N6-Methyladenosine (m6A) residues, which are found internally in viral and cellular mRNA populations at the sequences Apm6ApC and Gpm6ApC, have been proposed to play a role in mRNA processing and transport. We have developed a sensitive approach to analyze the level and location of m6A in specific purified cellular mRNAsin an attempt to correlate m6A location with function. Polyadenylylated mRNA is hybridized to cDNA clones representing the full size mRNA under study or fragments of it, and the protected RNA is digested andlabeled with polynucleotide kinase in vitro. After enrichment for m6A with anti-m6A antibody, the [32P]-pm6A is separated on TLC plates, and compared with the total amount of radiolabeled nucleotides. Using this combination of in vitro RNA labeling and antibody selection, we were able to detect m'A in purified stable mRNAs that cannot be readily labeled in cells with greater sensitivity than was possible by previous techniques. We applied this technique to bovine prolactin mRNA and showed that this mRNA contains m6A. Moreoverj all of the m'A residues in this message are found within the 3' two-thirds of the molecule and are highly concentrated (61%) within a sequence of 108 nucleotides at the 3' noncoding region of the message. The nonrandom distribution of m6A in a specific cellular mRNA, as demonstrated for bovine prolactin, will have to be taken into account when designing a model for m6A function.The most prevalent internal methylated nucleoside in eukaryotic mRNA is N6-methyladenosine (m6A). This modified nucleoside is found in RNAs of higher eukaryotic organisms (1-6), plants (7-9), and viruses (3,(10)(11)(12), and occurs at two specific sequences: . The high degree of sequence specificity, together with the fact that m6A is conserved during processing of the heterogeneous nuclear RNA in the nucleus (11,16,18,19), argues for an important biological function. In viral RNA, internal m6A is distributed in a nonrandom fashion. In Rous sarcoma virus, these methylated nucleosides are concentrated in a 3500-nucleotide region of the 10,000-nucleotide RNA genome (12). The highest level of internal m6A residues in simian virus 40 (SV40) nuclear RNA is found in RNA transcripts from two specific regions of the viral genome (20).The m6A-containing oligonucleotides of late SV40 16S and 19S mRNAs were further localized op the viral genome (15). The specificity of m6A location in particular regions of viral RNA implies that, apart from sequence specificity, other features of the RNA may influence the location of internal methylation and is suggestive of a biological role for m6A.Further indication of the role of m6A was obtained by inhibition studies. Inhibition of internal methylation of mRNA by cycloleucine in B77 avian sarcoma virus results in accumulation of genome-length RNA and decrease in the levels of spliced viral mRNA (21). Cycloleucine treatment of SV40-infected BSC-1 cells results in reduction of the amount of m6A residues as well as inhibition of SV40 mRNA production, but almost no ...

A gene coding for bovine growth hormone was isolated from a bovine genomic library. The nucleotide sequence of the coding regions of the gene was found to be identical with that of a nearly full-length growth hormone cDNA clone. The gene sequence is approximately 1800 bp in length and contains four intervening sequences. The second intervening sequence of 227 nucleotides does not contain a repetitive element similar to that observed in the rat growth hormone gene. A comparison of the 5' and 3' flanking and untranslated regions of the bovine, human and rat growth hormone genes revealed many areas of highly conserved sequence. Especially noteworthy was the observation that all three genes had a 38 nucleotide homologous sequence within their 5' flanking regions located about 100 bp upstream from their transcription initiation sites.

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A tentative structure and composition of a surfactant, BL-86, produced by Bacillus licheniformis 86 is described. The surfactant is a mixture of lipopeptides with the major components ranging in size from 979 to 1091 Da and varying in increments of 14 Da. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the surfactant. There are 7 amino acids per molecule. The peptide portion is composed of the following amino acids: glutamic acid or glutamine (glx), aspartic acid or asparagine (asx), valine, leucine, and isoleucine at a ratio of 1.0:1.0:1.4:3.0:0.6, respectively. The leucine is present as both the D and L isomers at a ratio of about 2:1, respectively. Forty percent of the molecules contain L-valine instead of L-isoleucine. The glx and asx are present as a combination of L-glutamic acid and L-asparagine and/or L-glutamine and L-aspartic acid. The N-terminus of the peptide is blocked, most likely by a peptide bond to the lipid portion. An ester carbonyl structure is present, which could be a part of a lactone ring connecting the beta position of the lipid to one of the carbonyl groups in the peptide. The lipid portion is composed of, on average, 8-9 methylene groups, and contains a mixture of linear and branched tails. Results of DCI-MS and FAB-MS analyses, as well as surface tension measurements, of purified BL-86 HPLC fractions support the proposed composition.

Partial protection of (C57BL/6 X DBA/2)F1 mice against infection with Schistosoma mansoni has been achieved by immunization with small amounts (0.2 to 2.0 microgram) of crude cercarial sonicate adsorbed on aluminium hydroxide gel adjuvant (alum). A decrease of 34-90% in the adult worm burden, of the immunized mice, as compared to that of untreated mice or those injected with adjuvant alone, has been found in five experiments by liver perfusion six weeks after percutaneous challenge infection. High titers of anti-cercarial IgE antibodies have been found in the sera of the immunized mice by two independent techniques, radioimmunoassay and degranulation of rat basophilic leukemia cells (RBL-2H3), determined by 3H-serotonin release. By counting the live worms in the lungs of immunized and uniummized mice on days 4-7 after infection it was observed that the schistosomula were killed before they reached the lungs, probably at the skin. Mice immunized with the same amounts of cercarial antigen in Freund's complete adjuvant were not protected against infection with S. mansoni. These animals developed high titer of total anti-cercaria antibodies (determined by radioimmunoassay) but had low levels of antiparasite IgE. The possible role of IgE in protective immunity is discussed.