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Plasmids

Plasmids

Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...

DNA-only Transposons

DNA-only Transposons

DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Genomic DNA in Eukaryotes

Genomic DNA in Eukaryotes

Eukaryotes have large genomes compared to prokaryotes. To fit their genomes into a cell, eukaryotic DNA is packaged extraordinarily tightly inside the nucleus. To achieve this, DNA is tightly wound around proteins called histones, which are packaged into nucleosomes that are joined by linker DNA and coil into chromatin fibers. Additional fibrous proteins further compact the chromatin, which is recognizable as chromosomes during certain phases of cell division.

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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

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Centromeric Sequences in Ogataea polymorpha Genome Enable Development of Stable Multigene Expression Plasmid Tools.

Yanfei Cheng¹, Yuanyuan Shi^1,2, Ning Sun^1,2

¹Department of Microbial Physiological & Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

Microbial Biotechnology

|November 12, 2025

Summary

Researchers identified unique centromeres in Ogataea polymorpha yeast, creating stable centromeric plasmids for biotechnology. These plasmids enable precise gene expression and metabolic engineering, expanding genetic tool diversity.

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Area of Science:

Molecular Biology
Yeast Genetics
Biotechnology

Background:

Centromeres are crucial for accurate chromosome segregation during cell division.
Centromeric plasmids offer stability and precise distribution, making them valuable genetic tools.
Ogataea polymorpha is a non-conventional yeast with significant biotechnological potential.

Purpose of the Study:

To locate and characterize centromeric regions in Ogataea polymorpha.
To construct and evaluate the stability of O. polymorpha centromeric plasmids.
To explore the application potential of these plasmids in biotechnology.

Main Methods:

Chromatin immunoprecipitation sequencing (ChIP-seq) using native Cse4 to identify centromeric regions.
Sequencing and characterization of centromeric DNA.
Construction and functional validation of centromeric plasmids.

Main Results:

Centromeric regions on seven O. polymorpha chromosomes were identified.
Unique centromeric sequences, distinct from other eukaryotes, were characterized for chromosomes 1, 2, and 5.
O. polymorpha centromeres were classified as small regional centromeres, lacking functional long terminal repeats (LTRs).
Novel, highly stable, and compatible O. polymorpha centromeric plasmids were successfully constructed.
Validated application potential for multigene or multicopy expression, demonstrated by uricase and triterpene squalene production.

Conclusions:

Elucidates the structural features of O. polymorpha centromeres, revealing their unique nature.
Provides the first stable centromeric plasmids for O. polymorpha, expanding centromere diversity.
Offers a powerful genetic toolbox for advanced metabolic and physiological engineering in yeast.