BIOL 3030 Exam 2

Growth Requirements for Bacteria

minimal media (nutrients required for growth), typically a carbon source (sugar), some salts and trace elements

Steps of standard bacterial techniques

Screening for Auxotrophs

screening for auxotrophs

bacteria is placed on a medium with leucine, replicated with a velvet surface, placed onto new plates, and leucine auxotrophs are recovered + cultured

Main ways that genetic material can be shared between bacteria

conjugation, transformation, transduction

conjugation

cytoplasmic bridge forms between donor and recipient cell, dna replicates and transfers from one cell to the other, and a cross over in the recipient, results in a recombinant chromosome

transformation

starts with dna fragments, naked dna taken up by recipient cell, cross over in bacterium leads to creation of recombinant chromosome

transduction

a virus attaches to the bacterial cell, injects its DNA, and replicates DNA, the cell lyses

the virus infects a recipient cell, carrying bacterial DNA with it, creating a recombinant chromosome

Experiments that led to the discovery of genetic material being shared between bacteria

cytological markers

Plasmid

small circular double-stranded DNA, physically separate from cell’s main chromosomal DNA

Significance of F’ Plasmids

modifed fertility factor formed when F’ plasmid is off of bacterial chromosome

carries parts of host bacterial DNA

how transduction experiments can be used to map phage genomes

bacteria are infected with phage, then the chromosome is fragmented, some of the bacterial genes become incorporated into some phages, cell lysis releases transducing phages, if this happens, recombination may take place and produce a transuced bacterial cell

mendel’s second law

independent assortment, pairs of alleles segregate independently of each other (di-hybrid cross)

evidence of crossing over

why some gene loci do not assort independently, leading to ratio deviations

genetic linkage: genes are physically located close to each other on the same chromosome

difference between linked, non-linked, and completely linked genes

linked: recombination frequency between 0-50%
unlinked: recombination frequency 50%, genes on different chromosomes or same but far apart
completely linked genes: recombination frequency is 0% (or nearly so)

linked recombination frequency

recombination frequency between 0-50%

unlinked recombination frequency + gene location

50%, genes on different chromosomes or same but far apart

completely linked genes recombination frequency

recombination frequency is 0% (or nearly so)

cis/coupling, trans/repulsion in terms of gene configuration

w+m+/w m | w+ m/ w m+

gene mapping in humans

• no controlled human test crosses

• determined x-linkage from pedigrees

• autosomal disease genes harder to map

• human genome alone cannot identify location of disease associated genes

crossing over in mitosis used for gene mapping

• x-linked recessive- short bristles

• yellow body

twin spots- adjacent regions of mutant bristles and yellow body

experiments regarding DNA as the genetic material

• Fredrick Griffith’s Transformation Experiments

• Hershey-Chase 1952

chemical components of DNA

Nucleotide (sugar + base + phosphate group), Deoxyadenosine 5’ (monophosphate)

chemical components of RNA

Nucleotide (sugar + base + phosphate group), Uridine 5’ (monophosphate or uridylic acid)

similarities between DNA and RNA

both have nucleotide and a monophosphate (potentially)

differences between DNA and RNA

uridylic acid in RNA

Chargraff’s rules, relation to base pairing

Purines : Pyrimidines = 1 : 1 (A + G = T + C)

A : T = 1 | G : C = 1

3D structural characteristics of DNA

• double helical structure, two distinctive regularities of .34 nm and 3.4 nm along the axis of the molecule

• antiparallel chains associate by base-pairing

Basic differences in viral, bacterial, and eukaryotic genomes

virus

DNA/RNA, single or double stranded, circular or linear

bacteria

tend to be circular and double stranded DNA, less info than eukaryotes

mtDNA

typically circular double stranded, size varies greatly between species, no introns (exceptions)

cpDNA

circular, double stranded, larer than mtDNA, but size is less variable between species, introns present (exceptions)

how to interpret gel electrophoresis DNA fragmentation

experimental evidence that supports semi-conservative model of DNA replication

importance of 3’-OH group in formation of phosphodiester bonds during replication

the primary chemical required to extend a DNA strand and form the phosphodiester bond

key components needed for replication to occur

• single stranded DNA template

• a primer

• dNTP’s and DNA polymerases

• in vitro, must add divalent cations (Mg^2+, Ca^2+)

steps of DNA replication

1. double stranded DNA unwinds at the replication origin

2. produces single stranded templates for the synthesis of new DNA

• replication bubble forms, usually with a replication fork at each end

3. the forks proceed around the circle

4. eventually two circular DNA molecules are produced

why does the anti-parallel nature of DNA result in leading and lagging strands

action of telomerase

synthesizes DNA on complementary strand

why is telomerase necessary in eukaryotes

maintain telomeres (end sequences) and reassures no replication

Polymerase Chain Reaction (PCR)

PCR: in vitro amplification of specific DNA fragments

• relies on temperature changes to promote steps

• thermostable DNA polymerase

DNA replication and Polymerase Chain Reaction (PCR) similarities/differences

similarities: both require dNTPs, and a template, both use DNA polymerase

differences: PCR is temperature reliant, even for polymerase

steps of recombination at the molecular level

cross over in prophase 1 of meiosis, breakage + reunion of homologous chromosomes

chiasmata

heteroduplex region

the “hybrid” segment of the homologs

RNA

Composed of nucleotides, Ribose, no 2’-OH group, A,C,G,U, Single stranded, Multiple secondary structures, easily degraded

DNA

Composed of nucleotides, deoxyribose, 2’-OH group, A,C,G,T, double stranded, double helix, stable

structural/functional differences of DNA/RNA

Deoxyribose/Ribose, 2’-OH group/no group, T vs U base, Double/Single Stranded, Double Helix/Multiple types of secondary structure, Stable/Easily Degraded

RNA types

rRNA, mRNA, snRNA, miRNA, siRNA

ribosomal RNA (rRNA)

bacterial and eukaryotic cell, located in cytoplasm, structural/functional components of the ribosome

messenger RNA (mRNA)

bacterial and eukaryotic cell, located in nucleus + cytoplasm, carries genetic code for proteins

transfer RNA (tRNA)

bacterial and eukaryotic cell, located in cytoplasm, incorporates amino acids into poly peptide chain

small nuclear RNA (snRNA)

eukaryotic cell, located in nucleus, processing of pre-mRNA

microRNA (miRNA)

eukaryotic cell, located in cytoplasm, inhibits translation of mRNA

small interfering RNA (siRNA)

triggers degradation of other RNA molecules

initiation

holo enzyme= sigma factor + RNA polymerase
- binds to promoter consensus sequences, unwinding begins

elongation

new nucleotides added to 3’ end of RNA
DNA unwinds at front of transcription bubble and rewinds at end

rho-dependent termination

rho binds to the rut site, moves towards 3’ end
RNA poly pauses at terminator sequence, rho catches up and used helicase activity to unwind hybring of DNA/RNA and end transcription

rho-independent termination

inverted repeat sequence transcribed into RNA, pauses RNA poly, inverted repeats fold into a hairpin loop and separate from template

consensus sequence, examples

most commonly encountered nucleotides at each site
- pyrimidines : Y , purines : R, N : no particular base

basic differences in eukaryotic transcription vs in E.coli

RNA polymerase 1

present in all eukaryotes, transcribes large rRNAs

RNA polymerase 2

present in all eukaryotes, transcribes pre-mRNA

RNA polymerase 3

present in all eukaryotes, transcribes tRNAs, small rRNAs

RNA polymerase 4

present in plants, transcribes some siRNAs

RNA polymerase 5

present in plants, transcribes some RNAs taking part in forming heterochromatin

eukaryotic processing of pre-mRNA message

spliceosome

ribonuclear protein (snRNPs with snRNAs), catalyze reactions that break bonds between exon and intron

- “U[X]” is spliceosome related

difference of origin between siRNAs and miRNAs

mRNA, transposon, or virus/ RNA transcribed from distinct gene