Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Selfish conflict underlies RNA-mediated parent-of-origin effects.

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.

Higher evolutionary dynamics of gene copy number for Drosophila glue genes located near short repeat sequences.

BACKGROUND: During evolution, genes can experience duplications, losses, inversions and gene conversions. Why certain genes are more dynamic than others is poorly understood. Here we examine how several Sgs genes encoding glue proteins, which make up a bioadhesive that sticks the animal during metamorphosis, have evolved in Drosophila species. RESULTS: We examined high-quality genome assemblies of 24 Drosophila species to study the evolutionary dynamics of four glue genes that are present in D. melanogaster and are part of the same gene family - Sgs1, Sgs3, Sgs7 and Sgs8 - across approximately 30 millions of years. We annotated a total of 102 Sgs genes and grouped them into 4 subfamilies. We present here a new nomenclature for these Sgs genes based on protein sequence conservation, genomic location and presence/absence of internal repeats. Two types of glue genes were uncovered. The first category (Sgs1, Sgs3x, Sgs3e) showed a few gene losses but no duplication, no local inversion and no gene conversion. The second group (Sgs3b, Sgs7, Sgs8) exhibited multiple events of gene losses, gene duplications, local inversions and gene conversions. Our data suggest that the presence of short "new glue" genes near the genes of the latter group may have accelerated their dynamics. CONCLUSIONS: Our comparative analysis suggests that the evolutionary dynamics of glue genes is influenced by genomic context. Our molecular, phylogenetic and comparative analysis of the four glue genes Sgs1, Sgs3, Sgs7 and Sgs8 provides the foundation for investigating the role of the various glue genes during Drosophila life.

Tigerfish designs oligonucleotide-based in situ hybridization probes targeting intervals of highly repetitive DNA at the scale of genomes.

Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.

Cloning and expansion of repetitive DNA sequences.

Repeat and structure-prone DNA sequences comprise a large proportion of the human genome. The instability of these sequences has been implicated in a range of diseases, including cancers and neurodegenerative disorders. However, the mechanism of pathogenicity is poorly understood. As such, further studies on repetitive DNA are required. Cloning and maintaining repeat-containing substrates is challenging due to their inherent ability to form non-B DNA secondary structures which are refractory to DNA polymerases and prone to undergo rearrangements. Here, we describe an approach to clone and expand tandem-repeat DNA without interruptions, thereby allowing for its manipulation and subsequent investigation.

X Chromosome-Specific Repeats in Non-Domestic Bovidae.

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies.

Dimeric structures of DNA ATTTC repeats promoted by divalent cations.

Structural studies of repetitive DNA sequences may provide insights why and how certain repeat instabilities in their number and nucleotide sequence are managed or even required for normal cell physiology, while genomic variability associated with repeat expansions may also be disease-causing. The pentanucleotide ATTTC repeats occur in hundreds of genes important for various cellular processes, while their insertion and expansion in noncoding regions are associated with neurodegeneration, particularly with subtypes of spinocerebellar ataxia and familial adult myoclonic epilepsy. We describe a new striking domain-swapped DNA-DNA interaction triggered by the addition of divalent cations, including Mg2+ and Ca2+. The results of NMR characterization of d(ATTTC)3 in solution show that the oligonucleotide folds into a novel 3D architecture with two central C:C+ base pairs sandwiched between a couple of T:T base pairs. This structural element, referred to here as the TCCTzip, is characterized by intercalative hydrogen-bonding, while the nucleobase moieties are poorly stacked. The 5'- and 3'-ends of TCCTzip motif are connected by stem-loop segments characterized by A:T base pairs and stacking interactions. Insights embodied in the non-canonical DNA structure are expected to advance our understanding of why only certain pyrimidine-rich DNA repeats appear to be pathogenic, while others can occur in the human genome without any harmful consequences.

Structure, Topology, and Stability of Multiple G-quadruplexes in Long Telomeric Overhangs.

Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.

Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Diversity and Evolution of Highly Repetitive DNA Sequences Constituting Chromosome Site-Specific Heterochromatin in Two Gerbillinae Species.

Constitutive heterochromatin, consisting of repetitive sequences, diverges very rapidly; therefore, its nucleotide sequences and chromosomal distributions are often largely different, even between closely related species. The chromosome C-banding patterns of two Gerbillinae species, Meriones unguiculatus and Gerbillus perpallidus, vary greatly, even though they belong to the same subfamily. To understand the evolution of C-positive heterochromatin in these species, we isolated highly repetitive sequences, determined their nucleotide sequences, and characterized them using chromosomal and filter hybridization. We obtained a centromeric repeat (MUN-HaeIII) and a chromosome 13-specific repeat (MUN-EcoRI) from M. unguiculatus. We also isolated a centromeric/pericentromeric repeat (GPE-MBD) and an interspersed-type repeat that was predominantly amplified in the X and Y chromosomes (GPE-EcoRI) from G. perpallidus. GPE-MBD was found to contain a 17-bp motif that is essential for binding to the centromere-associated protein CENP-B. This indicates that it may play a role in the formation of a specified structure and/or function of centromeres. The nucleotide sequences of the three sequence families, except GPE-EcoRI, were conserved only in Gerbillinae. GPE-EcoRI was derived from the long interspersed nuclear elements 1 retrotransposon and showed sequence homology throughout Muridae and Cricetidae species, indicating that the repeat sequence occurred at least in the common ancestor of Muridae and Cricetidae. Due to a lack of assembly data of highly repetitive sequences constituting heterochromatin in whole-genome sequences of vertebrate species published to date, the knowledge obtained in this study provides useful information for a deep understanding of the evolution of repetitive sequences in not only rodents but also in mammals.

Short Interrupted Repeat Cassette (SIRC)-Novel Type of Repetitive DNA Element Found in Arabidopsis thaliana.

Short interrupted repeat cassette (SIRC)-a novel DNA element found throughout the A. thaliana nuclear genome. SIRCs are represented by short direct repeats interrupted by diverse DNA sequences. The maxima of SIRC's distribution are located within pericentromeric regions. We suggest that originally SIRC was a special case of the complex internal structure of the miniature inverted repeat transposable element (MITE), and further MITE amplification, transposition, and loss of terminal inverted repeats gave rise to SIRC as an independent DNA element. SIRC sites were significantly enriched with several histone modifications associated with constitutive heterochromatin and mobile genetic elements. The majority of DNA-binding proteins, strongly associated with SIRC, are related to histone modifications for transcription repression. A part of SIRC was found to overlap highly inducible protein-coding genes, suggesting a possible regulatory role for these elements, yet their definitive functions need further investigation.

Evolution of the spinach sex-linked region within a rarely recombining pericentromeric region.

Sex chromosomes have evolved independently in many different plant lineages. Here, we describe reference genomes for spinach (Spinacia oleracea) X and Y haplotypes by sequencing homozygous XX females and YY males. The long arm of 185-Mb chromosome 4 carries a 13-Mb X-linked region (XLR) and 24.1-Mb Y-linked region (YLR), of which 10 Mb is Y specific. We describe evidence that this reflects insertions of autosomal sequences creating a "Y duplication region" or "YDR" whose presence probably directly reduces genetic recombination in the immediately flanking regions, although both the X and Y sex-linked regions are within a large pericentromeric region of chromosome 4 that recombines rarely in meiosis of both sexes. Sequence divergence estimates using synonymous sites indicate that YDR genes started diverging from their likely autosomal progenitors about 3 MYA, around the time when the flanking YLR stopped recombining with the XLR. These flanking regions have a higher density of repetitive sequences in the YY than the XX assembly and include slightly more pseudogenes compared with the XLR, and the YLR has lost about 11% of the ancestral genes, suggesting some degeneration. Insertion of a male-determining factor would have caused Y linkage across the entire pericentromeric region, creating physically small, highly recombining, terminal pseudoautosomal regions. These findings provide a broader understanding of the origin of sex chromosomes in spinach.

Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

A complete telomere-to-telomere assembly of the maize genome.

A complete telomere-to-telomere (T2T) finished genome has been the long pursuit of genomic research. Through generating deep coverage ultralong Oxford Nanopore Technology (ONT) and PacBio HiFi reads, we report here a complete genome assembly of maize with each chromosome entirely traversed in a single contig. The 2,178.6 Mb T2T Mo17 genome with a base accuracy of over 99.99% unveiled the structural features of all repetitive regions of the genome. There were several super-long simple-sequence-repeat arrays having consecutive thymine-adenine-guanine (TAG) tri-nucleotide repeats up to 235 kb. The assembly of the entire nucleolar organizer region of the 26.8 Mb array with 2,974 45S rDNA copies revealed the enormously complex patterns of rDNA duplications and transposon insertions. Additionally, complete assemblies of all ten centromeres enabled us to precisely dissect the repeat compositions of both CentC-rich and CentC-poor centromeres. The complete Mo17 genome represents a major step forward in understanding the complexity of the highly recalcitrant repetitive regions of higher plant genomes.

Characterization of repetitive DNA on the genome of the marsh rat Holochilus nanus (Cricetidae: Sigmodontinae).

Repetitive DNA are sequences repeated hundreds or thousands of times and an abundant part of eukaryotic genomes. SatDNA represents the majority of the repetitive sequences, followed by transposable elements. The species Holochilus nanus (HNA) belongs to the rodent tribe Oryzomyini, the most taxonomically diverse of Sigmodontinae subfamily. Cytogenetic studies on Oryzomyini reflect such diversity by revealing an exceptional range of karyotype variability. However, little is known about the repetitive DNA content and its involvement in chromosomal diversification of these species. In the search for a more detailed understanding about the composition of repetitive DNA on the genome of HNA and other species of Oryzomyini, we employed a combination of bioinformatic, cytogenetic and molecular techniques to characterize the repetitive DNA content of these species. RepeatExplorer analysis showed that almost half of repetitive content of HNA genome are composed by Long Terminal Repeats and a less significant portion are composed by Short Interspersed Nuclear Elements and Long Interspersed Nuclear Elements. RepeatMasker showed that more than 30% of HNA genome are composed by repetitive sequences, with two main waves of repetitive element insertion. It was also possible to identify a satellite DNA sequence present in the centromeric region of Oryzomyini species, and a repetitive sequence enriched on the long arm of HNA X chromosome. Also, comparative analysis between HNA genome with and without B chromosome did not evidence any repeat element enriched on the supernumerary, suggesting that B chromosome of HNA is composed by a fraction of repeats from all the genome.

Assembly of the Complete Mitochondrial Genome of Pereskia aculeata Revealed That Two Pairs of Repetitive Elements Mediated the Recombination of the Genome.

Pereskia aculeata is a potential new crop species that has both food and medicinal (antinociceptive activity) properties. However, comprehensive genomic research on P. aculeata is still lacking, particularly concerning its organelle genome. In this study, P. aculeata was studied to sequence the mitochondrial genome (mitogenome) and to ascertain the assembly, informational content, and developmental expression of the mitogenome. The findings revealed that the mitogenome of P. aculeata is circular and measures 515,187 bp in length with a GC content of 44.05%. It contains 52 unique genes, including 33 protein-coding genes, 19 tRNA genes, and three rRNA genes. Additionally, the mitogenome analysis identified 165 SSRs, primarily consisting of tetra-nucleotides, and 421 pairs of dispersed repeats with lengths greater than or equal to 30, which were mainly forward repeats. Based on long reads and PCR experiments, we confirmed that two pairs of long-fragment repetitive elements were highly involved with the mitogenome recombination process. Furthermore, there were 38 homologous fragments detected between the mitogenome and chloroplast genome, and the longest fragment was 3962 bp. This is the first report on the mitogenome in the family Cactaceae. The decoding of the mitogenome of P. aculeata will provide important genetic materials for phylogenetic studies of Cactaceae and promote the utilization of species germplasm resources.

Long interspersed nuclear element 1 and B1/Alu repeats blueprint genome compartmentalization.

The organization of the genome into euchromatin and heterochromatin has been known for almost 100 years [1]. More than 50% of mammalian genomes contain repetitive sequences [2,3]. Recently, a functional link between the genome and its folding has been identified [4,5]. Homotypic clustering of long interspersed nuclear element 1 (LINE1 or L1) and B1/Alu retrotransposons forms grossly exclusive nuclear domains that characterize and predict heterochromatin and euchromatin, respectively. The spatial segregation of L1 and B1/Alu-rich compartments is conserved in mammalian cells and can be rebuilt during the cell cycle and established de novo in early embryogenesis. Inhibition of L1 RNA drastically weakened homotypic repeat contacts and compartmental segregation, indicating that L1 plays a more significant role than just being a compartmental marker. This simple and inclusive genetic coding model of L1 and B1/Alu in shaping the macroscopic structure of the genome provides a plausible explanation for the remarkable conservation and robustness of its folding in mammalian cells. It also proposes a conserved core structure on which subsequent dynamic regulation takes place.

A new type of tandem repeats in Myotis petax (Chiroptera, Vespertilionidae) mitochondrial control region.

BACKGROUND: Tandem repeats in mitochondrial DNA control region are known to different animal taxa, including bat species of the family Vespertilionidae. The long R1-repeats in the bat ETAS-domain are often presented in a variable copy number and may exhibit both inter-individual and intra-individual sequence diversity. The function of repeats in the control region is still unclear, but it has been shown that repetitive sequences in some animal groups (shrews, cats and sheep) may include parts of ETAS1 and ETAS2 conservative blocks of mitochondrial DNA. METHODS AND RESULTS: Analysis of the control region sequences for 31 Myotis petax specimens allowed the identification of the inter-individual variability and clarification of the composition of the R1-repeats. The copy number of the R1-repeats varies from 4 to 7 in individuals. The specimens examined do not exhibit a size heteroplasmy previously described for Myotis species. The unusual short 30 bp R1-repeats have been detected in M. petax for the first time. The ten specimens from Amur Region and Primorsky Territory have one or two copies of these additional repeats. CONCLUSIONS: It was determined that the R1-repeats in M. petax control region consist of parts of the ETAS1 and ETAS2 blocks. The origin of the additional repeats seems to be related to the 51 bp deletion in the central part of the R1-repeat unit and subsequent duplication. Comparison of repetitive sequences in the control region of closely-related Myotis species identified the occurrence of incomplete repeats also resulting from the short deletions, but distinct from additional repeats of M. petax.

RNA-mediated heterochromatin formation at repetitive elements in mammals.

The failure to repress transcription of repetitive genomic elements can lead to catastrophic genome instability and is associated with various human diseases. As such, multiple parallel mechanisms cooperate to ensure repression and heterochromatinization of these elements, especially during germline development and early embryogenesis. A vital question in the field is how specificity in establishing heterochromatin at repetitive elements is achieved. Apart from trans-acting protein factors, recent evidence points to a role of different RNA species in targeting repressive histone marks and DNA methylation to these sites in mammals. Here, we review recent discoveries on this topic and predominantly focus on the role of RNA methylation, piRNAs, and other localized satellite RNAs.

A Guide to Sequencing for Long Repetitive Regions.

Full-length analysis of genes with highly repetitive sequences is challenging in two respects: assembly algorithm and sequencing accuracy. The de Bruijn graph often used in short-read assembly cannot distinguish adjacent repeat units. On the other hand, the accuracy of long reads is not yet high enough to identify each and every repeat unit. In this chapter, I present an example of a strategy to solve these problems and obtain the full length of long repeats by combining the extraction and assembly of repeat units based on overlap-layout-consensus and scaffolding by long reads.