Stepwise evolution of two giant composite LTR-retrotransposon-like elements DA and Xiao

<p>Abstract</p> <p>Background</p> <p>We recently discovered two composite long terminal repeat (LTR)-retrotransposon-like elements which we named DA (~300 kb) and Xiao (~30 kb), meaning big and small in Chinese respectively. Xiao and DA (three types of DA identified) were found to have been derived from several donor sites and have spread to 30 loci in the human genome, totaling to 5 Mb. Our bioinformatics analyses with the released human, chimp, rhesus macaque, orangutan, and marmoset genomic sequences indicate that DA and Xiao emerged ~25 million years (Myr) ago.</p> <p>Results</p> <p>To better understand the evolution of these two complex elements, we investigated various internal junctions of DA and Xiao as well as orthologous genomic sites of the 30 DA/Xiao loci in non-human primates including great apes, lesser apes, Old World monkeys, New World monkeys, and a prosimian. We found that Xiao and type I DA first emerged in the genome between 25 and 18 Myr ago, whereas type II and Type III DAs emerged between 14 and 7 Myr ago. Xiao and DA were most active in great apes, with their amplification peaking during 25-14 and 14-7 Myr ago, respectively. Neither DA nor Xiao seem to have been active in the human and chimp genomes during last 6 Myr.</p> <p>Conclusion</p> <p>The study has led to a more accurate age determination of the DA and Xiao elements than our previous bioinformatics analyses, and indicates that the amplification activity of the elements coincided with that of group I HERV-Es during evolution. It has also illustrated an evolutionary path with stepwise structural changes for the elements during past 25 Myr, and in doing so has shed more light on these two intriguing and complex elements that have reshaped our genome.</p

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

<p>Abstract</p> <p>Background</p> <p><it>Manduca sexta, Heliothis virescens</it>, and <it>Heliconius erato </it>represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date.</p> <p>Results</p> <p>We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the <it>H. erato </it>genome is somewhat more abundant in repeat elements and simple repeats than those of <it>M. sexta </it>and <it>H. virescens</it>. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of <it>H. virescens </it>being the most conserved as a typical lepidopteran, whereas both genomes of <it>H. erato </it>and <it>M. sexta </it>appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences.</p> <p>Conclusion</p> <p>The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes.</p

Reaction Mechanism of (6-4) Photolyase

The (6-4) photolyase catalyzes the photoreversal of the (6-4) dipyrimidine photoproducts induced in DNA by ultraviolet light. Using the cloned Drosophila melanogaster (6-4) photolyase gene, we overproduced and purified the recombinant enzyme. The binding and catalytic properties of the enzyme were investigated using natural substrates, T[6-4]T and T[6-4]C, and the Dewar isomer of (6-4) photoproduct and substrate analogs s5T[6-4]T/thietane, mes5T[6-4]T, and the N-methyl-3'T thietane analog of the oxetane intermediate. The enzyme binds to the natural substrates and to mes5T[6-4]T with high affinity (KD approximately 10(-9)-10(-10) M) and produces a DNase I footprint of about 20 base pairs around the photolesion. Several lines of evidence suggest that upon binding by the enzyme, the photoproduct flips out of the duplex. Of the four substrates that bind with high affinity to the enzyme, T[6-4]T and T[6-4]C are repaired with relatively high quantum yields compared with the Dewar isomer and the mes5T[6-4]T which are repaired with 300-400-fold lower quantum yield than the former two photoproducts. Reduction of the FAD cofactor with dithionite increases the quantum yield of repair. Taken together, the data are consistent with photoinduced electron transfer from reduced FAD to substrate, in a manner analogous to the cyclobutane pyrimidine dimer photolyase

Non-alignment comparison of human and high primate genomes

Compositional spectra (CS) analysis based on k-mer scoring of DNA sequences was employed in this study for dot-plot comparison of human and primate genomes. The detection of extended conserved synteny regions was based on continuous fuzzy similarity rather than on chains of discrete anchors (genes or highly conserved noncoding elements). In addition to the high correspondence found in the comparisons of whole-genome sequences, a good similarity was also found after masking gene sequences, indicating that CS analysis manages to reveal phylogenetic signal in the organization of noncoding part of the genome sequences, including repetitive DNA and the genome "dark matter". Obviously, the possibility to reveal parallel ordering depends on the signal of common ancestor sequence organization varying locally along the corresponding segments of the compared genomes. We explored two sources contributing to this signal: sequence composition (GC content) and sequence organization (abundances of k-mers in the usual A,T,G,C or purine-pyrimidine alphabets). Whole-genome comparisons based on GC distribution along the analyzed sequences indeed gives reasonable results, but combining it with k-mer abundances dramatically improves the ordering quality, indicating that compositional and organizational heterogeneity comprise complementary sources of information on evolutionary conserved similarity of genome sequences

Human basal-like breast cancer is represented by one of the two mammary tumor subtypes in dogs.

BackgroundAbout 20% of breast cancers in humans are basal-like, a subtype that is often triple-negative and difficult to treat. An effective translational model for basal-like breast cancer is currently lacking and urgently needed. To determine whether spontaneous mammary tumors in pet dogs could meet this need, we subtyped canine mammary tumors and evaluated the dog-human molecular homology at the subtype level.MethodsWe subtyped 236 canine mammary tumors from 3 studies by applying various subtyping strategies on their RNA-seq data. We then performed PAM50 classification with canine tumors alone, as well as with canine tumors combined with human breast tumors. We identified feature genes for human BLBC and luminal A subtypes via machine learning and used these genes to repeat canine-alone and cross-species tumor classifications. We investigated differential gene expression, signature gene set enrichment, expression association, mutational landscape, and other features for dog-human subtype comparison.ResultsOur independent genome-wide subtyping consistently identified two molecularly distinct subtypes among the canine tumors. One subtype is mostly basal-like and clusters with human BLBC in cross-species PAM50 and feature gene classifications, while the other subtype does not cluster with any human breast cancer subtype. Furthermore, the canine basal-like subtype recaptures key molecular features (e.g., cell cycle gene upregulation, TP53 mutation) and gene expression patterns that characterize human BLBC. It is enriched in histological subtypes that match human breast cancer, unlike the other canine subtype. However, about 33% of canine basal-like tumors are estrogen receptor negative (ER-) and progesterone receptor positive (PR+), which is rare in human breast cancer. Further analysis reveals that these ER-PR+ canine tumors harbor additional basal-like features, including upregulation of genes of interferon-γ response and of the Wnt-pluripotency pathway. Interestingly, we observed an association of PGR expression with gene silencing in all canine tumors and with the expression of T cell exhaustion markers (e.g., PDCD1) in ER-PR+ canine tumors.ConclusionsWe identify a canine mammary tumor subtype that molecularly resembles human BLBC overall and thus could serve as a vital translational model of this devastating breast cancer subtype. Our study also sheds light on the dog-human difference in the mammary tumor histology and the hormonal cycle

Frequent Alteration of the Tumor Suppressor Gene APC in Sporadic Canine Colorectal Tumors

Sporadic canine colorectal cancers (CRCs) should make excellent models for studying the corresponding human cancers. To molecularly characterize canine CRC, we investigated exonic sequence mutations of adenomatous polyposis coli (APC), the best known tumor suppressor gene of human CRC, in 23 sporadic canine colorectal tumors, including 8 adenomas and 15 adenocarcinomas, via exon-resequencing analysis. As a comparison, we also performed the same sequencing analysis on 10 other genes, either located at human 5q22 (the same locus as APC) or 18q21 (also frequently altered in human CRC), or known to play a role in human carcinogenesis. We noted that APC was the most significantly mutated gene in both canine adenomas and adenocarcinomas among the 11 genes examined. Significantly, we detected large deletions of ≥10 bases, many clustered near the mutation cluster region, as well as single or two base deletions in ~70% canine tumors of both subtypes. These observations indicate that like in the human, APC is also frequently altered in sporadic colorectal tumors in the dog and its alteration is an early event in canine colorectal tumorigenesis. Our study provides further evidence demonstrating the molecular similarity in pathogenesis between sporadic human and canine CRCs. This work, along with our previous copy number abnormality study, supports that sporadic canine CRCs are valid models of human CRCs at the molecular level

Using a Pericentromeric Interspersed Repeat to Recapitulate the Phylogeny and Expansion of Human Centromeric Segmental Duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome

Using BAC-end sequences, a sparse marker map and the sequences of the human, dog and cow genomes, an accurate and detailed sub-gene level map of the sheep genome has been constructed

Mechanistic enzymology of a methyltetrahydrofolate: Corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum of the acetyl-CoA pathway

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N\sp5-methyl group from (6S)-methyltetrahydrofolate (CH\sb3-H\sb4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP) in the acetyl-CoA pathway of anaerobic CO\sb2 fixation. This methyl transfer is similar to the first half reaction of cobalamin-dependent methionine synthase and a N\sp5-methyltetrahydromethanopterin:coenzyme M methyltransferase in methanogens. Studying the MeTr reaction is important to further our understanding of CO/CO\sb2 fixation under anaerobic conditions, and of one-carbon transfer reactions in eucaryotes and in methanogens. In order to elucidate the enzymatic mechanism of MeTr, presteady-state kinetics, steady-state kinetics, binding studies, spectroscopic and redox titration experiments were performed. Presteady-state data indicate that the Co(I)-C/Fe-SP performs a direct S\rm\sb{N}2 displacement of the methyl group of CH\sb3-H\sb4folate to form H\sb4folate and methyl-Co(III) without the intermediacy of Co(II). The kinetic mechanism involves a ternary complex in which substrates bind in a random order and independent of each other. Product inhibition experiments indicate a rapid equilibrium random Bi Bi mechanism suggesting substrate binding and product release are fast. The kinetic constants were: k\rm\sb{cat}=13.5\pm0.9s\sp{-1} (at 25\sp\circC), K\rm\sb{m} for the C/Fe-SP = 69.0 $\pm$ 8.6 $\mu$M and K\rm\sb{m} for (6S)-CH\sb3-H\sb4folate = 5.0 $\pm$ 0.6 $\mu$M. Methyl transfer to the C/Fe-SP is pH dependent. Both the forward and reverse reactions were fast at low pH with pK\rm\sb{a}\u27s ranging from 5.0 to 5.7. A different pH profile was obtained when free cobalamin was the methyl acceptor. The kinetic pK\rm\sb{a} values closely matched the pK\rm\sb{a} for the N\sp5 group of CH\sb3-H\sb4folate (pK\rm\sb{a} = 5.0); however, the experimental evidence indicates that the pH dependence appears to result from a pH-dependent MeTr conformational change (pK\rm\sb{a} = of 5.1). The conformational change is catalytically competent (k 40 s\sp{-1}) and partially rate-limiting. The hydrophobic interactions between MeTr and the C/Fe-SP appeared to increase the reduction potential of Co(II)/(I) couple by 50 mV and accelerate the reaction. A reaction scheme is proposed which involves two partially rate-limiting steps. At high pH, the conformational change and the interconversion of the ternary complexes (chemical step) control the rate and, at low pH, the interconversion of the ternary complexes primarily controls the rate. Substrate binding and product release are fast