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At least 19 recordsLinked to original sources
PubMed2026

Developmental block in ruminant embryos: Mechanisms, molecular insights and potential interventions.

Developmental block remains one of the major hurdles that makes it hard to develop embryos in vitro more efficiently. In ruminants, it is predominantly observed during the 8-16 cell stage, coinciding with the maternal-to-zygotic transition (MZT) and embryonic genome activation (EGA). In addition, reducing maternal transcripts and initiating embryonic transcription correctly is a major reason for developmental arrest. A broad array of molecular mechanisms has been implicated, encompassing incomplete epigenetic regulation, mitochondrial dysfunction, oxidative stress, improper cell cycle progression, and dysregulated apoptosis. During this process, several key genes, including ZAR1, NPM2, DPPA3, DNMTs, Cyclin B1, BCL2, and antioxidant enzymes (SOD1, GPX1, and CAT) have been recognized as essential regulators of the block. External factors, especially poor in vitro culture conditions, high oxygen levels, and the secretion of harmful metabolites, make developmental failure even worse. Recent research has underscored the significance of antioxidant supplementation, epigenetic modulators, and enhanced culture systems in mitigating developmental barriers. Therefore, the current review summarises the contemporary insights into the factors and molecular mechanisms responsible for ruminant embryonic developmental block, focusing on MZT, oxidative stress, and epigenetic regulation. It also addresses potential strategies to enhance the developmental competence of ruminant embryos in vitro.

Embryo↗
PubMedDate not supplied

Molecular mechanisms in genetic transformation.

Molecular mechanisms of recombination of genetic material are most readily studied in microorganisms; a great deal is known about recombination of transforming DNA, introduced into the cell from the medium, with DNA inside bacteria. At present it is doubtful that this type of transformation will be of use in improving crop plants. However, a new technique of making recombinant DNA molecules from two or more highly divergent organisms may turn out to have great practical importance for agriculture.

Animals↗
PubMed2026

Analysis of the molecular mechanism underlying di(2-ethylhexyl) phthalate-induced bladder carcinogenesis via network toxicology and molecular docking approaches: An observational study.

This study aims to investigate the toxicity of di(2-ethylhexyl) phthalate (DEHP) and the potential molecular mechanisms of DEHP-induced bladder cancer (BLCA) using network toxicology and molecular docking strategies. The toxicity of DEHP was assessed using Prox-II software, and potential targets for DEHP-induced BLCA were identified by integrating data from ChEMBL database, Search Tool for Interactions of Chemicals, SwissTargetPrediction, GeneCards, Therapeutic Target Database, Online Mendelian Inheritance in Man, and The Cancer Genome Atlas. STRING database and Cytoscape were employed to construct target networks and determine core targets. The expression levels of core targets were analyzed using R. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on potential and core targets. Molecular docking was carried out using CB-Dock 2 to verify the interactions between DEHP and core targets. A total of 105 potential targets related to DEHP-induced BLCA were identified, from which 7 core targets were selected: cyclin-dependent kinase 1, interleukin 6, cyclin-dependent kinase 2, cyclin B1, Erb-B2 receptor tyrosine kinase 2, cyclin B2, and B-cell lymphoma 2. IL-6 and B-cell lymphoma 2 showed downregulated expression in tumor tissues, while cyclin-dependent kinase 1, cyclin-dependent kinase 2, cyclin B1, Erb-B2 receptor tyrosine kinase 2, and cyclin B2 were upregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these targets were enriched in cell signaling and cancer-related pathways. Molecular docking confirmed that DEHP interacts with these core targets. DEHP may promote the development of BLCA by interacting with key proteins and signaling pathways. This study provides a theoretical basis for understanding the molecular mechanisms of DEHP-induced BLCA and offers references for future prevention and treatment strategies.

Diethylhexyl Phthalate↗
PubMedDate not supplied

[Molecular mechanisms of spontaneous transversions and transitions].

Molecular mechanisms of spontaneous base substitutions during template directed nucleic acid synthesis have been considered. For this purpose calculations of interaction energy in various coplanar base pairs with approximately such glycoside bond positions as for Watson--Crick base pairs have been carried out by means of atom-atom potential function method. A number of this energy local minima which could be realized in double helix during template synthesis have been found. The calculation results permit to present purine-purine pair formation with transition of one of nucleotides to syn-conformation as the main transversion pathway and formation of wobble pairs UG and AC as the transition pathway. A possible contribution of interactions between base pairs and enzyme to fidelity of template synthesis have been evaluated. Calculation results are in accord with experimental data on spontaneous mutation frequencies of errors in nucleic acid synthesis in vitro.

DNA↗
PubMed1978

Experimental evidence for a unifying concept of the molecular mechanisms of the cellular aging and the cellular neoplastic transformation.

Experimental data supporting a Unifying Concept of the molecular mechanisms of cellular aging and cellular neoplastic transformation of dividing cells in mass culture and clonal culture systems will be described. The Unifying Concept combines the hitherto antithetically presented Differentiation Theory, the Mutation Theory, the Error Catastrophy Theory and the Degradation Deficiency Theory with a newly worked out Virus Theory of the cellular aging and the cellular neoplastic transformation. Quantitative in vitro studies of embryonic fibroblast cell systems of two closely related inbred rat strains L.BN and Lewis were undertaken. The data obtained from the experimental analysis of the molecular mechanisms of the cellular aging and the cellular neoplastic transformation demonstrate, that the cellular aging of dividing cells is a Three-Stage-Differentiation Sequence under the control of three different genetic programs. The genetic constitution of the senescent cell regulates the expression of virogenes and oncogenes of the endogenous RNA tumor viruses of the C-type, resulting either in the cellular degeneration of the senescent cell under the control of the virogenes or in the cellular neoplastic transformation regulated by the oncogenes.

Aging↗
PubMed2026

Microbial partnerships and molecular mechanisms in plant stress physiology for climate-resilient and sustainable farming.

Plant-microbial partnerships and their underlying molecular mechanisms are indispensable, natural drivers of improved nutrient acquisition and stress tolerance in the face of climate-driven environmental challenges. Modern multi-omics tools, when coupled with artificial intelligence and synthetic biology, enable the precise design of targeted bioinoculants and synthetic microbial consortia. Translating these advanced microbiome-based strategies into scalable, field-level agricultural applications provides a sustainable path toward securing global food production while maintaining soil health. Global climate change imposes multifaceted abiotic and biotic stresses on crops, disrupting physiological and molecular processes and threatening agricultural productivity. Plant-associated microbes represent an underexplored yet powerful ally in enhancing crop resilience. This review presents current knowledge of plant-microbe interactions and the molecular mechanisms governing plant stress physiology, with an emphasis on climate-resilient and sustainable farming. Hence, ever-changing environmental cues pose a significant burden on agricultural productivity, and plant-associated microbial communities modulate a cascade of physiological and molecular responses, including production of phytohormones, signaling, regulation of reactive oxygen species homeostasis, and activation of plant immune responses to help plants withstand stress and enhance productivity. Moreover, root exudates, phytohormones, and quorum sensing mediate the central communication networks, facilitating plant-microbe cross talk. Additionally, the advances in OMICs approaches aid in disentangling the molecular underpinnings of these interactions by providing mechanistic insights and potential candidate gene targets for crop improvement and stress resilience. In the post-genomic era, integrating artificial intelligence and big data analysis to optimize microbiome-based strategies for sustainable agriculture is a new frontier for disentangling plant-microbe symbiosis to improve soil health, enhance crop yields, and improve stress tolerance. Thus, by integrating the ecological, physiological, and molecular perspectives, this review highlights the transformative potential of harnessing plant-microbe symbiosis for climate-resilient and sustainable agriculture.

Stress, Physiological↗
PubMed2026

Ossicle occurrence characteristics and related molecular mechanisms in the sea cucumber Apostichopus japonicus.

To investigate the morphogenetic pattern and molecular mechanism of ossicle formation in the sea cucumber Apostichopus japonicus, this study systematically examined the morphological development and temporal sequence of spicules using the NaClO maceration method, in-situ squash preparation and microscopic observation. Comparative transcriptome sequencing was performed between doliolaria and pentactula larvae to screen differentially expressed genes (DEGs) related to ossicles formation, followed by pathway enrichment analysis. The function of the candidate key gene papilin-like was verified using siRNA-mediated gene silencing. The results were as follows: 1) Ossicles of A. japonicus first appeared at the late auricularia stage, initiating as X-shaped ossicles at the base of the oral tentacles. The number of X-shaped ossicles increased dramatically during the doliolaria stage. X-shaped ossicles were gradually replaced by table-shaped and rosette-shaped ossicles at the pentactula stage, suggesting that X-shaped ossicles may differentiate into these two ossicle types. The morphology of table-shaped ossicles showed a "simple-complex-simple" pattern with development. 2) Key genes related to ossicles formation, including CA1, COL1A2, and papilin-like, were identified by transcriptome analysis. After papilin-like knockdown, abnormal morphologies were observed in table-shaped ossicles of 1-year-old A. japonicus, such as spine-like protrusions on the outer margin of the disc and loss of table legs, confirming its crucial roles in maintaining ossicle morphology. This study clarified the morphological development pattern of ossicles in A. japonicus and identified a key regulatory gene (papilin-like) involved in ossicle morphogenesis, providing preliminary insights into the underlying molecular regulatory mechanism. These findings enrich our understanding on ossicles formation in echinoderms, and provide important morphological and molecular biological information for further studies on the developmental mechanism of ossicles in A. japonicus.

Animals↗
PubMed2026

Transcriptomic insights into the molecular mechanism of antifouling agent-induced settlement inhibition in the Pacific oyster Crassostrea gigas.

Marine biofouling remains a persistent challenge to maritime industries and marine ecosystems worldwide. In this study, we systematically evaluated the acute toxicity, settlement inhibitory efficacy, and underlying molecular mechanisms of an N-oleyl-1,3-propanediamine-based antifouling agent using pediveliger larvae of the Pacific oyster Crassostrea gigas. The 96 h-LC50 of the agent was determined to be 0.81 mg/L, and exposure to 1.68 mg/L achieved complete larval settlement inhibition without inducing significant acute toxicity. Transcriptomic analysis identified 791 differentially expressed genes, dominated by downregulated genes associated with ribosomal function, translation, cell adhesion, and cytoskeletal organization. The agent exerts its inhibitory effect primarily through the global suppression of protein synthesis, disruption of cell-substrate adhesion and cytoskeletal integrity, and induction of proteotoxic stress responses. These findings reveal a multi-pathway molecular mechanism underlying antifouling agent-induced settlement inhibition in oyster larvae and provide key molecular biomarkers to support the development of eco-friendly antifouling technologies.

Animals↗
PubMed2025

A cross-species multi-omics analyze uncovers conserved molecular mechanisms underlying age-related erectile dysfunction.

BACKGROUND: The urgent need for new treatments is driven by the challenging clinical situation of age-related erectile dysfunction (ARED). AIM: To clarify the conserved molecular mechanisms of ARED across species using multi-omics. METHODS: Rat and mouse models with ARED were developed to facilitate the extraction of mRNA and proteins from the corpus cavernosum for high-throughput sequencing. Bioinformatics techniques were employed to analyze differentially expressed genes and to conduct analyses using the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and protein-protein interaction networks. Verification of the results was carried out using immunofluorescence, hematoxylin-eosin staining, and Masson staining. OUTCOMES: The multi-omics profiles of ARED rats and mice were analyzed and validated across species. RESULTS: In both species, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses of transcriptomic and proteomic data revealed that differentially expressed genes were predominantly enriched in pathways associated with alterations in extracellular matrix composition, downregulation of mitochondrial activity, and disruption of protein homeostasis. Immunofluorescence analysis demonstrated an upregulation of reactive oxygen species expression, coupled with a downregulation of Aldh18a1, collagen, and collagen I expression in the corpus cavernosum of mice and rats with ARED. CLINICAL IMPLICATIONS: To offer a novel approach for enhancing the erectile function in patients with ARED. STRENGTHS AND LIMITATIONS: The primary strength of this study lies in its utilization of cross-species multi-omics sequencing, which has elucidated the conserved molecular mechanisms underlying ARED. However, a significant limitation is the absence of subsequent validation in patients with ARED. CONCLUSIONS: Cross-species multi-omics comparisons present a potentially innovative approach for elucidating the underlying mechanisms and identifying preventive and therapeutic targets for ARED.

aging↗
PubMed2025

The molecular mechanism of cuproptosis and research progress in pancreatic diseases.

PURPOSE: Cuproptosis has been proven to be a novel mode of cell death, distinct from other types of cell death such as necrosis, ferroptosis, pyroptosis, and apoptosis. This study aims to systematically review the molecular mechanisms of cuproptosis in recent years and its research progress in pancreatic diseases. METHODS: By searching PubMed and Web of Science databases, 113 key literatures were included for thematic analysis, covering the molecular mechanism of cuproptosis and its role in the occurrence and development of pancreatic cancer, acute and chronic pancreatitis, diabetes, pancreatic cyst, pancreatic injury and pancreatic neuroendocrine tumor. RESULTS: Cuproptosis refers to the accumulation of copper ions in cells, which leads to instability of ferritin and aggregation of acylated proteins, resulting in oxidative stress-related cell death. Recent studies have shown that cuproptosis plays an important role in the occurrence and development of various pancreatic diseases, such as pancreatic cancer, acute and chronic pancreatitis, diabetes, pancreatic cysts, pancreatic injuries and pancreatic neuroendocrine tumor. The inducers of cuproptosis, such as disulfiram, chloroquinolones, and perilla phenols, alleviate pancreatic cancer by promoting cell cuproptosis. Copper chelators such as tetraethylenepentamine and tetrathiomolybdate promote the recovery of pancreatic injury by inhibiting cell cuproptosis. CONCLUSIONS: Cuproptosis plays a crucial role in the pathogenesis of pancreatic diseases. Further research on the cuproptosis pathway may become a potential target for the treatment of pancreatic diseases.

Animals↗
PubMed2026

Integrative machine learning and transcriptomic analysis reveals molecular mechanisms underlying low survival rate in larval Chinese Bahaba (Bahaba taipingensis).

Chinese Bahaba (Bahaba taipingensis) is a Class I protected marine fish endemic to China. Low larvae survival during artificial breeding severely hinder population recovery. To investigate the molecular mechanism of high mortality in larval fish, this study performed RNA-seq on liver from naturally deceased (ND) and mass-dead (MD) individuals, combined with least absolute shrinkage and selection operator (LASSO) regression and random forest (RF) algorithms to screen for core signature genes. A total of 873 differentially expressed genes (DEGs) were identified, including 112 upregulated and 761 downregulated genes. GO and KEGG enrichment analyses revealed significant enrichment in amino acid metabolism disorders, one‑carbon folate pool impairment, PPAR signaling abnormalities, ECM-receptor interaction, focal adhesion pathway, indicating widespread metabolic suppression accompanied by extracellular matrix remodeling and signaling disturbances in the livers of MD fish. MAD pre-filtering combined with dual machine learning algorithms yielded 18 robust core signature genes, among which SLC38A4, MMP1, FADD, FKBP5, and APOB were consistently identified as high-frequency core genes by both algorithms. SLC38A4 exhibited the highest importance score in the RF model and was significantly downregulated, making it the primary molecule distinguishing ND from MD phenotypes. ROC curve analysis showed that both models achieved an AUC of 1.000 (95% CI lower bound: 0.610), confirming the precise discriminatory ability of the core genes. GSEA further demonstrated significant enrichment of this core gene set in ND samples. This study provides the first systematic elucidation of the molecular mechanisms underlying liver dysfunction in low survival rate B. taipingensis, characterized by amino acid transport impairment, metabolic reprogramming, and structural remodeling, offering theoretical foundations for health assessment, early mortality risk warning, and artificial breeding conservation of this species.

Animals↗
PubMed1977

What's wrong with the theoretical research on molecular mechanisms of the chemical cancerization process.

The theoretical research on molecular mechanisms of chemical cancerization process is essentially a statistical problem in nature. Failing to have this notion in mind, many researchers in this field have engaged sterilizing controversies on the existence or the non existence of a correlation between the carcinogenic potency of a substance and some of its molecular properties. The following report is an example showing how the statistical concept and methods are necessary to resolve these problems.

Carcinogens↗
PubMed2025

Genetic effects on chromatin accessibility reveal the molecular mechanisms of complex traits in maize.

Cis-regulatory elements (CREs) are critical for modulating gene expression and phenotypic diversity in maize. While genome-wide association study (GWAS) hits and expression quantitative trait loci (eQTLs) are often enriched in CREs, their molecular mechanisms remain poorly understood. Characterizing CREs within accessible chromatin regions (ACRs) offers a powerful approach to link noncoding variants to chromatin structure alterations and phenotypic variation. Here, we generated ATAC-seq profiles from seedling leaves of 214 maize inbred lines, identifying 82 174 consensus ACRs. Notably, 39.55% of these ACRs exhibited significant population-wide chromatin accessibility variation. By mapping chromatin accessibility quantitative trait loci (caQTLs), we discovered 27 004 loci, including 1398 predicted to disrupt transcription factor (TF)-binding sites. Integration with multi-omics data revealed 7405 caACR-target gene pairs and linked 56 caACRs to GWAS signals for 51 agronomic traits, with significant enrichment in flowering-related pathways. Functional candidates such as ZmZIM30 - putatively regulated by caACRs - emerged as key regulators of flowering time. At the fad7 locus associated with linolenic acid content, allelic variants overlapping a caQTL showed differential chromatin accessibility. Our study provides a high-resolution cis-elements of maize leaves, deciphers the genetic basis of chromatin accessibility variation, and bridges noncoding caQTLs to molecular mechanisms underlying GWAS hits.

Zea mays↗
PubMedDate not supplied

[Molecular mechanisms of 2-aminopurine and 2,6-diaminopurine induced mutations].

Molecular mechanisms of base pair substitutions induced by 2-aminopurine and 2,6-diaminopurine have been proposed by calculations of nonbonded interaction energy of this substances with nucleic acid bases. The calculation results suppose that the main pathways of the transitions are wobble pair formations of these analogues with cytosine. The possibility of the transversions as a result of incorporation of 2-aminopurine and 2,6-diaminopurine in DNA have been considered. The influence of nucleotide sequences and enzymes of nucleic acid synthesis on induced mutation frequencies have been discussed.

2-Aminopurine↗
PubMed2024

Transcriptome analysis under pecan scab infection reveals the molecular mechanisms of the defense response in pecans.

Pecan scab, caused by the fungal pathogen Venturia effusa, is the most devastating disease of pecan (Carya illinoinensis) in the southeastern United States. Resistance to this pathogen is determined by a complex interaction between host genetics and disease pathotype with even field-susceptible cultivars being resistant to most scab isolates. To understand the underlying molecular mechanisms of scab resistance in pecan, we performed a transcriptome analysis of the pecan cultivar, 'Desirable', in response to inoculation with a pathogenic and a non-pathogenic scab isolate at three different time points (24, 48, and 96 hrs. post-inoculation). Differential gene expression and gene ontology enrichment analyses showed contrasting gene expression patterns and pathway enrichment in response to the contrasting isolates with varying pathogenicity. The weighted gene co-expression network analysis of differentially expressed genes detected 11 gene modules. Among them, two modules had significant enrichment of genes involved with defense responses. These genes were particularly upregulated in the resistant reaction at the early stage of fungal infection (24 h) compared to the susceptible reaction. Hub genes in these modules were predominantly related to receptor-like protein kinase activity, signal reception, signal transduction, biosynthesis and transport of plant secondary metabolites, and oxidoreductase activity. Results of this study suggest that the early response of pathogen-related signal transduction and development of cellular barriers against the invading fungus are likely defense mechanisms employed by pecan cultivars against non-virulent scab isolates. The transcriptomic data generated here provide the foundation for identifying candidate resistance genes in pecan against V. effusa and for exploring the molecular mechanisms of disease resistance.

Carya↗
PubMed1979

Dynamic cooperativity of molecular processes in active streaming, muscle contraction, and subcellular dynamics: the molecular mechanism of self-organization at the subcellular level.

Life phenomena are a kind of ordered dynamics appearing in macroscopic systems, living systems. Schrödinger has proposed a molecular mechanism for the organization of life phenomena, i.e., 'order-from-order' mechanism where ordered dynamics are composed of molecular dynamics having order as the ordered dynamics of a watch is caused by orderly movements of its mechanical elements. However, neither evidence supporting the 'order-from-order' mechanism has been found in living systems nor the reason why molecular dynamics acquire order instead of disorder has been elucidated for more than 30 years. The latter is quite anomalous from the point of views of thermodynamics, which is based on disordered behaviors of molecules. In this paper, we verify from studies of a streaming system reconstituted from rabbit skeletal F-actin and HMM that one life phenomenon, active streaming, is caused by the 'order-from-order' mechanism. This is also the case for muscle contraction. Moreover, it is probable that this mechanism generally works at the subcellular level, not only in biological motilities but also in life phenomena at biomembranes. We also clarify that dynamic cooperativity among molecule gives rise to order in molecular dynamics. Hence, dynamic cooperativity is the key mechanism for life phenomena caused by the 'order-from-order' principle at the subcellular level. To produce dynamic cooperativity it is necessary for component molecules or elements to have three states, i.e., inactive (stable) state 0, energized or energy storing (quasi-stable) state 1, and active (unstable) state 2. Each molecule performs elementary cycle 0 yields 1 yields 2 yields 0 repeatedly by using free energy at the molecular level. In a state far from thermodynamic equilibrium dynamic cooperativity is yielded in 2 yields 0 due to a kind of triggering action of neighboring elements and breaks thermodynamic detailed balance. In addition, dynamic cooperativity gives component molecules long-range interactions which depend on the structure of organelles or molecular assemblies. Dynamic cooperativity is able to decrease entropy production and will give a high efficiency in chemo-mechanical conversions. Great progress would be achieved in the understanding of the molecular mechanisms and thermodynamic principles of energy transformations in biological systems, if molecular dynamics during transformation could be directly observed. This is not only because physical changes accompanied by specific movements of macromolecules are essentially involved but also because such molecular movements play a substantial role in energy transformation. Entirely new ideas will be needed for this purpose although high voltage electron microscopy or X-ray diffraction, for instance, is now expected as to be one of the possible tools in the future. Fortunately even at present it is possible to obtain important information on molecular dynamics from biochemical and physiological data, if analyses are properly performed...

Actomyosin↗
PubMed2026

Study of NSD2 using a dTAG system reveals its molecular mechanism and oncogenic implications in t(4;14) multiple myeloma.

The histone H3 lysine 36 dimethylation (H3K36me2) methyltransferase NSD2 is deleted in Wolf-Hirschhorn syndrome and is aberrantly expressed in 10% to 15% of patients with multiple myeloma (MM) because of a t(4;14) translocation. Although NSD2 is thought to be a primary driver in MM, the exact molecular mechanisms by which it regulates transcription remain unclear. We applied the degradation tag (dTAG) system to acutely degrade NSD2 and used this, in combination with time-resolved thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), to identify 307 transcriptional targets of NSD2. Reconstitution with either wild-type NSD2 or a catalytically inactive mutant (NSD2Y1179A) showed that NSD2's transcriptional effects are almost exclusively dependent on its SET domain activity. Mechanistically, H3K36me2 deposition by NSD2 antagonizes H3K27me3 levels, and treatment with 2 distinct Polycomb repressive complex 2 inhibitors demonstrated that approximately half of the NSD2 target genes are regulated in an H3K27me3-dependent manner. Cleavage under targets and tagmentation (CUT&Tag) analysis showed that upon NSD2 depletion, there was an increase in H3K27me3 that occurred at genome-wide intergenic regions rather than at the promoters or gene bodies of NSD2 target genes. These data suggest that NSD2, via H3K36me2, antagonizes H3K27me3 deposition likely at distal regulatory elements, including enhancers, creating a chromatin landscape favorable for target gene transcription. Importantly, NSD2 target genes were enriched for key oncogenic pathways, and 24 transcription factors (TFs) implicated in neurodevelopment and acute leukemia, consistent with its role in Wolf-Hirschhorn syndrome and MM. Eight of these TFs are known oncogenic drivers in acute leukemia or MM, highlighting a novel molecular mechanism for NSD2's role in t(4;14) MM.

Histone-Lysine N-Methyltransferase↗
PubMed1979

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Bacteriophage phi X 174↗