HeT-A_pi1, a piRNA target sequence in the Drosophila telomeric retrotransposon HeT-A, is extremely conserved across copies and species

The maintenance of the telomeres in Drosophila species depends on the transposition of the non-LTR retrotransposons HeT-A, TAHRE and TART. HeT-A and TART elements have been found in all studied species of Drosophila suggesting that their function has been maintained for more than 60 million years. Of the three elements, HeT-A is by far the main component of D. melanogaster telomeres and, unexpectedly for an element with an essential role in telomere elongation, the conservation of the nucleotide sequence of HeT-A is very low. In order to better understand the function of this telomeric retrotransposon, we studied the degree of conservation along HeT-A copies. We identified a small sequence within the 3' UTR of the element that is extremely conserved among copies of the element both, within D. melanogaster and related species from the melanogaster group. The sequence corresponds to a piRNA target in D. melanogaster that we named HeT-A_pi1. Comparison with piRNA target sequences from other Drosophila retrotransposons showed that HeT-A_pi1 is the piRNA target in the Drosophila genome with the highest degree of conservation among species from the melanogaster group. The high conservation of this piRNA target in contrast with the surrounding sequence, suggests an important function of the HeT-A_pi1 sequence in the co-evolution of the HeT-A retrotransposon and the Drosophila genome.     

A method to find palindromes in nucleic acid sequences

Various types of sequences in the human genome are known to play important roles in different aspects of genomic functioning. Among these sequences, palindromic nucleic acid sequences are one such type that have been studied in detail and found to influence a wide variety of genomic characteristics. For a nucleotide sequence to be considered as a palindrome, its complementary strand must read the same in the opposite direction. For example, both the strands i.e the strand going from 5'' to 3'' and its complementary strand from 3'' to 5'' must be complementary. A typical nucleotide palindromic sequence would be TATA (5'' to 3'') and its complimentary sequence from 3'' to 5'' would be ATAT. Thus, a new method has been developed using dynamic programming to fetch the palindromic nucleic acid sequences. The new method uses less memory and thereby it increases the overall speed and efficiency. The proposed method has been tested using the bacterial (3891 KB bases) and human chromosomal sequences (Chr-18: 74366 kb and Chr-Y: 25554 kb) and the computation time for finding the palindromic sequences is in milli seconds.     

HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.

Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.     

Fast-evolving noncoding sequences in the human genome

Background

Gene regulation is considered one of the driving forces of evolution. Although protein-coding DNA sequences and RNA genes have been subject to recent evolutionary events in the human lineage, it has been hypothesized that the large phenotypic divergence between humans and chimpanzees has been driven mainly by changes in gene regulation rather than altered protein-coding gene sequences. Comparative analysis of vertebrate genomes has revealed an abundance of evolutionarily conserved but noncoding sequences. These conserved noncoding (CNC) sequences may well harbor critical regulatory variants that have driven recent human evolution.

Results

Here we identify 1,356 CNC sequences that appear to have undergone dramatic human-specific changes in selective pressures, at least 15% of which have substitution rates significantly above that expected under neutrality. The 1,356 'accelerated CNC' (ANC) sequences are enriched in recent segmental duplications, suggesting a recent change in selective constraint following duplication. In addition, single nucleotide polymorphisms within ANC sequences have a significant excess of high frequency derived alleles and high F _ST values relative to controls, indicating that acceleration and positive selection are recent in human populations. Finally, a significant number of single nucleotide polymorphisms within ANC sequences are associated with changes in gene expression. The probability of variation in an ANC sequence being associated with a gene expression phenotype is fivefold higher than variation in a control CNC sequence.

Conclusion

Our analysis suggests that ANC sequences have until very recently played a role in human evolution, potentially through lineage-specific changes in gene regulation.     

The role of HeT-A and TART retrotransposons in Drosophila telomere capping

Drosophila telomeres contain multiple copies of HeT-A and TART retrotransposons. These elements specifically transpose to chromosomal ends, compensating for loss of terminal nucleotides that occurs at each cycle of DNA replication. We have investigated the role of these sequences in the formation of telomere–telomere attachments induced by mutations in the UbcD1 gene. We have constructed UbcD1 mutant males carrying terminally deleted X chromosomes devoid of both HeT-A and TART sequences. Cytological analysis of larval neuroblasts from these males revealed that telomeres lacking HeT-A and TART and normal telomeres that contain these sequences participate in telomeric fusions with comparable frequencies. These results indicate that the UbcD1 substrate(s) binds chromosomal termini in a sequence-independent manner. Previous studies have shown that the telomere-capping protein HP1 also binds telomeres lacking HeT-A and TART. Taken together, these findings strongly suggest that the assembly of DNA–protein complexes that protect chromosome ends from fusions do not require specific terminal sequences.     

Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

The genomic organization of HeT-A retroposons inDrosophila melanogaster

Members of theDrosophila HeT-A family of transposable elements are LINE-like retroposons that are found at telomeres and in centric heterochromatin. We recently characterized an active HeT-A element that had transposed to a broken chromosome end fewer than mine generations before it was isolated. The sequence arerangement of this element, called 9D4, most likely represents the organization of an actively transposing member of the HeT-A family. Here we assess the degree of divergence among members of the HeT-A family and test a model of telomere length maintenance based on HeT-A transposition. The region containing the single open reading frame of this element appears to be more highly conserved than the non-coding regions. The HeT-A element has been implicated in theDrosophila telomere elongation process, because frequent transpositions to chromosome ends are sufficient to counter-balance nucleotide loss due to incomplete DNA replication. The proposed elongation model and the hypothetical mechanism of HeT-A transposition predict a predominant orientation of HeT-A elements with their oligo (A) tails facing proximally at chromosome ends, as well as the existence of irregular tandem arrays of HeT-A elements at chromosome ends resulting from transposition of new HeT-A elements onto chromosome ends with existing elements. Twenty-nine different HeT-A fragments were isolated from directional libraries that were enriched in terminal DNA fragments. Sequence analyses of these fragments and comparisons with the organization of the HeT-A element, 9D4, fit these two predictions and support the model ofDrosophila telomere elongation by transposition of HeT-A elements.     

Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres.

In most eukaryotes telomeres are extended by telomerase. Drosophila melanogaster, however, lacks telomerase, and telomere-specific non-LTR retrotransposons, HeT-A and TART, transpose specifically to chromosome ends. A Drosophila strain, Gaiano, that has long telomeres has been identified. We extracted the major Gaiano chromosomes into an Oregon-R genetic background and examined the resulting stocks after 60 generations. In situ hybridization using HeT-A and TART sequences showed that, in stocks carrying either the X or the second chromosome from Gaiano, only the Gaiano-derived chromosomes display long telomeres. However, in stocks carrying the Gaiano third chromosome, all telomeres are substantially elongated, indicating that the Gaiano chromosome 3 carries a factor that increases HeT-A and TART addition to the telomeres. We show that this factor, termed Telomere elongation (Tel), is dominant and localizes as a single unit to 69 on the genetic map. The long telomeres tend to associate with each other in both polytene and mitotic cells. These associations depend on telomere length rather than the presence of Tel. Associations between metaphase chromosomes are resolved during anaphase, suggesting that they are mediated by either proteinaceous links or DNA hydrogen bonding, rather than covalent DNA-DNA bonds.     

Human Mitochondrial DNA Variation and Evolution: Analysis of Nucleotide Sequences from Seven Individuals

We have analyzed nucleotide sequence variation in an approximately 900-base pair region of the human mitochondrial DNA molecule encompassing the heavy strand origin of replication and the D-loop. Our analysis has focused on nucleotide sequences available from seven humans. Average nucleotide diversity among the sequences is 1.7%, several-fold higher than estimates from restriction endonuclease site variation in mtDNA from these individuals and previously reported for other humans. This disparity is consistent with the rapidly evolving nature of this noncoding region. However, several instances of convergent or parallel gain and loss of restriction sites due to multiple substitutions were observed. In addition, other results suggest that restriction site (as well as pairwise sequence) comparisons may underestimate the total number of substitutions that have occurred since the divergence of two mtDNA sequences from a common ancestral sequence, even at low levels of divergence. This emphasizes the importance of recognizing the large standard errors associated with estimates of sequence variability, particularly when constructing phylogenies among closely related sequences. Analysis of the observed number and direction of substitutions revealed several significant biases, most notably a strand dependence of substitution type and a 32-fold bias favoring transitions over transversions. The results also revealed a significantly nonrandom distribution of nucleotide substitutions and sequence length variation. Significantly more multiple substitutions were observed than expected for these closely related sequences under the assumption of uniform rates of substitution. The bias for transitions has resulted in predominantly convergent or parallel changes among the observed multiple substitutions. There is no convincing evidence that recombination has contributed to the mtDNA sequence diversity we have observed.     

Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus.

To learn more about the mechanism of de novo telomere synthesis, we have characterized the sequence and structure of newly synthesized telomeres from Euplotes crassus. E. crassus is a particularly useful organism for studying telomere synthesis because millions of telomeres are made in each cell at a well-defined time during the sexual stage of the life cycle. These newly synthesized telomeres are approximately 50 bp longer than mature macronuclear telomeres. We have investigated the structure of the newly synthesized telomeres and have found that they are much more heterogeneous in length than mature telomeres. Most of the heterogeneity is present on the G-rich strand, indicating that the length of this strand is rather loosely controlled. In contrast, the length of the C-rich strand is much less variable, suggesting that synthesis of this strand is the more precisely regulated step in telomere addition. The G-rich strand exhibits variability both in the total number of G4T4 repeats and in the identity of the terminal nucleotide. In most cases, the G-rich strnd extends beyond the C-rich strand to leave a 3' overhang. While the size of this overhang is variable, the median length is 10 nucleotides. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model to describe the various steps involved in de novo telomere synthesis.     

Improving Hidden Markov Models for classification of human immunodeficiency virus-1 subtypes through linear classifier learning

Profile Hidden Markov Models (pHMMs) are widely used to model nucleotide or protein sequence families. In many applications, a sequence family classified into several subfamilies is given and each subfamily is modeled separately by one pHMM. A major drawback of this approach is the difficulty of coping with subfamilies composed of very few sequences.Correct subtyping of human immunodeficiency virus-1 (HIV-1) sequences is one of the most crucial bioinformatic tasks affected by this problem of small subfamilies, i.e., HIV-1 subtypes with a small number of known sequences. To deal with small samples for particular subfamilies of HIV-1, we employ a machine learning approach. More precisely, we make use of an existing HMM architecture and its associated inference engine, while replacing the unsupervised estimation of emission probabilities by a supervised method. For that purpose, we use regularized linear discriminant learning together with a balancing scheme to account for the widely varying sample size. After training the multiclass linear discriminants, the corresponding weights are transformed to valid probabilities using a softmax function.We apply this modified algorithm to classify HIV-1 sequence data (in the form of partial-length HIV-1 sequences and semi-artificial recombinants) and show that the performance of pHMMs can be significantly improved by the proposed technique.     

Cloning and characterization of Leishmania donovani telomeres

We describe here the cloning and sequence characterization of the absolute termini of several telomeres from the human parasite Leishmania donovani using a vector-adapter protocol. The 3' protruding strand of L. donovani telomeres terminates with the sequence 5'-GGTTAGGGT-OH 3'. This single-stranded sequence is adjacent to tandemly repeated blocks of double-stranded sequence consisting of variable numbers of the hexameric repeat 5'-TAGGGT-3', variable numbers of an octameric repeat 5'-TGGTCATG-3', and a single 62-bp sequence, in that order. A number of additional, more chromosome-internal, nonrepeated sequences were found adjacent to the telomere sequences. Hybridization analyses indicated that some of these telomere adjacent sequences are found on all L. donovani chromosomes, some are more abundant on certain subsets of chromosomes, and some are unique to individual chromosomes.     

Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms

Drosophila telomeres contain arrays of the retrotransposonlike elements HeT-A and TART. Their transposition to broken chromosomal termini has been implicated in chromosome healing and telomere elongation. The HeT-A element is attached by its 3' end, which contains the promoter. To monitor the behavior of HeT-A elements, we used the yellow gene with terminal deficiencies consisting of breaks in the yellow promoter region that result in the y-null phenotype. Attachment of the HeT-A element provides the promoterless yellow gene with a promoter that activates yellow expression in bristles. The frequency of HeT-A transpositions to the yellow terminal deficiency depends on the genotype of the line and varies from 2 x 10(-3) to less than 2 x 10(-5). Loss of the attached HeT-A due to incomplete replication at the telomere leads to inactivation of yellow expression, which is restored by attachment of a new HeT-A element upstream of yellow. New HeT-A additions occur at a frequency of about 1.2 x 10(-3). Short DNA attachments are generated by gene conversion using the homologous telomeric sequences as templates. Longer DNA attachments are generated either by conventional transposition of an HeT-A element to the chromosomal terminus or by recombination between the 3' terminus of telomeric HeT-A elements and the receding end of HeT-A attached to the yellow gene.     

Genes encoding the small subunit of RUBISCO belong to two highly conserved subfamilies in Nicotianeae

Summary The sequences of seven complementary DNAs or genes encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase oxygenase (RUBISCO) in several Nicotianeae were examined. Two new SSU genes isolated fromNicotiana sylvestris were included. Both sequence comparisons and Southern analyses with specific probes reveal that SSU genes fall into two homogeneous subfamilies that are highly conserved in Nicotianeae and are also present in other Solanaceae. Additional criteria such as number of introns and level of expression fitted to this classification. Homogeneity must have been maintained by gene conversion and/or an unusually high fidelity of DNA replication, whereas traces of slippage-stranded DNA mispairing and/or transposition probably explain local changes. Taken as a whole, these results show that the divergence between the two subfamilies predated the divergence between genera inside the Solanaceae, but that Nicotianeae retained the most simple SSU gene family structure.