Mechanism of suppression in Drosophila: a change in tyrosine transfer RNA

The mechanism of suppression of the vermilion locus in Drosophila melanogaster is examined. The suppressor locus, su(s)², is shown to control directly the amount of a specific tyrosine transfer RNA which occurs in the adult fly. Wild-type flies have three chromatographic forms of tyrosine tRNA but flies that are homozygous for the suppressor gene su(s)² contain little or none of the second chromatographic form. The isoacceptor patterns of tRNA for leucine, phenylalanine and serine are identical in the suppressor mutant and wild-type fly. Genetic data show that the phenotypic expression of su(s)² and the altered chromatographic pattern of tyrosine tRNA are recessive and that both map at the same position on the left tip of the X chromosome. Furthermore, another suppressor of vermilion was induced by ethyl methane sulfate, su(s)^e1, that is at the same locus as su(s)² and that produces the same change in tyrosine tRNA as su(s)².     

Role of modifications in tyrosine transfer RNA. II. Ribothymidylate-deficient tRNA

The ability of suppressor tyrosine transfer RNA to participate in protein synthesis was studied in an E. coli mutant unable to synthesize ribothymidylate. Sequence analysis has shown that uracil replaces thymine in the common TψCG sequence. This transfer RNA is able to support protein synthesis at a rate equivalent to that observed with normally methylated transfer RNA.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Mechanism of suppression in Drosophila. VII. Correlation between disappearance of an isoacceptor of tyrosine tRNA and activation of the vermilion locus.

The possibility that tyrosine tRNA modifies the catalytic activity of tryptophan oxygenase that is produced by the vermilion mutant (v) in Drosophila melanogaster is reconsidered. Dietary conditions can modify the ratio of the two major isoacceptors of tyrosine tRNA: one condition allows 85--90% to exist as the second isoacceptor, and another condition allows less than 5% to exist in this form. The function lacking in the vermilion mutant is partially restored when the second isoacceptor of tRNATyr is reduced to low levels (less than 40%), but the function is greatly reduced when this isoacceptor is present as 50% or more of the total. These data support the hypothesis that tRNATyr may be associated with and regulate tryptophan oxygenase. The corresponding isoacceptor of tRNATyr found in a suppressor mutant, su(s)2, should not have any effect on the function of the vermilion gene, and, indeed, it did not. The tRNAs for tyrosine, aspartic acid, and histidine all have one isoacceptor that contains nucleoside Q and all undergo parallel changes in flies raised on the various diets. It appears that these dietary changes affect the ability to synthesize or modify Q or to remove or insert it into tRNA.     

A doubtful relationship between tyrosine tRNA and suppression of the vermilion mutant in Drosophila.

The conditions under which Drosophila melanogaster are grown markedly influence the amount of the hypermodified nucleoside Q found in certain tRNAs. This effect on Q biosynthesis was found in both the wild-type and the suppressor of sable [su(s)2] mutant. Suppressed vermilion flies [su(s)2v; bw] with 78% of the tyrosine tRNA in the Q-lacking (gamma) form had brown eyes indistinguishible from su(s)2v; bw flies with only 6% of the tyrosine tRNA in the gamma form. The proposal that this tRNA is a specific inhibitor or tryptophan pyrrolase in vermilion flies, and that its absence in su(s)2 flies is the mechanism of suppression is not consistent with these results. In addition, the su(s)2 locus does not seem to be primarily responsible for controlling Q biosynthesis as previously suggested.     

RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies

Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHVΔB2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHVΔB2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHVΔB2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHVΔB2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHVΔB2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.     

Total synthesis of a tyrosine suppressor transfer RNA gene. XIV. Chemical synthesis of oligonucleotide segments corresponding to the terminal regions.

Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described. These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G. (1976) J. Biol. Chem. 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end. The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G. (1976) J. Biol. Chem. 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end. The syntheses used protected mono- and oligonucleotides and stepwise condensation methods. A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures.     

Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in DROSOPHILA: Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.     

Alteration of tyrosine isoaccepting transfer ribonucleic acid species in wild-type and asporogenous strains of Bacillus subtilis.

The relative amounts of two isoacceping species of tyrosine transfer ribonucleic acid, tRNATyrI and tRNATyrII, determined from reversed phase 5 profiles of tyrosyl-tRNA, prepared from Bacillus subtilis strain W168, were growth phase and medium dependent. The growth phase-dependent alterations in the relative amounts of tRNATyr species were also demonstrated in 11 asporogenous strains of B. subtilis. The proportion of tRNA-Tyr species and the extent of the alteration in their relative amounts during the transition from the exponential to the stationary phase of growth of these strains was not directly correlated with the formation of spores by strain W168 grown in various media or the stage at which the asporogenous strains are blocked in the process of sporulation.