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#yeast genetics

12 public questions tagged with this topic.

Eukaryotes gene regulation

Deletion construct showing >100% activity suggests presence of:

Promoter deletion analysis uses reporter fusions to map cis-elements. Wild-type promoter driving luciferase set as 100% activity. Progressive 5' deletions may reduce activity if activator site removed, indicating positive element. Unexpected increase above 100% suggests removed fragment contained repressive sequence or competing site, unmasking strong enhancer in retained portion. In GAL analyses, deleting Mig1 binding sites elevated expression in glucose while retaining UASG produced hyperactive constructs. Therefore constructs exceeding wild-type infer presence of enhancer module now freed f

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 7: Promoter Deletion Analysis – Interpreting Enhancer Activity

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Eukaryotes gene regulation

Which enzyme removes acetyl groups during repression?

Histone acetylation neutralizes lysine epsilon-amino positive charge weakening DNA-histone interaction relaxing chromatin structure facilitating transcription factor access. Reversal mediated by histone deacetylases HDACs divided into Rpd3, Hda1, Sir2 families uses zinc or NAD as cofactors restoring positive charge promoting nucleosome compaction and repressive chromatin assembly. During glucose repression Tup1-recruited HDACs Rpd3L, Hda1 deacetylate H3K9, H3K18, H4K16 at GAL promoters preventing SAGA acetyltransferase Gcn5 access. Similarly at Saccharomyces telomeres Sir2 NAD-dependent HDAC d

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 4: Histone Deacetylase HDAC Function in Repression

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Eukaryotes gene regulation

UASG in GAL genes functions as:

UASG contains four 17-base-pair palindromic sites for Gal4 dimer located 200-400 nucleotides upstream of GAL1/GAL10 shared TATA box. Functionally analogous to metazoan enhancer, UASG activates heterologous promoters irrespective of orientation, requires Mediator and SAGA for looped communication with basal promoter via DNA bending. When fused to CYC1 minimal promoter driving reporter, UASG confers strong galactose inducible expression, classic enhancer test. Although yeast lacks distal enhancer complexity, UASG exemplifies enhancer concept in simple eukaryote, demonstrating activator recruitme

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 7: UASG as Enhancer Element in Yeast GAL System

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Eukaryotes gene regulation

Galactose induces GAL gene expression by inactivating which protein?

Galactose sensing and induction depend on Gal3 protein, paralog of Gal1 galactokinase lacking kinase activity but preserving ligand-binding pocket. In presence of galactose and ATP, Gal3 undergoes conformational change promoting high-affinity interaction with Gal80 repressor both cytoplasmic and nuclear fractions, sequestering Gal80 away from Gal4-bound promoters. Gal3-Gal80 sequestration frees Gal4 activation domain recruiting coactivators initiating transcription of GAL1, GAL10, GAL7 genes required for galactose catabolism via Leloir pathway. Gal1 itself contributes to maintenance after init

Ref: NCBI Bookshelf, Yeast Gene Regulation: Galactose Inactivation of Gal80 by Gal3

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Eukaryotes gene regulation

Gal4 activation is blocked by which masking protein?

Gal4 contains Zn2C6 binuclear cluster DNA-binding domain recognizing UASG as dimer plus activation domains. Without galactose, Gal80 repressor binds Gal4 activation domain region 851-874, occluding surfaces for Mediator subunit Med15, SAGA acetyltransferase, and TFIIH recruitment. Masking retains Gal4 on DNA but creates inert complex unable to stimulate initiation. Gal80 does not evict Gal4, only blocks polymerase II holoenzyme recruitment. Upon galactose sensing, Gal3 sequesters Gal80, freeing activation domain to drive multiple transcription rounds and chromatin remodeling at GAL promoters e

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 7: Gal80 Masking of Gal4 Activation Domain

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Eukaryotes gene regulation

Mig1 represses GAL1 transcription by recruiting which complex?

Mig1 imposes repression by recruiting general co-repressor complex Cyc8-Tup1, yeast equivalent of metazoan TLE/Groucho corepressors. Mig1 C-terminal repression domain interacts with Tup1 WD40 repeat domain assembled with Cyc8 tetratricopeptide motifs stabilizing tetrameric complex. Assembly subsequently attracts histone deacetylases Rpd3L complex containing Hos2, Hda1, inducing deacetylation of H3K9, H3K18, H4K16 at GAL promoters, increasing nucleosome affinity and masking activation domains. Deacetylation reduces accessibility for acetyltransferase Gcn5 within SAGA complex, preventing Gal4 co

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 7: Mig1 Interaction with Tup1-HDAC Complex

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Recombination

In mating type switching, donor locus is

In budding yeast, chromosome III harbors two silent donor repositories: HML containing alpha information near left telomere and HMR containing a information near right telomere, each flanked by homology boxes X and Z1 shared with MAT. During switching, MAT is recipient receiving new genetic identity, while HML or HMR acts as template providing sequence without being altered itself, analogous to SDSA. Choice of donor regulated by recombination enhancer controlling accessibility; MATa cells preferentially recombine with HML alpha, MAT alpha cells with HMR a, ensuring productive switching rather

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 21: Silent Loci as Donors for MAT Switching

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Recombination

Mating type switching occurs via which recombination pathway?

Mating type conversion involves copying information from silent donor into broken MAT locus. After HO induced break, resected MAT end invades HML or HMR sharing Z1 and W homology regions forming D-loop. Repair synthesis copies Ya or Yα sequence from donor, then nascent strand dissociates from donor and anneals back to second end of MAT, filling gap without establishing stable double Holliday junction or crossover between chromosome III regions. This synthesis-dependent strand annealing mechanism results in non-crossover gene conversion, preserving donor cassette unchanged while converting reci

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 18: Mating Type Switching via SDSA Mechanism

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Recombination

HML and HMR loci are

Yeast possesses three mating type related loci on chromosome III but only MAT is transcriptionally active. HML alpha and HMR a loci are embedded in telomere proximal heterochromatin enriched for Sir2–Sir4 deacetylase complex that removes histone acetylation and establishes hypoacetylated nucleosomes silencing transcription. Rap1 and Abf1 recruit Sir proteins spreading silent chromatin across silencers E and I flanking cassettes. This silencing prevents co-expression of both mating programs simultaneously avoiding sterile phenotype, while allowing them to serve as unexpressed templates for reco

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: Silent HML HMR Loci and Heterochromatin Silencing

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Recombination

HO endonuclease cleaves

Mating type switching requires precise double-strand break initiation at active MAT locus. HO endonuclease is highly specific endonuclease expressed transiently in mother cells during late G1. It recognizes long degenerate 24 base-pair site spanning Ya/Z1 region at MAT, cleaving to generate 4 nucleotide 3' overhangs. Following cleavage, resection proceeds and strand invades silent donor cassette. HML and HMR themselves resist HO cleavage because heterochromatin and Sir proteins protect and because cleavage site degraded after donor copying. Thus HO activity is restricted to MAT enabling contro

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 7: HO Endonuclease Cleavage at MAT Locus

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Recombination

Mating type switching in yeast occurs at which locus?

Saccharomyces cerevisiae haploids maintain mating type via single active MAT locus on chromosome III encoding regulators for a or alpha phenotype. Switching involves regulated replacement of MAT sequences. HML and HMR loci contain silent copies of both mating types embedded in heterochromatin near telomeres and not expressed. HO endonuclease induced double-strand break at MAT triggers recombination using HML/HMR as donor template copying opposite mating information into MAT. Therefore actual site of switching event where expression changes occurs at MAT, while silent cassettes serve merely as

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 18: Yeast Mating Type Switch at MAT Locus

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Restriction Enzyme and Vector

Which selectable marker suppresses yeast mutations?

In yeast genetics, suppressor tRNA markers are used to monitor nonsense mutation suppression. SUP4 encodes a tyrosine tRNA with anticodon mutated to recognize ochre stop codon UAA, suppressing ade2-101 and other ochre alleles, leading to phenotypic color change in adenine biosynthesis pathway. In YAC vectors, SUP4 is placed within cloning site; disruption by foreign DNA abolishes suppression. Auxotrophic markers URA3 and TRP1 provide selection for transformation, while LacZ and GFP are bacterial reporter genes. SUP4 thus acts as insertional marker that visibly reports insertion by altering sup

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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