62. Cultured animal cells were transfected with expression vector encoding either ß-galactosidase (β-gal) alone or expressing a fusion protein of β-gal and glucocorticoid receptor (GR). After transfection. cells were kept in presence or absence of Dexamethasone. Immunofluorescence with a labeled antibody specific for β -gal was used to detect the expressed protein incytoplasm or nucleus of transfected cells. Possible results of the experiments are:
A. Expression of ß-gal alone in the cytoplasm in both absence and presence of Dexamethasone.
B. Expression of ß-gal-GR in the cytoplasm ‘n the absence of Dexamethasone.
C. Expression of ß-gal alone in the nucleus both in the presence or absence of Dexamethasone.
D. Expression of ß-gal-GR in the nucleus in presence of Dexamethasone.
E Expression of ß-gal alone in both cytoplasm nucleus in presence or absence of Dexamethasone
F. Expression of ß -gal-GR in both cytoplasm and nucleus in presence of Dexamethasone.
Choose the correct combination of results from the following options.
(1) B, C and D     (2) A, B and D
(3) B, D and E     (4) A, B and F

Nuclear and Cytoplasmic Localization of β-galactosidase and β-gal–Glucocorticoid Receptor Fusion Proteins

Background on Experiment

Cultured animal cells were transfected with vectors expressing either $\beta$-galactosidase ($\beta$-gal) alone or a fusion protein of $\beta$-gal with glucocorticoid receptor (GR). Cells were treated with or without dexamethasone (a synthetic glucocorticoid). Localization of expressed proteins was detected by immunofluorescence using an antibody against $\beta$-gal.

Key Concepts from Literature

• $\beta$-gal alone: a cytoplasmic protein, does not enter the nucleus regardless of dexamethasone presence.

• GR protein: typically resides in the cytoplasm when unliganded, bound in complexes with chaperones.

• Upon ligand (dexamethasone) binding, GR translocates rapidly to the nucleus where it acts as a transcription factor.

• $\beta$-gal–GR fusion: expected to mimic GR behavior for localization due to presence of nuclear localization signals in the GR domain.

Interpretation of Options

• A: $\beta$-gal alone stays in cytoplasm regardless of dexamethasone — consistent with literature.

• B: $\beta$-gal–GR fusion is cytoplasmic without dexamethasone — consistent with unliganded GR localization.

• C: $\beta$-gal alone localizes in nucleus regardless of dexamethasone — inconsistent with literature.

• D: $\beta$-gal–GR fusion localizes to nucleus in presence of dexamethasone — consistent with ligand-induced GR nuclear translocation.

• E: $\beta$-gal alone localizes in both nucleus and cytoplasm regardless of dexamethasone — inconsistent.

• F: $\beta$-gal–GR fusion in both cytoplasm and nucleus in presence of dexamethasone — sometimes partial nuclear accumulation occurs, but predominant nuclear localization expected; partial cytoplasmic presence possible due to dynamic shuttling.

Most Supported Combination

Based on the above and published evidence :

• (A) $\beta$-gal alone in cytoplasm, no nuclear translocation

• (B) $\beta$-gal–GR in cytoplasm without ligand

• (D) $\beta$-gal–GR translocates to nucleus with dexamethasone

This fits option (2) A, B and D.

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