Unearthing LTR Retrotransposon gag Genes Co-opted in the Deep Evolution of Eukaryotes

LTR retrotransposons comprise a major component of the genomes of eukaryotes. On occasion, retrotransposon genes can be recruited by their hosts for diverse functions, a process formally referred to as co-option. However, a comprehensive picture of LTR retrotransposon gag gene co-option in eukaryotes is still lacking, with several documented cases exclusively involving Ty3/Gypsy retrotransposons in animals. Here, we use a phylogenomic approach to systemically unearth co-option of retrotransposon gag genes above the family level of taxonomy in 2,011 eukaryotes, namely co-option occurring during the deep evolution of eukaryotes. We identify a total of 14 independent gag gene co-option events across more than 740 eukaryote families, eight of which have not been reported previously. Among these retrotransposon gag gene co-option events, nine, four, and one involve gag genes of Ty3/Gypsy, Ty1/Copia, and Bel-Pao retrotransposons, respectively. Seven, four, and three co-option events occurred in animals, plants, and fungi, respectively. Interestingly, two co-option events took place in the early evolution of angiosperms. Both selective pressure and gene expression analyses further support that these co-opted gag genes might perform diverse cellular functions in their hosts, and several co-opted gag genes might be subject to positive selection. Taken together, our results provide a comprehensive picture of LTR retrotransposon gag gene co-option events that occurred during the deep evolution of eukaryotes and suggest paucity of LTR retrotransposon gag gene co-option during the deep evolution of eukaryotes.     

Grande retrotransposons contain an accessory gene in the unusually long 3′-internal region that encodes a nuclear protein transcribed from its own promoter

LTR retrotransposons are major components of plant genomes playing important roles in the evolution of their host genomes, for example, generating new genes or providing new promoters to existing genes. The Grande family of retrotransposons is present in Zea species and is characterized by an unusually long internal region due to the presence of a 7-kbp region between the gag-pol coding region and the 3′LTR. We demonstrate here that such unusual sequence is present in the great majority of Grande copies in maize genome. This region contains a gene, gene23, which is transcribed from its own promoter in antisense orientation to the gag-pol genes. The expression of gene23 is ubiquitous, and its promoter contains all the putative consensus sequences typical of eukaryotic promoters, being able to direct GUS expression in different plant species and organs. The coding region of gene23 is conserved in most Grande copies and encodes a protein rich in glycine, serine, and acidic amino acids that shows no significant similarity with any protein of known function. Nevertheless, the C- and N-terminal parts are rich in basic amino acids, and these are interspersed with other amino acids in its C-terminus, compatible with a putative DNA-binding function. It contains a nuclear localization signal KRKR motif in the N-terminus. Fusions to GFP demonstrate that this protein localizes in the nucleus. We discuss the possible origin of gene23 and the potential function of its encoded protein.     

Composition, arrangement and cleavage of the mouse mammary tumor virus polyprotein precursor Pr77gag and p110gag.

The amino acid sequence relationship between the nonglycosylated structural proteins of murine mammary tumor virus and the polyproteins from infected cells immunoprecipitated with an anti-p27 serum were examined using two-dimensional tryptic peptide mapping procedures. The proteins were labeled with ¹⁴C-lysine and ¹⁴C-arginine so that all but one of the tryptic peptides released from a protein could be detected. Previous studies have shown that immunoprecipitation of mammary tumor cells with anti-p27 serum results in the isolation of seven proteins in the molecular weight range of 34,000–160,000 daltons; and that cell-free translation using viral genomic RNA yields three p27-related proteins of 160,000, 110,000 and 77,000 daltons, similar to the three high molecular weight proteins detected in vivo. The proteins of lower molecular weight were thought to be cleavage intermediates of Pr77^gag. As judged from the peptide maps, Pr77^gag contained the complete sequences of the four major internal proteins of the virion (p27, pp21, p14 and p10) and possibly a fifth highly basic protein (p8) also found in virions. The putative cleavage intermediates, as expected, lacked some tryptic peptides that could be assigned to one or more of the major virion proteins and thus allow a scheme for the cleavage events to be constructed. p110^gag contained all the tryptic peptides found in Pr77^gag, plus some additional peptides. A minor virion protein p30 was found to include the peptides of p14 as well as some of the additional peptides present in p110^gag, suggesting a precursor-product relationship between the pr110^gag and p30. The data obtained from these studies lead us to propose that there are three protein precursors which include, at least in part, the gag gene region of the virion—p160 (potentially a gag/pol precursor), p110^gag and Pr77^gag—and that the arrangement of the virion proteins within the gag gene (pr77^gag) is p10-pp21-p27-p14.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

Grossly Defective nef Gene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4⁺ T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.     

skippy,a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

An eye imaginal disc-specific transcriptional enhancer in the long terminal repeat of thetom retrotransposon is responsible for eye morphology mutations ofDrosophila ananassae

Optic morphology (Om) mutations ofDrosophila ananassae are semidominant, neomorphic and nonpleiotropic, map to at least 22 loci scattered throughout the genome, and are associated with the insertion of thetom retrotransposon. Molecular and genetic analyses have revealed that eye morphology defects ofOm mutants are caused by the ectopic or excessive expression ofOm genes in the eye imaginal discs of third instar larvae. It is therefore assumed that thetom element carries tissue-specific gene regulatory sequences which enhance expression of theOm genes. In the present study, we examined whether or not the long terminal repeats (LTR) of thetom element contain such an eye imaginal disc-specific enhancer, usingD. melanogaster transformants containing alacZ gene ligated to thetom LTR. Analyses oflacZ gene expression in the eye imaginal discs of third instar larvae of 18 independently established transformant lines showed that thetom LTR was capable of enhancinglacZ expression in all the transformant lines, but the degree of enhancement varied between lines. In addition, the effect of thetom LTRlacZ gene evidently changed when thetom LTR construct was relocated to different chromosomal positions. On the basis of these findings, it is hypothesized that ectopic and excessive expression of theOm genes in the eye imaginal discs is induced by an eye imaginal disc-specific enhancer present in thetom LTR, the effect of which may be subject to chromosomal position effects.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.