bgl2 gene  (New England Biolabs)

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Post-translational modifications of Bgl2 and Scw4 in T, G and L pools. Glut—glutathionylation, P—phosphorylation. Modified amino acid residues are underlined and highlighted in bold.

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: Modification

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Analysis of extracts from Saccharomyces cerevisiae cell walls obtained with 0.1 M Tris for 3.5 h at 30 °C—T, with 6 M GuHCl for 2 h at 30 °C—G ( A – C ) and with water at 100 °C after removal of lipid component—L ( C , D ). PAGE stained with Coomassie G-250 ( A ) or with silver nitrate staining ( C ) and Western blot stained with antibodies against Bgl2 ( B , D ).

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Western blot analysis of Saccharomyces cerevisiae cell lysates obtained from WT-OE, N202-OE, and N284-OE strains. Samples were incubated for 15 min with (+) or without (−) endoglycosydase H (EndoH). Bgl2 bands are denoted by arrowheads; (*) indicates unglycosylated Bgl2. Staining with antibodies against Bgl2.

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: Western Blot, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Microscopy of structures formed in G pool extracted from Saccharomyces cerevisiae cell walls: TEM ( A – F ), immunofluorescence microscopy, staining with antibodies against Bgl2 ( G – I ). General view of jellyfish-like associates obtained from wt strain ( A , B , G , H ), fibrillar structure of their bodies ( D , E ). Control samples from bgl2Δ strain ( C , F , I ).

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: Microscopy, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Structural model of Bgl2 molecule without ( A ) and with glutathione ( B – D ). N-terminal is highlighted in green, C-terminal is highlighted in cyan. G32-S42 loop is shown in magenta. The glutathione molecule located in the cavity of Bgl2 greatly changes the structure ( B ). The external location of glutathione molecule does not contribute to significant changes in Bgl2 structure ( C , D ). Possible conformations of glutathione resulting from molecular dynamics simulation are shown as molecular surface (gray area). Axial ( A – C ) and frontal ( D ) views of the cartoon.

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Immunofluorescence microscopy of Saccharomyces cerevisiae cells and cell walls stained with antibodies against Bgl2. Scale bars correspond to 2 µm. Cells ( A – C ). CW with ( D , E ) and without ( F ) EDC crosslinking and boiled in 3% SDS. CW before ( G – I ) and after ( J – K ) Tris extraction. Control samples from bgl2Δ strain ( C , I , L ).

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: The hypothetical scheme of Saccharomyces cerevisiae cell wall segment with a microcompartment. We suppose that PTM-free Bgl2 enters the cell wall as a part of L pool. Depending on the degree of association with proteins in L pool, Bgl2 is directed to T or G pool. Probably, Bgl2, acquiring one or another set of PTMs, can migrate between T and G pools. In these two pools, at least some of the molecules are closely located, therefore, we suppose that they form the microcompartment. The localization of L pool could not be revealed. It is possible that proteins of L pool are dispersed rather than compactly located in the cell wall. Glut—glutathionylation, MultiP and MonoP—multi- and monophosphorylation, respectively.

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Saccharomyces cerevisiae strains used in present research.

Article Snippet: To obtain potential Bgl2 N-glycosylation site mutants, PCR mutagenesis of plasmid-encoded BGL2 gene [ ] was performed with Q5 site-directed mutagenesis kit (New England Biolabs, Moscow, Russia) according to manufacturer’s instructions.

Techniques: