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Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.

A simple and rapid solid-phase RNA sequencing method has been developed based on Peattie's direct chemical method. 3'-Terminally labeled RNA was immobilized on DEAE-cellulose sheets and followed by specific modification with dimethyl sulfate, diethylpyrocarbonate, hydroxylamine (at pH 10 for the uridine and pH 5.5 for the cytidine reaction), and cleavage reaction with aniline. RNA fragments were washed from the DEAE-cellulose sheets using salt solutions, precipitated with ethanol, and separated by 15% polyacrylamide gel electrophoresis. Due to the complete removal of the impurities normally present in the solution method, the higher resolution of the sequencing bands and lower background on the autoradiograph make this solid-phase technique more efficient. This solid-phase technique is much faster and more convenient than the original method.

Riboflavin deficiency leads to depressed mitochondrial fatty acid oxidation rates but increased activity of carnitine palmitoyltransferase (CPT). Starvation leads to increased CPT activity in ad libitum-fed, riboflavin-supplemented rats. The present studies examined the mechanism of the increase in CPT activity in riboflavin deficiency and whether it was additive to that seen in starvation. Rats were divided into three groups initially: riboflavin-sufficient, ad libitum-fed; riboflavin-deficient, ad libitum-fed; and pair-fed. These groups were subdivided after 5 wk into fed and 24- and 48-h starved groups. When riboflavin-deficient rats were starved for 24 or 48 h, there was only a 30-40% increase in hepatic CPT activity, in contrast to the ad libitum-fed, riboflavin-supplemented rats, in which activity increased twofold. CPT activity of pair-fed rats was similar to that of controls in the fed state and did not increase significantly with starvation. CPT translation, mRNA levels and transcription rates correlated with CPT activity, as did immunoreactive CPT. Concurrently, hepatic ketone production and plasma beta-hydroxybutyrate concentration increased during starvation in the control and pair-fed but not in the riboflavin-deficient rats. The results indicate that increased CPT activity in riboflavin deficiency and starvation results at least in part from increased synthesis. Furthermore, the data support previous work suggesting that the block in fatty acid oxidation occurs in the beta-oxidation pathway at the level of acyl-CoA dehydrogenases.

The nucleotide sequence of 5.8S rRNA from the Chinese silkworm Philosamia cynthia ricini has been determined by gel sequencing and mobility shift methods. The complete primary structure is (sequence in text). This is one of the largest known 5.8S rRNAs. As compared to Bombyx 5.8S rRNA, it is two nucleotides longer; two nucleotides near the 5'end and two nucleotides near the 3'end are different, and psi 61 of the Bombyx RNA sequence is an unmodified U in Philosamia RNA. The secondary structure of Philosamia 5.8S rRNA may differ from the Bombyx RNA structure by three additional base pairs at the 5'/3' ends.

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.

An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the "shifty" tRNAs proposed by Jacks et al. (Cell 55, 447-458, 1988) to carry out frameshifting may be hypomodified isoacceptors.