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Results for “molecular biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR, such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We expect that the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders and to manipulate other tandem repeats.

molecular biology↗

bioRxiv2020

Virus-host protein-protein interactions between human papillomavirus 16 E6 A1 and D2/D3 sub-lineages: variances and similarities

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers with type 16 being the most frequent. Compared to the European Prototype (A1, denoted "EP"), the Asian-American (D2/D3, denoted "AA") sub-lineage or "variant" is reported to have increased abilities to promote carcinogenesis. Few global interactome studies have looked at protein-protein interactions (PPIs) between host proteins and variants of the key transforming E6 protein. We applied a primary human foreskin keratinocyte model transduced with EP and AA variant E6 genes and co-immunoprecipitated expressed E6 proteins along with interacting cellular proteins to detect virus-host binding partners. We reasoned that, due to single nucleotide polymorphisms, AAE6 and EPE6 may have unique PPIs with host cellular proteins--conferring gain or loss of function--resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to each of AAE6 and EPE6, along with 13 E6 targets common to both AAE6 and EPE6. We also applied a more inclusive process based on pathway selection and discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets between the two sub-lineages. Interpretations for both approaches were made using databases such as UniProt, BioGRID and Reactome. Detected E6 targets are implicated in important hallmarks of cancer: deregulating Notch and other signaling, energetics and hypoxia, DNA replication and repair, and immune response. Validation experiments, such as reverse co-immunoprecipitation and RNA interference, are required to substantiate these findings. Here, we provide an unprecedented resource for new research questions in HR HPV biology. The current data also underline our labs driving hypothesis that E6, being a "master regulator" in HPV-positive cancers, is an excellent candidate for anti-cancer treatment strategies. Author SummaryChronic infection with high-risk human papillomavirus (HPV) type 16 is the most prevalent cause of cervical and other mucosal cancers. The E6 oncoproteins of the European Prototype (EP) and the Asian-American (AA) HPV variants differentially promote carcinogenesis. We looked at protein-protein interactions between host proteins and two key HPV variant E6 proteins of these strains to reveal how high risk HPVs cause cancer, based on the proteins they bind to in infected cells. Our methodology combined molecular biology and data mining techniques using widely available databases. We confirmed and discovered novel virus-host associations that explained how HPV AA and EP variants differ in their carcinogenic capabilities, and confirmed the candidacy of the E6 protein as a viable target for HPV therapies.

molecular biology↗

bioRxiv2025

Nicking Loop™: An infinite amplification of single-stranded circular DNA

Circular single-stranded DNA (CssDNA) offers unique advantages for molecular diagnostics and next-generation sequencing (NGS) due to its nuclease resistance and compatibility with rolling circle amplification. We present the Nicking Loop, a novel and versatile method for converting both single- and double-stranded DNA into CssDNA and enabling robust, unbiased amplification. This technique preserves the original DNA template composition and outperforms PCR in reproducibility and sensitivity, particularly for low variant allele frequencies. We demonstrate that Nicking Loop-amplified CssDNA is directly compatible with NGS platforms utilizing circular DNA as a sequencing template and introduce a novel loop-based indexing strategy that enables efficient sample multiplexing. Furthermore, we confirm the methods compatibility with nanopore sequencing technologies, highlighting its broad applicability across sequencing platforms. Our results establish Nicking Loop as an alternative to conventional linear library preparation, streamlining workflows while enhancing sequencing accuracy and efficiency. Beyond its immediate application in NGS, the method holds promise for broader use in molecular biology, e.g. in DNA data storage. This proof-of-concept study highlights the transformative potential of Nicking Loop in enabling a shift toward circular DNA libraries for new generation of assay preps and precision sequencing applications.

molecular biology↗

bioRxiv2021

Impact of cobas PCR Media Freezing on SARS-CoV-2 Viral RNA Integrity and Whole Genome Sequencing Analyses

SARS-CoV-2 whole genome sequencing is an important molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swab samples and shipment to reference laboratories is usually required since RNA sequencing is rarely available in routine clinical microbiology laboratories where initial diagnosis and support to outbreak investigations occur. The cobas PCR Media transport medium developed by Roche facilitates high throughput analyses on cobas multianalyzer PCR platforms. There is no data on the stability of SARS-CoV-2 RNA after freezing and thawing of clinical samples in this transport medium, but potential denaturing of the molecular template could impair test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral whole genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Genomic coverage and sequencing depth did not significantly differ between fresh and frozen samples (n=10). For samples with lower viral inoculum and PCR cycle threshold above 30, sequencing quality scores and detection of single nucleotide polymorphisms did not differ either. Freezing of cobas PCR Media does not negatively affect the quality of SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories. Those results support secondary use of diagnostic nasopharyngeal swab material for viral sequencing without requirement for additional clinical samples.

molecular biology↗

bioRxiv2024

The Genetic Puzzle of Multicopy Genes: Challenges and Troubleshooting

BackgroundStudies with multicopy genes impose challenges related to gene redundancy and sequence similarity among copies. Recent advances in molecular biology and genomics tools associated with dedicated databases facilitate their study. Thus, the present work emphasizes the need for rigorous methodologies and standardized approaches to interpret RT-qPCR results accurately. ResultsThe present work in Physcomitrium patens provides a comprehensive five-step protocol, using thiamine thiazole synthase (THI1) and sucrose 6-phosphate phosphohydrolase (S6PP) genes as proof of concept, to showcase a systematic workflow for studying multicopy genes. Beyond examining genes of interest, we highlight the critical role of choosing appropriate internal controls in the analytical process for accurately interpreting gene expression patterns. We emphasized the importance of identifying the most relevant orthologous gene, recognizing the inherent challenges in determining the most functional copy for subsequent studies. Our objective is to enhance comprehension of gene redundancy by dissecting multicopy genes genomic landscape and its characteristics. Furthermore, we address the decision-making process surrounding the quantification of expression levels of multicopy genes. ConclusionsThe study of multicopy genes discloses early events of functional adaptation. We emphasize the significance of multicopy genes in plant biology and provide a practical protocol for their study. Plant systems are strongly influenced by light/dark cycles, and the challenges inherent to this process are acknowledged. In conclusion, our comprehensive approach aims to advance the understanding of multicopy gene dynamics, offering practical methodologies and contributing valuable insights to the scientific community.

molecular biology↗

bioRxiv2020

Scalable, rapid and highly sensitive isothermal detection of SARS-CoV-2 for laboratory and home testing

Global efforts to combat the Covid-19 pandemic caused by SARS-CoV-2 still heavily rely on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. We present a rapid, robust, sensitive and versatile RT-LAMP based SARS-CoV-2 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay, we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed open source enzymes that can be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing.

molecular biology↗

bioRxiv2024

Time-course transcriptome data of silk glands in day 0-7 last-instar larvae of Bombyx mori (w1 pnd strain)

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

molecular biology↗

bioRxiv2023

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

Enveloped viruses carry one or multiple proteins with receptor binding functionalities. Functional receptors can either be glycans, proteinaceous or both, recombinant protein approaches are instrumental to gain more insight into these binding properties. Visualizing and measuring receptor binding normally entails antibody detection or direct labelling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here we report a suite of different fluorescent fusions, both N- and/or C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained a total of three or six fluorescent protein barrels and were applied directly to cells to determine receptor binding properties.

molecular biology↗

bioRxiv2019

Natural infection and vertical transmission of two flaviviruses (Yellow fever and Zika) in mosquitoes in primary forests in the Brazilian state of Rio de Janeiro (Diptera: Culicidae)

BackgroundZika virus (ZIKV) was recently introduced in the American continent, probably transmitted by Aedes aegypti and possibly by Ae. albopictus and Culex quinquefasciatus in urban environments. ZIKV represents a known public health problem as it has been involved in newborn cases of congenital microcephaly in South America since 2005. The transmission of this virus in forested areas of other countries and its relative ubiquity in relation to its vectors and reservoirs raises suspicions of its adaptation to non-human modified environments (i.e., natural forests reserve) or on this continent, similar to those seen for Yellow fever virus (YFV). The objective of this work was to have an epidemiological monitoring tool mapping insects as well as circulating arboviruses in wild areas with low human interference. This study was based on the history of the insect flavivirus spreading cycle.\n\nMethods/Principal FindingsUsing a previously described sensitive PCR-based assay to assess the conserved NS5 region of the Flavivirus genus, both YFV partial genome and ZIKV were found in pools of Aedes albopictus, a sylvatic mosquito adapted to human-modified environments, and in Haemagogus leucocelaenus, a sylvatic mosquito.\n\nConclusionsThis is the first report of natural infection by ZIKV in mosquitoes in a sylvatic environment on the American continent. The wide distribution of these mosquitoes is probably important in the transmission of ZIKV. Vertical transmission indicates a higher efficiency for the maintenance and transmission of the virus in nature as well as the presence of the ZIKV in permanent character in the forest areas as it occurs with the YFV thus making more difficult the prevention of new cases of Zika in humans.\n\nAuthor SummaryArboviruses are diseases transmitted by arthropod vectors, hence the origin of the term ARthropod BOrne VIRUS, which is adopted since 1942. This work had as objective to survey the circulating insects as well as to detect the presence of viruses in them. Arboviruses circulate between insects and vertebrate hosts, having importance for promoting diseases in humans and animals. The diseases most known at the time, due to the recent cases reported by South America, are Dengue, Zika, Yellow Fever and Chikungunya. For this study, we used appropriate traps to collect the insects and their eggs in wild areas where there is little human interference. After collection, mosquitoes and / or eggs were identified and separated as to the source and species. The eggs were kept in laboratory conditions for the hatching of new insects. All the insects obtained were separated into pools to be macerated and thus extract the RNA from the viruses to be studied. Using molecular biology techniques, in our case the RT-PCR (Reverse Transcriptase Polymerase Chain Reaction), we amplified the RNA and in sequentially, we performed the sequencing reaction. With sequencing, it is possible to identify which virus material is present since each virus has a characteristic arrangement. For the identification of the sequences, we need to use some computational programs that guarantee us the correct result.

molecular biology↗

bioRxiv2021

Short '1.2x genome' infectious clone initiates deltavirus replication in Boa constrictor cells

Human hepatitis D virus (HDV), discovered in 1977, represented the sole known deltavirus for decades. The dependence on hepatitis B virus (HBV) co-infection and its glycoproteins for infectious particle formation led to the assumption that deltaviruses are human-only pathogens. However, since 2018, several reports have described identification of HDV-like agents from various hosts but without co-infecting hepadnaviruses. Indeed, we demonstrated that Swiss snake colony virus 1 (SwSCV-1) uses arenaviruses as the helper for infectious particle formation, thus shaking the dogmatic alliance with hepadnaviruses for completing deltavirus life cycle. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2x genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used SwSCV-1 as the model to compare the ability of the previously reported "2x genome" and the "1.2x genome" plasmid constructs/infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2x and 1.2x genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. We hypothesize that duplicating the ribozymes facilitates the cleavage of genome multimers into unit-length pieces during the initial round of replication. The 1.2x genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions. IMPORTANCEHepatitis D virus (HDV) is a satellite virus infecting humans with strict association to hepatitis B virus (HBV) co-infection because HBV glycoproteins can mediate infectious HDV particle formation. For decades, HDV was the sole representative of deltaviruses, which had led to hypotheses suggesting that it evolved in humans, the only known natural host. Recent sequencing studies have led to the discovery of HDV-like sequences across a wide range of species, representing a paradigm shift in deltavirus evolution. Molecular biology tools such as infectious clones, which enable initiation of deltavirus infection without helper virus, are key to demonstrate that the recently found deltaviruses are capable of independent replication. Such tools will enable identification of the potential helper viruses. Here, we report a 1.2x genome copy strategy for designing plasmid-based infectious clones to study deltaviruses and to demonstrate that plasmid delivery into cultured snake cells sufficiently initiates replication of different deltaviruses.

molecular biology↗

bioRxiv2018

Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the gamma-toxin endonuclease

The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that gamma-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by gamma-toxin with standard molecular biology techniques such as Northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the gamma-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have employed the gamma-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of gamma-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic and biochemical studies.

molecular biology↗

bioRxiv2020

Optimal protocols for sequence-based characterization of the human vaginal microbiome

The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment, but also to the use of conflicting methodologies to study its microbial composition. Here we systematically assess best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region has the best theoretical properties, but limitations of current sequencing technologies mean that the V3-V4 region performs equally well. Both of these approaches present very good agreement with qPCR quantification of key taxa, provided an appropriate bioinformatic pipeline is used. Shotgun metagenomic sequencing presents an interesting alternative to 16S amplification and sequencing, but it is not without its challenges. We have assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and - use, reference database of vaginal taxa. This strategy performed as well as the best performing previously published strategies. Despite the many advantages of shotgun sequencing none of the shotgun approaches assessed here had as good agreement with the qPCR data as 16S rRNA gene sequencing. ImportanceThe vaginal microbiome has been connected to a wide range of health outcomes, from susceptibility to sexually transmitted infections to gynecological cancers and pregnancy outcomes. This has led to a thriving research environment, but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail for them to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.

molecular biology↗

bioRxiv2022

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites - a case study with Omicron Spike DNA

Site directed mutagenesis is an invaluable technique which enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as four other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2023

A Standardized Protocol for Sample Preparation for Scanning Electron Microscopy (SEM) to Visualize Extrachromosomal DNA (ecDNA)

Extrachromosomal DNA (ecDNA) are large ([~]kilo to megabase) acentric, atelomeric, circular DNAs that are established cytogenetic markers for malignancy, hard-to-treat tumors and drug resistance. Often referred to as double minute chromosomes, ecDNA have been studied since the 1960s, primarily through molecular biology techniques, such as cytogenetics and fluorescence microscopy. More recently, next generation sequencing technologies present novel opportunities for identifying ecDNA. However, none of these approaches adequately address the architecture, size and composition of ecDNA within single cells. Developing an approach to systematically visualize ecDNA, confirm their circular architecture and determine their ultrastructure and composition is an urgent, unmet need. This work presents a protocol for visualizing ecDNA at high resolution using scanning electron microscopy (SEM). To this end, we have optimized an end-to-end procedure that involves preparing, processing and visualizing metaphase spread samples. This protocol was tested on five human cancer cell lines (COLO320DM, NCIH716, NCIH2170, SKGT2, SNU16), four of which express ecDNA in various amounts and one amplifies DNA via a homogeneous staining region (HSR). This work presents a standardized approach to preparing samples and visualizing ecDNA using SEM. SignificanceExtrachromosomal DNA (ecDNA) are proposed to have unique molecular traits, which include acentric, atelomeric and circular DNA. Standardized, high resolution microscopy approaches are in high demand to better understand structure-function relationships of ecDNA.

molecular biology↗

bioRxiv2016

Control mechanisms for stochastic biochemical systems via computation of reachable sets

Controlling the behaviour of cells by rationally guiding molecular processes is an overarching aim of much of synthetic biology. Molecular processes, however, are notoriously noisy and frequently non-linear. We present an approach to studying the impact of control measures on motifs of molecular interactions, that addresses the problems faced in biological systems: stochasticity, parameter uncertainty, and non-linearity. We show that our reachability analysis formalism can describe the potential behaviour of biological (naturally evolved as well as engineered) systems, and provides a set of bounds on their dynamics at the level of population statistics: for example, we can obtain the possible ranges of means and variances of mRNA and protein expression levels, even in the presence of uncertainty about model parameters.

Systems Biology↗

bioRxiv2026

Simple Electroporation of Chlamydomonas reinhardtii Strains with an Intact Cell Wall

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI

molecular biology↗

bioRxiv2023

Fast, safe and high-throughput Real-Time PCR protocol for molecular sex identification in Salmo salar, applicable to historic scale collections

Conservation and management of wild species needs a detailed understanding of their population structure and dynamics. For instance, there are increasing evidence for sex-specific life history trajectories, even in species with limited morphological sexual dimorphism. In these species, identifying the sex of individuals in some or all life stages is a methodological challenge, which requires the development of new tools. Building on recent findings in molecular biology, we developed a new protocol for sex identification in Atlantic salmon (Salmo salar), aiming at the following criteria: accurate sex identification, fast diagnostic, applicable to large sample size, applicable to degraded DNA extracted from e.g. fish scales, and safe for the operator by limiting health and safety hazard. The protocol relies on multiplex Real-Time PCR using a set of specific primers. We tested its accuracy on a test set of 90 DNA samples from individuals of known morphological sex, and demonstrated its applicability on a large DNA sample from 384 individuals of unknown sex.

molecular biology↗

bioRxiv2024

The fate of artificial transgenes in Acanthamoeba castellanii

In this study, artificial transformation experiments were performed to investigate how the A. castellanii genome responds to foreign DNA presented in both circular and linear plasmid form. Nanopore sequencing was used as a high throughput method to screen for transgene DNA in the resulting transformant cultures, and candidate transgene integrations were identified. Molecular biology experiments were performed to validate the sequence data and provide additional context on the fate of transgenes. A method was devised to estimate the rate of read chimerism in nanopore sequencing runs and accurately account for the effects of read chimerism in identifying putative transgene integrations. Based on the experimental data in hand, a potential mechanism for transgene maintenance in A. castellanii is proposed, one in which incoming foreign DNA is tandemly duplicated and telomeres are added to the ends. This nascent linear molecule is maintained as a transgene-bearing minichromosome, while also allowing for chromosomal integration.

molecular biology↗