Molecular Biology Exam 1

Molecular biology can be defined as

• study of essential macromolecules present in cells, including DNA, RNA, and proteins; biological pathways between them

• information flow → different types of molecules in a defined order: central dogma

• conditions necessary for life, evolution

What is life? ““

Life is a chemical system capable of Darwinia evolution

Several requirements for life

• raw materials → H, O, N, and C

• Energy from the sun, transferred from other things and stored in chemical bonds

• Presence of a barrier/envelope to separate the life unit

• Catalysism → enzymes

• Biological information

adaptation

cell and organisms are senstive and respond to their external environment

Homeostasis

with a living cell, parameter such as pH temperature ion concentrations in bio molecule concentrations are all maintained within narrow limits by transport, a required substances across cell membrane and by a regulated internal metabolism

Transmission of genetic information

reproduction to produce new cells or organisms is essential for species to remain part of the biosphere for more than one generation.

Energy acquisition

to maintain both complexity and homeostasis, living systems, undergo a constant struggle to obtain energy from sunlight, the environment or other organisms

Interactions

each living cell interacts with other cells or organisms and with its environment

Growth, development, and death

each organism has a finite lifecycle

Complexity and organization

eukaryotic cells are subdivided into compartments called organelles that perform specialized tasks in the cell. Multicellular organisms consist of tissues, organs, and organ systems. Larger organizational categories are species populations, and ecosystems.

Szostak Experiment

• May be reproducing in a lab fundamental steps that led to the birth of the first living systems

• Showed that with an increased concentration of micelles (single layer of fatty acids) in solution, vesicles (double layered structures) can grow to the point of changing their shape, which become elongated

• Gentle stacking of the tube containing the solution made that produce new

• This mechanism could be the base of ancestral replication for the first living systems

Requirement for life: Catalysis

• Chemical reactions are at the base of most biological functions, they are based on energy transfer

• In living systems, catalysis is typically promoted by specialized proteins called enzymes

What is the link between molecular biology and evolution?

• The information in encoded biomoleecules such and varirations in the info, which is manifested as chemical changes in the biomolecules, can lead to the evolution of new species

• Changes in genomic material are called mutations, occur randomly

  • Mutations can be caused by

    • DNA replication (localized changes)

    • DNA damages (which cannot be repaired completely) (localized changes)

    • Chromosome recombination (larger change of gene material)

    • Transposition - adding wrong base layer change of gene material

    • RNA processing (larger change of gene material)

E Coli evolution example

• Ionizing radiation this lab bacteria showed that the bacteria was able to evolve out of dying from this

• 20 rounds of IR and each time produced survivors that eventually show resistance similar to bacteria that normally survives this

• 40-80 mutations

Prebiotic life

• first hundreds million years on Earth was prebiotic era

• Life was not present but chemical reactions were filled with methane, water, ammonia, carbon dioxide

  • common

  • generated the first organic molecules

  • polymers started to accumulate

• We can consider the first self replicating polymers as the first form of life → similar or progenitors of RNAs

Self replicating RNAs satisfy two requiremtns for life

• catalysis (for rep) → ribosymes

• infromation → in their base sequence

Why RNA and not DNA for first life?

• because of stuructural flexibity provided by the sugar → extra interactions, more possible sturctures that are not accessible to DNA molecules

  • Extra O allows for more reactivity

• RNAs could catalyze lots of reactions, not only the ones needed for replication

• The problem of boundary could be solved by experiemtns like Szostack that demonstrate that RNAs encolsed in lipid vesicles could replicate and propagate in the environment

Evidence for RNA as first life

• Discovery of RNA based enzymes, ribozymes (80s Cech and Altman)

• There are exceptions, such that RNAs composing ribosomes together with proteins

• Additionally, experimental evidence suggests that while today RNA catalysis is limited and biocatalysis is typically performed by polypeptides (proteins), RNAs could potentially catalyzea variety of additional chemical reactions

• its possible that RNA action was also at the bas eof the rise of complex polypeptides and molecular machines based on a combination of RNA and proteins progresivley started appearing

LUCA hypothesis

• last universal common ancestor

• single celled (unicellular) living system at the base of all life as we know it

• Bare min to be considered life

• simple metabolism

• RNA genetic material

• Primitive ribosome and protein biosynthetic apparatus

• Transcriptional machinery

• Genetic code

LUCA has several improvements over self-replicating polymer

• RNA for storing more complex information

• Basic protein synthesis machinery to sustain new protein-based catalysts

• More structured membranes

• Rudimental regulation of metabolism/cell processes

HOw can we get information about the likely design of LUCA?

Experimentally:

1. By determining in vitro what is the minimal set of genes (and proteins) to create a self-replicating single cell organism

2. By determining through genomics what are the sets of genes shared among all present-day organisms

   • Genes encoding from protein synthesis and RNA transcription

   • Notably, all modern organisms share the same genetic code, which therefore must have been also in use by LUCA

LUCA may have appeared

more than 3 billion years ago

• around 1 billion years ago some bacteria were engulfed by more complex organisms and an endosymbiotic relationship led to the evolution of mitochondria and chloroplasts, needed to sustain a more complex metabolism.

Charles Darwin concepts

1. resources are limited

2. competition for resources within a population

3. Individuals with traits (phenotypes) that offer a competitive advantage are more likely to survive and reproduce (Darwin observed and documented the inheritance of characters throughout generations)

How is natural selection linked to molecular biology and LUCA hypothesis?

• Darwin introduced the idea of branching evolution, which implies the presence of a common ancestor

• Evolution would progress through competition within populations and variations of traits

  • Dariwn could not know it, but observed phenotypical variations depend on genetics and particular genetic mutations

• When a sufficiently high number of mutations are accumulated it is typically because they provide a sele

When a sufficiently high number of mutations are accumulated it is typically because

they provide a selective advantage to the new organism. once a sufficiently high number of mutations is reached, we may be in presence of a new species

Why is evolution by natural selection important to us?

Because it proceeds for (relatively) small changes, so that many traits and mechanisms are in common across species.

• This in turn implies we can study human molecular mechanisms by investigating other organisms

  → Relevance of model organisms in contemporary science

  • yeast, e coli, mice, rats, possusm

Genetic information is stored in

highly defined and regulated portions of DNA known as genes

• Mendel started figuring this out in 1850s

  • started understanding basis of hereditary transfer of bio information

Biological information is present in

pairs, dervied one for each sex cell → gametes

pair is splite when gametes are formed, but it is recomposed as two are brought together

Mendel studies are popular now

• through the use of microscopes it became clear that DNA is organized in chromosomes and all somatic cells (all cell types except the sex cells or gametes) have the same number of chromosomes, while this number is effectively halved in gametes

• Later in 1908 Thomas Hunt Morgan would use Drosophilia to demonstrate that the particles of heredity were included in chromosomes

Mendel studied garden pea

• Pisum sativum, not the plant as a whole but on single traits

  • 7 traits and made sure he started his observation from purebred: by crossing two plants, the following generations had to consistently maintained only the version of the trait of the parent plants - not all on diff chromosomes

  • then classifed traits into two categs: dom and recessive

Determining what phenotypes were dominant or recessive was easy based on

the crossing of the parental generation (P) which was purebred. The phenotypes of F1 in case of a crossing between a dominnatn and recessive would show only the dom phenotype

Various forms of a gene

allele

cells with two alleles per gene are known as

while gametes are

diploid

haploid

When gametes are formed, there is an

equal segregation of alleles

law of segregation

Exceptions to Mendel’s Laws

• incomplete dominance

• codominanace

• linked genes

incomplete dominance

In this case, as long as the R allele is present, some level of red pigment is produceThe RR’ genotype will lead to an intermediate, pink phenotype

Codominance

In this case, both proteins encoded by the two alleles of a gene are functional They are co-expressed as in blood cells.

Linked genes

In contrast to the observation that led to the formulation of the 2nd law, if two genes are located on the samechromosome they may* segregate together

• *the further away they are, the higher the chance they will not be linked during gametes formation (meiosis)

Cytogenetics

Cytology + improved microscopy = cytogenetics (the study of chromosomes and their role in heredity)

• Robert Hooke → named cell

Chromosomes

• colored bodies

• First observed by Karl Wilheml and Wilhelm Hofmeister in 1842

• Nucleus discovered in 1833

  • One of the multiple compartments and organelles that characterize the eukaryotic cell

Cell go through a regulated

cell cycle and they grow asynchronously

The phases of the cycle are known as interphase are

G1, S, and G2

During the interphase, chromosomes

are decondensed and not visible

During S phase, each

chromosome is duplicated

Euk cells are typically diploid, where n is the

number of chromosomes and after the S phase they become tetraploid

During the S phase the chromosomes are

duplicated generating “sister chromatid pairs”, or pairs of identical chromosomes linked by a structure calledcentromere.

Mitosis is for

somatic cells, gens of diploid cells

Meiosis is for

Sex cells (gametes), gens of haploid cells

Mitosis steps

Prophase 1 - Formation of the centrosome, the nucleus disappears

Metaphase 1 - Alignment of chromosomes (metaphase plate)

Anaphase 1 - Separation of chromosomes at the centromere

Telophase 1 - Division of the whole cell, formation of new nuclei

Cytokinesis - The final separation of daughter cells following mitosis

Meiosis steps

prophase 1 - Two homo sister pairs form a tetrad (mitosis until they remain independent)

metaphase 1 - spindles have the 4 homos to metaphase plate

anaphase 1 - tetrad splits and two sets of sister chromatid pairs move to opposite poles

telophase 1 - same at mitosis

No S-phase between divisons

Prophase 2 - sister pairs are visible but ther are half as many in mitosis because the homo pair isn’t present (in other daughter cell from first division)

Metaphase II - same

Telophase II - haploid daughters → four n cells from single 2n cell

Tetrad

A structure formed in meiotic prophase I by the association of two homologous sister chromatid pairs

All sisters will stay stacked on top of each other

Non sex chromosomes

Autosomes

Determined structure of sex chromosomes

1905, Edmund Wilson - og called X and accesory chromosomes

In mammales XY

In insects XO

Thomas Hunt Morgan 1908 Drosophilia Experiment

• X-linkage of the white-eye allele

• The whole F1 has red eyes → red eye is dom

• The white eye trait comes back in F2, meaning is it heritable but it only shows up in males

  • because the gene is linked to the X chromo and in F2 no female fruit fly can be homozygous for “ww”. Those F2 females can only be WW or Ww.

Calvin Bridges adding to Thomas Hunt Morgan 1908 Drosophilia Experiment

• Bridges reinforced the concepy that genes are located on chromosomes and some are linked to the sex chromosomes

• Bridges used white-eyed females (XwXw) for his experiments, crossed with red-eyed males (XwY)

• Nondisjuction: abnormal segregation of chromosomes (0.1% of cases in this experiemnt)

• sex is determined only by the number of copies of the X chromosome, not by the presence or absence of Y chromosome

Different Genes Assort Independenetly during Gamete formation is not universally true becauase

• during meiosis , we separate DNA in chromosomes, therefore chromosomes segregate, not individual genes

• Those genes that are on a single chromosome (linked genes) segregate together (most occasiaons)

Crossing over can unlink originally linked genes

• chromosome recominbation or crossing over can unlink genes previoulsy located on the same chromosome

• Happens in prophase 1

• crossing over increaes the final variability within gametes

Mapping genes using recombination frequency

• the probability of a crossing-over determining the un-linking of two genes originally located on the same chromosome increases with the distance between the genes (loci)

  • genes located at short distance do not recombine often

The recombination frequency indicates the distance between

loci. This allows the use of the recombination frequency to map the relative position of genes within a chromosome

• Farther away, more likely to cross

Mendel’s laws

1. Allele Pairs Segregate during Gamete Formation

2. Different Genes Assort Independently during Gamete Formation

Molecular genetics

study of genes at molecular level

first form of molecular biology: investigation of how info is encoded in DNA and then utilized by a cell through the synthesis of other biomolecules (RNA and proteins)

DNA was recoginzed as the molecule that carries the info in most modern living systems

1940s

generally attributed to Oswald Avery, but based on experiment in 1928 by Fred Griffith

Fred Griffith Experiment

• virulent: smooth colonies

• Nonvirulent: rough colonies

• virulent strain killed and spread its DNA to the nonvirulent strain and allowed the nonvirulent strain to become virulent

• difference was a polysaccharide capsule present only on virulent strains

• BIG IDEA: understand that DNA is code for info

BEadle and Tatum 1940s

• X-rays → mutations that blocked specific metabolic steps.

• Mutants grew only with added nutrient.

• Conclusion: one gene → one enzyme (later one polypeptide).

• Importance: showed genes control proteins, which control traits/metabolism.

Based on these results, Beadle and Tatum proposed their famous theory one gene one enzyme - now polypetide

• each gene codes for a specific polypeptide with catalytic properties

Flow of info gos from

DNA to RNA to polypeptides

• exceptions RNA to RNA and RNA to DNA

Mutations that are apparently small

are at the base of several pathogies

• ex: sickle cell anemia

• neg AA → smaller AA