biol10005

Classification

TAXONOMY: description and naming of organisms, which leads to

CLASSIFICATION: recognizing groups of related organisms and naming the groups, which leads to…

PHYLOGENY: the evolutionary history of an organism.

Ultimately, we hope that our classifications reflect phylogeny.

Carl Linnaeus (1707-1778) formalized use of the “binomial”, where each individual organism is known by a Genus and species name.

Order of classification

  • Kingdom
  • Phylum
  • Class
  • Order
  • Family
  • Genus
  • Species

There are often intermediate taxa such as subclass, subgenus etc.

The 6 kingdoms

  • Bacteria
  • Archaea
  • Protista
  • Plantae
  • Fungi
  • Animalia
Epigenetics

Changes in gene expression that do not involve a change in DNA sequence.

Epigenetic modifications alter chromatin structure in a variety of ways without changing the DNA including:

  • DNA methylation
  • Histone acetylation
  • RNA interference

Methylation

  • In tissue-specific genes or time-specific genes the enzyme DNA methyltransferase adds a methyl (CH3) group to the 5’ carbon of cytosine
  • When the CpG islands are methylated a gene is switched off
  • DNA methylation is reversible

Methylation and Gene Expression

  • CpG islands are approx 1000-2000bp in length and a series of these CG dinucleotides are found in the ‘island’
  • CpG = dinucleotide of G and C, p = phosphodiester link between them
  • In housekeeping genes (switched on all the time) the CpG islands are unmethylated so the gene is continuously expressed

Chromatin modification of histones
Amino acids at the N terminal of the histone can be modified by:

  • Methylation (Me)
  • Acetylation (Ac)
  • P phosphate groups (phosphorylation) 

Histone modification is reversible

Unmethylated and Acetylated histones allow genes to be expressed.
Methylated and de-aceylated histones stop genes from being expressed. 

Example include

  • X-inactivation
  • genomic imprinting

Genomic Imprinting

  • some genes are affected by whom you inherit the gene from, mother or father
  • sex of transmitting parent produces observable differences in the phenotype
  • specific genes are differentially marked during parental gametogenesis
  • Reversible and reverses in gametogenesis
Evidence for genomic imprinting Triploids (3N) with:
2N paternal + 1N maternal are different from 2N maternal and 1N paternal Prader willi syndrome and Angelmann Syndrome
  • deletion of paternal 15q11-q13 causes PWS
  • deletion of maternal 15q11-q13 causes AS
  • same region but different outcome! (genomic imprinting)
Some genes in this region of chromosome 15 are only expressed only from the maternal or paternal chromosome. Described as monoallelic expression. Other causes Uniparental disomy (UPD)
  • chromosomes of a pair come from the same parent.
  • In PWS, the two chromosome 15s are maternal (missing a paternal copy of chromosome 15)
  • In AS, the two chromosome 15s are paternal (missing a maternal copy of chromosome 15)

A. Heterodisomy - non disjunction meiosis I B. Isodisomy - non disjunction meiosis II

Imprinting

  • Due to methylation
  •  If a male inherits a maternal imprint but passes to his daughter the imprint changes (therefore not inherited)
  • Wiped in primordial germ cells but re-established according to the sex of the parent
  • so new methylation pattern according to the sex of the parent (may be different from what parent had)
DNA Replication

DNA replication is semi-conservative that is where the new molecule of DNA has one strand which comes from the parent molecule and one strand which is newly synthesised.

Differences in DNA replication:

Diagram of DNA replication:

M: the leading strand is that which is synthesized continuously

Bottom strand: lagging strand is synthesized in a discontinuous fashion 

O: Okazaki fragment one of the RNA-primed short segments of DNA synthesized during replication of the lagging strand of the double helix.

R: DNA heliase an enzyme that uses energy from ATP to unwind the DNA, connected with topoisomerase (gyrase) is an enzyme which can relax DNA by cutting and reforming the polynucleotide backbones (hydrogen bonds)

P: single-strand binding protein one of the proteins that attaches to single stranded regions of DNA and prevents the reformation of base pairs, particularly in the region of the replication fork.

S: DNA polymerase I enzyme which adds nucleotides to gaps when primers removed

N: DNA polymerase III main polymerase which adds nucleotdes to template in a complementary way

O: RNA primer sequence of RNA to make a stretch of double stranded DNA on which the DNA pol III can work (primase is an enzyme which synthesises the RNA primer)

Q: ligase enzyme which joins sequences of DNA

Test Cross, Back Cross and Reciprocal Cross

Test Cross
A test cross is a cross to a homozygous recessive genotype.

It may be used to establish the genotype of an individual with the dominant phenotype.

        e.g: Genotype could be AA or Aa so crossing with aa will give us different results.

                AA x aa  = All Aa (All one phenotype)

                Aa x aa = 1 Aa: 1 aa (half one phenotype, half other)

The appearance of the recessive phenotype in the offspring of a test cross indicates that the unknown genotype was heterozygous.

A test cross can also occur with 2 or more genes involved.

Back Cross
A cross of an F1 to one of its parents 
OR
A cross of an F1 to an individual with an identical genotype to the parent

Useful because you to know the genotype of the parent and can use that as a ‘constant’.

Reciprocal Cross

1st:  Male Phenotype A x Female Phenotype B
2nd: Male Phenotype B x Female Phenotype A 

Usual test for sex linkage as if it is sex linked these crosses will produce different results. If autosomal will produce same results for both crosses.

Ribonucleic Acid

RNA is a usually single stranded, nucleic acid, composed of nucleotides. Its sugar is ribose and its bases are: adenine, uracil, cytosine & guanine.

Types of RNA

  1. messenger RNA (mRNA): formed during transcription from a DNA template
  2. ribosomal RNA (rRNA): rRNA and protein combine to form ribosomes
  3. transfer RNA (tRNA): carries amino acids to the ribosome in translation, anticodon matches with codon of mRNA, specific amino acid for each anticodon attached to 3’ end
Lecture 1 - Dictionary

DNA: DNA is a double stranded molecule. It is a polynucleotide. It is made up of many (poly) nucleotides linked together.

Genes: discrete hereditary factors that determine traits.

Genome: the total amount of genetic material in a chromosome set (in humans - one set of chromosomes). The genetic material is DNA.

Genotype: describes the genetic constitution of an organism. Comes from a parent if haploid or parents if diploid.

Phenotype: describes the morphological, biochemical and behavioural properties of an organism resulting from a specific genotype and its interaction with the environment.

Evolution of the Eukaryotic Cell

What do we know about the origin of the Eukaryotic cell?

  • 1.3 - 2 billion years before the present
  • mechanism of evolution is not known
  • There is good evidence for the origin of mitochondria and chloroplasts from primary endosymbiosis

The first Eukaryotic cells to evolve were protists and they are an extremely diverse group of organisms.

When do Eukaryotic cells first appear in the fossil record?

  • Precambrian acritarch fossils are the first known of eukaryotic cells – about 1.3 – 2 billion years old.
  • Multicellular (filamentous), eukaryotic organisms appear about 1.4 billion years ago
  • Early seaweed fossils – from 635 million years ago

Where did Eukaryotic cells come from?

It is generally thought that eukaryotes eveolved from prokaryotic organisms.

The nuclear membrane and endomembrane system of eukaryotes probably evolved from a prokaryote where invaginations of the bacterial cell membrane enveloped the nucleoid.

Mitochondria and plastids (or choloroplasts, but choloroplast usually refers to ‘green plastids’ reseverved for plants or green algae) of eukaryotes arose by endosymbiosis, which refers to an organism living inside another.

Plastids derive from cyanobacteria and mitochondria are descended from purple bacteria. Plastids and mitochondria are derived from endosymbiotic bacteria that have become organelles in eukaryotic cells.

Mitochondria –> animals & fungi

Chloroplasts (Plastids) –> green algae and plants 

What is the Key Evidence for the Endosymbiotic Origin of Mitochondria and Chloroplasts?

  1. Mitochondria and chloroplasts are semiautonomous, retaining their own genome (DNA, RNA). Their genomes resemble those of prokaryotes (i.e., purple bacteria, cyanobacteria)
  2. They also retain their own machinery for synthesizing proteins, including ribosomes.

  3. Their metabolism is like existing prokaryotic organisms (e.g., a cyanobacteria for chloroplast).

  4. ftsZ protein involved in division of prokaryotes and organelles

  5. Some chloroplasts still have the bacterial peptidoglycan wall between the inner and outer membranes.

Protists with primary chloroplasts have 3 genomes, while all protists with secondary chloroplasts have either 3 or 4 genomes.

Examples of Gene Mutations

Cystic Fibrosis
ΔF508 mutation accounts for 70% of mutations in Caucasian populations, mutation is deletion of 3 bases in DNA coding
There are currently 1913 mutations listed for CF in the database including:
nonsense, missense, frameshift, insertion/deletion, splicing 

beta-Thalassaemia
Missense mutants – changes amino acid
Nonsense mutants - introduces stop codon
Frameshift mutants - e.g. deletion of 2 bases at codon 8 
Splicing mutants - Single nucleotide changes create new splice site, extended exon 2 

Huntington Disease

  • Defect in gene is a triplet (3 base) repeat (CAG) <35 repeats normal (most 17-20)
  • 27-35 rare but unstable when transmitted via a male
  • 36 - 41 indeterminate (reduced penetrance)
  • 40-50 most adult cases
  • >50 generally juvenile onset
  • Earlier onset and more severe if inherited from father 
Summary of Protists (because I hate them)

Eukaryotes not belonging to plant, animal or fungal kingdoms and have a variety of ways of gaining nutrition

  • Can be uni/multicellular or colonial and have many cellular forms including flagellates and plasmodia
  • Majority are aquatic and have flagella or cilia
  • Gain nutrition by photosynthesis, parasitism, predation and absorption

First eukaryotic organisms were probably similar to modern day protists

  • The nuclear membrane and endomembrane system probably evolved from infolding of the bacterial cell membrance that enveloped the nucleoid. Plastids and mitochondria are derived from endosymbiotic bacteria that have become organelles in eukaryotic cells.

Major eukaryotic lineages, such as animals and fungi, arose from different protist lineages, but many more independent protist lineages exist

  • choanoflagellates - animals
  • slime moulds - fungi

Photosynthetic protists, commonly called algae, are diverse and not all related.  Primary plastids arose in the ‘green lineage’ which includes glaucophytes, red algae and green algae

  • Glaucophytes - photosynthetic flagellates with primitive chloroplast that have a peptidoglycan wall like bacteria
  • Red Algae - multicullular and lack flagella, contain chlorophyll a ad phycobilin pigments
  • green algae - unicellular, colonial and multicellular forms, contain chlorophyll a and b

Secondary plastid acquisitions created an enormously diverse new array of protists, some which abandoned photosynthesis to become heterotrophic or parasitic

  • Chromists (cryptomonads, coccolithophorids, diatoms, brown algae, oomycetes) 
  • characterized by flagellar architecture and secondary plastids, typically have one smooth posterior flagellum and one hairy anterior flagellum
  • most photosynthetic with chlorophyll a and c
  • oomycetes absorb food through hypae
  • cryptomonads clearly ingested eukaryotic cells, nucleomorph is relict nucleus of engulfed cell

Alveolates are unicells with distinctive vesicles, cortical alveoli, beneath the cell membrane

  • Dinoflagellates - 2 flagella, many photosynthetic containing chlorophyll a and c, some predatory
  • Apicomplexans - intracellular parasites of animals, cause diseases such as malaria, apical complex used to penetrate host cells
  • Ciliates - predatory unicells characterized by 2 types of nuclei and a covering of cilia

Euglenoids are closely related to flagellates that all have an anterior depression from which the flagella emerge

  • euglenoids are freeliving, some have cloroplasts and some engulf prey through anterior depression (gullet)
Cyanobacteria

Green and purple bacteria lack PSII, do not use H20 as a source of electrons and, therefore do not produce oxygen. With the evolution of cyanobacteria came PSII, which provided the mechanism for using H20 as a source of electrons and provided the earth with an atmosphere that includes oxygen.

Cyanobacteria contain chlorophyll a and water soluble pigments called phycobilins. Blue phycobilins, present in most species give them a blue-green appearance.

Cyanobacteria have specialised cells such as akinetes and heterocysts. An akinete is a spore that develops from a cell which becomes enlarged and filled with food reserves. The spore can remain dormant and then germinate to produce a new filament. A heterocyst is relatively colourless, has a thick, transparent cell wall, may be involved in asexual reproduction and is a sige of nitrogen fixation.

What characteristics do Cyanobacteria share with chloroplasts?

  1. Their photosynthetic thylakoids contain chlorophyll A, as do all chloroplasts.
  2. Their accessory pigments, phycocyanin and phycoerythrin, are found in the chloroplasts of several protists.
  3. Chloroplast genomes show they are related to Cyanobacteria
Detecting Variation

Variation can be at the phenotypic level through to the molecular level.

  • look for visible differences in the phenotype
  • chromosome differences eg., length of long arm of Y
  • immunological markers eg., blood groups
  • protein gel electrophoresis eg., esterases in Drosophila
  • SSLPs (Simple sequence length polymorphisms) or VNTR (variable number of tandem repeats)
  • STR or (short tandem repeats)
  • Single nucleotide polymorphisms (SNP)

Discontinuous Variation

  • mid digital hair present or absent
  • blue or green budgerigar
  • black or brown dog

Variable regions often in non-coding regions

Minisatellites
15 -100b (eg., tandem repeat of 18 bases) total length from 1-5kb.

Microsatellite
short tandem repeats (STR): 2 - 9 bases – can have several alleles according to how many repeats

Single nucleotide polymorphism (SNP)
1 base – can only have two alleles at a locus

VNTR (variable number of tandem repeats) 
Minisatellites

  • In non coding regions of the genome
  • Repeat sequence between 15bp -100bp long
  • But number of repeats varies from person to person so VNTR can be from 1 - 20kbs in length

Multilocus probe (found in more than one location)

–  minisatellites
–  eg., tandem repeat of 18 bases total length from 1-5kb.

Single locus probe
– minisatellites (as above)
– microsatellite - short tandem repeats (STR) - 2 - 4 bases

Application
Identification

  1. Paternity
  2. Forensic analysis – Crime
  3. Matching after a disaster
Independent Assortment

Aa;Bb can form 4 different gametes:

AB, Ab, aB & ab

If a large number of gametes are produced we expect a 1:1:1:1 ratio.

Number of gametes produced can be determined by the number of chromosomes involved. As the gametes produced will be of the form 2^n where n is the number of chromosomes. 
         e.g Aa;Bb;Cc;Dd will produce 2^4 = 16 gametes.

Normal phenotypic ratio of a dihybrid cross where both phenotypes show complete dominance is 9:3:3:1.

This can be calculated by multiplying the 2 mono-hybrid cross ratios together.
          e.g  (Aa x Aa)   x   (Bb x Bb)
                     (3:1)      x      (3:1)
                           = (9:3:3:1) 

Genes and Alleles
  • Genes can be found in alternative forms - alleles
  • The most frequently occurring allele in a population may be referred to as wild type (not used for human alleles)
  • Alleles arise as the result of mutation
  • After DNA replication the alleles on the two chromatids are identical
  • Homologous chromosomes have the same genes but may have different alleles

What does a gene do?

  1. A gene can code for an enzyme but other inherited traits related to other proteins eg. collagen
  2. A gene codes for protein but some proteins are made up of more than one unit for which there is a separate gene
  3. So a gene codes for a polypeptide eg. α globin or β globin but in addition genes code for RNA

What is meant by “polypeptide”?

A molecule  of many amino acids joined together by peptide bonds, a protein is a large polypeptide chain.

What is an enzyme?

A biological catalyst, usually a protein, which increases the rate of a reaction.

What is a protein?

A polypeptide chain

What are peptide bonds, and phosphodiester bonds?

Peptide bonds are formed when the acidic carboxyl group of an amino acid attaces to the amino group of another with the release of a molecule of water.

A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. In DNA and RNA, the phosophdiester bond is the linkage between the 3’ carbon atom of one sugar molecule and the 5’ carbon atom of another.

What is a metabolic block?

It is a block that occurs in the step by step sequences of a chemical process within a cell.

What is a prototroph? An auxotroph?

A prototroph is an organism that can synthesize all of its nutrients required for growth.

An auxotroph is an organism that is unable to synthesize a particular nutrient required for its growth.

Name a structural protein & a transport protein.

Structural protein: actin/collagens
Transport protein:  ???

Of what 2 polypeptides is haemoglobin composed?

alpha-globin and beta-globin

RNA processing in Eukaryotes

In prokaryotes the primary transcript is mRNA.

In eukaryotes the primary transcript is modified and processed into mRNA.

  1. The ends of the transcript are treated for stability and binding to the ribosome

  2. The mRNA is commonly spliced , the introns are removed and the exons (expressed sequences) joined up to make the final transcript 



Naming of Strands

  • Template strand is transcribed into a complementary mRNA sequence but the sequence on the mRNA is similar to the non template strand except U instead of A
  • The non template strand is called the coding strand and is often used when showing a sequence because it represents the mRNA sequence
  • Non template strand is referred to as the coding strand

Depending on which exons are cut out during splicing can produce different effects.

Alternative splicing of the same primary transcript can lead to different polypeptides from the same DNA sequence.

PCR

Polymerase Chain Reaction - a technique which mimics DNA replication

It can make millions of copies of a DNA sequence 

You need to insert:

  • sample of DNA
  • Adenine, Thymine, Cytosine & Guanine
  • deoxyribose sugar
  • an enzyme (Taq polymerase)

PCR:

  • Need to know something about sequence to design the primers
  • Uses heat to separate DNA strands
  • Can amplify regions up to approx 2kb
  • So sensitive you can use primers with the whole human genome and it can find and amplify a specific sequence you are interested in
Transcription

DNA repliction –transcription/processing–> mRNA –translation–> Polypeptide

Transcription

  • A gene consists of the coding sequence and regulatory sequences
  • Only one strand of the DNA double helix in a given gene codes for the polypeptide = template strand
  • only 1 strand is a template strand from which the mRNA is manufactured
  • Either strand may be a template but transcription to form new RNA occurs in a 5’ to 3’ direction
  • For a particular gene, it is always the same strand which is the template, and it depends on which way the mRNA is produced

Initiation - promoter recognition
RNA polymerase binds to promoter regions and this determines where RNA synthesis begins 

  • RNA polymerase binds and DNA is denatured in this region
  • DNA strands separate
  • First RNA nucleoside triphosphate is placed at the site

Elongation
Addition of complementary nucleotides 

  • RNA moves along DNA template strand adding bases 5’ 3’ to growing RNA strand
  • Bases are complementary to the DNA template

Termination
RNA polymerase reaches a chain termination sequence. This is a sequence which can form a loop.

  • RNA polymerase and mRNA are released