias

BACKGROUND/AIM: Defects in the steroid 5alpha-reductase type 2 (SRD5A2) activity cause decreased formation of dihydrotestosterone (DHT) from testosterone (T), resulting in defective masculinization of external genitalia; the T/DHT ratio is increased. We investigated 10 patients with elevated T/DHT ratios in whom mutations in the SRD5A2 and AR genes had been excluded to find out whether structural alterations of the SRD5A1 gene could contribute to their genital malformations. METHODS: Single-strand conformation polymorphism analysis and direct sequencing were used to detect variations in the SRD5A1 gene of the patients and of 49 adult fertile men who served as controls. RESULTS: The sequence analysis of exon 3 of the SRD5A1 gene indicated an adenine-to-guanine change (ACA vs. ACG), both triplets encoding the amino acid residue threonine. The ACG sequence was detected in 57% of all subjects and was equally distributed in patients and controls. The T/DHT ratio was significantly higher in controls with the ACG variant as compared with those having the ACA variant. However, no particular sequence aberration was found in the SRD5A1 genes of either group. CONCLUSION: Mutant SRD5A1 isoenzyme does not seem to play a crucial role in the development of hypospadias.     

Pharmacological profiling of the Dictyostelium adenylate cyclases ACA, ACB and ACG

Intracellular and secreted cAMPs play crucial roles in controlling cell movement and gene regulation throughout development of the social amoeba Dictyostelium discoideum. cAMP is produced by three structurally distinct ACs (adenylate cyclases), ACA, ACG and ACB, which have distinctive but overlapping patterns of expression and, as concluded from gene disruption studies, seemingly overlapping functions. In addition to gene disruption, acute pharmacological abrogation of protein activity can be a powerful tool to identify the protein's role in the biology of the organism. We analysed the effects of a range of compounds on the activity of ACA, ACB and ACG to identify enzyme-specific modulators. Caffeine, which was previously used to specifically block ACA function, also inhibited cAMP accumulation by ACB and ACG. IPA (2',3'-O-isopropylidene adenosine) specifically inhibits ACA when measured in intact cells, without affecting ACB or ACG. All three enzymes are inhibited by the P-site inhibitor DDA (2',5'-dideoxyadenosine) when assayed in cell lysates, but not in intact cells. Tyrphostin A25 [alpha-cyano-(3,4,5-trihydroxy)cinnamonitrile] and SQ22536 [9-(tetrahydro-2'-furyl)adenine] proved to be effective and specific inhibitors for ACG and ACA respectively. Both compounds acted directly on enzyme activity assayed in cell lysates, but only SQ22536 was also a specific inhibitor when added to intact cells.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development.

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.     

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.     

Inhibition of the reproduction of a herpes simplex I virus carrying mutations in the thymidine kinase and DNA polymerase genes

Sensitivity of Herpes Simplex Virus type I (HSV-I) mutants carrying genetic defect in the DNA polymerase and thymidine kinase genes to the action of some drugs was studied. TK- mutant of HSV-I was resistant to Ara-T and ACG and sensitive to PAA, Ara-A as well as to ribavirin and ADEA. PAAr mutant of HSV-I was resistant to PAA, Ara-A, ACG and sensitive to Ara-T, ribavirin and ADEA. A double mutant of HSV-I-TK-, PAAr was resistant to all drugs, except for ribavirin and ADEA. To inhibit reproduction of HSV with genetic defect, it is important using drugs of independent mode of action on the function of defective viral gene.