Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii.

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.     

Polyadenylated mRNA from the photosynthetic procaryote Rhodospirillum rubrum.

Total cellular RNA extracted from Rhodospirillum rubrum cultured in butyrate-containing medium under strict photosynthetic conditions to the stationary phase of growth has been fractionated on an oligodeoxythymidylic acid-cellulose column into polyadenylated [poly(A)+] RNA and poly(A)- RNA fractions. The poly(A)+ fraction was 9 to 10% of the total bulk RNA isolated. Analysis of the poly(A)+ RNA on a denaturing urea-polyacrylamide gel revealed four sharp bands of RNA distributed in heterodisperse fashion between 16S and 9S. Similar fractionation of the poly(A)- RNA resulted in the separation of 23, 16, and 5S rRNAs and 4S tRNA. Poly(A)+ fragments isolated after combined digestion with pancreatic A and T1 RNases and analysis by denaturing gel electrophoresis demonstrated two major components of 80 and 100 residues. Alkaline hydrolysis of the nuclease-resistant, purified residues showed AMP-rich nucleotides. Through the use of snake venom phosphodiesterase, poly(A) tracts were placed at the 3' end of poly(A)+ RNA. Stimulation of [3H]leucine incorporation into hot trichloroacetic acid-precipitable polypeptides in a cell-free system from wheat germ primed by the poly(A)+ RNA mixture was found to be 220-fold higher than that for poly(A)- RNAs (on a unit mass basis), a finding which demonstrated that poly(A)+ RNAs in R. rubrum are mRNAs. Gel electrophoretic analysis of the translation mixture revealed numerous 3H-labeled products including a major band (Mr, 52,000). The parent protein was precipitated by antibodies to ribulose bisphosphate carboxylase-oxygenase and comprised 6.5% of the total translation products.     

Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli

Two cDNA clones for mitochondrial adenylate kinase were isolated from a cDNA library of bovine liver poly(A)+ RNA by using synthetic oligodeoxynucleotides as probes. The clone containing a 0.9-kilobase insert had the reading frame for a 241-residue protein (AK2A), while the other clone containing a 1.6-kilobase insert had the frame for a 234-residue protein (AK2B). Nucleotide sequences of these two clones were the same in the 5' portion up to the coding sequence for the 233rd residue, but different in the remaining 3' portions. The reported amino acid sequence of mitochondrial adenylate kinase from bovine heart corresponded to AK2A. Neither AK2A nor AK2B had a cleavable NH2-terminal presequence as that found in other imported mitochondrial proteins. RNA blot analysis of poly(A)+ RNAs from bovine liver and heart revealed three species of mRNA with approximate sizes of 0.9, 1.4, and 1.7 kilobases. The 1.7- and 1.4-kilobase species were specific for AK2B, whereas the 0.9-kilobase species was specific for AK2A. In the liver, the 1.7-kilobase mRNA was more abundant, whereas in the heart the 0.9-kilobase mRNA was predominant. The 1.4-kilobase mRNA was present only in the heart. The AK2A- and AK2B-coding sequences were expressed in Escherichia coli cells under the control of trc promoter. Both the products reverted the temperature-sensitive phenotype of the adenylate kinase mutant of E. coli.     

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.     

Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells

A significant fraction of the polyadenylated mRNAs of HeLa cells contain an oligo(uridylic acid) [oligo(U)] sequence of 15-30 nucleotides. Several different experimental approaches were used to determine if these oligo(U)'s occupied similar sites within all mRNAs. In one approach, poly(adenylic acid)-containing mRNAs [poly(A+) mRNAs] averaging 2800 nucleotides in length were reduced to an average size of 500 nucleotides by controlled alkaline hydrolysis. Over 20% of the oligo(U)-containing fragments isolated from the hydrolysate retained a poly(A) sequence, showing that oligo(U)'s were not exclusively located near 5' ends of mRNA although 20% were apparently close to 3' ends. To confirm these observations, oligo(U)-containing mRNA [oligo(U+) mRNA] was exposed to the 3'-exonucleolytic activity of polynucleotide phosphorylase to produce fragments containing the 5' regions of mRNA. Each of a set of fragments of decreasing length generated by increased times of exposure of the mRNAs to the enzyme was found to have about the same oligo(U) content, including the shortest that averaged 550 nucleotides. These data not only eliminated an exclusive location for oligo(U) in either 3' or 5' ends of mRNA but also suggested that oligo(U)'s might be close to the 5' ends of some mRNAs. To verify this last observation, periodate-oxidized poly(A+) mRNA was labeled at the 5' caps and at 3'-adenosine residues by sodium [3H]borohydride reduction before it was nicked 3-5 times with alkali to produce 5' and 3' end-labeled pieces that could be separated with oligo(thymidylic acid)-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.     

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).     

Polypeptides synthesized in vitro by poly(A)+ and poly(A)- mRNAs of rat ascites hepatoma AH 7974 cells.

Polysomal RNA of rat ascites hepatoma AH 7974 cells was fractionated into poly(A)+ and poly(A)- mRNAs and these RNAs were translated into polypeptides in a protein-synthesizing system derived from wheat germ. The analysis of polypeptides synthesized in vitro by two-dimensional gel electrophoresis revealed that there are three classes of polypeptides. The first group can be synthesized equally by both poly(A)+ and poly(A)- mRNAs; the second and third groups are synthesized predominantly by poly(A)+ and poly(A)- mRNAs, respectively. These results suggest that the three classes of polypeptides can be characterized by the presence or absence of poly(A) tails of the corresponding mRNAs.