bio 207 lab
what packages DNA
chromosomes
how is DNA packages in chromsomes
can be tight or loose, dna is wrapped around histone proteins (loose packaging) all the way to a highly condensed chromsome (tight packaging)
when is loose packaging required
for DNA replication and DNA transcription
what is high level packaging used for
transmission of DNA into new cells (mitosis or meiosis)
what is the basic structure of eukaryotic chromsome
linear DNA molecule wrapped around various proteins
what is the end of a chromsome called? what about the region between these ends?
telomere and centromere
what is unique about the centromere
contains DNA that is packaged tightly and is identifiable as a constriction point on the metaphase chromosome
are centromeres always in the same place on a chromsome
no it varies, creating 3 different types of chromsomes
metacentric chromsome has
a centromere located exaclty between the telomeres
acrocentric chromsome has
a centromere located closer to one of the telomeres
telocentric chromosome has
a centromere literally right by the telomere hence the name
how else can we distinguish between eukaryotic chromsomes
their size
how does size vary between chromsomes
the amount of DNA (measured in base pairs) determines the chromsomes size (larger--> more DNA). no terminology used to classify this, size is relative
how do gametes and somatic cells differ
gametes are egg and sperm cells they are haploid (n) whereass somatic cells are body cells and they are diploid (2n)
pairs of the same chromosome in diploid cells are called
homologous chromosomes
what is a genome
all the genes present in a gamete
the C-value (c) is
the measure of the amount of DNA in a genome, it is expressed in units of mass (ug) or length(bp)
c value of haploid vs diploid (generic convention)
haploid: 1 , diploid: 2
karyogram
displays the chromosomes of a cell, typically the chromosomes are in metaphase arranged into groups based on their size and centromere postion.
what is the cell cycle process
where cells alternate between a growth phae (interphase) and dividing phases (mitosis and cytokinesis) to produce two new daughter cells.
does every cell have the same frequency of divison
no it is highly variable
can the growth phase and the dividing phase of a cell occur simulatenously
no, because during thr growth phase (transcription) the chromsome needs to be packaged loosley where as for the dividing phase (mitosisi) it needs to be packaged tightly. Also becuase the cell needs to store enough energy and make material for cell divison
what are the 3 phases of interphase
gap 1 (G1) phase, s-phase, gap 2 (G2) phase
what is the gist of interphase
the time during th ecell cycle where cells preform designated functions, mainaitn structural and metabolic functions, prepare to divide. makes several proteins to prepare for replication
what happens during G1
when the previous round of cell divison ends G1 begins, it performs cell specfic duties and builds new cellular components, DNA IS NOT MADE DURING THIS PHASE>
what happens during the s phase
it starts after G1 when DNA packaged as chromosomes begin to replicate. by the end of the s phase the single piece of DNA started with in each chromosome becomes replicated consiting of two identical DNA moleculues.
what are the two identical DNA molcules made in the s phase called
sister chromatids. these sister chromatids are held together by proteins located at the centromere throughout interphase and most of cell division
after the s phase is the G2 phase, what happens here
the cell carries on with cell specific duties/building cell stuff and also prepares for cell division (mitosis)
how does mitosis differ from cytokinesis
mitosis: division of the nucleus
cytokinesis: division of the CYTO plasm
after interphase we proceed to mitosis what happens here
mitosis consists of 4 stafes which contribute to the equal division of the nuclues of one cell into genetically identical daughter cells
we start mitosi with prophase what happens here (6)
this stage marks the beginning of mitosis.
Chromatin Condenses: Chromosomes become visible.
Nucleolus Disappears: Nucleolus fades.
Mitotic Spindle Forms: Microtubules form spindle apparatus.
Centrosomes Move: Move to opposite poles.
Asters Form: Star-shaped structures around centrosomes.
Nuclear Envelope Breaks Down: Prepares for chromosome alignment.
then metaphase (3)
Chromosomes Align: Chromosomes line up at the cell’s equator (metaphase plate- a location not a phsyical structure ). indepdent movement of chromosomes
Spindle Fibers Attach: Spindle fibers (microtubules) connect to the centromeres of chromosomes to pull then to the middle of cell.
Chromosomes Are Fully Condensed: Chromosomes are fully visible and tightly packed.
anaphase
microtubules contract and pull the sister chromatids of each replicated chromosome to opposite poles. chromatids gain the status of chromosomes as soon as they segregate from their sister
telophase
nuclear membrane reforms around chromsomes at each pole, chromsomes became less condesened/ more loosely packaged, results in two genetically equal nuclei- contain the same number/types of chromosomes. at the end of cytokineisis these become two cells
what is crossing over
The pairing of homologous chromosomes helps set up specific processes that allow genetic material to be exchanged between the non-sister chromatids in each chromosome pair.
what does crossing over produce
increased genetic variation, 4 chromatids containing different allelic combinations compared to each other and compared to the chromosomes from which they originated
what is intrachromosomal recombination
the shuffling of genetic material between homologous chromosomes which occurs during crossing over
which prophase takes the longest time to complete
prophase I because of the extra time it takes homologous chromosomes to pair and for crossovers to occur.
prophase I
Chromosome condensation: Chromosomes become visible as thickened structures.
Synapsis: Homologous chromosomes pair up to form tetrads (groups of four chromatids).
Crossing over: Homologous chromosomes exchange genetic material at the chiasmata, increasing genetic diversity.
Formation of the synaptonemal complex: A protein structure that helps align homologous chromosomes.
Nuclear envelope breakdown: The nuclear membrane dissolves, allowing chromosomes to move freely.
Outcome: Formation of tetrads, genetic recombination, and preparation for the next stages of meiosis.
metaphase I
Chromosome alignment: Homologous chromosomes (tetrads) align along the metaphase plate.
Independent assortment: The orientation of each homologous pair is random, leading to genetic variation.
Spindle attachment: Spindle fibers attach to the centromeres of each homologous chromosome.
Outcome: Homologous chromosomes are positioned for separation in the next stage (Anaphase I). Genetic diversity is increased due to independent assortment.
indepedent assortment
indepedent and random oreintation of each bivalent creating genetic difference between gametes
genetic variation caused by independent assortment is reffered to as
interchromosomal recombination
anaphase I
Separation of homologous chromosomes: The spindle fibers shorten, pulling each homologous chromosome to opposite poles of the cell.
Chromatids remain attached: Unlike in mitosis, the sister chromatids of each chromosome remain attached at this stage.
Disjunction: The homologous chromosomes are randomly distributed to each daughter cell.
Outcome: Homologous chromosomes are separated into two distinct groups, reducing the chromosome number by half in preparation for meiosis II.
telophase I
Chromosomes reach poles: The separated homologous chromosomes arrive at opposite poles of the cell.
Nuclear envelope reformation: A new nuclear membrane forms around each set of chromosomes.
Chromosomes may de-condense: In some cells, chromosomes may begin to uncoil slightly.
Cytokinesis: The cytoplasm divides, resulting in two non-identical daughter cells, each with half the original chromosome number.
Outcome: Two haploid daughter cells are formed, each with one set of chromosomes, preparing for meiosis II.
what are the 4 differences betwen mitosis and meiosis I
chromosome number, number of divisions, homolgous chromosome pairing, genetic variation, purpose
mitosis vs meiosis I: chromosome number
Meiosis I: Reduces chromosome number by half (haploid).
Mitosis: Maintains chromosome number (diploid).
mitosis vs meiosis I: number of divisions
Meiosis I: One division, results in two haploid cells.
Mitosis: One division, results in two diploid cells.
mitosis vs meiosis I: homologous chromosome pairing
Meiosis I: Homologous chromosomes pair and cross over.
Mitosis: No pairing or crossing over.
mitosis vs meiosis I: genetic variation
Meiosis I: Homologous chromosomes pair and cross over.
Mitosis: No pairing or crossing over.
mitosis vs meiosis I: purpose/outcome
Meiosis I: Produces gametes for reproduction. 2 non identical, haploid cells
Mitosis: Used for growth, repair, and asexual reproduction. 2 identical diploid cells.
meiosis II. defintion, key events, outcome
definition: second divison f meiosis, where sister chromatids are sepearted, resulting in four non identical haploid cells
key events: similar to mitosis, but starts with haploid cells
outcome: four genetically diverse haploid daughter cells (gametes)
prophase II
Chromosomes condense: Chromosomes become visible again.
Nuclear envelope dissolves: The nuclear membrane breaks down.
Spindle fibers form: Spindle apparatus begins to form in each haploid cell.
Outcome: Chromosomes are prepared for alignment in Metaphase II.
metaphase II
Key Events:
Chromosomes align: Chromosomes line up along the metaphase plate.
Spindle attachment: Spindle fibers attach to the centromeres of each chromosome.
Outcome: Chromosomes are positioned for separation in Anaphase II.
anaphase II
Key Events:
Sister chromatids separate: The centromeres divide, and sister chromatids are pulled to opposite poles.
Spindle fibers shorten: The chromosomes move toward opposite ends of the cell.
Outcome: Sister chromatids are separated, becoming individual chromosomes.
telophase II
Chromosomes reach poles: The separated chromosomes arrive at opposite poles.
Nuclear envelope reforms: New nuclear membranes form around the chromosomes.
Cytokinesis: The cytoplasm divides, resulting in four non-identical haploid cells.
Outcome: Four genetically diverse haploid daughter cells, each with half the original chromosome number.
meiosis I vs meiosis II
Meiosis I: Reduces chromosome number, includes homologous chromosome separation, and crossing over.
Meiosis II: Separates sister chromatids, results in four haploid cells, no chromosome number reduction.
what are precursors used for
microoragnissm may obtain an organic compound they require from the environment or synthesize it from simple nutrients, these simple nutrients are precursors
what is the process of syntehsis of precursors
precursor--> intermediate--> organic compound (end product)
what is this series of chemical reactions called
the biosynthetic pathway, each pathway has several steps (steps faciliated by specific enzyme or protein) and each cell has many different pathways (serves to produce different productions for different functions)
who proposed the one gene-one enzyme hypothesis in 1941
beadle and tatum
what is the one gene-one enzyme hypothesis
each step in a biochemical pathway is catalyzed by an enzyme most enzymes are made of polypeptides (chain of aa that may/maynot function as an enzyme on their own), and each polypeptide is encoded by a gene
if the enzyme is consisted of a single polypeptide what is it called
a monomer
a homodimer
enzyme that contains two copies of the polypeptide
what is the complication of beadle and tatums hypothesis
some enzmes conssits of two different polypeptide, such that each polypepetide is encoded by different genes
what is a heterodimer
enzymes that consists of two different polypeptides
does a single gene stiill encode a single polypeptide in heterodimers
yes
cm (complete media)
cm: contain all the organic and in organic molecules required by an organism to grow, including the end products of biosynthetic ex. LB (lysogeny broth)
(mm) minimial media
contain only a carbon source, an energy source, and salts, the most basic nutrients required for a micororganism to grow
defined vs complex medium
defined: every chmeical and its amount is known
complex: undefined, contain compoenents that cannot be precisley measured, the lab used this because it is easier and cheaper
what do organissms need to do in mm
must synthesize all complex organic compounds that they need using many different biosynthetic pathways
wild type and prototrophic organism
has all the genes present to encode their enzymes so they can function properly (can grow in the wild). prototrophic means they can grow on mm and CM.
what is a mutated version of gene called
a mutant allele, where alleles refer to different forms of a gene this constracts with the orignal/wild type allele
what happens with the cells with the mutant allele
they can either not make a protein or make a dysfunctional protein
what is a detrimental about having a mutant allele
the cell cannot grow since the biosynthetic pathway is blocked, the reactions in the pathway can only proceed to the point of the blocked step
auxotrophs
microorganism that carry this muation and are unable to grow on MM since the pathway that is blocked leads to product essential for growth
auxotrophic cells can only grow on
CM
how can we identify the blocked steps in a biosynthesis pathway
through a biochemical pathway analysis
breif explanation of a biochemical pathway analysis
1. grow unknown auxotrophic strain on MM supplemented with various organic molecules that occur within the biosyntheic pathway
2. if the auxotroph cannot grow on a MM plate + a specific intermediate then the blocked step is downstream of that particular intermediate
hence if you add it C and it can grow the end product that means the function from C--> D is working so the blockage is upstream but if you add C and it doesnt grow the function from C-->D is not working
an auxotrophic is placed on a MM + glucose plate and MM+ glucose-6-Phosphate plate which is the procceeding intermediate of the serine biosyntehtic pathway. the strain only grows on the MM+glucose-6-phopshate plate. where is the blcokage
there is a blocked step at the conversion of glucose to glucose-6-phosphate probably because the enzyme responsible for this conversion is likely deficient or nonfunctional due to a mutation in the gene that encodes this enzyme
how can we conclusively determine which gene is mutant in an auxotroph?
a transformation resuce experiment
transformation r
systematically providing an auxotrophic strain with wild type alleles of the genes known to function in the relevant biosyntheic pathway using genetically engineered plasmids (small self replicating cirlce of DNA that occur naturally in e.coli) and contain genes otuside of the main chromosome. the gnees in question can be inserted into the ecoli using transformation
transformation resuce experiment gist
inserting one type of plasmid with that wildtype allele of one of the genes in question, growing those transfromed cells on an MM plate and looking for growth. if the cell grows on the mm plate they have been rescued since they have been given the wil type allel of the gene in which their mutation occurs and now can make all enzymes needed. if it cant grow on the mm the wild type allele of a gene recieved is different that the gene that is muated hence still have a defcient enzyme
proceed to. asecond transformation usign a wild type alle of a different gene till a rescue occurs
how are dna fragments generated
by digesting larger dna molecules with restriction endonucleases (restriction enzymes
are all restrction enzymes the same
no theres different ones which recognize different specific sequences of DNA and cuts the DNA at only that sequence
how can the dna fragments be reconnected
using the enzyme DNA ligase that joins.ligates the dna fragments together potentiall in new unique pairings
restriction enzyme cuts where
cuts the sugar phosphate backbones of DNA at a specific sequence- the restriction endonucleases recongition site
dna ligase
joins the sugar phosphate backbone of DNA
what is similar about restriction enzymes and DNA ligase
both are heat sensitive (beging to denature at room temp)
has optimal conditons (pH, salt conc etc.) for effectiveness the correct buffer must be used
why do we make dna fragments
these fragments typically contain genes which are usually the target of DNA manipulations.
to manipulate genes they need to be isolated and then copied numerous times, how is this done
inserting the restriction fragment containing the target gene into a palsmid, which is a small, circular DNA molecule capable of replicating inside a bacterial cell
explain cloning
isolate gene--> incorportaing it into a plasmid--> insert plasmid into bacterial cell (for replication)--> increases the # of copies of the gene the organism contains
how does subcloning differ
sub cloning simply consists of moving a piece of DNA from one plasmid (vector to vector), it is not isolated from a novel source like regular cloning
sticky ends are what
overhanding single stranded regions at the ends of DNA molecule after it has been cut by certain restriction enzymes, they can stick to other dna mocleule that was cut with the same restriction enzyme
where is the lac Z gene from
orignally siolated from wild type Ecoli, it is the first gene in the lac operon
what protein does the LacZ encode
the protein B-galactosidase, which is an enzyme that catalyzes the hydolytic cleavage of lactose into glucose and galactose
what does the protein b-galactosidase do which marks its prescence physically
it also cleaves the colourless substrate X-gal which is a chemical analouge of lactose to form an insoluble blue coloured product.
say you add X gal to an agar plate how can we detect B-galactosidase is present
blue colony--> B-g is present and functional
white colony-> B-g absent
B-galactosidase can be seperated into two polypeptide parts what are they
a-fragment (alpha fragment) the smaller fragment
w-fragment (omegae fragment) the larger fragment
they can both be encoded seperately
what encodes the alpha fragment of b-g
a small part of the lacZ called LacZ' (lac Z primer) that is niether the enitre lac operon nore the entire lac Z gene, only the promoter and the first part of the coding region of lac Z ( has the N terminal (amino group at the end NH2)) not active by it seld
what encodes the omega fragments of b-g
only uses the last part of the LacZ as the coding region, the lacZ' part is deleted. also not active by itseld and contain the c terminal (carboxyl group COOH)