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Results: 5

1.
Figure 5

Figure 5. From: Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

A proposed model for how Snt309p modulates interactions of Prp19p with other components in the Prp19p-associated complex.

Hau-Ren Chen, et al. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5406-5411.
2.
Figure 2

Figure 2. From: Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

(A) Far Western blotting of the Prp19p-associated complex isolated from the wild-type (WT) and ΔSNT309 extracts by using 32P-labeled Prp19p as a probe. Prp19p was tagged with the HA-epitope and the complex was isolated by chromatography on an anti-HA antibody-conjugated Sepharose column. (B) Western blotting of the Prp19p-associated complex isolated as in A by using antibodies against Ntc85p, Prp19p, Ntc30p, Snt309p, and Ntc20p. (C) Western blotting of the Prp19p-associated complex immunoprecipitated by the anti-HA antibody in wild-type and ΔSNT309 extracts, in which Ntc20p was tagged with the HA-epitope.

Hau-Ren Chen, et al. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5406-5411.
3.
Figure 1

Figure 1. From: Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

The splicing activity of the ΔSNT309 extract was complemented by the Prp19p-associated complex and partially complemented by recombinant Snt309p. The splicing reactions were carried out in the wild-type (WT) extract (lane 1) and in ΔSNT309 extracts (lanes 2–9) with the addition of the Prp19p-associated complex (lane 3) or of His.Snt309p (lanes 4–9). The amounts of His.Snt309p added are, for lane 4, 150 ng; for lane 5, 50 ng; for lane 6, 15 ng; for lane 7, 5 ng; for lane 8, 3 ng; and for lane 9, 1.5 ng. M, mock-treated; +CPX, the Prp19p-associated complex added.

Hau-Ren Chen, et al. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5406-5411.
4.
Figure 4

Figure 4. From: Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

Spot assay of ΔSNT309 cells. ΔSNT309 or wild-type cells carrying different plasmids were grown until A600 reached ≈0.5 and then diluted in series by one order. From each diluted culture, 5 μl was spotted on plates. (A) ΔSNT309 cells were temperature-sensitive in growth (−/−) but grew better when carrying a PRP19-containing plasmid either on 2μ- or centromere-based vector (2μ/− and CEN/−). ΔSNT309 cells carrying a 2μ-based SNT309-containing plasmid grew well in the presence of another plasmid carrying PRP19 on CEN or 2μ plasmid, or under the GPD promoter control (CEN/2μ, 2μ/2μ, and GPD/2μ). (B) Wild-type strains carrying PRP19 on CEN or 2μ plasmid, or under the GPD promoter control (CEN, 2μ, and GPD) grew well at all temperatures. (C) ΔSNT309 cells carrying GPD–PRP19 on TRP1 plasmid and the SNT309 gene on URA3 plasmid grew less well by 1–2 orders on 5-fluoroorotic acid plates than when the PRP19 gene was on a 2μ- or CEN-based plasmid. Note that 0, −1, −2, −3, and −4 indicate no, 10−1, 10−2, 10−3, and 10−4 dilutions, respectively.

Hau-Ren Chen, et al. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5406-5411.
5.
Figure 3

Figure 3. From: Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

(A) Western blot analysis of splicing extracts prepared from strains overproducing Snt309p alone (lanes 1), both Snt309p and Prp19p (lane 5), or Prp19p alone (lane 6). Lane 2 is the cell pellet from the preparation of the splicing extract of lane 1. Lane 3 represents the total cell lysates from the strain overproducing Snt309p alone. Equivalent amounts of cell extracts were loaded on each lane. Ex, splicing extract; P, cell pellet; T, total lysates; M, molecular mass marker. (B) Complementation of the ΔSNT309 extract (Ext) by Prp19p–Snt309p binary complex. (Lane 1) Wild-type (WT) extract; (lanes 2–6) ΔSNT309 extracts. Fractions used for complementation (Comp): the Prp19p-associated complex (Cp; lane 3); affinity-purified fractions (AF Fr) overproducing Prp19p and Snt309p (lane 4), overproducing Prp19p only (lane 5), and overproducing no protein (lane 6). (C) Reconstitution of the Prp19p-associated complex. ΔSNT309 extracts (lanes 2–6) were added to an anti-HA antibody column-purified fraction from extracts overproducing both Snt309p and Prp19p (lanes 3 and 6), Prp19p only (lane 4), or normal extracts (lane 5), and then precipitated with the anti-Ntc20p antibody (lanes 1–5) or nonspecific (N) antibody (lane 6). Lane 1, wild-type extract.

Hau-Ren Chen, et al. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5406-5411.

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