Bio115 Exam 3 flashcards

• Initiation
• Unwinding
• Elongation
• Termination

The lagging-strand in the 5' to 3' direction and breaks hydrogen bonds while moving along the replication fork

relieves torsional strain that builds up ahead of the replication fork due to unwinding

sysnthesizes short strands of RNA including a 3'OH group for replication to begin

Just one

• 5' to 3': polymerase activity: adds nucleotides (primary)
• 3' to 5': exonuclease activity for error correction

• 5' to 3': polymerase activity: adds nucleotides
• 3' to 5': exonuclease activity
• 5' to 3': exonuclease activity - remove RNA primers
• Not as efficient as DNA polymerase III

links together DNA at nicks/Okazaki fragments by forming a phosphodiester bond between adjacent nucleotides

Binds to origin and separates strands of DNA to initiate replication

Attach to single-stranded DNA and prevent secondary structures from forming (stabilizes)

• Replication forks meet
• A termination protein (Tus in E. coli) binds to specific sequences to block helicase

Origin of replication found in yeast

Easier to break - only 2 H-bonds compared to 3 for C-G base pairs

In eukaryotes, an ORC binds to origins and unwinds the DNA in this region

In eukaryotes, it is the regulation of precise replication once per cell cycle from a large number of origins

Telomerase is a ribonucleoprotein which maintains telomeres by extending the DNA and filling in the gap due to removal of the RNA primer after replication

Prophase I, after DNA replication

• Holliday
• Double-strand break(*)

• Helix-turn-helix
• zinc fingers
• leucine zipper

negative inducible

negative repressible

region 3 and 4 pair resulting in termination of transcription

binding to mRNA to create double-stranded RNA blocking the ribosome-binding site

blocks the ribosome binding site by conformation change

induce cleavage

Methylation, acetylation, and phosphylation

acetyl transferase

bind to sites in DNA and reposition nucleosomes allowing transcription factors to bind to promoters and initiate transcription

is associated with the repression of transcription in vertebrates and plants

bind to sites on DNA and stimulate transcription specific to a gene or subset of genes

do not directly block RNA polymerase, but instead compete with activators or interfere with the basal transcription apparatus

can operate at distant promoters by way of DNA looping out

block the action of enhancers

Several eukaryotic genes respond via consensus sequences to extreme heat producing heat-shock proteins

be spliced in multiple ways generating different proteins in different tissue or at different times in development

1.0

0.5

tra pre-mRNA to be spliced at a downstream 3' site resulting in tra protein which leads to female fruit flies

ribonucleases

specialized complexes in which RNA molecules are degraded or sequestered for later release

• Cleavage of mRNA leading to degradation using Dicer, siRNAs and RISC
• Inhibition of translation using Dicer, miRNAs and RISC
• Transcriptional silencing using RITS, siRNA and methylation
• Degradation of mRNA (not via cleavage) using Dicer and RISC

• Selective cleavage and trimming of amino acids from the ends
• Acetylation
• Addition of phosphate groups
• Addition of carboxyl groups
• Addition of methyl groups
• Addition of carbohydrates

the transcription factor is not produced and the heart doesn't develop

• Base substitution
• Base insertion
• Base deletion

transition mutations

• Missense
• Nonsense
• Silent mutations

a missense mutation that alters the amino acid, but does not change its function

• Wobble
• Unequal crossing over
• Deamination
• Depurination

a chemical with a structure similar to any of the four standard bases

• EMS: alkylation
• Nitrous acid: deamination
• Hydroxylamine: hydroxylation

molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange

1927: showed mutations in fruit flies inducible by radiation

alter base structure, break phosphodiester bonds, and even cause double-strand breaks

less energy, but still mutagenic

can be caused by UV light; create covalent bonds between bases which block replication

eukaryotic system of eta polymerase that can bypass pyrimidine dimers

• Mismatch repair
• Direct repair
• Base excision
• Nucleotide excision

Replication errors, including mispaired bases and strand slippage

Pyrimidine dimers; other specific types of alterations

Abnormal bases, modified bases, and pyrimidine dimers

DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers

• Detects 3D distortion
• cuts out distored part with exonuclease
• fill in using original strand as template with DNA polymerase
• repairs nicks with DNA ligase
• Methylation at GATC sequence on old strand for differentiation

removes nucleotides usually at end of DNA strand

replaces nucleotides

seals nick in sugar-phosphate backbone

• Converts modified nucleotides to original form
• e.g. O6-Methyltransferase removes methyl group restoring base to guanine
• e.g. photolyase uses light to break covalent bonds that link pyrimidine dimers

an enzyme that uses light energy to break covalent bonds in pyrimidine dimers

• Excises modified bases and then replaces entire nucleotide
• DNA glycosylase recognizes and removes damaged base
• AP endonuclease cleaves phosphodiester bond on 5' site and removes sugar
• DNA polymerase adds new nucleotide to 3'
• DNA ligase fixes nick in sugar-phosphate backbone

removes nucleotide usually in middle of DNA strand

• Enzyme complex recognizes 3D distortion
• DNA strand is separated and stabilized with binding proteins
• An enzyme cleaves the strand on both sides of the damage
• Part of damage strand is removed
• Gap filled by DNA polymerase
• Sealed by DNA ligase

A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.

Two DNA strands are connected thru covalent bonds. Not much know about this.

• Detection
• Excision
• Polymerization
• Ligation

Damaged section of DNA is recognized

DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed

DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides

DNA ligase seals the nices in the sugar-phoshate backbone

How detection and excision are accomplished

autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers

Created first recombinant DNA molecule

Set of molecular techniques for locating, isolating, altering, and studying DNA segments

• Find gene
• Separate gene
• Make copies of gene
• Insert gene into plasmid without degredation
• Induce bacteria to take up plasmid
• Select bacteria that take up plasmid

• Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
• Produced naturally by bacteria to defend against viruses

Cut outside recognition sequence

• Cuts within recognition sequence
• Used in molecular genetic work
• Names indicate original bacteria
• More than 800 isolated

• Palindromic recognition sequence
• Fragment end either cohesive or blunt

Staggered cut -> sticky ends

Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C

Standard technique for separating molecules on basis of size/electrical charge

polysaccharide isolated from seaweed

DNA fragments move to positive pole with smaller fragments moving faster

fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest

Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe

• A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
• Size of mRNA molecule
• Relative abundance of mRNA
• Tissue in which MRNA is transcribed

• Transfer of proteins from gel to a membrane
• Probe is usually an antibody
• Determine size of protein
• Pattern of protein's expression

create identical copies of a piece of DNA

DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell

• Origin of replication
• selectable marker
• one or more unique restriction sites where DNA can be inserted

• Plasmid, e.g. pUC19
• Bacteriophage
• Cosmid
• BAC
• YAC
• Retroviral vectors
• Transposons
• Expression

The capacity of bacterial cells to take up DNA from the environment

Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection

Originally contructed from F plasmids

DNA molecule that has a yeast ORI, pair of telomeres, and a centromere

Plasmid that can be used to introduce DNA into plants

Vector that allows the production of protein (i.e. transcription and translation)

• 1) Heat to 90-100 C for denaturation
• 2) Cool to 30-65 C for primers to anneal
• 3) Heat to 60-70 C for DNA synthesis
• Repeat

• Requires knowledge of at least part of sequence for primers
• Taq polymerase is poor at proofreading
• Fragments larger than 50Kb cannot be isolated

Detects presence of a particular sequence, e.g. HIV

Clone all sequences in an organism into vectors

Collection of clones containing all the DNA fragments from one source

Set of bacterial colonies or phages containing fragments in a DNA library

isolation of mRNA using oligo(dT) chains

• oligo(dT) act as primers
• reverse transcriptase for DNA strand
• Rnase digests most of RNA strand
• Remaining RNA act as primers for second DNA strand

A type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue

Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation

No OH groups

Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence

allows sequencing of entire genomes in a couple months

• PCR used to amplify STR loci each with large numbers of alleles which assort independently
• Difference in number of tandem repeats have no phenotypic consequence
• Probability of two randomly selected people having the same DNA profile is less than 1 in 10 billion

• DNA extracted from tissue samples
• PCR primers for specific STR loci used to amplify fragments
• DNA from sample is compared with reference DNA
• Usually use genomic DNA, but mitochondrial DNA can be used as well

• Function -> gene
• Frequently used in less complex organisms to discover new genes

• 1. Isolate mutants that have phenotypic mutation.
• 2. Map the mutations.
• 3. Sequence the gene to find the mutation.
• 4. Clone the gene using molecular techniques.
• 5. Further genetic, molecular genetic, and biochemical experiments can further define a gene's function in that process.

• gene -> function
• Frequently used in mice to see if genes discovered in simpler organisms
• have a similar phenotype in mammals

• 1. Begin with a gene with known sequence.
• 2. Induce a mutation in that gene.
• 3. Look to see what effect these mutations have on the phenotype of the organism

An organism with an added transgene

non-innate DNA added to an organism

Mouse in which known gene has been disabled via homologous recombination

wildtype gene is replaced with known mutant gene

• Target "normal" gene disabled by inserting neo+ gene in the middle and a tk+ gene is added at the end
• Disabled gene is transferred to embryonic mouse stem cells to undergo recombination with normal cells resulting in some neo+ tk- cells
• Cells grown in antibiotic, and only recombinated ones survive
• Surviving cells injected into early mouse embryo resulting in variegated mouse
• Variegated progeny interbred resulting in some homozygous mice for the knocked-out gene

• Short sequence of nucleotides removed and replaced by synthetic sequence containing mutated bases
• Requires flanking restriction sites that are nowhere else in DNA

• Oligonucleotide created that differs from target sequence by single nucleotide
• Two sequences pair
• Oligonucleotide used as primer which yields molecule with single mismatched pair
• DNA transferred back to bacteria where about half are repaired
• Bacteria then screened for altered sequence

• Often used for making small changes in DNA sequences already cloned into plasmids
• Can't be used in multicellular organism: long, noncircular DNA, multiple ori, etc.

Can be delivered to cell by injecting or soaking to turn down expression without inducing mutation

Can be cloned into vectors and used to make transgenic animals

• siRNAs could be used against RNA viruses, such as HIV
• siRNAs could be used to treat genetic diseases, high cholesterol and cancer

Used in delivery of siRNA to lower cholesterol thru silencing in monkeys

Somatic

It is the field of genetics that attempts to understand the content, organization, function, and evolution of genetic information contained in whole genomes

Haemophilus influenza

A map-based method

A whole-genome shotgun technique using computers

A site in the genome at which individual members of a species differ in a single base pair

Because there is no selective pressure to weed out the mutations.

It is the set of SNPs and other genetic variants found on a particular part of a chromosome.

Africans, as this is consistent with many other studies that suggest humans first evolved in Africa.

A continuous stretch of DNA

A field that fuses molecular genetics and computer science

• ab initio approach: scans the sequences looking for characteristics such as an open reading frame
• comparative approach: Looks for similarities between a new sequence and sequences of all known genes

A frame which includes a start and stop codon in the same reading frame

Basic Local Alignment Search Tool, is a program to determine whether a similar gene sequence has already been found in the same or another species

• Orthologs are homologous genes found in different species that evolved from the same gene in a common ancestor.
• Paralogs are homologous genes in the same organism that arise by duplication of a single gene

A region in a protein that has a specific function or shape

An array of numerous microscopic DNA fragments/probes used to find complementary sequences corresponding to known genes

A gene that researchers attach to a regulatory sequence of another gene of interest that allows for visual identification (e.g. Green Fluorescent Protein, GFP)