UNIT 3/7/10 - Genetics Flashcards
Explain why the four haploid cells produced by meiosis are not genetically identical
independent assortment of maternal and paternal homologues chromosomes
crossing over of segments of individual maternal and paternal homologous chromosomes
State and define two methods of exposing a chromosome mutation by karyotyping the chromosomes set of a fetus
amniocentesis - passing a needle through the mothers abdominal wall using ultrasound to guide the needle, withdrawal of a sample of amniotic fluid from the amniotic sac of a developing fetus
chorionic villus sampling (cvs) - cells are sampled from the placenta, specifically the chorion by entering through the vagina (can be done on an earlier stage)
List and define the types of gene mutations depending on their sensuality
- missense mutations - beneficial mutations changing the gene sequence to create new variations of a trait
- nonsense mutations - detrimental mutations truncating the gene sequence to abrogate the normal function of a trait
- silent mutations - have no effect on the functioning of the specific feature
Define point mutations and what they may involve
changes to one base in the DNA code
- substitution of a base (e.g ATG -> ACG)
- insertion of a base (e.g. ATG -> ATCG)
- deletion of a base(e.g. ATG -> AG)
State by which parent the gender of offspring in humans is determined
male partner
Explain how the male embryos differentiate from female embryos
in the seventh week of pregnancy, in male embryos the prime male-determining gene (SRY) , located on Y chromosome starts to develop events leading to the growth of male genitalia
because the SRY codes for - testis-determining factor (TDF) which initiates the production of relatively low level of testosterone which at this stage inhibits the development of female genitalia
in the absence of Y chromosome the embryonic gonad tissue forms an ovary and later under the influence of such ovary the female reproductive structures develop
Define a karyogram
representation of the chromosomes found in a cell
State meaning of the “sex-linked trait”
locus of the gene is ether on the X chromosome or the Y chromosome
Distinguish between a dominant and recessive allele
dominant alleles are always expressed in the phenotype while recessive ones have an effect on the phenotype only when present in homozygous state
Define a test cross
testing a suspected heterozygote plant/animal by crossing it with a known homozygous recessive bc an recessive allele can be masked and otherwise it is often impossible to tell whether an organism is AA or Aa unless they produce an offspring that have the recessive trait
State the brief purpose of DNA profiling
using DNA to reveal its owner identity
State the brief purpose of gene sequencing
mapping DNA by finding where every A,T,C,G is
State the two main ways in which gamete production is able to generate genetic variety in offspring
crossing over during prophase I
random orientation during metaphase I
State what does F1 and F2 refer to
F1 - first filial generation, is a result of offspring of the cross between the parents who are distinctly different
F2 - refers to the second filial generation, are the offspring of the F1 generation
List an example of autosomal disorders
- Huntington’s disease
- cystic fibrosis
List an example of sex-linked disorders
- colour-blindness
- hemophilia
Define a monohybrid cross
only examines one genetic trait and its purpose is to see what kinds of offspring two parents with different alleles can produce
Define polygenic inheritance
involving two or more genes influencing the expression of one trait
most human traits
Define continuous variation
an array of possible phenotypes can be produced
fyi produces a bell-shaped ditribution curve
present in e.g. colour of skin in humans
Define genetics
a study of inheritance and of variation in the inherited characteristics that chromosomes control
Define chromosome in terms of genetics
linear series of genes
Define a gene
a heritable factor consisting of a length of DNA and influencing a specific characteristic
but also
specific length of the DNA double helix, hundreds of thousands of base pairs long, which codes for a specific protein
State the number of chromosomes in humans
46
Define locus (pl loci)
corresponding position of a gene on a chromosome
Define an allele
one specific form of a gene differing from other alleles by or a few bases
one of two or more versions of DNA sequence (a single base or a segment of bases) at a given genomic location
Define a genome
total of all the genetic information
Define recombinant chromosomes
consisting of genetic material from both homologues
State the two types of chromosomes present in humans
- pair of sex chromosomes
- 22 pairs of autosomal chromosomes
Define fertilization
union of gametes from two different parents
State when synapsis happens, define it
during prophase I, always in autosomes but often sex-chromosomes remain unpaired
the joining of homologous chromosomes from each parent during the first stage of cell division
Define the synaptonemal complex
protein-RNA complex connecting the chromosomes
Define mutations
change in the amount or chemical structure of DNA which may result in a change in the characteristics of an organism
State two reasons for why mutations might occur
- spontaneously - result of errors in normal cell processes
- induced - by environmental agents (mutagens)
State and define the types of mutations
somatic mutations - occurring in body cells in multicellular organisms (can cause cancer in the immediate descendants of that cells)
germ line mutations - occurring in the cells of gonads (can cause genetic mutations in offspring)
State the types of gene mutations
- missense mutations
- nonsense mutations
- silent mutations
List and define the chromosome mutations
- polyploidy - presence of more than two sets of chromosomes in a cell.
- aneuploidy - gain/loss of single chromosomes, usually in meiosis (plants tolerate it animals not really).
List the types of aneuploidy chromosome mutations
- monosomy (2n-1)
- trisomy (2n+1)
- nullisomy (2n-2)
List an example of monosomy, lethality and their characteristics
monosomy is lethal in animals esp in human uterus
only viable monosomy in humans - Turner syndrome is produced by karyotype XO
characteristics: normal intelligence, some impairment in cognitive function and secondary sex characteristics failing to develop (eg. breasts)
List examples of trisomy and their characteristics
XXY - Klinefelter syndrome - sterile male, mentally retarded
XYY - Jacob syndrome - fertile, no extra Y in gametes, X pairs with one of the Ys
XXX - metafemale - fertile, no extra X in gametes -> two Xs pair
Trisomy 21 - Down syndrome - characteristic facial features, short statue, heart defects, susceptibility to respiratory infection, mental retardation
Define a nondisjunction
failure of a pair of chromatids to separate and go to opposite poles during the division of the nucleus
State the reason of sickle cell anemia
gene mutation, amino acid change (Glu->Val) which alters the structure of haemoglobin causing it to form insoluble fibrous strands, which cannot carry oxygen as effectively as normal ones
Define the sickle cell trait
one abnormal allele of the hemoglobin beta gene, no severe symptoms bc the alleles are codominant and thus produce both types of hemoglobin
List the conclusions stemming from Mendel’s investigation of the inheritance of a single contrasting characteristic
- within an organism the breeding factors controlling characteristics are - “tall” “short”
- there are two factors in each cell
- one factor comes from each parent
- the factors separate in reproduction and either can be passed on to an offspring
- two copies of the factors separate from each other in the formation of gametes
State the Mendel’s first law of segregation
characteristics of an organism are controlled by pairs of alleles which separate in equal numbers into different gametes as a result of meiosis
Define the genotype and list its two types
genetic constitution (allele make-up) of an organism
- heterozygous - having two different alleles of a gene
- homozygous - having two identical alleles of a gene
Define phenotypes
way in which the genotype is expressed
characteristics of an organism
State which genotype corresponds to the recessive characteristic phenotype
homozygous (tt)
State which genotype corresponds to the dominant characteristic phenotype
either homozygous for dominant allele (TT) or heterozygous for dominant allele (Tt)
Define incomplete dominance
a cross between organisms with two different phenotypes producing an offspring with a third phenotype that is a blending of the parental traits
Define codominance
cross between organisms with two different phenotypes producing offspring with a third phenotype which both of the parental traits appear together
Define multiple alleles
more than two alleles exist for a particular locus
Define sex linkage
special case of linkage occurring when a gene is located on a sex chromosome, usually the X chromosome
Define a carrier
individual with a recessive allele of a gene that does not have an effect on their phenotype
State with reason whether a recessive allele present on the X chromosome will be apparent in the phenotype of a male
yes
because the X and Y chromosome contain non-corresponding alleles on the other
Define DNA
the genetic material occurring in the chromosomes of the nucleus (and in small amount in mitochondria and chloroplasts)
State two types of nucleic acid
DNA - deoxyribonucleic acid
RNA - ribonucleic acid
Define RNA
nucleic acid occurring in the nucleus and cytoplasm (particularly in the ribosomes) involved in “reading” the DNA information
State and define what are the nucleic acids made of
nucleotides
consisting of a nitrogenous base, pentose sugar (ribose or deoxyribose) and a phosphate
State the two types of nitrogenous bases and their names
purines - two-ringed : adenine, guanine
pyrimidines - single-ringed : cytosine, thymine (DNA), uracil (RNA)
Distinguish between deoxyribose and ribose
Deoxyribose sugar is found only in DNA. It differs from ribose sugar thats found in RNA by the lack of a single oxygen atom.
Ribose has OH in bottom right. Deoxyribose only has H.
State the reaction needed to form a nucleotide
condensation reaction of the three components (nitrobase, pentose sugar, phosphoric acid) to form a nucleotide and two molecules of water
State the components of a DNA molecule
two polynucleotide strands paired together and held by hydrogen bonds between the nitrogenous bases
State the paired bases, bond between them and the name of the pairing
adenine = thymine = uracil
guanine (triple hydrogen bond) cytosine
complementary base pairing
State and define the characteristics of a DNA molecule
complementary - sequence of one strand determines the sequence of the other strand
antiparallel - the strands run in opposite directions to each other i. e. top-unattached 5-prime carbon at the top, 3-prime at the bottom of each
State the name of the bond joining the nucleotides within each strand of DNA
covalent phosphodiester bond
State the directionality of the polynucleotide DNA strand
from the 5’ end (with the phosphate group) to the 3’ end with the (-OH group)
State and define the organization of the eukaryotic DNA
supercoiled in nucleosomes
a nucleosome consists of a molecule of DNA wrapped around a core of eight histone proteins (octamer)
State the reason why a nucleosome can get DNA to wrap around
the negatively charged DNA associates with positively charged amino acids on the surface of the histone proteins
State the function of nucleosomes
- supercoiling DNA
- promoting gene expression/silencing a gene in transcription and translation by marking particular genes
Describe the process of supercoiling the chromosome
DNA complexed with eight histone proteins (octamer) forming nucleosomes -> nucleosomes are linked together by additional histone proteins (H1 histone) to form a string of chromosomes -> the string is coiled to form a solenoid structure -> condensation into a 30 nm fibre -> forming of the loops -> compression and folding around a protein scaffols to form chromatin -> chromatin supercoils during cell division to form as chromosome
State and define the types of DNA sequences
Highly repetitive sequences - 45% of the genome, does not have any coding function, satellite DNA, transposable elements
protein-coding genes - 1-2% of the genome, provides base sequence essential to produce proteins
structural DNA - highly coiled DNA, no coding function, occurs around the centromere and at the telomeres
Define transposable elements of the DNA
able to move from one genome location to another
Define satellite DNA
tandemly repetitive DNA sequences (e.g. STRs)
structural component of heterochromatin and centromeres,
commonly used for DNA profiling
Define telomeres
regions of repetitive DNA at the end of a chromosome
protects against chromosomal deterioration during replication
Define introns
non-coding sequences within genes
removed by RNA splicing prior to the formation of mRNA
Define non-coding RNA genes
codes for RNA molecules that are not translated into protein
like genes for tRNA
Define gene regulatory sequences
sequences involved in the process of transcription
includes promoters, enhancers and silencers
List the non-coding DNA types
- satellite DNA
- telomeres
- introns
- non-coding RNA genes
- gene regulatory sequences
Define the DNA replication and state the phase it takes place in
duplication of DNA by making a copy of an existing molecule
interphase S phase
State and define the characteristic of the DNA replication and what does it depend on
replication is semi-conservative
as half of the pre-existing molecule is always conserved
depends on the complementary base pairing
State and define the three alternative models of DNA replication
- conservative - parental double helix remains intact, all new copy is made
- semi-conservative - two parental strands separate, each functions as a template for a complementary strand (the correct one)
- dispersive - each strand of both daughter molecules contains a mixture of old and newly synthesize parts
State the experiment serving as evidence for semi-conservative replication of DNA
Meselson and Stahl
State and describe the steps of DNA replication
- Unwinding of the DNA molecule
helicase enzyme unwinds the double helix by breaking the hydrogen bonds creating “replication forks”
- Making new DNA strands
DNA primase adds a short RNA primer to the exposed 5’ - 3’ strand
lining up of the new nucleotides with appropriate complementary bases opposite the exposed template strands
linking of the phosphate of the new nucleotide to the sugar of the nucleotide before it by a covalent bond with the help of DNA polymerase III enzyme
synthesis of the lagging strand in fragments (Okazaki fragments)
RNA primers between fragments are removed and replaced by DNA by DNA polymerase I
fragments are joined together by DNA ligase
Rewinding the DNA molecule
State the direction of the DNA replication
5’ - 3’ direction
meaning new nucleotides are added to the C3 hydroxyl group such that the strands grow for the 3’ end
thus the DNA polymerase moves in the 3’ - 5’ direction
State the form of the nucleotides combined by the action of DNA polymerase
deoxynucleotide triphosphates
phosphate groups linked by energy-containing bonds.
Define a replication fork
uncoiled region of DNA only in the region of active DNA replication
State the ways and consequence of DNA synthesis during the replication
because of the anti-parallel nature
- continuous way - creates a leading strand
- fragments - creates a lagging strand
Define topoisomerases and list an example
enzymes that regulate the overwinding or underwinding of DNA
the winding problem of DNA arises due to the intertwined nature of its double-helical structure
e.g. gyrase
Describe the overwinding problem of DNA
during DNA replication and transciption, DNA becomes overwound ahead of a replication fork
the topoisomerases bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA
which allows the DNA to get untangled or unwound at the end of the processes and the DNA backbone is resealed again
Define the leading and lagging DNA strand
leading strand - the new strand of DNA that is synthesised continuously in the same direction as the unzipping
lagging strand - the new strand of DNA that is synthesized in the opposite direction to the unzipping
made by joining the Okazaki fragments together
State the function of helicase in DNA replication
- unwinds the DNA at the replication fork
- breaks the hydrogen bonds between the bases
- requires ATP
State the function of DNA polymerase III in DNA replication
adds deoxynucleotide triphosphates to the 3’ end using complementary base pairing to the template strand
State the function of DNA gyrase in DNA replication
removes the supercoiling created by the action of helicase
State the function of DNA ligase in DNA replication
joins the Okazaki fragments together
State the function of DNA polymerase I in DNA replication
- removes the RNA primer
- replaces it using deoxynucleotide triphosphates and complementary base pairing
State the function of DNA primase in DNA replication
adds nucleoside triphosphates on the lagging strand to form an RNA primer using complementary base pairing to the template strand
State the function of single stranded binding proteins in DNA replication
prevents the separated DNA strands from re-joining
Define the dideoxynucleosides, state their function
the ddNTPs
lack the 3’-hydroxyl group necessary for forming a phosphodiester bond
they prevent further elongation of a nucleotide chain and effectively terminate the replication
the resulting length of a DNA sequence will reflect the specific nucleotide position at which the ddNTP was incorporated
Define transcription
the genetic information of DNA is transcribed into a molecule of mRNA by complementary base pairing
Define translation
a process of protein production using mRNA as a guide
Define the central dogma (DNA -> protein)
information passes from genes on the DNA to an RNA copy
the RNA copy then directs the production of proteins at the ribosome by controlling the sequence of amino acids
State the general parts of protein synthesis and where do they occur
- transcription - nucleus
- amino acid activation - cytoplasm
- translation - ribosomes in the cytoplasm
Define the amino acid activation
amino acids are combined with short lengths of different tRNA molecules involved in protein synthesis
Describe the transcription process
- Initiation - RNA polymerase binds to the promoter, DNA strands unwind, polymerase initiates RNA synthesis at the start point on the template strand
- Elongation - polymerase moves downstream unwinding the DNA and elongating the RNA transcript 5’ -> 3’ , in the wake of transcription the DNA strands reform a double helix
- Termination - RNA transcript (mRNA molecule) is released and the polymerase detaches from the DNA
Define the promoter region in the DNA transcription
a specific sequence of DNA bases at the start of a gene to which RNA polymerase binds (non-coding)
Define the terminator region in the DNA transcription
a specific sequence of DNA bases marking the end of the transcription (non-coding)
Distinguish between the anti-sense and sense strand DNA
sense strand - side of the double helix that is a gene,
carries the promoter sequence of bases to which RNA polymerase binds and begins transcribing the anti-sense strand,
has the same sequence as the mRNA (T instead of U),
carries the terminator sequence too
anti-sense - complementary sequence of bases to the gene,
is transcribed into mRNA by RNA polymerase, has the same base sequence as the tRNA (T instead of U),
is read in the 3’ to 5’ direction but mRNA synthesis occurs in the 5’ to 3’ end
State the function of the 5’ cap and poly-A tail
- facilitating the export of mature mRNA from the nucleus
- protection of the mRNA from degradation by hydrolytic enzymes
- helping ribosomes attach to the 5’ end of the mRNA once the mRNA reaches the cytoplasm
Define the RNA processing in post-transcriptional modification
addition of the 5’ cap and poly-A tail
Define an intron
unit of non-coding gene sequence in a split gene
Define an exon
section of genes in a split gene that carries meaningful information
State the processes involved in the post-transcriptional modification
introns are removed, remaining coding portions of mRNA are spliced together, mRNA is passed out into cytoplasm, to ribosomes where it is involved in protein synthesis
Name an enzyme splicing the pre-mRNA
spliceosome
Define a codon
sequence of three bases on the mRNA coding for one of the 20 amino acids and used in the construction of the polypeptide chains
Explain why the codon code is described as degenerative
most amino acids have three or two similar codons coding for them
there are 64 possible different triplet combination (more codons than amino acids)
Explain why the genetic code is described as universal
all living organisms on Earth share the same genetic code book
Explain why the genetic code is described as a triplet code
three bases code for one amino acid
State and define the two steps of protein synthesis
- amino acid activation - combining the amino acids with short lengths of a different sort of transfer tRNA in the cytoplasm
- translation - protein production using mRNA as a guide in the ribosomes
State the structure the tRNA molecule folds into and list its four key regions with their function
cloverleaf structure
- acceptor stem (3’ - ACC) - carries and amino acid
- anticodon - associates the mRNA codon via complementary base pairing
- T arm - associates with the ribosome via the E, P and A binding sites
- D arm - associates with the tRNA activating enzyme responsible for adding the amino acid to the acceptor stem
Describe the steps of the amino acid activation
- entering of the active site of the specific synthetase by the amino acid and the appropriate tRNA
- energy is used (ATP) to catalyze the covalent bonding of the amino acid to its specific tRNA
- the tRNA is released by the synthetase
State the function of an aminoacyl-tRNA synthetase
catalyzes an ATP-requiring reaction where the carboxyl group of the amino acid becomes attached to the 3’ end of the tRNA
Distinguish between the two ribosome types (not the 70s and 80s ones)
free ribosomes - in the cytoplasm, synthesize proteins for use within the cell
bound ribosomes - attached to endoplasmic reticulum, synthesize proteins for secretion from the cells or for lysosomes
Describe the structure of a ribosome
- two subunits: large and small
- three binding sites for tRNA on the surface of the ribosome
- binding site for mRNA on the surface of the ribosome
List the names and functions of the binding sites of the ribosomes
- A site (Aminoacyl - tRNA binding site) - holds the tRNA carrying the next amino acid to be added to the polypeptide chain
- P site (Peptidyl-tRNA binding site) - holds the tRNA carrying the growing polypeptide chain
- E site (Exit site) - site from which tRNA that has lost its amino acid is discharged
Explain how the two amino acids will combine together
to form a dipeptide by one amino group of one amino acid reacting with the carboxyl group of the other forming a peptide linkage
List the three phases of translation
- the initiation phase
- the elongation phase
- the termination phase
Describe the initiation phase of translation
- small ribosomal unit binds to mRNA molecule
- an initiator tRNA (carrying the amino acid methionine) with the anticodon UAC, base - pairs with the start codon AUG,
- the large ribosomal subunit arrives and is brought together by the proteins called initiation factors
- GTP is hydrolyzed providing the energy for the assembly, the tRNA is in the P site, the A site is available to the tRNA bearing the next amino acid.
List and describe the elongation phase of translation
Codon recognition - base pairing of the incoming aminoacyl tRNA anticodon with the complementary mRNA codon in the A site, hydrolysis of GTP
Peptide bond formation - catalysis by the rRNA molecule of the large ribosomal subunit, of a peptide bond between the amino group of the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site thus attaching it to the amino acid on the tRNA in the A site
Translocation - translocation of the tRNA in the A site to the P site by the ribosome, simultaneous moving of the empty tRNA from the P site to the E site and there its release, moving along of the mRNA to its bound tRNAs to bring the next codon to be translated into the A site
Describe the termination phase of the translation process
- reaching of the stop codon on mRNA,
- accepting of the “release factor” by the site of the ribosome
- hydrolysis promotion by the release factor of the bond between the tRNA in the P site and the last amino acid of the polypeptide thus freeing the polypeptide from the ribosome
- dissociation of the two ribosomal subunits and the other components
Define the release factor of the translation
a protein shaped like a tRNA instead of an aminoacyl tRNA
Define a polysome
mRNA, several ribosomes and their growing protein chains
State when is the post-translation modification needed
when the proteins are in the form of inactive precursors
State the reason for the regulation of transcription
prevention of the production of unwanted proteins/enzymes.
prevention of the energy and amino acids being used up unnecessarily
State the three main methods of transcription regulation
- nucleosomes
- regulatory proteins called transcription factors produced by regulatory genes
- methylation of DNA
State the two ways in which nucleosomes regulate the transcription
- acetylation of histone proteins - permits the transcription
- methylation of histone proteins - physically impedes the binding of transcription factors or causes the chromatin to bind tightly together so that the genes cannot be transcribed
Explain why the nucleosomes are so prone to chemical modification
because of the easily accessible amino acids in the histone tails protruding towards from a nucleosome
Explain why the acetylation of histone tails permits transcription
because it promotes loose chromatin structure and thus accessible for transcription
Distinguish why acetylation makes DNA less coiled and methylation more coiled
Adding an acetyl group to the tail (acetylation) neutralizes the charge, making DNA less tightly coiled and increasing transcription
Adding a methyl group to the tail (methylation) maintains the positive charge, making DNA more coiled and reducing transcription
Distinguish between a “switched on” gene and a “switched off” one
..
State the positioning and function of the CpG islands
blocking transcription of a gene (methylation)
are often close to the promoter region at the 5’ end of the gene
State the element required for the eukaryotic RNA polymerase to initiate transcription
the transcription factors
Define asymmetric segregation of cellular determinants
the assymetric localisation of cytoplasmic molecules (proteins or mRNAs) within a cell before it divides
it is a result of inductive signals coming from other cells or their external environment
Define and distinguish polycistronicity from monocistronicity
in eukaryotic cells - transcriptional unit has one or more genes - polycistronic
in prokaryotic cells - transcriptional unit has only one gene - monoisotopic
Define DNA sequencing
the process by which the base order of a nucleotide sequence is elucidated
usually involves the use of chain-terminating dideoxynucleosides
Define annealing
cooling the sample to 55 degrees to allow primers to anneal - designate the sequence to be copied
Define genetic engineering
a process in which genes from one organism are transferred to the set of genes (the genome) of another unrelated organism
List the simplified steps of genetic engineering
- Identification of the gene that codes for the protein of interest
- Remove a gene from the donor
- Insert a gene into a host
- Grow the altered bacteria on a large scale to make the protein product
- Isolate protein
State the function of restriction enzymes
occur naturally in bacteria where they protect against the activity of viruses by cutting up viral DNA that enters the bacterium
otherwise the viral DNA might take over the host cell
State the genetic engineering role of restriction enzymes
cutting at particular base sequences into two types blunt ends and sticky ends to the cut fragments
Define a sticky end
single-stranded fragments of DNA, without its complementary sequence opposite
sticky bc if created by any one particular restriction enzyme they have complementary base sequences so that they can be used to link together pieces of DNA by hydrogen bonding
Define electrophoresis
process used to separate particles including biologically important molecules such as DNA, RNA, proteins and amino acids
State the factors on which the electrophoresis is dependent on
size of the molecules
electric charge
State the material on which the electrophoresis is carried out
agarose gel or on polyacrylamide gel (PAG)
Explain the mechanism behind electrophoresis
DNA molecules carry negative charges so they move during the same direction during gel electrophoresis but not at the same rate
small fragments will move faster and so further than the large ones
Define PCR and state its full name
polymerase chain reaction
technique that can be used to amplify small quantities of DNA, without the risk of using up a limited sample
List the stages of PCR
- denaturing the DNA sample using heat
- annealing each DNA strand with a primer at a slightly cooler temp
- adding free nucleotides and the DNA polymerase
- DNA polymerase binds to the primers and synthesizes a complementary strand of DNA using the free nucleotides
Define a primer
short strand of DNA providing a starting sequence for DNA extension
Define DNA cloning
process of making large quantities of a desired piece of DNA using a vector by which a gene is transferred to a receiving organism
Define a vector in relation to DNA cloning and list two types of it
self-replicating DNA molecule used to transmit a gene from one organism to another
can be a: plasmid or bacteriophage
State the natural function of ligase
occurs naturally in the nuclei of organisms where it repairs DNA damaged in replication
by catalyzing the joining of sugar-phosphate backbones of adjacent DNA strands (annealing) after their sticky ends have aligned by complementary base pairing
Explain the ligation of vector
- gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases (because the sticky ends of the gene and the vector overlap via complementary base pairing)
- the gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
- DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond
Describe the gene cloning in vivo using plasmids
- prepare isolated plasmids as vectors, restriction enzyme used to cut open the plasmid.
- both carry sticky ends (cut by the same restriction enzyme)
- open plasmid and the gene-containing fragments combine
together by ligase. - recombinant plasmid is introduced into a bacterial cell by adding the DNA into the bacterial culture -> some bacteria will take up the plasmid from solution by transformation
Describe the common features of a typical plasmid vector
promoter region
- transcription initation
recognition site
- for endonuclease
recombination site
- for endonuclease
antibiotic resistance gene
- to select transgenic cells
replication origin
- plasmid synthesis
repressor sequence
- in some plasmids only
Describe the use of restriction enzyme and PCR in gene cloning
PCR is used to produce multiple copies of the DNA fragment or gene of interest
the ends of the fragments have the same restriction site as the cloning vector
the plasmid and the DNA fragments are cut with the same restriction enzyme so that they can combine through sticky ends ligation and introduced into bacterial host cells
the plasmid will also contain an antibiotic resistance gene that allows only cells with a plasmid to survive when the antibiotic is present
State the function of reverse transcriptase in genetic engineering
collection of mature mRNA from eukaryotic cells and production of DNA from it so that it can be incorporated into the bacterial DNA for protein production
bc bacteria themselves cannot remove introns
Define Complementary DNA
made from mRNA, lacks introns and can be used for protein expression in bacteria
Outline the production of human insulin using recombinant DNA technology
..
State the cause of pituitary dwarfism and how it is treated
failure to secrete sufficient human growth hormone (hGH) in children and young people
genetically modified E. coli is used for hGH production and then injection into patients
List the reasons why it is much more difficult to manipulate genes in eukaryotes
- eukaryotes do not have plasmids (with the exception to yeast)
- eukaryotes are diploid -> two alleles of each gene must be engineered into nucleus
- transcription of eukaryotic DNA to mRNA is much more complicated
- vectors have difficulties in penetrating cell walls
Describe the production of transgenic plants
formed using tumor-forming Agrobacterium
Agrobacterium invades broad-leaved plants forming a tumor (the gene for that forming is naturally in a plasmid in the bacterium)
genes can be added to the Ti plasmid, using the restriction enzymes and ligase, and then be placed back into the Agrobacterium as the recombinant plasmid
a host crop plant is infected by the modified bacterium and thus after regeneration of the plant there is the plant with a new trait
Name some of the improvements in plants through genetic engineering
herbicide resistance in crop plants;
rice varieties that contain β carotene (vitamin A precursor)
tomato varieties able to ripen on the plant and develop full flavor, without rotting quickly when picked and marketed as red fruit;
resistance to insect pets;
Define DNA profiling and state what principle is it based on
exploitation of the genetic engineering technique to identify a person or an organism from a sample of their DNA
a unique, personal sequence of nucleotides in our DNA
especially as the STRs (microsatellites/short tandem repeats) are repeated up to 100 times in our DNA with half of them coming from our mother and half from our father
Outline the steps of DNA profiling
- the DNA is extracted from sample of cells and then separated and purified.
- this sample is then copied through PCR to obtain sufficient DNA to analyze.
- the DNA is cut into small, double-stranded fragments using a restriction enzyme.
- the DNA fragments are separated by gel electrophoresis into bands.
- the gel is treated to split DNA into single strands and then a copy is transferred to a membrane.
- the selected, radioactively labelled DNA probes are added to the membrane to bind to particular bands of DNA.
- the membrane is overlaid with X-ray film which becomes selectively fogged by emission from the retained labelled probes.
8.the x-ray film is developed, showing the position of the bands to which probes have attached and thus the profile will have an appearance of a bar code.
List the application of DNA profiling
- forensic investigations: identification of criminals, rape crime, corpse
- paternity determination
- studies of wild animals
Define gene therapy and how can it work.
the introduction of genes into an afflicted individual for therapeutic purposes
- through providing a correctly working version of a faulty gene
- by adding a novel gene to perform a corrective role
- gene expression blockage to control cellular activity
State what does gene therapy require
a gene delivery system
so a way to transfer the gene to the patient’s cell using infectious agents such as a virus (transfection)
State the therapy implemented for Familial hypercholesterolemia (FH)
FH is the genetic disease which the membrane receptors for LDLs are defective and low density lipoproteins (LDLs) accumulate in the blood, leading to atherosclerosis and coronary heart disease
it is treated through gene therapy involving engineering a healthy human gene into a sample of liver cells that genetically corrected cells may be re-established in the liver and restore correct functioning
Define cloning
a group of genetically identical individuals (or cells)
Outline how can cloning happen
Naturally:
- asexual reproduction - producing genetically identical progeny in plants, animals and Protoctista’s.
- sexual reproduction - the fertilized egg cell divides to form a blastocyst and if it divides into two separated masses of cells before it embeds in the wall then both cell masses go on to form embryos and they become genetically identical twins.
Artificially:
- by single-cell cultures (cloning plants)
- by embryo splitting
- by somatic cell nuclear transfer (SCNT)
Outline the process of cloning plants though single-cell cultures
differentiated cells are taken from the root of the carrot and incubated in culture medium and then they grow into normal adult plants each genetically identical to the parent plant
thus the differentiation in plant cells does not involve irreversible changes in the DNA and thus they can give rise to all the specialized cell types
Describe the process of cloning by embryo splitting
- an egg is fertilized in vitro and allowed to develop into multicellular embryo.
- the individual cells can be separated from the embryo while they are still pluripotent and transplanted into surrogate mothers.
State the functions of cloning by embryo splitting
because of their same characteristics as their parents.
- high value livestock
- isolation of stem cells and thus tissue and cell engineering
Describe the cloning by nuclear transfer
isolated, fully differentiated cell is induced to be domrant though semistarvation
then its converted to the embryonic state by fusion with egg cell (different) from which the nucleus has been removed
State and define the purposes of using differentiated cells to generate cloned embryos
Reproductive cloning:
if the embryo is implanted into the uterus of a surrogate a new cloned organism will develop
Therapeutic cloning:
embryonic cells can be induced to differentiate to create specific tissues or organs for transplantation
State the reason why cloned animals of the same species do not always look or behave identically
random mutations which occur during the development or environment
random chromosome X inactivation for example and more
Define DNA sequencing
process by which the base order of a nucleotide sequence is elucidated.
usually involving the use of chain-terminating dideoxynucleosides
List and expand on the issues associated with the Genetically Modified Crops
new allergens could be created -
For instance, during laboratory testing, a gene from the Brazil nut was introduced into soybeans. It was found that people with allergies to Brazil nuts could also be allergic to soybeans
cross-breeding - GM crops can cross-breed with the surrounding vegetation transferring undesired characteristics
cross-contamination - plants bioengineered to produce pharmaceuticals (such as medicines) may contaminate food crops
biodiversity - growing GM crops on a large scale might affect the balance of wildlife and the environment
Define a genetically modified organism (GMO)
organism whose genetic material has been altered using genetic engineering techniques (recombinant DNA technology) use DNA molecules from different sources which are combined into one molecule to create a new set of genes with the desired characteristics
Describe gene therapy using retroviral vector
retrovirus rendered harmless acts as a vector exploiting its ability to insert DNA transcript of its RNA genome into the chromosome DNA OF ITS HOST CELL the cell will possess the gene product if expressed
Describe the symptoms and the genetic cause of cystic fibrosis and it’s inheritance
Symptoms:
Disruption of all glands (pancreas, sweat, intestinal), infertility
Genetic cause:
autosomal recessive, on chromosome 7 a mutation of deltaF508 (which accounts for around 70% of all defective CF genes) its a deletion of the 508th triplet in the DNA code for the chloride transport protein CFTR - which results in the missing phenylalanine amino acid and thus the CFTR protein cannot carry out its function of regulating chloride ion balance in the cell
Inheritance:
autosomal recessive pattern => affected people are homozygous recessive for the mutation, heterozygous are carriers
Describe the symptoms and the genetic cause of Huntington Disease and it’s inheritance
Symptoms:
long hunting protein is cut into smaller toxic fragments, which accumulate in nerve cells and eventually kill them, becomes apparent in mid-adulthood with jerky, involuntary movements and loss of memory, reasoning and personality
Cause:
autosomal dominant mutation of the HTT gene caused by a trinucleotide repeat expansion on the short arm of chromosome 4,
in the mutation (mHTT) the number of CAG repeats increases from 6-30 to 36-125, the more repeats the more severe the disease because the repeats result in the production of an abnormally long version of the huntingtin protein
Inheritance:
autosomal dominance pattern, affected are either homozygous or heterozygous for the mutant allele
