DNA Sequence Research Articles

William Bateson, black slavery, eugenics and speciation: The relative roles of politics and science

The peace of the world is challenged by societal confrontations that can often be labeled "racial" or "ethnic." Emblematic of this is discrimination based on skin colour. William Bateson's background suggests sympathy with the black emancipation movement. Yet the movement's success is attributed more to battles between political figures than between scientists with contending views on the biology of racial differences. However, in the long term, Bateson's contributions to slavery and eugenic issues may be seen as no less important than those of politicians. Mendel's discovery of what we now know as "genes" languished until seized upon by Bateson in 1900. For six exhausting years he struggled to win scientific acceptance of these biological character-determining units. Later, he pressed the Mendelian message home to the general public, opposing simplistic applications of Mendelian principles to human affairs, and arguing that minor genic differences that distinguished "races" - e.g. skin colour - do not initiate new species. Bateson praised the "physiological selection" speciation hypothesis of Darwin's young research associate, George Romanes. This enthusiasm was rekindled by Robert Lock and formulated in modern terms with C. R. Crowther. Thus, the spark that initiates a divergence into two species can be non-genic. This normal form of hybrid sterility, based on genome-wide DNA sequence differences, operates on, but has not succeeded in dividing, the human species. It should not be labeled "idiopathic," and be clearly distinguished both from pathological sterility and undiagnosed sterilities that may prove to be pathological. We are one reproductively isolated population, the human species.

Mechanistic Insight Into the Conformational Changes of Cas8 Upon Binding to Different PAM Sequences in the Transposon-Encoded Type I-F CRISPR-Cas System.

The INTEGRATE system is a gene-editing approach that offers advantages over the widely used CRISPR-Cas9 system. It does not introduce double strand breaks in the target DNA but rather integrates the desired DNA sequence directly into it. The first step in the integration process is PAM recognition, which is critical to understanding and optimizing the system. Experimental testing revealed varying integration efficiencies of different PAM mutants, and computational simulations were carried out to gain mechanistic insight into the conformational changes of Cas8 during PAM recognition. Our results showed that the interaction between Arg246 and guanine at position (-1) of the target strand is critical for PAM recognition. We found that unfavorable interactions in the 5'-AC-3' PAM mutant disrupted this interaction and may be responsible for its 0% integration efficiency. Additionally, we discovered that PAM sequences not only initiate the integration process but also regulate it through an allosteric mechanism that connects the N-terminal domain and the helical bundle of Cas8. This allosteric regulation was present in all PAMs tested, even those with lower integration efficiencies, such as 5'-TC-3' and 5'-AC-3'. We identified the Cas8 residues that are involved in this regulation. Our findings provide valuable insights into PAM recognition mechanisms in the INTEGRATE system and can help improve the gene-editing technology.

Competitive Binding Strategy for Reliable Signal-off Surface Enhanced Raman Scattering Sensing in Protecting Apples from Patulin without External Interference

Patulin is a toxin that can be found in damaged or moldy fruits, especially apples. Very high detection sensitivity can be achieved using the SERS (Surface Enhanced Raman Scattering) technique, which provides information about molecular vibrations. However, unexpected desorption of target toxins from the surface of the SERS substrate and interference from other substances pose a major challenge. Here we investigate a competitive binding strategy for reliable SERS analysis of PAT. Core-Raman molecule-shell (CMS) structures and programmable, specific DNA interactions are utilized to regulate Raman signal intensity without affecting the embedded Raman molecules. Specfically, aptamer-functionalized CMS structures are combined with metal-organic scaffold nanoparticles that possess complementary DNA sequences. In the presence of PAT, binding of the aptamer to the toxin leads to degradation of the hybrid nano assembly, which can be measured by a change in the SERS signal. This SERS strategy enables turn-off sensing in apple juice over a wide range (10pg/mL to 1μg/mL), with a detection limit as low as 0.02ng/mL. Analysis of real samples gives results consistent with high-performance liquid chromatography (HPLC) test. The excellent selectivity and sensitivity of the SERS sensor demonstrate its potential for a wide range of SERS sensor applications.

The impact of DNA point mutations and epigenetic modifications on the nucleosome wrapping energy

Nucleosome positioning and mobility, which plays an important role in gene expression and regulation, is in turn modulated by DNA sequence and its underlying mechanical properties. The free energy, required to deform a 147 bp linear DNA fragment into a nucleosomal configuration, is a way to quantify DNA mechanical propensity for nucleosome formation. This work explores how such energy, referred to as the nucleosome wrapping energy, is altered by DNA sequence mutations and epigenetic modifications. The nucleosome wrapping energy is computed using a newly developed computational method based on minimising the cgNA+ coarse-grain model energy with elastic constraints on phosphate positions. We find that the effect of sequence mutations and epigenetic modifications is highly dependent on the positions along the sequence of the modified bases. Most point mutations as well as cytosine methylation and hydroxymethylation, depending on their positions, can lead both to an increase and decrease in the nucleosome wrapping energy. We use the data of mutation-caused energy changes to identify sequence patterns in specific positions, leading to the smallest and highest wrapping energy values. Furthermore, we construct sequences corresponding to extremely low and high nucleosome wrapping energies, compared to energy distribution for human genome sequences. We believe that our findings are helpful for better understanding the role of DNA sequence and its epigenetic modifications in nucleosome positioning. Furthermore, our constructed minimum energy sequence could be used as a candidate for producing stable synthetic nucleosomes.

Design of a Golden Gate Cloning Plasmid for the Generation of a Chimeric Virus-Like Particle-Based Subunit Vaccine Against Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is a pervasive pathogen in the swine industry, leading to a spectrum of disorders known as porcine circovirus associated diseases (PCVAD). The PCV2 Cap protein contains critical antigenic epitopes and is the primary target for vaccine development. Current vaccines include inactivated viral particles and virus-like particles (VLPs), with experimental vaccines exploring various innovative approaches. This study introduces a novel Golden Gate assembly-based, Escherichia coli expression destination plasmid, pPRSVCP-E18, designed for the in-frame insertion of short peptide-coding DNA sequences into a highly antigenic site of Papaya ringspot virus (PRSV) coat protein (CP). We successfully cloned four PCV2 Cap peptides into this plasmid, expressed the chimeric CP proteins in E. coli under flask and bioreactor conditions, and assembled them into VLPs. These VLPs, when adjuvated and administered to BALB/c mice, elicited a specific IgG response against the PCV2 Cap peptides. Our findings demonstrate the plasmid's high efficiency for Golden Gate cloning and its potential in developing subunit vaccines against PCV2, contributing to the sustainable control of PCVAD in the swine industry.

Whole exome sequencing revealed ultra-rare genetic variations in juvenile myoclonic epilepsy.

Juvenile Myoclonic Epilepsy (JME) is the most common adolescent and adult-onset genetic generalized epilepsy. In this study, we aimed to identify all rare variants present in exons and exon-intron junctions in patients who met the criteria of JME, determine potentially pathogenic variants, and find the assumed genotype/phenotype correlation between the identified variants and the JME clinical features. Whole Exome Sequencing (WES) was performed for ten JME patients from different families. Validation, co-segregation and mode of inheritance were determined using Sanger DNA sequencing. Predictable damaging variants were found in six families with positive co-segregation. Eight variants in eight genes (SCN1B, KCNQ2, CACNA1I, GABRA3, BSN, RYR3, SEZ6, and RYR2) and one novel variant in (TNR) gene were found to be associated with JME. All these genes play key roles in the interactions between neurons, neurotransmitter release, and maintenance of the balance between neuronal excitation and inhibition. Since the identified genes are involved in the molecular mechanisms underlying seizures, such variants can potentially be epileptogenic. In conclusion, the identified variants that co-segregate with JME symptoms and likely contribute in creating the adequate genetic background for the JME phenotype.

Phylogenomic analyses unravel the tangled evolutionary history of Genisteae (Fabaceae)

Genisteae, a tribe in the subfamily Papilionoideae (Fabaceae), is characterized by the production of quinolizidine alkaloids that confer pest resistance in most of its members. Many relationships at the generic level remain unresolved due largely to a lack of modern attempts to reconstruct the phylogeny. Previous studies with limited taxon sampling and only a few molecular loci indicated the presence of three clades within the tribe: the Lupinus clade, the Cytisus-Genista complex and the Argyrolobium group. There are also two historical genera, Teline and Chamaecytisus, that have been reclassified over the years with some controversy. Species from Teline are currently classified in Genista, and Chamaecytisus species are placed inside of Cytisus. Sellocharis is another genus with vague placement inside of Genisteae near Anarthrophyllum, based mostly on morphology and cytology. Representative taxa from 24 of the 25 genera of Genisteae, along with species of historical genera Teline and Chamaecytisus, were sampled and utilized in a phylogenomic investigation using both plastid and nuclear data to resolve relationships at the generic level. Whole genomic DNA was sequenced and complete plastomes were assembled and annotated. Low-copy nuclear genes were retrieved from the genomic DNA sequences using a mapping-assembly-scaffold approach. Phylogenetic analyses using maximum likelihood, Bayesian and coalescence methods resulted in fully resolved and strongly supported trees for both nuclear and plastid data that show four major clades inside of Genisteae: Cytisus-Genista complex, Lupinus clade, Argyrolobium group and the novel Anarthrophyllum group. The resulting phylogenetic trees also supported the transfer of the Argyrolobium group from Crotalarieae to Genisteae, and the reclassification of Teline inside of Genista, both of which were previously suggested in literature. The phylogenetic trees also determined a placement for Sellocharis in the new Anarthrophyllum group. Although both nuclear and plastid trees were congruent with respect to the monophyly and relationships among the four major clades, incongruence was detected within some of the major clades and the potential causes are discussed.

First report of Hepatozoon and Lankesterella spp. infections in wild rodents from Pakistan, and their potential impact on blood parameters and oxidative stress markers in vital organs.

Pakistan is home to a rich rodent fauna, yet no investigations have been conducted on the presence of Haemogregarines in these animals, leaving their epidemiology and genetic diversity in this subtropical region unexplored. In this study, blood samples from four wild rodent species, Rattus (R.) rattus (n = 122), Mus (M.) musculus (n = 64), Rattus norvegicus (n = 57), and Dryomys nitedula (n = 1), were collected between May 2022 and July 2023 from three districts in Punjab and three in Khyber Pakhtunkhwa. PCR targeting a fragment of 600 base pairs from the 18S rDNA gene revealed a 2.86% (7/244) prevalence of Haemogregarines in the rodents. DNA sequencing and BLAST analysis confirmed the presence of Hepatozoon spp. and Lankesterella spp. in the blood samples. Phylogenetic analysis indicated genetic diversity in the Pakistani sequences, which clustered with sequences found in reptiles, amphibians, birds, and mammals from various countries. The prevalence of the parasite varied among rodent species, with R. rattus showing the highest infection rate, followed by R. norvegicus and M. musculus. Female rodents were more frequently infected than males. Infected R. rattus exhibited significant disruptions in white blood cells, red blood cells, and platelet counts. Oxidative stress markers indicated elevated superoxide dismutase in the kidney, catalase in the heart, and malondialdehyde in the liver and lungs of infected rodents compared to uninfected ones. This study is an important contribution towards science as it the first report of Haemogregarines and Lankesterella spp. infections among Pakistani rodents.

Characterizing the evolutionary dynamics of cancer proliferation in single-cell clones with SPRINTER.

Proliferation is a key hallmark of cancer, but whether it differs between evolutionarily distinct clones co-existing within a tumor is unknown. We introduce the Single-cell Proliferation Rate Inference in Non-homogeneous Tumors through Evolutionary Routes (SPRINTER) algorithm that uses single-cell whole-genome DNA sequencing data to enable accurate identification and clone assignment of S- and G2-phase cells, as assessed by generating accurate ground truth data. Applied to a newly generated longitudinal, primary-metastasis-matched dataset of 14,994 non-small cell lung cancer cells, SPRINTER revealed widespread clone proliferation heterogeneity, orthogonally supported by Ki-67 staining, nuclei imaging and clinical imaging. We further demonstrated that high-proliferation clones have increased metastatic seeding potential, increased circulating tumor DNA shedding and clone-specific altered replication timing in proliferation- or metastasis-related genes associated with expression changes. Applied to previously generated datasets of 61,914 breast and ovarian cancer cells, SPRINTER revealed increased single-cell rates of different genomic variants and enrichment of proliferation-related gene amplifications in high-proliferation clones.

Construction of a high-density genetic map and QTL mapping of growth traits in kiwifruit

The trunk diameter (TD) and annual branch diameter (ABD) are closely associated with the growth vigor, abiotic resistance and fruit yield of kiwifruit. These traits are quantitative traits that are controlled by more than one quantitative trait loci (QTLs). However, QTLs for TD and ABD in kiwifruit remain unelucidated. In this study, we generated a cross of the diploid species Actinidia chinensis ‘Hongyang’ (♀) × ‘Boshanbiyu’ (♂) and performed restriction-site associated DNA sequencing on the parents and 173 F1 progeny to identify single-nucleotide polymorphisms (SNPs) in the population. A high-density linkage map spanning 2,548.97 cM was developed using 6,506 bin markers. This bin map consisted of 29 linkage group (LG), with an average genetic distance of 0.39 cM. A total of 8 QTLs were identified: four for TD and four for ABD, with the variance explained by these QTLs ranging from 4.43 % to 16.11 %. A total of 358 TD genes and 181 ABD genes were extracted from the mapped QTLs, and analyzed by KEGG enrichment. In addition, we developed a TD marker from LG23 and an ABD marker from LG10 which were significantly correlated with growth traits. The TD marker is associated with Actinidia22292, a gene involved in protein polar localization, while the ABD marker is linked to Actinidia40478, an Ein3-binding F-box protein. The two SNP markers developed in this study were further validated in a biparental population, indicating their potential use in predicting seedling growth vigor. In summary, these genetic findings deepen our understanding of the genetic basis of growth traits in kiwifruit, and provide valuable information to facilitate kiwifruit breeding.

Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification.

Polymerase Chain Reaction (PCR) requires thermal cycling to melt DNA and proceed through the subsequent cycles of DNA synthesis needed for exponential amplification. Previously, we engineered a superhelicase, with enhanced processivity and speed, to replace this traditional PCR melting step with enzymatic DNA unwinding while retaining desired PCR characteristics, such as multi-kb amplicon size and applicability to cloning and gene editing outcome assessment. This isothermal amplification method is named SHARP (SSB-Helicase Assisted Rapid PCR) because single-stranded DNA binding protein (SSB) and superhelicases are added to standard PCR reagents. Here, we show that SHARP can be effective for DNA sequences that PCR is unable to amplify or that produce side products of. SHARP is demonstrated to be capable of amplifying up to six identical repeats of the Widom 601 nucleosome positioning sequence and up to 35 identical repeats of ankyrin sequence. We also show that a sequence with 91% AT-content can be amplified using SHARP and that the amplification product can be validated using single-molecule optical tweezers experiments.

The Species of the mali-Group of Aphelinus (Hymenoptera: Aphelinidae), with Descriptions of Three New Species, DNA Sequence Data and One Newly-Recorded Species from China

Seventeen species of the mali-group of Aphelinus (Hymenoptera: Aphelinidae) are reviewed worldwide, including three new species, and five previously described species in China. The mali-group was defined by a combination of characters: head and body dark with parts of metasoma pale; fore wing with a complete row of setae inside the linea calva, or also only with a few setae in the angle between the linea and marginal vein; legs with mesocoxae, metacoxae and metatibia dark, metafemur pale. In this paper, three new species, Aphelinus tuberocephalus Wang & Huang, sp.n., Aphelinus hainanensis Wang & Huang, sp.n. and Aphelinus ruellia Wang & Huang, sp.n., are described and illustrated, with notes on one species, Aphelinus coreae Hopper & Woolley, new to China. A revised key to species of the mali-group is provided. Mitochondrial (COI) partial sequences were obtained successfully for A. tuberocephalus, A. hainanensis, A. ruellia and A. coreae.

Hormone response elements for the thyroid receptor-α include specific distal 5'-flanking DNA.

Optimal gene transcription is achieved through precise interactions between transcription factors and their DNA binding sites. We provide evidence that conserved distally located 5'-flanking sequences interact directly with the intrinsically disordered amino-terminal region of the thyroid receptor-α (TRα) to control transcriptional activity. Simulated modeling and dynamics with multiple ChIP-seq-derived sequences consistently reveal specific lysine/arginine-DNA minor groove interactions. The impact of these interactions is to distort DNA structural conformations, and these are also revealed with atomic force microscopy. The importance of the 5'-flanking DNA is further emphasized with reporter gene assays and comparisons with canonical response elements. Overall, the study reveals the inadequacy of current definitions of the DNA hormone response element (HRE) and suggests that future descriptions of the HRE include the conserved distal DNA sequences. The broad impact of this study is further underscored by the common occurrence of Lys/Arg-rich motifs within the intrinsically disordered regions of nuclear receptors.

DNA sequence and lesion-dependent mitochondrial transcription factor A (TFAM)-DNA-binding modulates DNA repair activities and products.

Mitochondrial DNA (mtDNA) is indispensable for mitochondrial function and is maintained by DNA repair, turnover, mitochondrial dynamics and mitophagy, along with the inherent redundancy of mtDNA. Base excision repair (BER) is a major DNA repair mechanism in mammalian mitochondria. Mitochondrial BER enzymes are implicated in mtDNA-mediated immune response and inflammation. mtDNA is organized into mitochondrial nucleoids by mitochondrial transcription factor A (TFAM). The regulation of DNA repair activities by TFAM-DNA interactions remains understudied. Here, we demonstrate the modulation of DNA repair enzymes by TFAM concentrations, DNA sequences and DNA modifications. Unlike previously reported inhibitory effects, we observed that human uracil-DNA glycosylase 1 (UNG1) and AP endonuclease I (APE1) have optimal activities at specific TFAM/DNA molar ratios. High TFAM/DNA ratios inhibited other enzymes, OGG1 and AAG. In addition, TFAM reduces the accumulation of certain repair intermediates. Molecular dynamics simulations and DNA-binding experiments demonstrate that the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in certain sequence motifs enhances TFAM-DNA binding, partially explaining the inhibition of OGG1 activity. Bioinformatic analysis of published 8-oxodG, dU, and TFAM-footprint maps reveals a correlation between 8-oxodG and TFAM locations in mtDNA. Collectively, these results highlight the complex regulation of mtDNA repair by DNA sequence, TFAM concentrations, lesions and repair enzymes.

Nucleotide Transformer: building and evaluating robust foundation models for human genomics.

The prediction of molecular phenotypes from DNA sequences remains a longstanding challenge in genomics, often driven by limited annotated data and the inability to transfer learnings between tasks. Here, we present an extensive study of foundation models pre-trained on DNA sequences, named Nucleotide Transformer, ranging from 50 million up to 2.5 billion parameters and integrating information from 3,202 human genomes and 850 genomes from diverse species. These transformer models yield context-specific representations of nucleotide sequences, which allow for accurate predictions even in low-data settings. We show that the developed models can be fine-tuned at low cost to solve a variety of genomics applications. Despite no supervision, the models learned to focus attention on key genomic elements and can be used to improve the prioritization of genetic variants. The training and application of foundational models in genomics provides a widely applicable approach for accurate molecular phenotype prediction from DNA sequence.

Bead‐Based DNA Synthesis and Sequencing for Integrated Data Storage Using Digital Microfluidics

Bead‐Based DNA Synthesis and Sequencing for Integrated Data Storage Using Digital Microfluidics

Mahonia nanlingensis (Berberidaceae), a new species from China

Mahonia nanlingensis (Berberidaceae), a new species from Nanling Mountains in China, is described and illustrated. It is distinguished morphologically from allied species by its 7–9 fine-toothed leaflets, the rounded or cordate apex of the outer sepals, narrowly incised apex of petals, truncate stamen anthers and broadly discoid stigma. Phylogenetic analysis based on chloroplast and nuclear ribosomal DNA (nrDNA) sequences further supports its recognition as a new species. Due to habitat disturbance and medicinal use, M. nanlingensis is assessed as “Vulnerable” (VU) according to the IUCN Red List categories and criteria.

Bead-Based DNA Synthesis and Sequencing for Integrated Data Storage Using Digital Microfluidics.

DNA is considered as a prospective candidate for the next-generation data storage medium, due to its high coding density, long cold-storage lifespan, and low energy consumption. Despite these advantages, challenges remain in achieving high-fidelity, fully integrated, and cost-efficient DNA storage system. In this study, a homemade digital microfluidic (DMF)-based compact DNA data storing pipeline is orchestrated to complete the entire process from the synthesis to the sequencing. The synthetic half employs phosphoramidite chemistry on 200 nm magnetic beads (MBs), where the dimethyltrityl protecting group is removed by droplet manipulation of trichloroacetic acid. The sequencing counterpart relies on pyrophosphate releasing originated from polymerase-catalyzed primer extension, which leads to photon-countable chemiluminescence (CL) signal in 2.5-μL drops of trienzyme cascading reactions. Further by DNA denaturation, repeated pyrosequencing plus plurality voting can improve the nucleobase accuracy beyond 95 %. As a proof-of-concept trial, semantic information is saved in DNA via the Huffman coding algorithm plus the Reed-Solomon error-correction, and then robustly retrieved from this streamlined platform. As a result, it took a net total of approximately 6.5 h to writing and reading 8 bytes of data, that equal to a storaging speed of 49 min/byte, much quicker than the previously reported 2.8-4.2 h/byte. This bead-based miniaturized device promises an unattended protocol for achieving high-throughput, full-packaged, and above all, neatly precision DNA storage.

Spherical Nucleic Acid Probes on Floating-Gate Field-Effect Transistor Biosensors for Attomolar-Level Analyte Detection.

Field-effect transistor (FET) sensors are attractive for the label-free detection of target biomolecules, offering ultrahigh sensitivity and a rapid response. However, conventional methods for modifying biomolecular probes on sensors often involve intricate and time-consuming procedures that require specialized training. Herein, we propose a simple and versatile approach to functionalize floating-gate (FG) FET sensors by exploiting the strong binding ability of polyvalent interactions and the three-dimensional structure of densely functionalized spherical nucleic acids (SNAs). Crucially, the SNAs can be easily deposited onto a dielectric layer under mild conditions, ensuring stable immobilization of the probes. Further, the SNAs show efficient and robust immobilization on various dielectric layers including Y2O3, Ta2O5, and HfO2, forming conjugates that resist denaturation by various agents. By modifying the DNA sequence within the SNAs, we achieved highly sensitive FG-FET biosensors for DNA, adenosine triphosphate, and viral nucleic acids at the attomolar level. For clinical samples detection, unamplified enterovirus 71 RNA at levels as low as 0.13 copies μL-1 was detected within 100 s. Moreover, the sensor attained 100% accuracy for analyte detection in both positive and negative samples. Our findings provide a general and simple method for fabricating FET-based biochemical sensors and demonstrate that the SNA-modified FG-FET biosensor is a versatile and reliable integrated platform for ultrasensitive biomarker detection.

A redescription of the Pacific Bounty hunter, Pacificana cockayni Hogg, 1904 and an attempt to reunite with its family

ABSTRACT The male of the Bounty Island endemic Pacificana cockayni Hogg, 1904 is described for the first time and the female is redescribed. We attempted to place the monotypic genus Pacificana in an appropriate family based on cytochrome c oxidase subunit 1 DNA sequence data, however, our phylogenetic analysis was inconclusive. Morphologically, P. cockayni shares characters with the New Zealand genera Pakeha Forster & Wilton, 1973 (Cycloctenidae) and Otira Forster & Wilton, 1973 (Amaurobiidae) plus two Australian amaurobiid genera, Storenosoma Hogg, 1900 and Oztira Milledge, 2011. We note that while other spider species recorded from the Bounty Islands are found on other sub-Antarctic islands, Pacificana is not. We have not suggested a family placement until the placement of Otira, Oztira, Pakeha, and Storenosoma has been resolved by further, more extensive phylogenetic analysis.