e2_p2 Flashcards
Co-transformation of linked genes
genes located close together are often transferred as a unit to recipient cell = co-transformation
co-transformation of 2 genes at frequency greater than product of the single-gene transformation frequencies implies the 2 genes are located close together Genes that are far apart are less likely to be transferred together.
Co-transformation is used to map gene order
Hfr cell
Cells with the F plasmid integrated into the bacterial chromosome
When an Hfr cell undergoes conjugation, the process of transfer of F factor is initiated in the same manner as in an F+ cell
However, because F factor is part of the bacterial chromosome transfer from an Hfr cell also includes DNA from the bacterial chromosome
F factor & conjugation
F (fertility) factor is a conjugative plasmid transferred from cell to cell by conjugation.
It is an episome.
The F plasmid is a low-copy-number plasmid (1-2 copies per cell) and ~100 kb in length
It replicates once per cell cycle and segregates to both daughter cells in cell division.
E.Coli as a model organism
1 circular DNA molecule
4.6 million bp
haploid
divides once every 20-60 min (depending on culture media)
Abnormalities of Structure : Insertion
Non-Reciprocal translocation
Segment from one chromosome is inserted into a different chromosome - requires 3 chromosome breaks (rare)
A chromosomal duplication is usually caused by
abnormal events during recombination
Telomerase activity
different level of activity in different cells
no activity in most somatic cells
Moderately repetitive
Found a few hundred to few thousand times
Genes for rRNA & histones
Sequences that regulate gene expression and translation.
Transposable elements.
Unique (or non-repetitive sequences)
Found once or a few times in the genome.
Includes structural genes as well as intergenic areas.
In humans, makes up roughly 41% o the genome.
Highly repetitive
Found 10s of 1000s to millions of times
Each copy is relatively short (a few nucleotides to several hundred in length)
Some sequences are interspersed throughout genome.
-Example : Alu family in humans
Other sequences are clustered together in tandem arrays -> these are commonly found in the centromeric regions
Alu family in humans
highly repetitive sequences
Approximately 300 bp long
Represents 10% of the human genome
Found every 5000-6000 bp
3 implications of Hfr cell conjugation
- Recipient usually does not receive the entire chromosome, nor F plasmid (so remains F-)
- Genes proximal to 5’ end of F integration site transferred most frequently.
- Homologous recombination between ‘donated’ DNA & recipient. If donor is wild & recipient is mutant, DNA donation can be tested.
If donor is wild-type and recipient is mutant, DNA donation can be tested (how?)
We can map the relative position of genes versus F integration site.
Genes close to integrated F factor are always transferred, but the further away from F position, the less likely.
How many minutes does mixing of Hfr donor and F- recipient need to last until a certain gene is transferred?
->Every gene has unique ‘minute’ location on genome, (complete genome transfer 100 min)
Mapping Problem
The distance between genes is determined by comparing their times of entry during an interrupted mating experiment.
The approximate time of entry is computed by extrapolating the time back to the x-axis.
Interrupted Mating Experiment
Mix togetehr Hfr donor cells and F- cells
After different periods of time, take some and place in a blender (breaks about the cells).
Plate on growth medium to detect gene transfer.
DNA replication - circular DNA
-bidirectional replication of a circular chromosome in ecoli, etc
replication begins at the point of origin (oriC) and proceeds in both directions at the same time (bidirectional)
replicon = individual unit of replication
DNA replication - linear DNA
Large team of enzymes coordinates replication.
Replication of linear DNA in a eukaryotic chromosome is initiated at many sites along the DNA.
Multiple initiation -> reduces total replication time
IN eukaryotic cells, origins of replication are ~40,000 BP apart, which allows each chromosome to be replicated in 15 to 30 min.
Chromosome do not replicate simultaneously, and so complete replication of all chromosomes in eukaryotes takes from 5 to 10 hours.
Organization of eukaryotic chromosomes
Eukaryotic species contain 1 or more sets of chromosomes. Each set is composed of several different linear chromosomes.
Genes are interspersed throughout the chromosomes.
Each chromosome contains a single, linear molecule of DNA.
Typically 10s to 100s of millions of base pairs and a few hundred to several thousand genes.
Abnormalities of Chromosome Structure
Chromosome reakage followed by rejoining of inappropriate chromosome ends.
May occur because of ‘inappropriate’ recombination between repeated DNA or copy number variants (CNV’s)
Balanced rearrangements
Unbalanced rearrangements
Some rearrangements are capable of passing through cell division unaltered, others are unstable.
Exceptions to trisomy being unviable
13,18,21,X
Exceptions to monosomy being unviable
X
DNA replication in bacteria
The origin of replication in E.Coli is termed ori C : origin of chromosomal replication
DnaA proteins : bind to DnaA boxes and to each other at oriC
DnaB/helicase : travel along the DNA in the 5’-> 3’ direction and unwinds DNA
DNA repair mechanism steps
- damage to DNA distorts configuration of molecule
- an enzyme complex recognizes the distortion
- The DNA is separated and SSB proteins stabilize the single strands
- An enzyme cleaves the strand on both sides of the damage.
- The damaged part is removed.
- The gap is filled in by DNA polymerase and sealed by DNA ligase.
Gene Conversion
Genetic recombination can cause 2 different alleles to become identical alleles. = gene conversion
Gene conversion can occur in one of two ways:
1)DNA mismatch repair
2)DNA gap repair synthesis
DNA mismatch repair
DSB occurs within recessive gene.
A region adjacent to DSB is digested away hich eliminates the recessive b allele.
Strand invasion causes D-loop formation.
DNA gap repair synthesis
uses the strands from the dominant B allele to fill in the region
The intertwined strands are resolved
Mismatchhed bases are standard bases. How does repair system determine what partner to eliminate?
In bacteria, old strand methylated and new strand methylation lags behind moving replication fork.
Mismatch repair system preferentially excises nucleotides from the non-methylated daughter strand.
Huntington
Incidence : 3-7/100,000
-neuronal loss & brain atroph
-abnormal cellular metabolism before MRI changes
-progressive dementia
-involuntary movements (chorea)
-psychiatric disturbances, personality changes
-average onset of 35-45
-death usually within 10 years of onset
Deamination is
a spontaneous chemical change in which a DNA base loses an amino group
many cytosines in the human genome are methylated in CpG islands
repair machinery recognizes only uracil as base error, but not thymine
if uracil is recognized and cut out -> depyrimidation
Molecular mechanism for trinucleotide expansion
- DNA polymerase slips off template DNA & newly replicated DNA strand forms a secondary structure called a hairpin loop
- DNA polymerase reattaches and begins replication making 2nd copy of repeat region. Then secondary structure expands out to linear DNA & repair of this region leads to expansion.
