MOL. BIO LEC 2 Flashcards
● Structural components that forms the outer edge of the DNA double helix composed of alternating sugar and phosphate groups
● Arranged at specific distances from one another in the double helix
● 2 regions formed in the helix by the backbones: major groove & minor groove
SUGAR - PHOSPHATE GROUP
● Often found in nature
● Base modifications have significant effects on phenotype
● Some result from DNA damage, naturally modified for specific functions or to affect gene expression
● Used by bacteria & viruses as a primitive immune system
Modified nucleotides
● Macromolecule made of nucleotides bound together by the phosphate & hydroxyl groups on their sugars
NUCLEIC ACID
DNA is oriented in ___________
5’ to 3’ direction
It is the process by which DNA makes a copy of itself during cell division
DNA REPLICATION
- every double helix in the new generation of an organism consists of 1 COMPLETE “OLD” strand and 1 COMPLETE “NEW” strand wrapped around each other
- Key to maintaining the sequence of the nucleotides in DNA through new generations
SEMI-CONSERVATIVE
Demonstrated the mechanism of semiconservative replication using the technique of equilibrium density centrifugation on a cesium gradient
MATTHEW MESELSON & FRANKLIN STAHL
POLYMERASE
- 1st purified enzyme shown to catalyze DNA replication in prokaryotes
- Followed by DNA polymerase II & |||
DNA POLYMERASE I (POL I)
POLYMERASE
- Main polymerizing enzyme during bacterial replication
- Functions as a multi-subunit HOLOENZYME
DNA POLYMERASE III (POL III)
- Addition of labeled nucleotides at nicks (single-strand breaks) in DNA
- Often used in vitro as a method to introduce labeled nucleotides into DNA molecules, which will be used for DNA detection in hybridization analyses
NICK TRANSLATION
- Type of DNA polymerase that can synthesize polynucleotide chains WITHOUT A TEMPLATE
- How?
▪ By adding nucleotide to the end of a DNA strand in the absence of H-base pairing with a template
- How?
- Used in the laboratory to generate 3’-end labeled DNA species
Terminal transferase
POLYMERASE CHARACTERISTIC:
⮚ Processivity (staying with the template longer to make longer products)
⮚ Fidelity (faithful copying of the template)
⮚ Substrate specificity (affinity for altered nucleotides)
It is the process by which cellular DNA is maintained
DNA METABOLISM
- Endonucleases that RECOGNIZE SPECIFIC BASE SEQUENCE AND BREAK OR RESTRICT THE DNA POLYMER at the sugar-phosphate backbone
- Originally isolated from bacteria, as part of a primitive immune system Named for the organism from which they were isolated:
- BamHI (from Bacillus amyloliquefaciens H)
- HindIII (from Haemophilus influenzae Rd)
- Smal (from Serratia marcescens Sbb)
RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- NUCLEASE & METHYLASE activity in a single enzyme
- Bind to host-specific DNA sites of 4-6 bp separated by 6-8 bp & containing methylated adenines
Example: Ecok from E. coli K 12, recognizes the ff site:
TYPE I RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Used most frequently in the laboratory
- DO NOT have inherent methylation activity
- Bind as simple dimers to symmetrical 4-8 bp DNA recognition sites (palindromic)
- Cleave DNA directly at the binding site, producing fragments of predictable size
TYPE II RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Resemble Type I enzymes in their ability to both methylate & restrict (cut) DNA
- Have multiple subunits, including helicase (unwinding) activity
- Recognition sites are asymmetrical & the cleavage of the substrate DNA occurs 24-26 bp from the site to the 3’ side
Example: Pstill from Providencia stuartii, recognizes the ff site & cuts DNA 25-26 bp 3’ to the recognition site
TYPE III RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Similar subunit structures & enzyme requirements
- CUTTING & METHYLTRANSFERASE functions
Example: BseMII from Bacillus sterothermophilus, with target sequence:
TYPE IV RESTRICTION ENYMES
CUTS 1 strand of DNA 10 bp ff the recognition sequence.
RESTRICTION ENDONUCLEASE FUNCTION
ADDING METHYL GROUPS methyl groups to both of the adenine residues in the target sequence
METHYLATION FUNCTION
- Catalyzes the formation of a phosphodiester bond between
adjacent 3’-hydroxyl & 5’-phosphoryl nucleotide ends
(SEALS) - Discovered in 5 different laboratories in 1967
DNA LIGASE
o Degrade DNA from free 3’-hydroxyl or 5’- phosphate ends
o Used under controlled conditions, to
manipulate DNA in vitro
o Different substrate requirements & will therefore degrade specific types of DNA ends
EXONUCLEASES
degrades single-stranded DNA (ssDNA) from the 3’-hydroxyl end into mononucleotides
EXONUCLEASE I FROM E.coli
removes 5’ mononucleotides from the 3’ end of double-stranded DNA (dsDNA) in the presence of Mg2+ & Mn2+
EXONUCLEASE III FROM E. coli
digests ssDNA from either the 5’-phosphate or 3’-hydroxyl end
EXONUCLEASE IV FROM E.coli
TYPES OF HELICASE
- interconvert topological isomers or relax super twisted DNA ((eukaryotes)
TOPOISOMERASE
TYPES OF HELICASE
- (type II topoisomerase): untangles DNA through double-strand breaks & separate linked rings of DNA (prokaryotes)
GYRASES
Catalyze the ADDITION OF METHYL GROUPS to nitrogen bases, usually adenines & cytosines in DNA strands
METHYLTRANSFERASE
- It is the mixture & assembly of new genetic combinations
- Occurs through the molecular process of crossing-over or physical exchange between molecules
RECOMBINATION
The one that holds a new combination of DNA sequences is called the _______
RECOMBINANT MOLECULE or ORGANISM
Each generation of sexually reproducing organisms is a NEW COMBINATION of the parental genomes → generates genetic diversity
MENDEL’S LAW
MENDEL’S LAW 3 WAYS OF Recombination in Sexually Reproducing Organisms
CROSSING - OVER
RANDOMLY ASSORTED
FERTILIZATION
- Beginning of meiosis: duplicated chromosomes line up & recombine by crossing over or breakage & reunion of the 4 DNA duplexes
- Recombined duplexes are randomly assorted into gametes
- Gamete will merge with the gamete from the other parent carrying its own set of recombined chromosomes
- Controlled mixing of genes
- Single genes can be altered, replaced, deleted, or moved into new genomes
- Implications:
1. Laboratory
2. Treatment & prevention of disease (gene therapy)
RECOMBINANT DNA TECHNOLOGY
RECOMBINATION IN ASEXUAL REPRODUCTION 3 WAYS:
CONJUGATION
TRANSDUCTION
TRANSFORMATION
- Transfer of genetic information by PHYSICAL ASSOCIATION OF CELLS
- 2 types (or sexes) of participating bacteria: F+ and F-
- Conjugating cells must be in physical contact with each other for successful transfer of the F+ phenotype
CONJUGATION
- Extrachromosomal circle of dsDNA or plasmid carrying the
genes coding for construction of the mating bridge - After mating: both bacteria are F+
F FACTOR
o Strains with chromosomally embedded F factors
o Used in first mapping studies
High-frequency recombination (Hfr) bacteria
- Transfer of genetic information from 1 cell to another through a VIRAL INTERMEDIATE
- 1960s, studied by Francois Jacob & Elie Wollman
- Viral intermediate: BACTERIOPHAGE (bacterial viruses)
TRANSDUCTION
- Transfer of genetic information among cells WITHOUT PHYSICAL ASSOCIATION, such that a new phenotype is produced in the recipient cells
- A bacterium takes up a piece of DNA floating in its environment (often DNA that has been shed by other bacteria)
TRANSFORMATION
These are small, usually circular dsDNAs, often carrying genetic information, that replicates INDEPENDENTLY or in SYNCHRONY with host cell replication
- Most are 2,000-100,000 bp (2-100 kilobase pairs) in size
- Can carry limited amount of genetic information
- DNA duplex is compacted, or supercoiled & can be relaxed by nicking or by local unwinding of the double helix
PLASMIDS
PLASMID CLASSIFICATIONS:
- Include F factor & some of the R plasmids
- CARRY GENES for their own transfer & propagation & self-transmissible
- Occur in small numbers (1 or 2 copies per chromosome equivalent)
LARGE PLASMIDS
PLASMID CLASSIFICATIONS:
- More numerous in the cell (20 copies per chromosomal equivalent)
- DO NOT CARRY GENES directing their maintenance
- Rely on high numbers for distribution into daughter cells at cell division or uptake by host cells in transformation
SMALL PLASMIDS
PLASMID CLASSIFICATIONS:
- Class of plasmids carrying resistance to BACTERIOCINS (toxic proteins manufactured by bacteria)
COLLICINOGENIC
