Hygromycin B resistance as dominant selectable marker in yeast

Agrobacterium tumefaciens-mediated transformation with plasmids carrying the hygromycin B resistance gene hph frequently is being used for inserting genes into fungal spores and mycelial cells and for conducting insertional mutagenesis to identify genes connected to a particular phenotype. In this article, we report that stable hygromycin B resistance can develop spontaneously in germinating conidia from Monilinia fructicola and that the mutants exhibit altered phenotypes. One spontaneously developing hygromycin B-resistant colony developed per 2.5 × 105 germinating conidia. Mutants grew significantly slower on potato dextrose agar, were 2.4- to 3.1-fold more sensitive to demethylation inhibitor fungicides, lacked melanization, and did not produce spores. The mode of action of hygromycin B resistance in the mutants seemed to be different from the hph transgene-mediated hygromycin B resistance based on different phenotypic characters. The ability of M. fructicola and possibly other fungi to spontaneously develop hygromycin B resistance associated with an altered phenotype may interfere with the selection of true transformants if hygromycin B is used as selective agent. This is particularly confounding if the hph gene is used as selectable marker in insertional mutagenesis experiments conducted for the identification of genes involved in melanization, sporulation, or fungicide resistance.

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Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

Plant Methods ◽

10.1186/s13007-019-0526-5 ◽

2019 ◽

Vol 15 (1) ◽

Author(s):

Xinjia Yang ◽

Jialin Peng ◽

Junmin Pan

Keyword(s):

Resistance Genes ◽

Selectable Marker ◽

Ectopic Expression ◽

Nuclear Gene ◽

Expression Vectors ◽

Cross Resistance ◽

Hygromycin B ◽

Selectable Markers ◽

Hygromycin B Resistance ◽

Transgenic Strains

Abstract Background Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae. Results C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide. Conclusions This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

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The hygromycin B-resistance-encoding gene as a selectable marker for stable transformation of Trypanosoma brucei

Gene ◽

10.1016/0378-1119(91)90159-9 ◽

1991 ◽

Vol 105 (2) ◽

pp. 255-257 ◽

Cited By ~ 26

Author(s):

Lee Mary Gwo-Shu ◽

Lex H.T. van der Ploeg

Keyword(s):

Trypanosoma Brucei ◽

Selectable Marker ◽

Stable Transformation ◽

Hygromycin B ◽

Hygromycin B Resistance ◽

Encoding Gene

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Insertional mutagenesis in Coprinus cinereus : use of a dominant selectable marker to generate tagged, sporulation-defective mutants

Current Genetics ◽

10.1007/s002940050512 ◽

1999 ◽

Vol 36 (6) ◽

pp. 371-382 ◽

Cited By ~ 59

Author(s):

W. Jason Cummings ◽

Martina Celerin ◽

Jennifer Crodian ◽

Linda K. Brunick ◽

Miriam E. Zolan

Keyword(s):

Insertional Mutagenesis ◽

Selectable Marker ◽

Coprinus Cinereus ◽

Dominant Selectable Marker

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The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4011-4019.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4011-4019

Author(s):

J A Nelson ◽

P B Savereide ◽

P A Lefebvre

Keyword(s):

Chlamydomonas Reinhardtii ◽

Selectable Marker ◽

Marker Gene ◽

Small Subunit ◽

Direct Selection ◽

Nuclear Transformation ◽

Ribulose Bisphosphate Carboxylase ◽

Bisphosphate Carboxylase ◽

Dominant Selectable Marker ◽

Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

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Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2929-2931.1984 ◽

1984 ◽

Vol 4 (12) ◽

pp. 2929-2931 ◽

Cited By ~ 1

Author(s):

K Blochlinger ◽

H Diggelmann

Keyword(s):

Selectable Marker ◽

Dna Transfer ◽

Direct Selection ◽

Regulatory Sequences ◽

Hygromycin B ◽

Dominant Marker ◽

Dna Coding ◽

Sarcoma Virus ◽

Moloney Sarcoma Virus ◽

Selection For

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

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