scholarly journals CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

2018 ◽  
Author(s):  
Florian Nadler ◽  
Felix Bracharz ◽  
Johannes Kabisch
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Gene Dosage ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Gene Copy ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Copy Numbers ◽  
Gene Copy Numbers

1.AbstractThe Gram-positive bacteriumBacillus subtilishas long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacteriumBacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host’s intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold highergfp(green fluorescent protein) expression and up to 1.3-fold highermPLC(mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-bornegfpexpression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

Organization of regionally expressed silkmoth chorion genes

1986 ◽  
Vol 6 (9) ◽  
pp. 3215-3220
Author(s):  
A K Hatzopoulos ◽  
J C Regier
Keyword(s):  
Gene Expression ◽  
Follicle Cells ◽  
Gene Copy ◽  
Late Period ◽  
Chorion Genes ◽  
Copy Numbers ◽  
Total Dna ◽  
Dna Strand ◽  
Gene Copy Numbers ◽  
Silkmoth Chorion

We described the organization of two silkmoth chorion genes, called E1 and E2, whose expression is largely restricted in time to the very late period of choriogenesis and in space to one of two major subpopulations of follicle cells. Using E1 and E2 clone cDNAs as probes, we showed that gene copy numbers per haploid genome remain constant throughout silkmoth development despite major changes in total DNA content per nucleus. Furthermore, gene copy numbers are the same in both cellular regions of the choriogenic follicle despite differences in nuclear size and levels of E gene expression. Southern analysis indicated between two and four copies each for E1 and E2 genes. Analysis of chromosomal clones showed that single copies of E1 and E2 are separated by about 7.5 kilobases and are transcribed from the same DNA strand. Two distinct pairs of cloned E1 and E2 genes were characterized. No other chorion genes were in their immediate vicinity.

scholarly journals The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression.

1985 ◽  
Vol 101 (5) ◽  
pp. 1749-1756 ◽  
Author(s):  
D E Muscarella ◽  
V M Vogt ◽  
S E Bloom
Keyword(s):  
Ribosomal Rna ◽  
Gene Dosage ◽  
Regulation Of Gene Expression ◽  
Nucleolar Organizer ◽  
Nucleolus Organizer ◽  
Rrna Genes ◽  
Gene Copy ◽  
Multiple Generations ◽  
Copy Numbers ◽  
Gene Copy Numbers

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.

scholarly journals Measuring the buffering capacity of gene silencing in Saccharomyces cerevisiae

2021 ◽  
Vol 118 (49) ◽  
pp. e2111841118
Author(s):  
Kenneth Wu ◽  
Namrita Dhillon ◽  
Kelvin Du ◽  
Rohinton T. Kamakaka
Keyword(s):  
Gene Silencing ◽  
Gene Dosage ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Levels ◽  
Copy Numbers ◽  
Upstream Activating Sequence ◽  
Functional Consequences ◽  
Gene Copy Numbers ◽  
Silent State

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

scholarly journals Organization of regionally expressed silkmoth chorion genes.

1986 ◽  
Vol 6 (9) ◽  
pp. 3215-3220 ◽  
Author(s):  
A K Hatzopoulos ◽  
J C Regier
Keyword(s):  
Gene Expression ◽  
Follicle Cells ◽  
Gene Copy ◽  
Late Period ◽  
Chorion Genes ◽  
Copy Numbers ◽  
Total Dna ◽  
Dna Strand ◽  
Gene Copy Numbers ◽  
Silkmoth Chorion

We described the organization of two silkmoth chorion genes, called E1 and E2, whose expression is largely restricted in time to the very late period of choriogenesis and in space to one of two major subpopulations of follicle cells. Using E1 and E2 clone cDNAs as probes, we showed that gene copy numbers per haploid genome remain constant throughout silkmoth development despite major changes in total DNA content per nucleus. Furthermore, gene copy numbers are the same in both cellular regions of the choriogenic follicle despite differences in nuclear size and levels of E gene expression. Southern analysis indicated between two and four copies each for E1 and E2 genes. Analysis of chromosomal clones showed that single copies of E1 and E2 are separated by about 7.5 kilobases and are transcribed from the same DNA strand. Two distinct pairs of cloned E1 and E2 genes were characterized. No other chorion genes were in their immediate vicinity.

Relative gene copy numbers in various patterns of cervical cancer growth.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18029-e18029
Author(s):  
Natalya N. Timoshkina ◽  
Natalia A. Petrusenko ◽  
Vera P. Nikitina ◽  
Diana A. Spiridonova ◽  
Ekaterina V. Verenikina ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Copy Number ◽  
Gene Dosage ◽  
Total Sample ◽  
Gene Copy ◽  
Relative Copy Number ◽  
Genomic Changes ◽  
Copy Numbers ◽  
Cyp1a1 Gene ◽  
Gene Copy Numbers

e18029 Background: Numerous studies on cervical cancer confirm an important role of specific genomic changes in the onset and development of cervical intraepithelial neoplasia and their effect on the progression of cervical cancer. Solid tumors are characterized by genomic changes leading to a change in the DNA sequence copy number. The purpose of the study was to reveal changes in the relative copy number of the ESR1, ESR2, GPER1, STS, SULT1A1, SULT1E1, CYP1A1, CYP1A2 genes responsible for the reception and metabolism of estrogens in cervical tissues in endophytic and exophytic patterns of tumor growth in order to find predictive markers of malignancy. Methods: The study included 40 patients aged 28-65 years with cervical cancer of endophytic (n = 20) and exophytic (n = 20) growth patterns. Eligibility criteria included a morphologically confirmed cervical squamous cell cancer T1b-2aN0M0, stage I-II. The Thermo Scientific GeneJET FFPE DNA Purification Kit was used for the DNA extraction from FFPE blocks of tumor and healthy tissues. DNA concentrations were measured on the Qubit 2.0 fluorimeter (Invitrogen, USA) using the Quant-iT dsDNA High-Sensitivity (HS) Assay Kit (Invitrogen, USA). Results: The relative copy number of the GPER1, SULT1A1, CYP1A1 genes in tumor samples increased (p < 0.05) compared with normal tissues in the total sample of patients diagnosed with cervical cancer. In contrast to the total sample, an increase in the SULT1A1 gene dosage did not reach a statistically significant level in any group (p = 0.242 and p = 0.157); the copy number of the GPER1 locus significantly increased only in the group with the endophytic growth pattern (p = 0.040), as well as the CYP1A2 gene dosage (p = 0.025). Patients of 36-55 years with endophytic tumors showed a statistically significant (p < 0.05) increase in the GPER1 and CYP1A1 gene copy numbers with the rates of 41.7% and 66.7%, respectively, as well as an increased amplification of the CYP1A2 gene in 41.67% of patients. In women of 56-75 years with endophytic tumors, an increase in the copy numbers of the ESR2, GPER1, SULT1A1 genes was observed with a frequency of 50%, 100% and 75%, respectively. Patients aged 20-35 and 36-55 years with exophytic tumors showed a statistically significant (p < 0.05) increase in the CYP1A1 gene copy numbers in 33.33% and 45.45%, respectively. Conclusions: The results suggest the use of the GPER1, SULT1A1 and CYP1A1 gene copy numbers as biomarkers of cervical tumors.

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