We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species. A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced. Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease. This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species. Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences. These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin. The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca. Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes. These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway. The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants.

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

Human T cell leukemia/lymphotropic virus (HTLV) is a complexstudies have demonstrated the presence of p30, p13 and p12 9 kb human retrovirus with at least eight alternatively spliced in addition to the previously recognized Tax, Rex and p21 rex mRNAs expressed from the 3Ј or pX region of the genome.proteins. 3,5,7 A unique splice site has also been detected 17 Cell linesHAM) 2 is a complex 9 kb human retrovirus. The degree of complexity of the viral genome has recently been recognized Several previously described IL-2-dependent and IL-2-indewith the detection by several groups of at least eight alternapendent HTLV-I-infected T cell lines were utilized in these tively spliced mRNAs expressed from the 3Ј or pX region of studies. They included the IL-2-independent lines C10/MJ the genome. 3-6 These mRNAs allow for the expression of established from a patient with ATL (who subsequently novel proteins from the previously recognized pX open readdeveloped TSP/HAM), 11 NS1 established from a healthy seroing frames I and II in addition to Tax, Rex and p21 rex encoded positive individual, 12 C8166-45 established from a patient from orf III and IV. The alternatively spliced messages include:with ATL and containing defective proviruses 13 and LAF, the doubly spliced pX-tax/rex with the coding capacity for established from a patient with TSP/HAM. 5 The IL-2-depenTax, Rex and p21 rex ; the singly spliced pX-orf I encoding a dent cell lines, N1185 and N1186, were established by coprotein, p12, and the doubly spliced pX-rex-orf I coding for cultivation of PBMCs from two patients with ATL with cord p12 and with the potential for encoding a 152 amino acid blood cells. 4 The cells were maintained in RPMI with 10% protein (Rex-orf I or Rof), the singly spliced pX-orf II encoding FCS with the addition of Il-2 (20 units/ml, Boehringer p13; the doubly spliced pX-tax-orf II encoding a larger protein Mannheim, Indianapolis, IN, USA) for the IL-2-dependent (p30, Tax-orf II or Tof) which utilizes the initiation codon of cell lines. Tax; and the singly spliced pX-p21 rex , a monocistronic message encoding p21 rex of unknown function. In vitro transfection RNA preparation

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This study provides the first report in which spliceosomal intron losses within a single-copy gene create functional polymorphic alleles in a population. 4f-rnp has previously been shown to be a nuclear gene that is localized on the X chromosome in D. melanogaster and to have eight short spliceosomal introns. An insect species survey was done via polymerase chain reaction (PCR) amplification and sequencing of a 1028-bp gene fragment spanning introns 4-8, which are located in the 3' half of the gene. The results show that 4f-rnp and (thus far) introns 7 and 8 are at least as old as order Odonata (dragonflies), an early-diverging insect line. Unexpectedly, several species within the dipteran family Drosophilidae were found to contain two differently sized 4f-rnp gene sequence variants, owing to precise in-frame intron losses. Results of single-male D. melanogaster PCR analyses show that the two gene size variants are allelic and that the intron loss mechanism appears to be biased toward the 3' end of the gene. A stable potential stem-loop has been identified in D. melanogaster, predicted to fold the 4f-rnp mRNA 3' terminus into a natural primer for subsequent reverse transcription into cDNA. When results are displayed in a phylogenetic context, multiple independent intron loss events are identified. These observations support a model in which frequently occurring cDNAs have led to numerous independent intron losses via homologous recombination/gene conversion during 4f-rnp gene evolution. The results provide insights into the evolution of intron loss and may lead to improved understanding of the dynamics of this process in natural populations.