Mosquito mitochondrial transfer RNAs for valine, glycine and glutamate: RNA and gene sequences and vicinal genome organization.

We report the sequences of 3 transfer RNAs from mosquito (Aedes albopictus) mitochondria, those for valine (anticodon UAC), glutamic acid (anticodon UUC) and glycine (anticodon UCC), as well as sequences for the corresponding genes and for some neighboring mitochondrial genes. TRNAval is notable for its high level of psi, tRNAglu for its low level of G and C, and tRNAgly is notable in that it appears as two species widely separated in gel electrophoresis, differing only in modification status. TRNAglu is the first sequenced insect mitochondrial tRNA that would be expected to engage in U.R wobble (where U is a modified U in the first position of the anticodon, and R is G or A in the third position of codons), if the insect system followed the modified wobble rules proposed for mammalian and fungal mitochondria; and the sequence determined does fit the proposal. The gene for tRNAval follows immediately that for 12S ribosomal RNA. The gene for tRNAglu occurs in a cluster of 6 tRNA genes that is separated from the gene for tRNAgly by a short reading frame. Features of the DNA sequences are discussed with reference to Drosophila, and mammalian, mitochondrial genome organization.

The 3' end of large ribosomal subunit RNA from mosquito mitochondria: homogeneity of transcribed moieties.

3' Terminal sequences of mosquito mitochondrial 16 S ribosomal RNA, which is post-transcriptionally polyadenylated, have been examined with the aim of determining the degree of homogeneity of transcribed moieties. 3' End-labeled samples were subjected to oligonucleotide fingerprint analysis and to ladder gel analysis after primary and secondary nuclease digestion; and products of reverse transcriptase reactions were characterized using 16 S RNA as template and selected oligodeoxynucleotides as primers. The results indicated a remarkable degree of homogeneity compared to homologous mammalian mitochondrial systems, and suggested differences in modes of expression of insect, versus mammalian, mitochondrial genomes.

Sequences of the coding and flanking regions of the large ribosomal subunit RNA gene of mosquito mitochondria.

We have sequenced a 1.6 kbp region of the mosquito (Aedes albopictus) mitochondrial genome containing the large ribosomal subunit ("LSU") RNA gene, and have located the ends of the gene by S1 protection analysis and by comparison with RNA sequences. The gene is preceded by a tRNAval gene and followed by genes for tRNAIeuUAG (rather than tRNAleuUAA, as in mammalian mitochondria) and an extended reading frame homologous to mammalian URF1. It is approximately 1335 residues long and is very low (17%) in G + C. The 5' half is even lower in G + C (9%), and shows little apparent homology to other LSU RNA classes. The 3' half is relatively rich (26%) in G + C and has many stretches of homology to prokaryotic and mammalian mitochondrial LSU RNA.

Sequence and structure of a serine transfer RNA with GCU anticodon from mosquito mitochondria.

We have determined the primary sequence and modification status of a transfer RNA from mosquito mitochondria whose GCU anticodon indicates that it is a serine tRNA (tRNASerGCU), and have obtained information on higher order structure using partial digestion with nucleases S1 and T1 under non-denaturing conditions. Although its primary sequence homology to mammalian mitochondrial tRNASerGCU is modest (46%), the mosquito tRNA resembles its mammalian mitochondrial counterpart in that a plausible secondary structure configuration includes a drastically abbreviated D arm and a sex base-pair anticodon stem. Other unusual features include a ribose-methylated cytidine residue at the end of the anticodon stem, and the likely occurrence of a psi residue between the amino acid arm and arm IV.

Sequence and structure of a methionine transfer RNA from mosquito mitochondria.

We have sequenced a methionine tRNA from mosquito mitochondria, and examined its structure using nucleases S1 and T1 under non-denaturing conditions. The sequence is highly homologous to a putative initiator methionine tRNA gene from Drosophila mitochondria. Its anticodon stem contains a run of three G-C base pairs that is characteristic of conventional initiator tRNAs; however, nuclease S1 analysis suggested an anticodon loop configuration characteristic of conventional elongator tRNAs. We propose that this tRNA can assume both initiator and elongator roles.

A cluster of four transfer RNA genes in mosquito mitochondrial DNA.

We have sequenced a 342 base-pair segment of mosquito (Aëdes albopictus) mitochondrial DNA. This segment contains genes corresponding to two of the more unusual of the mosquito mitochondrial tRNAs that we sequenced earlier, tRNAargUCG and tRNAserGCU, as well as genes for tRNAalaUGC and tRNAasnGUU and a portion of a gene that is homologous to mammalian mitochondrial unidentified reading frame 3. There are differences in gene order and directions of transcription between the mosquito mitochondrial sequence on the one hand, and corresponding Drosophila and mammalian mitochondrial sequences on the other, that indicate the occurrence of multiple discrete transpositional events during mitochondrial DNA evolution.

A major lysine tRNA with a CUU anticodon in insect mitochondria.

We have sequenced a lysine tRNA from mosquito mitochondria that has the anticodon CUU. The preponderance of AAA lysine codons in insect mitochondrial genes, the parsimonious organization of the genomes, and the fact that this tRNA is a major component of the mosquito mitochondrial tRNA complement, lead us to suggest that the CUU anticodon recognizes AAC and AAA codons.

Sequences of three transfer RNAs from mosquito mitochondria.

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNAArgUCG, tRNAAspGUC, and tRNAIleGAU, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNAArgUCG and tRNAAspGUC determined similarly, and a bovine mitochondrial tRNAIleGAU determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to "conventional" (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.

The 3'-terminal region of mosquito mitochondrial small ribosomal subunit RNA: sequence and localization of methylated residues.

The 3'-terminal 101 residues of the small ribosomal subunit (SSU) RNA of mosquito cell mitochondria have been determined. This stretch includes the four methylated residues of the molecule: an m4C, an m5C, and two m26A residues. The m26A's occur in a typical m26A "arm," and the methylated Cs in the unique subsequence G x m4C . C . m5C . A, which is homologous in position to a conserved methylated GCCCG subsequence of other SSU RNA classes. There is fairly good overall homology between the mosquito mitochondrial sequence and corresponding regions of other SSU RNA classes, except that a domain of 50-100 residues, previously considered universal, is absent. Comparison with mammalian mitochondrial sequences revealed a marked preponderance of transitional base substitutions, supporting earlier evidence that the 3'-terminal region of SSU RNA is under special structural constraints. The extreme 3' end of the mosquito sequence is heterogeneous, three-fourths of the molecules ending in ... GA and one-fourth ending in ... GAA. Evidence is presented indicating that some, at least, of the 3'-terminal A residues may be added post-transcriptionally, as occurs in mammalian mitochondrial systems. Taken together, the results provide modest support for the monophyletic evolutionary origin of insect and mammalian mitochondria from a primitive procaryotic ancestor.

Methylation patterns of mycoplasma transfer and ribosomal ribonucleic acid.

The methylation patterns of transfer and ribosomal ribonucleic acid (RNA) from two mycoplasmas, Mycoplasma capricolum and Acholeplasma laidlawii, have been examined. The transfer RNA from the two mycoplasmas resembled that of other procaryotes in degree of methylation and general diversity of methylated nucleotides, and bore particular resemblance to Bacillus subtilis transfer RNA. The only unusual feature was the absence of m5U from M. capricolum transfer RNA. The methylation patterns of the mycoplasma 16S RNAs were also typically procaryotic, retaining the methylated residues previously shown to be highly conserved among eubacterial 16S RNAs. The mycoplasma 23S RNA methylation patterns were, on the other hand, quite unusual. M. capricolum 23S RNA contained only four methylated residues in stoichiometric amounts, all of which were ribose methylated. A. laidlawii 23S RNA contained the same ribose-methylated residues, plus in addition approximately six m5U residues. These findings are discussed in relation to the phylogenetic status of mycoplasma, as well as the possible role of RNA methylation.

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.

Selective membrane toxicity of the polyene antibiotics: studies on natural membranes.

The effect of polyene antibiotics on Candida albicans, human erythrocytes, and Acholeplasma laidlawii was studied. The results sustain the observations made with lecithin-sterol liposomes. The distribution of double bonds in the membrane sterol nucleus appears to be of major importance in conferring polyene susceptibility; those sterols with the ergosterol nucleus are far more effective than those with a nucleus similar to cholesterol. Different polyenes vary in their membrane selectivity. The clinical implications of these observations are discussed.

Selective membrane toxicity of the polyene antibiotics: studies on lecithin membrane models (liposomes).

In the absence of sterol, amphotericin B at 5 x 10(-6) M caused maximum marker release from the saturated dipalmitoyl lecithin liposomes, minimum release from the unsaturated dioleoyl lecithin liposomes, and an in-between response from egg lecithin liposomes. Nystatin at 2.5 to 4.0 x 10(-5) M induced appreciable marker release from all three types of sterol-free liposomes. The amphotericin B- and nystatin-induced permeability changes in dipalmitoyl lecithin liposomes were drastically suppressed by the incorporation of cholesterol or stigmasterol (with identical Delta5 sterol nuclei), but were unaffected by the incorporation of ergosterol or 5,7-cholestadien-3beta-ol (with identical Delta5,7 sterol nuclei). The nystatin sensitivity of dioleoyl lecithin liposomes remained low after the incorporation of cholesterol or stigmasterol, but was greatly enhanced by the incorporation of ergosterol or 5,7-cholestadien-3beta-ol. Digitonin, a compound known to interact specifically with membrane sterol, induced marker release from liposomes in proportion to the amount of either cholesterol or ergosterol incorporated; epicholesterol did not sensitize to digitonin. These results lead to the following conclusions: (i) polyene-induced permeability alteration in model membrane systems is effected by the composition of membrane phospholipid fatty acyl chains; (ii) the distribution of double bonds in the sterol nucleus is related to the selective toxicity of the polyenes toward natural sterol-containing membranes; and (iii) polyenes differ in membrane selectivity.