What are bacterial terminator sequences?
rho factor used
inverted repeat sequences transcribed form hairpin loops
Most bacterial transcripts are said to be collinear, what does this mean?
DNA directly encodes them.
one-to-one correspondence between of bases between the gene and the mRNA transcribed from the gene
What are homologs?
genes related to a second gene by descent from a common ancestral DNA sequence
What singles the beginning of DNA replication?
Helicase bindind to DNA and breaking the hydrogen bonds between the two strands to separate them.
Who discovered transposable elements?
What are class 2 TEs?
-aka DNA transposons
-encode the protein transposase, required for insertion and excision
-never use RNA intermediaries
What interferes with L1 activity?
siRNAs interfere with L1 activity and are derived from the 5' UTR of L1 LTRs
What is the Medaka fish example of?
Example of how TEs affect gene regulatory regions and phenotypes
Tol2 transposon either hops out of fish cleanly (results in albinoism) or takes with it genetic material (pigment alterations).
How were full length cDNA sequences selected for?
-Biotin capping the 5' end
-Washing the cDNAs with an RNA digestign enzyme like RNase I
-Only cDNAs with biotin cap (full cDNAs) could be recovered
Beta-thalassemia is a result of ___
an insertion (point) mutation
What is eukaryotic termination?
-for RNA pol 1:
similar to Rho-dependent termination of bacteria
-for RNA pol 2:
complex, mRNA might be synthesized well beyond coding region.
cleaved by a complex and then adenylated
for RNA pol 3:
similar to Rho-independent termination
What is the difference in bacterial/ eukaryotic promoters?
Bacteria- 3 elements
pribnow box (TATAA site)
consensus sequence TTGCCA
upstream element (AT-rich)
Eukaryotes- 7 elements
enhancer sequences (bind with activators to alter DNA structure and facilitate RNA pol 2 binding
need transcription factor
In humans, what are the only active class of transposons (jumping genes)
The non-LTR class of retrotransposons (class 1 TEs)
What are nonautonomous TEs?
-TEs that require another TE to move
-They either lack the gene for transcriptase or retrotransposase, so must borrow from a nearby TE
-ex: McClintock's DS element
one form of leukemia is caused by ____
translocation (chromosomal) mutation
What characterizes class 2 TEs?
-inverted repeats on their ends to be recognized by transposase (ex: ACGCTA, TAGCGT)
-short direct repeats that flank inverted repeats and play a role in insertion. They are footprints left behind and sometimes alter gene expression. They are not actually part of TE, because they're left behind
What were the genetic crosses that McClintock performed?
What was the hypothesis of phenotype of the resulting progeny?
What actully occurred?
What was the conclusion?
C'C'bzbz- - x CCBzBzDsDs
To yield heterozygots:
all progeny would be colorless
many kernels were colorless but some cells were purple or brown resulting in spots/streaks of color
A breakage at the Ds locus caused some cels to lose the C' and Bz that would yield colorless or brown phenotypes. Depending on when the breakage occured during development, there was more or less spotting
What are autonomous TEs?
-TEs that can move on their own
-ex: McClintock's AC elements
What are the minor/major grooves of the DNA double helix? What is their role?
Major/minor groove: caused by the asymmetric spacing of the antiparallel helix backbone
Their Role: role in binding of proteins that regulate gene transcription
Besides epigenetic silencing, how do TEs silence themselves?
siRNA, allows TEs to mediate their own silencing
siRNA: prevent transposition. double stranded RNA
How do siRNAs interefere with L1 activity?
5' UTR of L1 promoter encodes a sense promoter that transcribes L1 gene and an antisense promoter that transcribes an antisense RNA
The result is homologous sequence and hybridization and a double stranded RNA that can serve as a substrate for RNAi.
some cancers are caused by ____
insertion (chromosomal) mutation
What differentiates DNA from RNA?
DNA doesn't have a 2' OH group on the sugar molecule
Who is Kornberg?
-enzymologist and nobel prize winner
-described DNA replication as a tape recording of instructions for completing a task
-discovered that DNA polymerase catalyzes DNA synthesis by mixing necessary ingredients for e-coli DNA synthesis in a test tube
How were okazaki fragments discovered?
-Pulse chase experiment
- DNA exposed to isotopic-nucleotides (pulse) and then regular nucleotides for varying periods of time (chase)
-short chases resulted in most radioactivity in "slow" DNA
-increasing length of chases resulted in radioactivity increase in "fast" DNA but not "slow" DNA
-shows that in short period of time okazaki fragments form but with more time they elongate as DNA replication continues
What are class 1 TEs? How do they differ from class 2 TEs?
-move through the action of RNA intermediaries
-do not encode RNA transcriptase, rather produce RNA transcripts and rely on reverse transcriptase enzymes to reverse transcribe RNA into DNA, which is inserted into target site
What are chemical mutations?
Free radicals (oxidizing agents) may modify nucleotides
Example: Dioxin intercalates between base pairs, disrupting DNA helix integrity
Example: benzo[a]pyrene induces lesions at guanine sites in P53, lung cancer!
Why are RNA primers required at the origin of replication?
Because DNA can't bind de novo and can only add deoxyribonucleotides to the 3' OH group of an existing chain
Human chromosomes range from _ o _ in length
50 to 250 million base pairs
What are transposons?
-DNA sequences that move from one location on the genome to another
What are the essential chemical ingredients in PCR?
- DNA polymerase
-essential salts (like Mg)
primers with exposed 3' OH groups
What is the central dogma of molecular biology?
First step transcription
Second step translation
Cri-du-chat syndrome is caused by ___
deletion (chromosomal) mutation
What is an example of a mutation "hot spot"?
regions of DNA with large numbers of trinucleotide repeats.
This could lead to DNA polymerase slippage and alteration of the repeat sequence
What is the length of a turn of DNA?
What is the width of the DNA double helix?
How many bases are in 1 turn?
length: 3.4 nm (34 A)
width: 2 nm (20 A)
1 turn: 10 nucleotide bases
What is the Ames test?
Test to determine if a chemical compound or any of the digested material derived from it is a mutagen
What is a protein domain?
a region tha can adopt a 3D structure
What are two families of non-LTR retrotransposons?
-Line1 (L1) (LONG! LESS!)
-6 kb in length
-less copies in humans but longer so 15-17% of human genome
-only a few hundred nucleotides
-more copies in humans
What is cDNA?
complimentary DNA synthesized from mRNA using reverse transcriptase
What is the role of DNA polymerase I?
Removes the RNA primer after DNA polymerase III has begnun and replaces it with nucleotides
What are two ways in which sequences are fixed
proofreading- correction during replication
mismatch repair-correction after replication
What is the role of DNA polymerase III?
does most of the elongation work
adds nucleotides one by one to the 3' end of a growing single strand
In what direction does DNA synthesis occur?
5' to 3' direction ONLY
What are the bonds that link the backbone of DNA and what are they made up of?
Phosphodiester bonds of the DNA backbone are made up of 5 carbon sugars and phosphate groups
What is a protein motif?
-short, conserved region of a protein
-10 to 20 contiguous residues
What are the sequences at the start and end of splice sites?
What adds new nucleotides to an DNA strand during DNA replication?
What are the two types of class 1 TEs?
Class 1 TEs: retotransposons
Type 1: LTR retrotransposons
-Have long terminal repeats on both ends
Type 2: non-LTR retrotransposons
-do not have long terminal repeats at both ends
What are orthologs?
genes in different species that evolved from a common ancestral gene
What results in sickle-cell anemia?
A point mutation in a nucleotide converts the sixth amino acid from glutamic acid into valine
How is genetic information encoded in DNA?
genetic information is encoded in the sequence of bases attached to the 1' carbon of the sugar molecule of the DNA backbone
What are connected to the carbons of the sugar molecule in DNA?
1' carbon: OH group is replaced by nucleotide bases
2': in RNA, has an OH group. In DNA, it does not
3': a phosphate group attaches here
4': attached to the 5' carbon
5' carbon: 5' phosphate group
What is an example of environmental mutation?
UV mutations induce hydrolysis of cytosine causes it to mispair to adenine
(creates a C-T fingerprint type mutation)
UV also induces covalent bonds between adjacent pyrimidines
What were some other discoverys by McClintock?`
-Additional discoveries showed an element Ac that was necessary for phenotypic effects of Ds
-McClintock had trouble mapping Ac and Ds, noting that they changed locations
T/F: Scientists have identified a polymerase that can add nucleotides to the 5' end
Why is maize ideal for genetic analysis?
Each kernel is a unique embryo so many different phenotypes can be scored on a single ear of corn
How were TEs discovered?
-studying phenotypic system of maize (triploid endosperm that is the colored protein coating and diploid zygote
-Four maize genes studied: C' (dom, colored inhibitor)/ C (rec, color shown), Bz (dom, color purple)/ bz (rec, color brown), Ds (location on Ch9 where breakage occurs), As (unknown factor that impacts Ds expression)
Ds observed due to unexpected phenotypes in maize genetic crosses
What is the difference between purines and pyrimidines?
Purines: composed of two rings (guanine and adenine)
pyrimidines: composed of one ring (thymine and cytosine)
What is a single-nucleotide polymorphism?
A single base pair alteration that is common in populations
Basically, any location where at least two sequences are found and are prevalent in at least 1% of population
How are complimentary bases connected?
Complimentary nucleotides are hydrogen bonded to each other.
Where does translation occur?
When does it begin?
Once initiation factor proteins bind to small subunit of ribosome
Methionine carrying tRNA binds to mRNA near AUG start
Methionine is the first amino acid encoded in any new protein
methionine not removed in MK case
cystic fibrosis is a result of ___
deletion (point) mutation
Optiz Kaveggia syndrome is a result of _____
An insertion (chromosomal mutation)
What is the role of DNA ligase
seals the bond between two adjacent nucleotides.
This "nick" is due to removal of RNA primer and left behind by DNA polymerase I and RNase H.
Which complimentary nucleotides form 2 hydrogen bonds and which complimentary nucleotides form 3?
2 hydrogen bonds: AT
3 hydrogen bonds: GC (hence why they form a stronger bond)
What is topoisomerase?
reduces torsional strain caused by DNA unwinding
What are two types of sponatenous mutations?
loss of an amino group in a nucleotide
typically if cytosine, repair mechanism fixes this unless cytosine is methylated. Then it is read as a thymine
(CpG islands frequent targets for methylation and mutation)
purine is removed so polymerase can't determine what nucleotide to add to new strand.
generally polymerase adds an adenine (sometimes guanine)
What is the result of an expanding trinucleotide repeat?
The normal number of repeated trinucleotides is expanded
results in fragile X, huntington's disease
What catalyzes splice reactions and what type of reactions are involved?
transesterification bonsd guanine and adenine
3' and 5' end of intron connected via transesterification as well
What is a transversion mutation?
What is DNA cloning?
-DNA clonin involves the cloning of expressed DNA genes via a reversal of the central dogma of molecular biology
-An mRNA transcript is transcribed into DNA via reverse transcriptase
What is the difference between a non-sense and a mis-sense mutation?
non-sense: changes AA sequence to premature stop codon
mis-sense: changes AA sequence
What are the differences between eukaryotic and prokaryotic replication?
-many replication origins
-DNA organized into nucleosomes with histones
-different set of polymerases (polymerasa beta and polymerase gamma instead of polymerase III)
-13 polymerases discovered to date
-chromosomes are linear
-gap in newly synthesized DNA ends protected by telomeres
-single replication origin (oriC)
-chromosomes are circular
where does the splicing process occur?
in spliceosomes in nucleus ?
What experimental evidence proved the existence of reverse transcriptase?
1. The DNA polymerase only incorporated deoxyribonucleotides, not ribonucleotides, into its product.
2. The product itself "behaved" like DNA--in other words, it was sensitive to treatment by deoxyribonucleases but not ribonucleases.
3. The RNA itself was the template, as shown by the fact that treatment of virions with ribonucleases destroyed the ability of the polymerase to incorporate radioactively labeled nucleotides.
How do TEs drive the evolution of genomes?
-help repair double-strand breaks
-translocation of gene sequences
What is gene amplication?
An example of copy number variants
It's a result of the number of tandem copies of a locus being increased
in some breast cancers
Transposable elements make up how much of human and maize genome
-50% of human genome
-90% of maize genome
What is meme?
searching for common motifs in unaligned sequence
What epigenetic defense mechanisms can silence TEs?
chromatin becomes so constricted in some areas that transcription factors can't bind
DNA methylation (heterochromatin)
In Maize for example: wild-type sequences are methylated. In mutated maize, transposons are transcribed.
Used in gene regulation: cause histone modification and DNA methylation, bind to mRNA
What is exon shuffling? Why does it occur?
Exon shuffling occurs becaue TEs do not always excise perfectly from sequence. The result is that two previously unrelated exons are are juxtaposed, potentially creating new gene products
What is oriC?
The replication origin
a base-pair sequence of nucleotides
What is a transition mutation?
Mutation of purine to purine or pyrmidine to pyrmidine
A-> G (purine)
What are the 3 types of RNA polymerases?
RNA Pol 1: ribosomal RNAs
RNA Pol 2: responsible for mRNA synthesis from template DNA
RNA Pol 3: responsible for small RNA, tRNA synthesis
Role of RNA primase?
inserts starter of RNA nucleotides at trancription start site
also attaches RNA primers to lagging strand to yield okazaki fragments
What is the role of RNase H?
Removes the RNA primer after DNA polymerase III has begnun and replaces it with nucleotides
What is the usefulness of cDNA libraries?
can be used to identify genes that are expressed differently in different types of tissues or different stages of development
cDNA libraries give a snapshot of gene activity since only genes that are expressed and transcribed into mRNA can be cloned
What is prosite?
a database of protein patterns that can be searched by either regular expression patterns orsequence profiles
What is phi blast
searching a specific protein sequence pattern with local alignments surrounding the match
What is the difference between the 5' and 3' end of the DNA double helix?
5' end: has a free 3' phosphate group (negatively charged)
3' end: has a hydroxyl group on the 3' carbon of the sugar
How does replication of the lagging strand occur?
RNA primase creates an RNA primer with 3' OH so that DNA replication can begin.
DNA polymerase III adds deoxynucleotides in the 5' to 3' direction following the 3' end of the primer
DNA polymerase I replaces DNA polymerase III and replaces RNA with DNA after exonuclease removes RNA primer
DNA ligase binds 3' OH of growing strand with 5' phosphate of the existing strand by eliciting formation of a phosphodiester bond.
What are paralogs?
genes related by diplication within a genome
What is the difference between the leading and lagging strand?
DNA polymerase can only add new nucleotides to a free 3' end of a growing chain.
Leading strand: replication occurs continuously in the 5' to 3' direction
lagging strand: to provide a 3' OH, RNA primase attaches to the DNA and synthesizes a short RNA primer. DNA polymerase III then adds deoxynucleotides to the 3' end of the primer. DNA polymerase I replaces the RNA with DNA. The enzyme DNA ligase forms a phosphodiester bond betwen the 3' OH of the growing strand and the 5' phosphate in front of it.
What is the role of SSBPs?
singal stranded binding proteins keep separated DNA strands from reattaching
What do jumping genes do?
-May result in mutation when they insert into a gene
-Example: L1 into Factor VIII results in hemophilia
-Example 2: L1 in APC genes in colon cancer
-L1 transposes in mammalian somatic cells, which could play a role in disease development.
Where does helicase split the DNA molecule apart?
At the start of the replication fork, typically areas rich in A and T because they're only connected by two hydrogen bonds
What are the 4 steps to replication?
3. primer synthesis
What is a silenced TE?
Why are some TEs silenced?
-Silenced TE: does not produce a phenotypic effect (opposed to L1, most TEs appear to be like this)
-Inactive due to:
- mutations that affect ability to move around chromosomes
- kept inactive by epigenetic defense mechanisms