1
Q
3 laws of inheritance (mendel)
A
1) Law of Dominance
2) Law of Segregation
3) Law of Independent Assortment
2
Q
Law of dominance
A
allels can be dominant or recessive
3
Q
Law of Segregation
A
separation of alleles @ the level of the gametes
- Two alleles of a pair segregate or separate during gamete formation such that a gamete receives only one of the two factors
4
Q
Law of Independent Assortment
A
The alleles of two different genes get sorted into gametes independently of one another (Mendel had selected traits that were on different chromosomes –> law not true when different genes are present on the same chromosome (passed on linked as a linkage group))
5
Q
homologous chromosomes
A
chromosome from mom and chromosome for dad - code the same genes but may have different alleles on both
6
Q
linkage group
A
if you have a particular trait, there may be another trait that is always associated with it
- competes with the Law of Independent Assortment
- different genes on the same chromosome can be passed on as a linkage group
7
Q
Thomas Hunt Morgan
A
mutation recognized as mechanism for variation in populations
8
Q
physical underpinning of the unit of inheritance
A
chromosomes
9
Q
Genetic material in all
organisms
A
DNA
10
Q
Building block for DNA
A
nucleotide
11
Q
Nucleotide structure
A
- Phosphate
- Sugar (deoxyribose)
- Nitrogenous base (pyrimidine/purine)
12
Q
pyrimidines
A
Thymine and Cytosine
13
Q
purines
A
Adenine and Guanine
14
Q
DNA structure
A
- sugar-phosphate backbone (phosphodiester bonds between hydroxyl on C3 of sugar and phosphate group on C5 of phosphate)
- anti-parallel strands
- H-bonding between complimentary bases
15
Q
chargaffs rule
A
16
Q
The Watson-Crick-Franklin Proposal
A
- DNA is composed of two chains of nucleotides.
- These two chains form a spiral pair of right-hand helices.
- The two chains are antiparallel, they run in opposite directions.
- The sugar-phosphate backbone is the exterior of the molecule, and the bases are interior.
- Bases are perpendicular to sugar-
phosphate backbone. - DNA chains are held together by
hydrogen bonds between bases (A pairs with T via 2 hydrogen bonds; Gand C pair via 3 hydrogen bonds) - Double helix width 2nm (diameter)
- Pyrimidines always paired with
purines. - Only A-T and C-G pairs fit within
double helix.
10.Molecule has a major groove and a minor groove.
11.Complete turn is 10 base pairs. - Complementary base sequences on each of the 2 strands.
17
Q
allele
A
18
Q
crossover
A
19
Q
During cell division, the nuclear material is organized into visible “threads” called
A
chromosomes (chromatids are not visible)
20
Q
transposons
A
genetic elements that can move from one genome site to another
21
Q
5’ end of dna
A
- where the free phosphate is (lecture)
- has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus
22
Q
supercoiled dna
A
- negative supercoiling: underwound (less than 10 base pairs in the complete turn) - compacts DNA and important in replication and transcription
- positive supercoiling: overwound (more than 10)
23
Q
topoisomerases
A
change the level of DNA supercoiling
- Type 1: transient break in 1 strand of DNA duplex
- Type 2: transient break in both strands of DNA duplex (can also interlink or separate circular DNA)
24
Q
DNA denaturation
A
DNA strands coming apart after applying heat
- heating causes an increase in UV light absorbance (absorbance depends on amount of base pairing)
- Melting temperature (Tm): halfway through shift in absorbance from low to high
- higher GC content (3 hydrogen bonds; stronger interaction) = higher Tm (more heat needed to separate)
25
DNA renaturation (reannealing)
Single-strand DNA reassociates
26
Nucleic acid hybridization
Complementary strands of
nucleic acids from different
sources --> hybrid molecules
- DNA sequencing, cloning,
amplification
27
PCR
Denature @ 95 - Anneal @ 68 - Elongate @ 72
- need primers and dNTPs
28
3 different types of DNA sequences with different reannealing rates
- Highly repeated fraction
- Moderately repeated fraction
- Non-repeated fraction
29
Highly repeated DNA sequences
(Tandem repeats)
Short sequences (few 100 nucleotides) + clustered + repeat over
and over again (arranged end-to-end ie. tandem); 1-10% of total DNA
Further subdivided:
- Satelite DNAs: short sequences that evolve rapidly
- Minisatelite DNAs: unstable, variable in population, DNA fingerprinting
- Microsatelite DNAs: aka short tandem repeats; small clusters, implicated in genetic disorders
30
Fluorescence in situ hybridization
(FISH)
- used to determine location of a gene/dna sequence on a chromosome/in genome
- denaturing dna and using fluorescent labelled probe to anneal to it and identify location
31
Moderately Repeated DNA Sequences
- variability in how many are present in the genome (20% to greater than 80% of total DNA depending on organism) and how frequently they are repeated (from a few times to tens of thousands of times)
- Some sequences code for gene products like RNAs or histones
- Most sequences lack coding function - interspersed throughout the genome (grouped into SI and ES (short interspersed elements or lines long interspersed elements)
32
Non-repeated DNA Sequences
- include genes with Mendelian patterns of inheritance, localize to particular site on particular chromosome
- DNA sequences that code for all proteins other than histones (globins, actins, myosins, collagens, tubulins, integrins, and most other proteins in a eukaryotic cell)
- less than 1.5% of human genome
- sequences not present in multiple copies (single copy)
33
polyploidization
more than 2 sets of chromosomes (as compared to haploid/diploid)
- common in plants (getting FULL set of chromosomes from both mom and dad - no meiosis)
- in animals: single celled embryo accidentally undergoes chromosome duplication and retains the DNA
34
Gene duplication
duplication of one or more copies of a gene or region of a chromosome - can occur through unequal crossing over
35
Unequal crossing over
Homologous chromosomes misalign and exchange genetic material at non-identical positions (results in one chromatid gaining extra genetic material (duplication) and the other losing it (deletion)) - role in evolution of multigene families (for example, protein families like alpha and beta tubulins)
36
transposition
particular dna sequences (transposable elements/transposons) could move around genome and insert into target sites randomly
- transposase: enzyme encoded in transposon that cuts the transposon and helps in its target site insertion
37
eukaryotes have
transposons (cut and paste) and retrotransposons (copy and paste)
38
retrotransposons
- get rna (transcription by RNA polymerase) -->
- get cDNA (reverse transcription to form single stranded cDNA) -->
- get double stranded DNA
39
human genome
20,000 genes
40
Alternative splicing
a single gene can encode a number of related proteins
41
explanation for small number of protein-Coding Genes in the Human Genome
- Alternative splicing: a single gene can encode a number of related proteins
- MicroRNAs: Noncoding RNAs (not coding for proteisn) that can have gene regulatory functions (can act on genes to produce different but related proteins)
- Proteins work together as complex networks rather than individually
42
Comparative Genomics
- if It’s conserved it must be important
- best way to identify functional sequences --> compare genomes of different organisms
43
Intergenic genome
Majority of genome lies between protein coding genes
44
Exons
The coding regions of a gene that are spliced together to form the final mRNA and translate into proteins.
45
An event in which offspring are produced that have twice the number of chromosomes in each cell as their diploid parents is called
Both polyploidization and whole-genome duplication
46
nucleosome
[organization of the chromosome]
DNA wrapped around histone
- nucleosomes further coil into a chromatin fiber, which further condense to form looped domains, which pack into chromosomes
47
nucleosomes
DNA + histone octamer (core complex)
- 146 bp supercoiled DNA wraps around histone twice
Histone octamer
- 4 histone heterodimers: 2 (H3+H4) + 2 (H2A + H2B)
Histone H1 (linker histone)
- binds linker DNA (connecting diff nucleosomes to each other to form the chromatin fiber)
- can be modified
48
histone structure
- globular region (histone fold) - alpha helices of proteins
- histone tail
- minor groove of dna faces histone
49
histone tail
- N-terminus of histone
- projects beyond wrapped dna helix too
- subject to modification
50
chromatin
- 30 nm chromatin fiber loop diameter (can be 80-100nm too)
- proteins involved in the nuclear scaffold might be creating dna loops (?)
- loops maintained by COHESION (also holds replicated dna molecules together during mitosis)
51
packing ratio of DNA
10,000:1
- chromosome length = 1 um
- length of contained dna = 1 cm
52
Euchromatin
loosely packed chromatin (interphase)
53
Heterochromatin
Tightly packed chromatin
- Constitutive heterochromatin
- Facultative heterochromatin
54
Constitutive heterochromatin
- always condensed; silent DNA (cuz so packed, its not available for the transcription machinery to get in there and transcribe genes - therefore FEW GENES)
- highly repeated sequences
- in centromeres, telomeres, distal arm of Y chromosome
55
Facultative heterochromatin
- can switch between condensed and relaxed (can be inactivated - changes with time, varies from cell to cell)
- example: X chromosome (Barr body) - throughout a woman's lifetime, there will be one transcriptionally active X chromosome and one transcriptionally inactive X chromosome (inactive is Barr body)
56
barr body
- heterochromatic clump
- transcriptionally not active
x- inactivation process is random
57
Histone code hypothesis
the modification of histone tails decides whether the particular region of DNA that it is interacting with is euchromatin or heterochromatin
- modification happens at N-termini of H3 and H4
- different proteins interact with the histone tails after they are modified
- methylation of H3 is responsible for the formation of heterochromatin
- can be phosphorylation, acetylation, methylation, ubiquination
histone tail modifications can:
- recruit non-histone proteins
- alter interaction of neighbouring histones in their nucleosomes
58
Correlation Between Transcriptional Activity and Histone Acetylation
Removal of acetyl groups from
H3 and H4 histones converts euchromatin to heterochromatin (more condensed state - replication and transcription machinery has no access - gene does not get transcribed)
59
Karyotype
homologous chromosome pairs ordered according to size- pattern can be used to screen chromosomal abnormalities
60
Telomeres
protective structures located at the ends of chromosomes
- consist of repeated DNA sequences + protein caps
- TTAGGG
- Repeated ~ 500-5000 times in
humans (same sequence in all vertebrates; similar sequences in most other organisms)
61
Telomerase
solves the End-Replication Problem by adding repetitive nucleotide sequences (e.g., TTAGGG in humans) to the ends of telomeres - extends the telomere becoming shorter
- Reverse transcriptase that
synthesizes DNA from RNA
template
- Adds new repeat units to 3' end of
overhanging strand
- Unlike most reverse transcriptases, contains the RNA template it uses
- Once 3' end is lengthened, conventional DNA polymerase can
return 5' end of complementary
strand to previous length
62
telomere importance
- Required for complete
replications of chromosome
- Form caps that protect
chromosomes from nucleases
and other destabilizing
influences
- Prevent ends of chromosomes
from fusing with one another
63
centromere
- sites of concavity (indentation)
- tandemly repeated constitutive heterochromatin
- centromeric DNA is the site of microtubule attachment during mitosis
64
epigenetics
inheritance that is not encoded in DNA
- Example: X-Chromosome Inactivation: In females (XX), one X chromosome is randomly inactivated in each cell to equalize gene dosage with males (XY). This inactivation is maintained in all daughter cells throughout the individual’s lifetime.
- Epigenetic state may be reversed: X chromosomes reactivated prior to gamete formation (ensuring both X chromosomes are functional in the resulting egg cells)
- twins with different longevity and disease susceptibility (due to epigenetic changes accumulating)
65
interchromosomal interactions
distant genes can interact in response to physiological stimuli (like hormone treatment)
- target genes repositioned into close physical proximity - genes physically move to sites within nucleus called TRANSCRIPTION FACTORIES (have transcription mahcinery)
66
Topographically Associated Domains
DNA within region tends to
interact much more strongly
with DNA in same region
- 100s kb (1 kb = 1000 bases)
- nuclear compartmentalization example
67
transcription
Process by which RNA is formed from a DNA template
68
Translation
Process by which proteins are
synthesized in the cytoplasm
from an mRNA template
69
Messenger RNA (mRNA)
▫ Intermediate molecule between
a gene and a polypeptide
70
What kind of bond forms between the phosphate groups of the DNA backbone and the positively charged amino acid resides of the histones and helps to hold the molecules together?
Ionic bonds
(dna backbone is negative czu of phosphate group and histone has basic amino acids that give it positive charge)
71
what is the packing ratio of DNA in mitotic chromosomes?
10,000:1
72
What is chromatin that remains compacted during interphase called? This compacted, densely stained chromatin is found at the nuclear periphery.
Heterochromatin
Cuz (compacted chromatin fibres)
72
central dogma
DNA to RNA to protein
73
mRNA
formed after transcription from DNA template
- codes for proteins
74
rRNA
80% of the RNA (most common form of RNA)
- form the basic structure of the ribosome
- catalyze protein synthesis
75
tRNA
adaptors between mRNA and amino acids
- bring in nucleic acid building blocks as we synthesize protein
76
RNA strcuture?
- folding decides function
- folding depends on complementary base pairing
- create stem and loop configurations
- rna molecule looping in on itself
- uracil instead of thymine
- non-standard base pairing can happen (uracil base pairing with guanine) - not possible in DNA
- modified bases: methylate an adenosine or sum (room for regulation)
77
RNA polymerase
- present in eukaryotes and prokaryotes both
- DNA dependent (require a DNA template to build the RNA molecule)
- synthesizes COMPLEMENTARY rna strand
- addition of 20-50 nucleotides per sec (quick process)
- addition of new nucleotides - nucleotides added to the 3' end
78
RNA polymerase direction
- moves along the template strand of DNA in the 3' to 5' direction
- synthesizes a complementary RNA strand in the 5' to 3' direction
- adds nucleotides to the 3' end of the growing RNA strand (RNA strand elongates in the 5' to 3' direction)
79
stages of transcription
1) Initiation: RNA polymerase binds to promoter region of DNA sequence, unwinds DNA, begins RNA synthesis
2) Elongation: RNA polymerase moves along DNA synthesizing RNA
3) Termination: RNA polymerase encounters transcription stop signal in DNA
80
operon
groups of genes that are transcribed together (prokaryotic) - one initiation step, very long elongation step - just one mRNA molecule for multiple genes
81
prokaryotic transcription
- no physical separation of DNA,
RNA, ribosomes (all processes take place in cytoplasm)
- transcription and translation happen together
- operons: group of genes transcribed together into a single mRNA molecule
- only one RNA polymerase (core enzyme made up of 5 subunits + sigma factor that binds to promoter = holoenzyme)
- sigma factor dissociates after 10 Ns (conformation change - becomes a transcriptional elongation complex) - transcription stops with terminator sequence, completed RNA released, may require p factor to dissociate
82
promoter (prokaryote)
specific DNA sequence 35 bases upstream of gene
- Binds transcriptional machinery
- Location of transcription initiation
- specific promoter region in bacteria: TTGACA
83
RNA polymerase moves along the DNA template strand in a ____ direction
3' to 5'
84
RNA polymerase 1
synthesizes most & larger rRNAs (RNA part of ribosomal structure)
85
RNA polymerase 2
synthesizes mRNA + most small nuclear RNAs
86
RNA polymerase 3
small RNAs : tRNA + small rRNA
87
primary transcript
directly synthesized from the template DNA strand - includes introns (taken out during RNA splicing)
88
tarsncription bubble supercoiling
- positive supercoiling (overwound DNA) in the direction that RNA polymerase is moving
- behind the RNA polymerase we have underwound DNA (-ve supercoiling)
- transcription bubble is 15N
89
RNA polymerase
- has magnesium ion to neutralize phosphate of DNA
- multi-subunit complex
- RNA transcript exits via channel
- processive (continually synthesizes)
90
Preinitiation Complex
- RNA polymerase II
- General transcription factors (GTFs)
- Promoter
91
TATA box
promoter sequence that is 24-32 bases upstream/before initiation site
- binding site for transcription factors, particularly the TFIID, which facilitates the recruitment of RNA polymerase II to the gene's promoter
92
promoter sequences
- B recognition element (BRE): even more upstream than TATA (TFIIB)
- TATA box: 24-32 bases upstream (TBP [tata-binding protein] of TFIID)
- Initiator (INR): found at the initiation start point (TFIID)
- Downstream promoter element (DPE): within the gene itself (TFIID)
93
TFIID
Subunits:
- TBP subunit: TATA binding protein; recognizes TATA box
- TAF subunit: TBP associated factors; regulates DNA binding by TBP - mad eup of multiple polypeptdies (associated factors)
TAF help recognize sequence TBP binds sequence
94
TFIIB
positions RNA polymerase at the start site - recognition space for RNAP2
95
TFIIF
stabilizes RNA polymerase interaction with TBP and TFIIB - binds directly w RNA polymerase 2
96
TFIIE
attracts and regulates TFIIH
97
TFIIH
has kinase and helicase activity (unwinds DNA at transcription start point and phosphorylates RNAP2 at the c-terminal so complex can dissociate)
98
capping, polyadenylation, splicing _____
happen before export out of nucleus + while RNA is being synthesized enzymes responsible are present on the tail of RNAP2
99
RNA capping
Modification of 5' end with methylated guanine group after 25 nucleotides
100
polyadenylation
- Poly-A tail at 3' end
- Encoded in genome
- 50-250 adenosine
coded in the gene itself (at the end of the gene, there is a DNA template to create this string of adenosine nucleotides) - cap is added, but the polyadenylation is from the DNA template itself
101
capping and polyadenylation purposes
- Translation - play roles in the identification of the mRNA molecule
- Efficient export
- Stability
102
split gene
has both the coding and noncoding portions
103
difference in size between heterogenous nuclear DNA (hnDNA) and mature mRNA
true
104
hnRNA transcripts are processed ______
cotranscriptionally
105
why splicing
we can create different proteins from taking different combinations of the exons
106
proteins that help cellular RNA polymerases recognize promoters are called
transcription factors
107
types of transcription factors
regulate gene expression
General TFs: bind at core promoter sites in association with RNA polymerase
Sequence-specific TFs:
- Bind to various regulatory sites of particular genes
- Transcriptional activators – stimulate transcription/ expression of a gene
- Transcriptional repressors – inhibit transcription/ expression of a gene
108
Transcription Factor Structure
2 domains:
- DNA-binding domain: has alpha helix so TF can be inserted into major groove (motif)
- Activation domain: carries out function
will often dimerize with similar protein (as a form of activation - regulation of gene expression)
109
Oct4
- many transcription factors
- plays a role in regulating its own transcription
- TF combination decides extent of transcription
110
embryonic stem cells
- self-renewal
- pluripotent: can become any other cell
- turned fibroblasts into stem cells using transcription factors
111
most RNA in cell is _____
rRNA (build structure of ribosomes)
112
snoRNA
small nucleolar RNAs, help to process and chemically modify rRNA
113
rDNA
- regions of the genome that encode ribosomal RNA (rRNA) - DNA for ribosomal RNA
- genes organized into multiple tandem repeats (multiple copies of the same gene)
- rDNA arranged in gene clusters (in the nucleolus?)
114
Eukaryotic Ribosome Structure
Ribosome = rRNA + ribosomal proteins
4 distinct rRNAs in eukaryotic ribosome & 2 subunits:
- Small (40S)– 18 S (+ 33 ribosomal proteins)
- Large (60S)– 28S, 5.8S, 5S (+ 49 ribosomal proteins)
translational unit: 80S
115
Svedberg unit (S)
measure particle's size based on sedimentation rate - Larger number = larger particle size
116
Prokaryotic Ribosome Structure
3 distinct rRNAs in eukaryotic ribosome & 2 subunits:
- Small (30S)– 16 S
- Large (50S)– 23S, 5S
translational unit: 70S
117
Synthesis & Processing of Mammalian rRNA Cleavage
3 of 4 human rRNAs derived from single primary transcript called the 45S precursor (pre-rRNA) -->. cleavage of primary transcript by nucleases --> mature rRNA
- 28S, 18S, 5.8S
- Synthesized by RNA polymerase 1
5S: Synthesized by separate RNA
precursor outside of nucleolus - imported into nucleolus afterwards
- Via RNA polymerase 3
118
the corresponding segment of DNA from which the primary transcript is transcribed is called a _____
transcription unit
119
rRNA Nucleotide Modifications along with cleaving
- Methylation of nucleotides
- Conversion of uridine (uracil + ribose) --> pseudouridine
- modifications performed by snoRNPs (small nucleolar ribonucleoprotein particles = snoRNA + proteins)
- ~200 different snoRNAs – one for every site in the pre-rRNA that is modified
- conserved in evolution
120
snoRNAs that methylate
have Box D (5’-CUGA-3’)
121
snoRNAs that convert uridine to pseudouridine
have ACA sequence
122
Synthesis and Processing Eukaryote tRNAs
- genes scattered in small clusters throughout genome rather than tandem repeats
- RNA polymerase 3
- primary transcript undergoes modification - trimming of 5' and 3' ends
- Ribonuclease P cleaves at 5' end
- Large spacer segments interspersed with tRNA coding sequences
123
why only 20 amino acids if there are 64 codons possible
Redundant, Conservative, Unambiguous, Universal
124
Redundant
Most amino acids coded by more than one codon
125
Conservative
When multiple codons specify same amino acid, the first two bases identical
126
Unambiguous
One codon codes for only one amino acid
127
Universal
All codons specify same amino
acid across species
128
RNA polymerase movement
RNA polymerase moves along the template strand of DNA in the 3' to 5' direction, synthesizing a complementary RNA strand in the 5' to 3' direction.
- The template strand of DNA (aka antisense strand) serves as the guide for transcription.
- RNA is synthesized antiparallel to the template strand.
- The RNA sequence will be complementary to the template strand and identical (except for uracil replacing thymine) to the coding strand of DNA (also called the sense strand).
129
reading codon
- Codons read in the 5′ to 3′ direction during translation
- Start codon: AUG (Encodes methionine)
- Stop codons:
▫ UAA
▫ UAG
▫ UGA
130
anticodon
opposite base pair of the codon
131
tRNA
Match mRNA codon with amino
acid it codes for
- Each tRNA is linked to a specific
amino acid
- tRNA recognizes a particular
codon in the mRNA via region
known as the anticodon
- 73-93 NTs long
- D arm recognizes enzyme that attaches its amino acid - aminoacyl tRNA synthetase
- T arm recognizes ribosome
- 2 important regions: Anticodon loop (7 NTs) & [AA acceptor arm - 3’ C-C-A sequence]
132
wobble hypothesis
explains why multiple codons can code for a single amino acid - Non-Watson-Crick base pairing - Interchangeability of base in 3rd position
133
tRNA charging
aminoacyl-tRNA synthetase covalently links amino acids to 3’ end of tRNA (amino acid acceptor arm)
- enzyme unique to each amino acid
134
what is sometimes needed for the termination of bacterial transcription?
rho protein (p factor)
135
small subunit (40S)
decodes the genetic message
136
large subunit (60S)
catalyzes peptide bond formation
- contains peptidyl transferase to form peptide bonds
- tRNA in P site transfers aa to tRNA in A site
137
translation steps
1. Initiation
2. Elongation
3. Termination
138
Initiation of Translation in Eukaryotes
- Requires over 12 initiation factors (over 25 polypeptide chains)
- mRNA binds small & large subunits of ribosome in separate stages
- 43S complex: small subunit and its initiation factors + (tRNA + GTP + initiation factors)
1) 43S complex + 5’ end of mRNA --> 48S complex (initiation complex; attached to mRNA and searches for AUG)
2) Initiation factors dissociate once initiation complex finds start codon
3) Binding of large ribosomal subunit --> 80S complex
139
Three tRNA binding sites
- A (aminoacyl): Binds incoming aminoacyl-tRNA carrying new aa.
- P (peptidyl): holds the tRNA with the polypeptide
- E (exit): holds the tRNA without the aa which is then released by the ribosome
140
Binding 2nd aminoacyl-tRNA to A site requires GTPase
- Bacteria: elongation factor EF-Tu
- Eukaryotes: eEF1A
141
what enzyme is responsible for covalently linking amino acids to the 3' end of the cognate tRNA
aminoacyl-tRNA synthetase
BMSC 220 post midterm
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