Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: Yeast Strains

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Transformation Assay

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: The YPT31 and YPT32 genes encode two functionally homologous exocytic GTPases. ( A ) Comparison of the amino acid sequences of Ypt31 and Ypt32 proteins. The predicted protein sequence of Ypt32 was compared to Ypt31 using the MegAlign program (DNAStar Inc., Madison, WI, Clustal method with the PAM250 residue weight table). Identities are shaded with solid black, and residues conserved to within two distance units are shaded. Overall, the two proteins are 81.1% identical and 89.6% similar when compared using the bestfit program (Genetics Computer Group, Madison, WI). ( B ) Ypt31 and Ypt32 proteins belong to a subfamily of exocytic Ypt GTPases by phylogenetic analysis of the Ypt/rab family of small GTPases. The predicted amino acid sequence of Ypt31 and Ypt32 were compared to all other Ypt proteins, using the completed S. cerevisiae genome sequence and their human homologues. The analysis shows that Ypt/rab proteins fall into two functional subfamilies: those involved in endocytosis and vacuolar protein sorting ( Endocytosis-Vac .) and those involved in exocytosis. Sequences were aligned as above. The scale at the bottom indicates the number of substitutions between sequences. hum. , Homo sapiens ; H-ras , Harvey murine sarcoma virus ras protein; all other sequences, Saccharomyces cerevisiae. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers: Ypt31, U18778; Ypt32, X72834; rab11, X56740; Ypt7, X68144; rab7, U44104; Ypt1, X00209; rab1a, M28209; Sec4, M16507; Ypt6, U17244; rab6a, M28212; Ypt51, X76173; Ypt52, X76174; Ypt53, X76175; rab5b, X54871; and H-ras, X00740. ( C ) Requirement of YPT31 or YPT32 genes for cell viability. The YPT31 gene was precisely deleted using the HIS3 gene, and this strain was transformed with a URA3 -marked CEN vector containing the YPT31 gene under control of its own promoter (NSY301, first row ). This strain (NSY301) was subsequently deleted for the YPT32 gene using the KAN r gene as a dominant delectable marker (NSY302, middle row ). Finally, this strain (NSY302) was transformed with a second plasmid marked with LEU2 and carrying the YPT31 gene (NSY306, bottom row ). The three strains were grown in synthetic media maintaining selection for plasmids. Serial dilutions of cells were then spotted onto either SD or SD-FOA and grown at 26°C. Cells deleted for both genes do not grow on SD-FOA plates, indicating that they cannot lose the URA -marked YPT31 plasmid unless they carry also the LEU -marked YPT31 plasmid.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Sequencing, Functional Assay, Transformation Assay, Plasmid Preparation, Marker, Selection

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: The YPT32-A141D mutation is in a conserved residue and confers a tight conditional growth phenotype. ( A ) Comparison of the amino acid sequence of the α-helix 5 region in Ypt/rab and ras proteins. Arrow indicates conserved residue, which when mutated to Asp results in temperature-sensitive function in Ypt1 ( ypt1-A136D ), Ypt32 ( ypt32-A141D ), and Sec4 ( sec4-8 , sec4-G147D ). ( B ) The ypt32-A141D mutation confers a temperaturesensitive growth phenotype in cells deleted for the YPT31 gene. Serial dilutions of wild-type (NSY128), Δypt31 (NSY290), Δypt32 (NSY296), and Δypt31,ypt32-A141D (NSY313) cells were spotted on YPD plates and incubated at 26° or 37°C.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Sequencing, Incubation

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: Ypt31 protein is found at sites of polarized cell growth. ( A ) Characterization of anti-Ypt31 antibodies. Antibodies raised against Ypt31p react with Ypt31p, and to a lesser degree with Ypt32p, but not Ypt1 and Sec4 proteins. Bacterially purified Ypt1, Ypt31, Ypt32, and Sec4 proteins were analyzed by SDS-PAGE followed by either Coomassie staining (1 μg protein in each lane) or Western blotting (5 ng protein in each lane) using anti-Ypt1 or anti-Ypt31 antibodies. ( B ) Detection of Ypt31 and Ypt32 proteins in yeast cell extracts: Ypt31p is present in excess of Ypt32p in wild-type yeast cells. Wild-type (NSY128) or deletion strains ( Δypt31 , NSY 290 and Δypt32 , NSY296) transformed with the indicated plasmids were grown in synthetic medium maintaining selection for deletions and plasmids present. Cells were lysed by the addition of 2× Laemmli buffer and boiling for 5 min. 0.5 OD 600 U of lysate from each strain was then subjected to immunoblot analysis using affinity-purified anti-Ypt31 antibodies. ( C ) Localization of the Ypt31 protein in yeast cells using immunofluorescence microscopy. Diploid yeast cells (JK9-3d) were double stained with rhodamine-phalloidin (to visualize filamentous actin) and with anti-Ypt31 antibodies. Cells were also photographed using Nomarski optics to distinguish budded from unbudded cells ( top ). Arrowheads indicate polarized Ypt31 staining, which localizes to regions of cell growth in (from left to right) a cell in late G1, a cell in early S phase, and a cell undergoing cytokinesis. Bar, 10 μm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Purification, SDS Page, Staining, Western Blot, Transformation Assay, Selection, Affinity Purification, Immunofluorescence, Microscopy

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: ypt31-Δ/ypt32-A141D mutant cells exhibit a tight conditional secretory block of invertase transport downstream of the medial -Golgi. Wild-type (NSY349, left ) and mutant (NSY348, right ) cells were labeled for 7 min at 26°C. Cells were then chased at 26°C ( A ) or shifted to 37°C ( B ) with prewarmed media and chased for the indicated times. At each time point, cells were separated from the periplasmic fraction by spheroplasting, lysates were prepared, and samples were immunoprecipitated with antiinvertase antibodies. Equal portions of precipitate from each time point were then separated on 8% SDS–polyacrylamide gels. ( C ) Intracellular invertase from the indicated time point was divided into three equal portions and precipitated with antisera against invertase, α1,6-mannose, or α1,3-mannose residues as indicated. ER ( core ), Golgi ( outerchain ), and cytoplasmic ( cyto. ) forms of invertase are indicated in the left margin.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Blocking Assay, Labeling, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: ypt31-Δ/ypt32-A141D (NSY313) and sec4-G147D (PNY404) mutant cells both show defects in the trans -Golgi processing and transport of α-factor at the nonpermissive temperature. ( A ) The indicated strains ( wild-type is NSY128) were labeled for 7 min at 26°C, shifted to 37°C with prewarmed media, and chased for the indicated times. Cells were separated from medium by centrifugation and processed for immunoprecipitation with anti–α-factor antibodies. ( B ) Alternatively, cells were preshifted to 37°C for 2 min with prewarmed medium, labeled for 7 min and chased for the indicated times. Only mature peptide is shown, as higher molecular weight forms of α-factor behave similarly to those shown in A , except that both mutants secreted proportionally less high molecular weight α-factor after the preshift, and all strains (including wild-type) show translocation and processing defects. Positions of ER ( core ), cis -Golgi ( α16 ), medial -Golgi ( α1-3 ), and trans -Golgi ( mαf ) forms are noted in the left margin. Arrows in the right margin indicate secreted α-factor that was not fully processed in the trans -Golgi.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Labeling, Centrifugation, Immunoprecipitation, Molecular Weight, Translocation Assay

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: ypt31-Δ/ypt32-A141D mutant cells do not exhibit a major defect in targeting of CPY to the vacuole at the nonpermissive temperature. Extracts from the 37°C samples shown in Fig. were subjected to precipitation with anti-CPY antibodies and resolved on 8% SDS–polyacrylamide gels. Positions of the ER ( p1 ), Golgi ( p2 ), and vacuolar ( m ) forms of CPY are indicated in the left margin.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: ypt31-Δ/ypt32A141D mutant cells accumulate aberrant Golgi-like structures at the nonpermissive temperature. Wild-type and three strains mutated at the analogous conserved residue in YPT1 , YPT32 , and SEC4 were analyzed by electron microscopy. Electron micrographs of representative cells are shown for the following strains: ( A ) wildtype (NSY128), ( B ) ypt1A136D (NSY222), ( C ) ypt31Δ/ypt32-A141D (NSY348), and ( D ) sec4-G147D (PNY 404). Cells were grown at 26°C, shifted to 37°C for 2 h, and then processed for thin section electron microscopy. Arrows point to membranous structures unique to each strain. Arrowheads indicate ER. G , wild-type Golgi cisterna (shown in detail in Fig. A , second panel from left ); V , vacuole; n , nucleus. ( E ) Quantification of the distinct membranous structures that accumulate in the different mutant strains: small vesicles (50–80 nm), Golgi (cisternae or Berkeley bodies), and large vesicles (100–150 nm). Bars represent the mean number of structures in 30 cell sections. Data is normalized to density per cubic micrometer for vesicle populations and density per 10 μm 2 for cisternae and Berkeley bodies. Error bars represent one standard deviation (see Materials and Methods for details of the quantification procedure). The relatively high standard deviations are probably due to the aggregation of the aberrant membranes to one side of the cell, resulting in cell sections that are either rich in or devoid of the corresponding membranous structure. Bar, 1 μm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Electron Microscopy, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: ypt31-Δ/ypt32A141D mutant cells accumulate Golgi membranes at the permissive temperature and Berkeley bodies at the nonpermissive temperature. Electron microscopy of ( A ) wild-type (NSY128), ( B ) ypt31-Δ / ypt32-A141D mutant strain (NSY348) at 26°C, and ( C ) ypt31-Δ/ypt32A141D mutant strain (NSY348) at 37°C. Arrows indicate a single Golgi cisterna in a wild-type cell and regions of Golgi accumulation in mutant cells. v , vacuole; n , nucleus. ( D ) Quantification of cisternal profiles and Berkeley bodies in wildtype cells at 26°C and ypt31Δ / ypt32-A141D mutant (NSY348) cells at 26° and 37°C. The percentage of the total structures counted that are cisternal is indicated by the gray bar, and the percentage of Berkeley bodies is represented by the black bar. At the permissive temperature, the mutant shows a fivefold increase in the frequency of Golgi profiles, the majority of which (70% of total) are cisternal. After 2 h at 37°C, the number of Golgi profiles has doubled and the population is dominated by Berkeley bodies (60% of total). ( E ) Immunofluorescence microscopy using anti-Ypt1p antibodies . ( 1 ) wild-type cells (NSY128) grown at 26°C; ( 2 ) the ypt31-Δ/ypt32-A141D mutant (NSY348) grown at 26°C; or ( 3 ) shifted to 37°C, for 90 min; ( 4 ) the same cells shown in 3 photographed with Nomarski optics to show the contours of the cells. Bars: ( A–C ) 1 μm; ( E ) 10 μm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Electron Microscopy, Immunofluorescence, Microscopy

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: Details of Golgi structures in wild-type and ypt31-Δ / ypt32-A141D mutant cells. ( A ) Electron microscopy of wild-type (NSY128) cells. Wild-type Golgi is usually observed as an elongated or cup-shaped single cisternae , which are not always continuous ( third panel from left ) because of fenestration . ( B ) Enlarged ypt31-Δ / ypt32-A141D mutant (NSY348) Golgi structures. Long arrows indicate an example of stacked cisternae in the mutant strain. Short arrows indicate spherical and tear drop–shaped Berkeley bodies. The tear-shaped structure suggests that Berkeley bodies are derived from individual cup-shaped cisternae that fuse at the rim (see curved arrow for a possible intermediate in this process). Open arrow indicates swelling to 100 nm at the cisternal rim, which may represent an intermediate preceding vesicle formation by membrane fission. Arrowheads indicate rare 100nm vesicles seen in the vicinity of cisternae, for size comparison with structures that look like budding vesicles. Note also the increased length of cisternal profiles in the mutant strain compared to wild-type. Regions of four different cells are shown for each strain. Bars, 0.5 μm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Electron Microscopy, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: Ypt1p is present on aberrant Golgi membranes accumulated in the ypt31-Δ / ypt32-A141D mutant cells. ypt31-Δ/ypt32A141D mutant cells were grown at 26°C and shifted to 37°C for 1 h before fixation and processing for immunoelectron microscopy using anti-Ypt1p antisera. Gold particles are seen specifically labeling stacked cisternae ( long arrow ) and spherical structures ( short arrow ). Note a strand of ER that is unlabeled ( arrowhead ) and the lack of gold particles over mitochondria ( m ) and cytosol. Bar, 250 nm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Immuno-Electron Microscopy, Labeling

Journal: The Journal of Cell Biology

Article Title: Two New Ypt GTPases Are Required for Exit From the Yeast trans -Golgi Compartment

doi:

Figure Lengend Snippet: The ypt31-Δ / ypt32-A141D , sec1-1 triple mutant accumulates Golgi structures. Electron microscopy of ( A ) ypt31-Δ / ypt32A141D (NSY348) mutant, ( B ) the ypt31-Δ / ypt32A141D , sec1-1 triple mutant transformed with YPT31 on a single copy vector (phenotypically sec1-1 ), and ( C ) the ypt31-Δ/ypt32 - A141D , sec1-1 (NSY368) triple mutant. Arrows point to structures characteristic of each strain; arrowheads indicate ER that can be seen in continuity with the nuclear envelope in the triple mutant, but are also present in ypt31-Δ/ ypt32-A141D mutant cells. n , nucleus. Bar, 1 μm.

Article Snippet: YPT32 deletion was confirmed using the oligos YPT32 dv (GCA TGG TTC GCG CTG GAA AGA G) and HIS3 -1, or kan r -1 (GTG AGA ACT GTA TCC TAG CA) for deletion by HIS3 and kan r , respectively.

Techniques: Mutagenesis, Electron Microscopy, Transformation Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: Functional metagenomics identifies novel genes ABCTPP, TMSRP1 and TLSRP1 among human gut enterotypes

doi: 10.1038/s41598-018-19862-5

Figure Lengend Snippet: Bacterial strains and plasmids used in the present study.

Article Snippet: Transposon, EZ-Tn5TM , Tn5 TM Transposon Kan r , Epicentre Biotechnologies Madison, Wisconsins, USA.

Techniques: Clone Assay, Recombinant, Plasmid Preparation, Cloning

Journal:

Article Title: Cloning and Characterization of the Bile Salt Hydrolase Genes ( bsh ) from Bifidobacterium bifidum Strains

doi: 10.1128/AEM.70.9.5603-5612.2004

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: When appropriate, ampicillin (200 μg/ml) and kanamycin (40 μg/ml) were added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Origin or relevant characteristics Source or reference Bacterial strains B. bifidum ATCC 11863 Wild type for cloning ATCC B. bifidum ATCC 35914 Human feces ATCC B. bifidum ATCC 15696 Infant intestine ATCC B. bifidum ATCC 29521 Type strain; infant feces ATCC B. bifidum KL 301 Dairy isolate This study B. bifidum KL 306 Dairy isolate This study B. adolescentis ATCC 15703 Type strain; adult intestine ATCC B. longum ATCC 15707 Type strain; adult intestine ATCC B. infantis ATCC 15697 Type strain; infant intestine ATCC B. breve ATCC 15707 Type strain; infant intestine ATCC E. coli DH5α F − φ80d lacZ ΔM15 Δ( lacZYA-argF ) U169 endA1 recA1 hsdR17 (r K − m K + ) deoR GIBCO-BRL E. coli BL21(DE3) F − ompT hsdS B (r B − m B − ) gal dcm lacY1 (DE3) Novagen Plasmids pBR322 Ap r ; cloning vector Invitrogen pUC19 Ap r ; cloning vector Invitrogen pDrive Ap r Kan r ; U-overhang PCR cloning vector QIAGEN pET36b(+) Kan r ; E. coli expression vector Novagen pBSH14 pBR322 with 3.5-kb B. bifidum ATCC 11863 chromosomal insert; BSH + This study pBSH27 pBR322 with 7.5-kb B. bifidum ATCC 11863 chromosomal insert; BSH + This study pBSH274 pUC19 with 4.0-kb Pst I insert from pBSH27; BSH + This study pDB150 pDrive with 1.5-kb PCR product; BSH + This study pUCB150 pUC19 with 1.5-kb Hind III & Kpn I insert from pDB150; BSH + This study pDB095 pDrive with 950-bp PCR product; BSH + This study pUCB095 pUC19 with 950-bp Hind III & Kpn I insert from pDB095; BSH − This study pBSH36b pET36b(+) with 950-bp Nde I & Hind III insert from pDB095; BSH + This study pBCS pDB095 with Cys-1-Ser mutation; BSH − This study pBCT pDB095 with Cys-1-Thr mutation; BSH − This study Open in a separate window Bacterial strains and plasmids used in this study Enzyme purification.

Techniques: Plasmid Preparation, Clone Assay, PCR Cloning, Expressing, Mutagenesis

Journal: Microbial Cell Factories

Article Title: A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

doi: 10.1186/s12934-023-02262-4

Figure Lengend Snippet: Production and testing of precursor plasmid v1 and nChr 1 . (a) Incorporation of an initial, one-off, gene-landing pad into the precursor plasmid. Work on eDA83 was halted due to leaky I-SceI expression in E. coli . (b) Following deletion of I-SceI in eDA83 by insertion of Kan R (creating eDA110), Tel was inserted to yield eDA137, the precursor plasmid ( v 1) of nChr 1. The plasmid was linearized in vitro and used to transform (“L&T” in figure) CBS7435 K. phaffii cells creating strain yDA122. The agarose gel shows I-Sce I digestion of Tel -containing eDA137 ( versus control, i.e. no- Tel , eDA110). (c) Despite nChr 1 appearing stable in chromosome-loss assays performed on yDA122, WGS (and subsequently, colony PCR, see gel) revealed major chromosomal rearrangements as indicated in the schematic. Note, in yDA122, the loss of PCR-product 3 (of nChr 1), but retention of PCR-product 2 of the suspected translocation product nChr 1* (i.e. nChr 1(p):Chr 3(q))

Article Snippet: To achieve this, the 13.5-kb Sph I-digested and gel-extracted eDA83 was ligated with a 980-bp PCR-amplified Kan R (using oligos 321/322 and vector pUC57-Kan (Addgene) as a template).

Techniques: Plasmid Preparation, Expressing, In Vitro, Agarose Gel Electrophoresis, Translocation Assay

Journal:

Article Title: Differential Effects of Replacing Escherichia coli Ribosomal Protein L27 with Its Homologue from Aquifex aeolicus

doi: 10.1128/JB.183.22.6565-6572.2001

Figure Lengend Snippet: Strains and plasmids used in this work

Article Snippet: pREP4 , Kan r lacI , Qiagen.

Techniques: Plasmid Preparation

Journal: Microbial Biotechnology

Article Title: Engineering and application of LacI mutants with stringent expressions

doi: 10.1111/1751-7915.14427

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: pBAD18‐kan , P BAD ; pBR322 origin, Kan r , BioVector NTCC Inc., Beijing, China.

Techniques: Laser Capture Microdissection, Mutagenesis, Modification