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MCDB 101B > Cramming right before test > Flashcards

Cramming right before test Flashcards

1
Q

What are the steps involved in positional cloning?

A

• Region of interest narrowed by finding closely linked DNA markers.
• Candidate genes are located in the region of interest.
• Sequence and expression of
candidate genes are determined
in normal and diseased individuals.
• difference in sequence correlates with phenotype

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2
Q

What is positional cloning?

A

Is identifying the POSITION of a given gene

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3
Q

What are some limitations to Positional cloning?

A
  • crosses with two heterozygous parents wont be informative with heterozygous offspring
  • linkage map requires at least one parent to be double heterozygote
  • small families and few pedigrees with provide incomplete data
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4
Q

What is the equation for LOD?

A

LOD = log10[P(L)/P(NL)]

P(L) = probability of linkage
P(NL) = probability of no linkage
Odds = P(L)/P(NL)

LOD must be >3 to conclude genes are linked

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5
Q

What steps of gene expression are involved in regulation?

A
  • Transcription initiation
  • Transcript processing
  • Export from nucleus
  • Translation of mRNA
  • Protein localization
  • Protein modification
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6
Q

How are enhancers identified?

A

• Constructing a recombinant DNA molecule that has a putative enhancer sequence fused to a reporter gene such as the green fluorescent protein
(GFP)
• Generating a transgenic organism that has the recombinant DNA in its genome.
• test levels of GFP in organism without enhancer versus GFP levels of organism with enhancer

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7
Q

What is a mediator complex?

A
  • a complex of more than 20 proteins
  • bridge RNA pol II at the promoter and activator or repressor proteins at the enhancer
  • doesn’t bind DNA directly
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8
Q

What are the functional domains of activator proteins?

A
  • DNA binding domain: binds to specific enhancer
  • Activation domain: binds to other proteins (basal factors or coactivators)
  • Dimerization domain: SOME activators also have a domain that allows them to interact with other proteins
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9
Q

What are the two functions of corepressors?

A
  • Prevent RNA pol II complex from binding the promoter

* Modify histones to close chromatin structure

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10
Q

What are indirect repressors?

A
  • proteins that interfere with the function of an activator
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11
Q

What are the different functions of an indirect repressor?

A
  • Competition due to overlapping binding sites
  • Repressor binds to activation domain (quenching)
  • Binding to activator and keeping it in cytoplasm
  • Binding to activator and preventing homodimerization
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12
Q

How are transcription factors identified?

A

by fusing GFP to promoter region of gene and observing the effects of GFP expression in organisms with mutations in activator and repressor genes

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13
Q

What is the Max network?

A

Family of related “basic helix-loop-helix” DNA binding proteins that bind DNA as dimers
- Family is Max, Myc, and Mad proteins

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14
Q

What is the Max protein?

A
  • Max protein is a member of the Max network.
  • present in all cells
  • can form homodimers or heterodimers with other family members (Mad and Myc)
  • other family members can only form heterodimers with Max (they are present at different levels in different cell types)
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15
Q

What is the Myc protein?

A
  • Activator protein in the Max network

- can only form dimers with Max

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16
Q

What is the Mad protein?

A
  • repressor protein in the Max network

- can only form dimers with Max

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17
Q

How is Cell-type specific transcription is achieved

A

by changes in transcription factors
• Allosteric interactions (ex. steroid hormone receptor binds to enhancer only when bound to steroid hormone)
• Modification of transcription factors (ex. phosphorylation)
• Transcription factor cascades

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18
Q

How is gene expression regulated by hormones?

A
  • rapid
  • NR (nuclear receptor) is sequestered in the cytoplasm
  • Binding of membrane permeable steroid hormone to the NR causes a conformational change and forms dimers
  • NR-hormone complex translocates into the nucleus where it binds to Hormone Receptor enhancer elements
  • Recruits coactivators and basal factors
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19
Q

What is Chromatin immunoprecipitation-sequencing (ChIP-Seq)?

A

tool for finding all target genes of a particular transcription factor
within the entire genome of a particular type of cell

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20
Q

what are the steps of Chromatin immunoprecipitation-sequencing (ChIP-Seq)?

A
  • Crosslink DNA and protein component of chromatin
  • Fragment DNA
  • Allow an antibody specific to the protein of interest to bind
  • Purify complexes with antibody, protein of interest and DNA fragments
  • Sequence DNA
21
Q

How does an insulator sequence function?

A
  • Human insulators bind CTCF proteins to form loops called
    topologically associating domains (TADs)
  • enhancers will only be able to activate promoters that are located on the same loop
22
Q

How is mRNA translation regulated in response to nutrients?

A
  • 4E-BP1 binds to initiation factor eIF4E, blocks initiation
  • Presence of nutrients and growth factors in the environment leads to phosphorylation of 4E-BP1
  • Once phosphorylated, 4E-BP1 inactivates and initiation can begin
23
Q

what is the structure of primary transcripts of miRNAs?

A

The primary transcripts have double-stranded stem loops

24
Q

What are the three proteins required for processing miRNA?

A
  • Drosha
  • Dicer
  • RISC (miRNA-induced silencing complex)
25
Q

What is the purpose of Drosha?

A

excises stem loop from primary miRNA (pri-miRNA) to generate pre-miRNA

26
Q

What is the purpose of Dicer?

A

process pre-miRNA to a mature duplex miRNA

27
Q

What are the two ways in which miRNAs can down-regulate expression of target genes?

A
  1. when complementarity is perfect, target mRNA is degraded

2. when complementarity is imperfect, translation of mRNA target is repressed

28
Q

How are siRNAs (small interfering RNAs) produced?

A

• sources of dsRNA are precursors of siRNAs
- transcription of both strands of endogenous genomic sequence
-arise from exogenous virus
• dsRNAs are processed by Dicer

29
Q

How do siRNAs function?

A
  • Form ribonucleoprotein complexes with Argonaute proteins

* Interfere with gene expression or may destroy viral mRNAs

30
Q

How do piRNAs function?

A
  • minimize transposable element mobilization
  • make complexes with Piwi proteins
  • Complexes modify histones to interfere with TE transcription or degrade TE RNAs
31
Q

What are the 5 substages of Prophase I (meiosis)?

A

Leptotene, zygotene, pachytene, diplotene, and diakinesis

32
Q

What happens during the leptotene phase?

A

DSB appear

33
Q

What happens during the zygotene phase?

A

strand invasion and D-loop

34
Q

What happens during the pachytene phase?

A

double Holliday structure or

SDSA

35
Q

What happens during Diplotene phase?

A

resolution of double Holliday

structure in NCO or CO pathways

36
Q

What happens during the diakinesis phase?

A

Chromosomes are pulled apart

37
Q

What is an SRY gene?

A

SRY is a gene that determines whether a fetus will develop testes or ovaries. The SRY gene is usually only present on the Y chromosome. However, people who have two X chromosomes but an SRY gene on one of them will still develop testes. On the other hand, people who are born with a Y chromosome that lacks the SRY gene will develop ovaries.

38
Q

What are some features of the Y chromosome?

A
  • Male Specific Y (MYS): sends embryo down male pathways plus a few genes needed for testes development
  • Mostly highly condensed heterochromatin
  • a few non-testes related genes scattered on the rest of the Y
  • PARs at tips homologous with tips of Y (allows pairing and recomb.)
39
Q

Which gene is responsible for the deactivation of the X chromosome?

A

The Xist gene, located in the X inactivation center (XIC). Xist is upregulated on the inactive X chrom. and completely shut off in the active X chrom.

40
Q

What does the Xist gene do to promote X shut down?

A

The Xist gene promotes methylation of the DNA, starting at the X inactivation center (XIC) and spreading outward.

41
Q

What is the equation for calculating interference?

A

I = (1 - (obs# DCO / exp# DCO))(100)

42
Q

What is the SDSA pathway?

A

Recombination without crossing over: synthesis-dependent
strand annealing
Anticrossover helicase disentangles the invading strand from
the nonsister chromatid preventing Holliday junction
formation.

43
Q

How do you calculate the recombination frequency of unlinked genes in tetrads?

A

RF = (NPD + 1/2T)/total tedrads

44
Q

How do you calculate the recombination frequency of linked genes in tetrads?

A

RF = (1/2T + 3NPD)/Total

45
Q

How are mRNA introns spliced?

A
  1. The 5’ splice donor site (GU) is cut by spliceosome
  2. RNA folds over on itself, connecting to the donor site (A) in the middle of the intron (Lariat structure)
  3. The 3’ splice acceptor site (AG) is cut by spliceosome
  4. Mature mRNA is ligated together and intron is degraded
46
Q

What are the main Eukaryotic translation initiation factors?

A
  • Cap binding protein
  • eIF-4E
  • eIF-4A
  • eIF-4G
  • PolyA Binding Protein
47
Q

What does eIF-4E do?

A

The eIF-4E protein binds the 5’ cap in translation rounds following the pioneer round

48
Q

what does eIF-4A do?

A

RNA helicase activity, at cap

49
Q

what does eIF-4G do?

A

at cap, but binds to PABP

MCDB 101B (3 decks)

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