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allele
one of several alternative forms of a gene occupying a given locus on a chromosome
Locus
the position on a chromosome at which the gene for a particular trait resides; it may be occupied by any one of the alleles for the gene.
Genetic recombination
a process by which separate DNA molecules are joined into a single molecule, due to such processes as crossing over or a transposition.
what is recombination the result of
crossing over at the chiasma during meiosis and involves two fo the four chromatids
How many pairs of chromosomes do humans have
23
what would life be without recombination
chromosomes would carry a fixed set of allels that would persist unchanged through evolutionary time.
Does recombination allow alleles to mix
Yes, but some alleles always segregate together and never segregate together
What does genetic mapping measure
the physical distances between genes on a chromosome
does loci recombination for frequently when close or far apart
Frequency of recombination between two genes is proportional to their physical distance.
two loci far apart recombination occurs more frequently causing theme to segreate almost independently
two loci close together are rarely recombined
How do genes of different chromosomes recombinate
segregate independently resulting in 50% recombinant types during meiosis
How doe genes sufficiently far apart on the same chromosome recombinate
the probability of at least one crossover in the region is high and show 50% recombination.
How do adjacent genes recombinant
although the probability of recombination is reduced, but a recombination can occur in only some proportions of meiosis.
One gene : one enzyme hypothesis
summarizes the basis of modern genetics: that a gene is a stretch of DNA encoding one or more isoforms of a single polypeptide chain
Beadle and Tatum, 1941
One gene : One polypeptide hypothesis
a modified version of the not generally correct one gene: one enzyme hypothesis; the hypothesis that a gene is responsible for the production of a single polypeptide.
Why did Beatle and Tatum upgrade their hypothesis
not all proteins are enzymes
some proteins have multiple polypeptide units
alternative splicing and post-translational modifications
Heteromultimer
a molecular complex (such as a protein) composed of different subunits
Homomultimer
a molecular complex (such as a protein) in which the subunits are identical
Do all genes encode polypeptides
No, some genes encode structural or regulatory RNAs
Recessive mutations
due to a loss of function by the polypeptide product
dominant mutations
result from a gain of function
null mutation
tests whether a gene is essential - one that completely eliminates functionssile
silent mutations
have no phenotypic effect, either because the base change does not change the sequence or amount of polypeptide, or because the change in polypeptide sequence has no effect
Neutral substitutions
substitutions in a protein that cause changes in amino acids that do not affect activity.
A locus having many different mutant allets allows…..
the possibility of heterozygotes that represent any pairwise combination of alleles
ABO human blood group is an example of
a locus may have more than one wild type allele; a locus may have a polymorphic distribution of alleles with no individual alle considered to be a sole wild type
Chiasma
formed at prophase I of meiosis and is responsible for generating recombinant chromosomes.
Heteroduplex DNA
Recombination intermediate; depends on the complementarity of two DNA strands.
When the two DNA molecules from different homologous chromosomes interact, sections of single-stranded DNA from one chromosome pair with complementary single strands from the other chromosome. This creates a heteroduplex, meaning that the two strands of DNA in this region come from different sources (one from each homologous chromosome).
Independent segregation
ensures genetic variation because different traits can combine in many different ways leading to diverse offspring.
Example: Suppose we have two genes:
Gene for seed shape: R (round) and r (wrinkled)
Gene for seed color: Y (yellow) and y (green)
Since these genes are on different chromosomes, a parent with genotype RrYy can produce all four possible gametes in equal proportions:
RY, Ry, rY, ry
This happens because when homologous chromosomes separate, the R and r alleles do not influence whether Y or y gets into the same gamete.
Why is this phrase true?
The frequency of recombination between 2 genes is proportional to their physical distance.
When two genes are close together on a chromosome, crossing over (recombination) between them is rare because there is less physical space for a crossover to occur between them.
When two genes are farther apart, recombination happens more frequently because there is more space for crossover events.
This is why geneticists use recombination frequency to map genes—the farther apart two genes are, the higher the chance that recombination occurs between them.
Why is this phrase true?
For genes that are very far apart on a single chromosome, the frequency of the recombination is not proportional to their physical distance because recombination happens so frequently.
When genes are very far apart on the same chromosome, recombination occurs so frequently that it starts to behave as if the genes were on separate chromosomes.
In such cases, the maximum measurable recombination frequency approaches 50%, which is the same as independent assortment (as if the genes were on different chromosomes).
Beyond a certain distance, recombination events happen so often that we lose the ability to use recombination frequency to measure physical distance accurately.
How is genetic code read
in triplet codons; non-overlapping starting at fixed point
acridines
mutagens that act on DNA to cause the insertion or deletion of a single base pair.
They are useful in defining the triplet nature of the genetic code via their ability to cause frameshift mutations in the 1960’s.
When triple mutants were constructed (+++) or (- - - ) combinations show the wild type phenotype implying the code is read in triplets
genetic code
the relationship between a sequence o DNA and the sequence of the corresponding polypeptide
coding region
in deciphered by a complex apparatus that interprets the nucleic acid sequence
codon
each trinucleotide sequence that represents one amino acid
A coding sequence being read in nonoverlapping triplets implies:
Each codon consists of three nucleotides and that successice codons are represented by successive trinucleotides.
An individual nucleotide is part of only 1 codon
A coding sequence being read at a fixed starting point implies:
assembly of a polypeptide must start at one end and work to the other, so that different parts of the coding sequence cannot be read independently.
Result of single point mutation or nucleotide subsitution
only will affect one amino acid due to only one codon changing
Result of an insertion or deletion
will change the triplet sets for the entire subsequent sequence - Framshift
What did scientist know when trying to decipher the genetic code
there were 4 nucleotides (ACGT) which let them know that a sinlge nucleotide couldn’t code for one amino acid
how many combinations for 3-nucleotide sequence
64 combinations , which is more than enough to code for 20 amino acids
What did Har Gobind Khorana do
Major contribution in synthesizing artificial RNA sequences and used them in experiments to determine which codons specify which amino acids.
His work built of Marshall Nirenberg who demonstrated that Poly-U RNA directs synthesis of amino acid Phenylalanine
This approach was extended and refined by synthesizing RNA molecules with specific repeating sequences and testing their effects in protein synthesis.
How did the experiment deciphering the genetic code using synthetic RNA work?
step 1: Because scientists know that mRNA is translated to proteins by ribosomes and tRNA cells, they were able to study the process outside of living cells via cell-free extracts from e.coli that contained machinery for protein synthesis (ribosomes, tRNA, and enzymes) but no natural mRNA
step 2: Adding synthetic RNA sequences with repeating nucleotide patterns like Poly-U (UUUU..) which produced phenylalanine, Poly-C(CCCC) which to to only proline ect. He then went further to produce repeating di-, tri-, and tetranucleotide sequences like (UCUC) which gave serine (UCU) and leucine CUC
Step 3: identify amino acids produced and helped assign specific codons to amino acids and was able to confirm and complete the full genetic code.
What is the effect of a mutation that adds or deletes 3 bases
No change in reading of triplets beyond the last site of mutation
Key findings of genetic code experiments
codons are 3 nucleotides long
Start and Stop codons were identifedd through phage experiments
the wobble hypothesis
What did Nirenberg and Matthaei
experiments with synthetic RNA and Poly-U and determined the first evidence that RNA codes for amino acids.
Khorona expanded on their idea with repeating sequences
What was the frameshift mutation by Crick and Benner
mutations in bacteriophage DNA to confirm the triplet nature of codons
introduced single nucleotide insertions and deletions and found that adding or deleting 1 or 2 nucleotides completely disrupted protein function
addition or deleting three nucleotides allowed the protein to still function suggested triplets.
Key findings
codons are 3 nucleotides long
each codon corresponds to a specific amino acids
the genetic code is redundant * multiple codons can code for the same amino acids
open reading frame
a sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon (ATG) and ending with a termination codon (TAA, TAA, TGA)
how many of the reading frames are normally read
1
closed (blocked) reading frame
a reading frame that cannot be translated into polypeptide because of the occurrence of termination codons.`
Unidentified reading frame (URF)
an open reading frame with an as yet undetermined function.
mRNA
what a typical bacterial gene is expressed as from transcription and then a peptide by translation
gene expression
the process by which the information in a sequence of DNA in a gene is used to produce an RNA polypeptide involving transcription and translation
what are the 3 areas that an mRNA consist of
5’ UTR (untranslated region)
coding region
3’ UTR (untranslated region)
intron
not represented in the polypeptide product
Splicing
the way introns are removed and togetherness of exons
RNA processing
modifications to RNA transcripts of genes. This may include alterations to the 3’ and 5’ ends and the removal of introns
pre-mRNA
the nuclear transcript that is processed by modification and splicing to give mRNA
exon
any segment of an interrupted gene that is represented in the mature RNA product.
Ribosome
a large assembly of RNA and proteins that synthesizes polypeptides under direction from an mRNA template
ribosomal RNAs (rRNAs)
a major component of the ribosome
transfer RNA (tRNA)
the intermediate in polypeptides synthesis that interprets the genetic code
each linked to an amino acid
has an anticodon sequence that is complementary to a triplet codon representing the amino acid.
cis acting mutation
not expressed as RNA or polypeptide and affects only contiguous stretch of DNA
trans-acting
all gene products (RNAs or Polypeptide) can act on any copy of a gene in the cell.
trans acting mutation
affects both alleles of a gene that it controls
restriction endonucleases
enzymes that recognize specific short DNA sequences and cleave the duplex
function as bacterial immune system, protecting against phage
used extensively during phage research in the 1950’s-1970s due to their simple genomes.
phage therapy
use bacteriophages to target and kill specific bacteria
what big deal happened in the 1970s
scientists successfully isolated restriction endonucleases leading to advancements in molecular cloning
nucleases
enzymes that cleave nucleic acids
endonuclease
exonuclease
phosphatases
How are restriction endonucleases used
used to cleave DNA into defined fragments
create sticky ends and blunt ends
what is HaeIII
an restriction endonuclease enzyme that cuts DNA into 4 base pair fragments with blunt ends
what is EcoRI
a restriction endonuclease that creates sticky ends and generates 4000 bp fragments
Restriction mapping
used to identify unknown DNA molecules by cutting with different enzymes and analyzing fragment patterns
generates reproducible cleavage patterns, allowing for molecule identification.
palindromic sites
are often recognized by restriction endonucleases and cut at or near the sequence
crucial for genetic engineering, cloning, and DNA analysis due to the predictability for consistent cutting
Cloning vector
A DNA molecule (often from plasmids or phages) used to propagate foreign DNA in a host cell.
contains selectable markers and replication origins for identification and maintenance in the host
usually a circular plasmid, modified for genetic engineering
some plasmids carry antibiotic resistance genes which help selection.
Types of cloning vectors
plasmid vectors
phage vectors
cosmids
yeast artificial hormones (YACs)
Shuttle Vectors
Expression Vectors
Plasmid vectors
high copy number ~ 10kb DNA insert limit
cosmids
hybrid of plasmids and phage DNA, with high copy number, 48kb
yeast artificial chromosomes (YAC)
used for very large fragments >1Mb
shuttle vectors
can function in multiple host cell types
expression vectors
contain promoters to drive transcription of cloned genes
Recombinant DNA
a DNA molecule from two or more different sources
Key cloning steps
DNA cleavage : use restriction enzymes to cut DNA at specific sites
ligation : join DNA fragments using DNA ligase
transformation: introduce recombinant DNA in host cells.
Subcloning
breaking a cloned DNA fragment into smaller pieces for further cloning
Multiple cloning sites
a DNA sequence containing multiple restriction for inserting foreign DNA.
Lac Z
encodes an enzyme that produces a color change when metabolizing sugar, often used as a marker in cloning.
also serves as a reporter gene
in example of blue glowing mouse, lacZ was expressed under the control of the promoter. When lacZ gene is transcribed and translated, the cell produces B-Galactosidase which cleaves X-gal producing the blue color.
if foreign DNA disrupts lacZ gene, B-Gala not produced and colonies are white.
transformation
uptake of exogenous DNA by a cell, leading to the acquisition of new genetic material
the process by which DNA is introduced to a host cell/combining pool of fragments
washing the bacteria in salt wash of CaCl2 or electroporation
vector
cloning vehicle
insert
molecule to be cloned
what 3 key sites does a plasmid contain
origin of replication
gene for ampicillin restistance
lacZ with an MCS
what are two common methods of transformation
salt wash of CaCl2
electroporation
what is the result of transformation
only a handful of bacteria that contains recombinant plasmid from the millions plated.
The plate contained ampicillin which would kill non recombinated plasmids.
how does blue/white selection work
If a fragment of DNA is cloned into the MCS the LacZ gene (blue) will be disrupted, inactivation it and the resulting B-gal will no longer be able to cleave X-gal, resulting in white bacterial colonies rather then blue.
phage vector
infects bacteria, via phage packaging, 20kb
BAC vector
based on F plasmid, ohysical 300kb
reporter gene
easily detected, under the control of the promoter of interest
pYac2
a cloning vector with features that allow replication and selection in both bacteria and yeast
also includes yeast features like centromere and telomeres
example of a shuttle vector as it can be propagated in more than one type of host cell (yeast and bacteria)
Because many types of cells cannot be transformed as easily as bacteria and yeast, what other methods are used?
viral vector that uses the viral infective process to enter a cell
liposomes can fuse with the plasma membranes and release their contents into the cell.
microinjection uses a very fine needle to puncture a cell membrane. A solution carrying DNA is injected into cytoplasm or directly into nucleus if large enough
gene gun to shoot very small particles into the cell by propelling them through the wall at high velocity.
autoradiography
a method of capturing an image of radioactive materials on film
what is the purpose of a probe
gives us the ability to identify a specific sequence using hybridization as long as we know a sequence to target.
can detect and bind to a sequence to which it is complementary
usually labeled with radioactive phosphorus
