Genetic conflicts during meiosis and the evolutionary origins of centromere complexity.

Centromeric DNA evolves rapidly, ranging in size and complexity over several orders of magnitude. Traditional attempts at studying centromeres have left unexplained the causes underlying this complexity and rapid evolution. Instead of directly studying centromeric DNA sequence, our approach has been to study the proteins that epigenetically determine centromere identity. We have discovered that centromeric histones (CenH3s) have evolved under positive selection in multiple lineages, suggesting an involvement in recurrent genetic conflict. Our hypothesis is that 'centromere-drive' is the source of this conflict. Under this model, centromeres compete via microtubule attachments for preferential transmission in female meioses occurring in animals and plants. Since only one of four meiotic products will become the egg, this competition confers a selfish advantage to chromosomes that can make more microtubule attachments, resulting in runaway expansions of centromeric satellites. While beneficial to the 'driving' chromosome, these expansions can have deleterious effects on the fitness of an organism and of the species. CenH3s as well as other heterochromatin proteins have evolved under positive selection to suppress the deleterious consequences of 'centromere-drive' by restoring meiotic parity.

Ribonuclease H evolution in retrotransposable elements.

Eukaryotic and prokaryotic genomes encode either Type I or Type II Ribonuclease H (RNH) which is important for processing RNA primers that prime DNA replication in almost all organisms. This review highlights the important role that Type I RNH plays in the life cycle of many retroelements, and its utility in tracing early events in retroelement evolution. Many retroelements utilize host genome-encoded RNH, but several lineages of retroelements, including some non-LTR retroposons and all LTR retrotransposons, encode their own RNH domains. Examination of these RNH domains suggests that all LTR retrotransposons acquired an enzymatically weak RNH domain that is missing an important catalytic residue found in all other RNH enzymes. We propose that this reduced activity is essential to ensure correct processing of the polypurine tract (PPT), which is an important step in the life cycle of these retrotransposons. Vertebrate retroviruses appear to have reacquired their RNH domains, which are catalytically more active, but their ancestral RNH domains (found in other LTR retrotransposons) have degenerated to give rise to the tether domains unique to vertebrate retroviruses. The tether domain may serve to control the more active RNH domain of vertebrate retroviruses. Phylogenetic analysis of the RNH domains is also useful to "date" the relative ages of LTR and non-LTR retroelements. It appears that all LTR retrotransposons are as old as, or younger than, the "youngest" lineages of non-LTR retroelements, suggesting that LTR retrotransposons arose late in eukaryotes.

Allogeneic bone marrow transplantation in beta-thalassaemia--single centre study.

OBJECTIVE: To evaluate outcome of allogeneic BMT in beta-Thalassaemia at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan from August 2001 to November 2003. METHODS: Nineteen patients with beta-Thalassaemia underwent allogeneic BMT/PBSC transplantation from HLA identical sibling donors. Patients were classified in three groups according to Pesaro (Italy) risk classification. Class-I (n = 9) and Class-II (n = 7) patients received conditioning with busulphan/cyclophosphamide, whereas Class-III (n = 3) patient received conditioning with hydroxyurea, azathioprine, fludarabine, along with Bu14 / Cy 200. Cyclosporine, prednisolone and methotrexate were given for GvHD prophylaxis. Stem cells dose infused was >4.0 x 10(8)/kg body weight of the patient. RESULTS: Engraftment was achieved in all Class-I patients, whereas in Class-II and Class-III , graft rejection was observed in one patient from each class. Median time to achieve absolute neutrophil recovery (> 0.5 x 10(9)/l) was 13 days, platelet count (> 20 x 10(9)/1) was 15 days and reticulocyte count (>0.5%) was 15 days. Acute GvHD was observed in 15 patients. One patient developed grade IV GvHD (liver and skin) and died within 30 days post BMT. Post transplant infectious complications were pseudomonas septicemia, disseminated fungal infection, CMV pneumonia and tuberculosis. Three patients died of these complications during post transplant period (31-90 days). Median stay in hospital was 25 days. CONCLUSION: Allogeneic BMT is the only curative therapy for beta-Thalassaemia patients, however the success rate can be increased if the patients are selected carefully and transplanted at an early age.

The centromere paradox: stable inheritance with rapidly evolving DNA.

Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.

Phylogenetic analysis of ribonuclease H domains suggests a late, chimeric origin of LTR retrotransposable elements and retroviruses.

We have conducted a phylogenetic analysis of the Ribonuclease HI (RNH) domains present in Eubacteria, Eukarya, all long-term repeat (LTR)-bearing retrotransposons, and several late-branching clades of non-LTR retrotransposons. Analysis of this simple yet highly conserved enzymatic domain from these disparate sources provides surprising insights into the evolution of eukaryotic retrotransposons. First, it indicates that the lineage of elements leading to vertebrate retroviruses acquired a new RNH domain either from non-LTR retrotransposons or from a eukaryotic host genome. The preexisting retroviral RNH domain degenerated to become the tether (connection) domain of the reverse transcriptase (RT)-RNH complex. Second, it indicates that all LTR retrotransposons arose in eukaryotes well after the origin of the non-LTR retrotransposons. Because of the younger age of the LTR retrotransposons, their complex structure, and the absence of any prokaryotic precursors, we propose that the LTR retrotransposons originated as a fusion between a DNA-mediated transposon and a non-LTR retrotransposon. The resulting two-step mechanism of LTR retrotransposition, in which RNA is reverse transcribed away from the chromosomal target site, rather than directly onto the target site, was probably an adaptation to the uncoupling of transcription and translation in eukaryotic cells.

Adaptive evolution of Cid, a centromere-specific histone in Drosophila.

Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.

Ten-kilodalton domain in Ty3 Gag3-Pol3p between PR and RT is dispensable for Ty3 transposition.

Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast Saccharomyces cerevisiae. In cells overexpressing Ty3 under the GAL1 upstream activation sequence, Ty3 RNA, proteins, and DNA are made. Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein. A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains. Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA. These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.

Dual recognition-incision enzymes might be involved in mismatch repair and meiosis.

Mismatch repair in many organisms depends on three proteins: the mismatch-recognition protein MutS, a nicking endonuclease MutH, and MutL, which acts as a scaffold between these. However, many genomes lack MutL but possess MutS. In one of these cases, in a coral mitochondrial genome, a gene is present that encodes a MutS protein fused to an HNH nicking endonuclease, potentially eliminating the requirement for MutL. Likewise, many prokaryotes could operate similarly, independently of MutL by encoding a fused MutS-Smr (MutS2) protein. Smr, which is proposed to be a nicking endonuclease, can also be found separately in many eukaryotes, where it might play a role in mismatch repair or meiotic chromosome crossing-over.

Poised for contagion: evolutionary origins of the infectious abilities of invertebrate retroviruses.

Phylogenetic analyses suggest that long-terminal repeat (LTR) bearing retrotransposable elements can acquire additional open-reading frames that can enable them to mediate infection. Whereas this process is best documented in the origin of the vertebrate retroviruses and their acquisition of an envelope (env) gene, similar independent events may have occurred in insects, nematodes, and plants. The origins of env-like genes are unclear, and are often masked by the antiquity of the original acquisitions and by their rapid rate of evolution. In this report, we present evidence that in three other possible transitions of LTR retrotransposons to retroviruses, an envelope-like gene was acquired from a viral source. First, the gypsy and related LTR retrotransposable elements (the insect errantiviruses) have acquired their envelope-like gene from a class of insect baculoviruses (double-stranded DNA viruses with no RNA stage). Second, the Cer retroviruses in the Caenorhabditis elegans genome acquired their envelope gene from a Phleboviral (single ambisense-stranded RNA viruses) source. Third, the Tas retroviral envelope (Ascaris lumricoides) may have been obtained from Herpesviridae (double-stranded DNA viruses, no RNA stage). These represent the only cases in which the env gene of a retrovirus has been traced back to its original source. This has implications for the evolutionary history of retroviruses as well as for the potential ability of all LTR-retrotransposable elements to become infectious agents.

Putative telomerase catalytic subunits from Giardia lamblia and Caenorhabditis elegans.

Eukaryotic chromosomes end in short nucleotide repeats that are added by the enzyme telomerase. The catalytic subunit of telomerase has been shown to be most closely related in sequence to reverse transcriptases encoded by eukaryotic retrotransposable elements. This raises the question as to whether the telomerase subunit was present in the first eukaryotes or was derived during early eukaryote evolution from the replication machinery of a retrotransposable element. We present the sequence of a putative telomerase catalytic subunit from the diplomonad parasite, Giardia lamblia. The G. lamblia subunit appears to have most of the characteristics of other sequenced telomerases, except that it lacks the conserved telomerase-specific 'T' motif previously identified in other eukaryotic genes. Searching genomic databases with the G. lamblia sequence, we also identified a potential telomerase catalytic subunit from Caenorhabditis elegans. The C. elegans subunit is uncharacteristically short, and lacks several motifs found in all other telomerases. The identification of a G. lamblia telomerase similar to that of most other eukaryotes suggests that telomerase dates back to the earliest extant marker of eukaryotic evolution. The atypical C. elegans telomerase, on the other hand, raises intriguing biochemical questions concerning sub-domains of the telomerase catalytic subunit previously considered indispensable. The enzymatic machinery for telomere formation in C. elegans is likely to differ substantially from that of other eukaryotes.

NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans.

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.

Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements.

The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X12-14aa-Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein-DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3' end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme, FokI, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.

The age and evolution of non-LTR retrotransposable elements.

A comprehensive phylogenetic analysis was conducted of non-long-terminal-repeat (non-LTR) retrotransposons based on an extended sequence alignment of their reverse transcriptase (RT) domain. The 440 amino acid positions used included a region proposed to be similar to the "thumb" of the right-handed RT structure found in retroviruses. All identified non-LTR elements could be grouped into 11 distinct clades. Using the rates of sequence change derived from studies of the vertical inheritance of R1 and R2 elements in arthropods as a comparison, we found no evidence for the horizontal transmission of non-LTR elements. Assuming vertical descent, the phylogeny suggested that non-LTR elements are as old as eukaryotes, with each of the 11 clades dating back to the Precambrian era. The analysis enabled us to propose a simple chronology for the acquisition of different enzymatic domains in the evolution of the non-LTR class of retrotransposons. The first non-LTR elements were sequence specific by virtue of a restriction-enzyme-like endonuclease located downstream of the RT domain. Evolving from this original group were elements (eight clades) that acquired an apurinic-apyrimidic endonuclease-like domain upstream of the RT domain. Finally, four of these clades have inherited an RNase H domain downstream of the RT domain. The phylogenies of the AP endonuclease and RNase H domains were also determined for this report and are consistent with the monophyletic acquisition of these domains. These studies represent the most comprehensive effort to date to trace the evolution of a major class of transposable elements.

The domain structure and retrotransposition mechanism of R2 elements are conserved throughout arthropods.

R2 elements are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. Partial sequence data from many species have previously suggested that these elements have been vertically inherited since the origin of this phylum. Here, we compare the complete sequences of nine R2 elements selected to represent the diversity of arthropods. All of the elements exhibited a uniform structure. Identification of their conserved sequence features, combined with our biochemical studies, allows us to make the following inferences concerning the retrotransposition mechanism of R2. While all R2 elements insert into the identical sequence of the 28S gene, it is only the location of the initial nick in the target DNA that is rigidly conserved across arthropods. Variation at the R2 5' junctions suggests that cleavage of the second strand of the target site is not conserved within or between species. The extreme 5' and 3' ends of the elements themselves are also poorly conserved, consistent with a target primed reverse transcription mechanism for attachment of the 3' end and a template switch model for the attachment of the 5' end. Comparison of the approximately 1,000-aa R2 ORF reveals that it can be divided into three domains. The central 450-aa domain can be folded by homology modeling into a tertiary structure resembling the fingers, palm, and thumb subdomains of retroviral reverse transcriptases. The carboxyl terminal end of the R2 protein appears to be the endonuclease domain, while the amino-terminal end contains zinc finger and c-myb-like DNA-binding motifs.

Modular evolution of the integrase domain in the Ty3/Gypsy class of LTR retrotransposons.

A phylogenetic analysis of the Ty3/Gypsy group of retrotransposons identified a conserved domain (GPY/F) present in the integrases of several members of this group as well as of certain vertebrate retroviruses. The analysis suggested an evolutionary scheme for the acquisition and loss of the GPY/F domain as well as the acquisition of a chromodomain module in the integrase encoded by this group of elements that may direct targeting specificity in the host genome.

Retrotransposable elements R1 and R2 in the rDNA units of Drosophila mercatorum: abnormal abdomen revisited.

R1 and R2 retrotransposable elements are stable components of the 28S rRNA genes of arthropods. While each retrotransposition event leads to incremental losses of rDNA unit expression, little is known about the selective consequences of these elements on the host genome. Previous reports suggested that in the abnormal abdomen (aa) phenotype of Drosophila mercatorum, high levels of rDNA insertions (R1) in conjunction with the under-replication locus (ur), enable the utilization of different ecological conditions via a population level shift to younger age. We have sequenced the R1 and R2 elements of D. mercatorum and show that the levels of R1- and R2-inserted rDNA units were inaccurately scored in the original studies of aa, leading to several misinterpretations. In particular, contrary to earlier reports, aa flies differentially underreplicate R1- and R2-inserted rDNA units, like other species of Drosophila. However, aa flies do not undergo the lower level of underreplication of their functional rDNA units (general underreplication) that is seen in wild-type strains. The lack of general underreplication is expected to confer a selective advantage and, thus, can be interpreted as an adaptation to overcome high levels of R1 and R2 insertions. These results allow us to reconcile some of the apparently contradictory effects of aa and the bobbed phenotype found in other species of Drosophila.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli.

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2'-phosphotransferase to transfer the splice junction 2'-phosphate from ligated tRNA to NAD, producing ADP ribose 1"-2" cyclic phosphate (Appr>p). We show here that functional 2'-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2'-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2'-phosphate. Analysis of the database shows that likely members of the 2'-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2'-phosphotransferase into Eubacteria, suggesting that the 2'-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2'-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2'-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

The RTE class of non-LTR retrotransposons is widely distributed in animals and is the origin of many SINEs.

RTE-1 is a non-long-terminal-repeat (non-LTR) retrotransposable element first found in the Caenorhabditis elegans genome. It encodes a 1,024-amino-acid open reading frame (ORF) containing both apurinic-apyrimidic endonuclease and reverse-transcriptase domains. A possible first ORF of only 43 amino acids overlaps with the larger ORF and may be the site of translation initiation. Database searches and phylogenetic analysis indicate that representatives of the RTE clade of non-LTR retrotransposons are found in the bovine and sheep genomes of mammals and in the silkmoth and mosquito genomes of insects. In addition, the previously identified SINEs, Art2 and Pst, from ruminate and viper genomes are shown to be truncated RTE-like retrotransposable elements. RTE-derived SINE elements are also found in mollusc and flatworm genomes. Members of the RTE clade are characterized by unusually short 3' untranslated regions that are predominantly composed of AT-rich trimer, tetramer, and/or pentamer repeats. This study establishes RTE as a very widespread clade of non-LTR retrotransposons. RTE represents the third distinct class of non-LTR retrotransposons in the vertebrate lineage (after Line 1 elements in mammals and CR1 elements in birds and reptiles).

Determining the evolutionary potential of a gene.

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base-substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta-galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.