Microbiology Unit 4
College: First year
Domain
cell type
kingdom
most general characteristics
species
most specific or unique characteristics
mycology
the scientific study of fungi
Characteristics of fungi
eukaryotic
nonphotosynthetic
unicellular or multicellular
asexual or sexual reproduction
not organized into tissues or organs
most are saprophytes
chitin cell wall
What are fungi?
yeast, molds, mushrooms, puffballs, bracket, chytrid molds
yeast
single-celled micrscopic fungi
used to manufacture drugs and fermented products such as alcohol
Fungi phylum suffix
-mycota
Fungi class suffix
-mycetes
Examples of fungi
zygomycota
zygomycetes
deuteromycota
Zygomycetes
causes zygomycosis
is an opportunistic infection caused by species of molds like rhizopus
Zygomycosis
fungal infection of the pulmonary, gastrointestinal, or cutaneous system
patients are normally immune-compromised to begin with
Deuteromycota
fungi imperfecti (most sexual phases have not been discovered)
contains most of the medically important fungi in this phylum
asexual vegetative fungal spore (susceptible to drying and chemicals)
Coccidioides immitis
dimorphic - depending on the temp. it can be found as a mold on the skin or a spherule inside the body
endemic in California (Valley Fever - 400 cases a year)
causes pneumonia (inside body) & disseminated cutaneous mycosis (on skin)
dermatophytes
skin loving fungi
mycosis
fungal infection
dermatophytes cause...
cutaneous mycoses
cutaneous mycoses
fungal skin infection
tinea =
cutaneous mycosis
(medical term! not scientific name)
tinea pedum
athletes foot
tinea manus/ manumm (UMLS)
there is a lesion on the hand
Diagnosing & treating tinea manus
take a skin scraping to look for fungal element & add 40% KOH to break down the keratin and reveal fungal elements
tinea cruris
jock itch
tinea barbae
barber's itch, beard fungal infection
tinea corporis
ringworm of the body
starts as a red spot but has an emerging margin because the mold grows in the center and the hyphae extend out triggering an inflammation response
tinea capitis
ringworm of the scalp
main dermatophytes
trichophyton
microsporum
epidermophyton
wood's lamp
used to find molds in the skin and hair
is a diagnostic UV light
the hyphae growing seem to "glow" in the dark
toxin
biological poison
Aspergillus fumigatus
produces aflatoxin, grows on peanuts or corn, & produces potent carcinogens
carcinogens
cancer causing agents
yeast
reproduce by budding
usually in a tiny % of normal flora
Candida albicans
causes oral mycoses (aka thrush) & primary cause of vaginitis due to fungi
Chalamydomonas nivalis
a green algae that looks red ("watermelon snow")
psychrophillic - extreme cold loving
photoflagellate
has a secondary red carotenoid pigment (astaxanthin) in addition to chlorophyll (green)
microscopic
photoflagellate
uses sunlight for energy & moves using a flagella
Algae is classified by...
shape, size & color
Astaxanthin
secondary red carotenoid pigment for chlamydomonas nivalis
Dinoflagellates
Gonyaulax tamarensis & pfiesteria piscicida
single celled organisms that live in silica and calcium based material
Phycoerythrins
red photosynthetic pigments
Dinoflagellates
live in marine habitats
cause of red tide
marine neurotoxin
Marine neurotoxin
will affect any filter feeders
will concentrate in the flesh of the clams or oysters
testing shellfish for red tide
Perforated plates of a dinoflagellate
causes parasitic lesions in the fish
Rhodophyta
plant-like macroscopic appearance
red algae
usually marine habitats
phycoerythrins used as a photosynthetic pigments
Chondrus crispus
commercially used red algae
used as a source of carregeenin
used in dairy products and agar used in bacteriology
Pterocladia lucida
a source of agar in new zealand
Mechanisms to get DNA to RNA to Protein
replication
transcription
translation
Each mechanism has
initiation
elongation
& termination
where do we find genes?
in chromosomes
Prokaryotic DNA
one chromosome in a circle
extrachromosomal DNA is in a plasmid
Eukaryotic DNA
a linear chromosome
extrachromosomal DNA in mitochondria and ribosomes
Replication
DNA dependent DNA synthesis
Transcription
DNA dependent RNA synthesis
what makes up the overall structure of nitrogen bases?
nitrogen
Pruines
A & G (stop sign and a house)
Pyrimidines
T & C (stop sign)
Purines and Pyrimidines form
hydrogen bonds
Base pairs that form 2 hydrogen bonds
A & T
Base pairs that form 3 hydrogen bonds (harder to pull apart)
G & C
Nitrogen Bases in RNA
A, G, C, & U
substitute uracil for thymine
Deoxyribose
can add three phosphate groups
has no oxygen on sugar
Phosphodiester bond
goes from 3' to 5'
makes the sugar phosphate backbone and gives strength to the DNA
linkage between sugars & phosphate
3'
the hydroxyl group
5'
phosphate groups
DNA is
semiconservative
has an antiparallel construction
Semiconservative
ladder is pulled apart & new nucleotides come in
Experiment of the possible models of replication was performed by:
"the most beautiful experiment in biology"
Matthew Meselson & Franklin Stahl in 1958
Antiparallel construction
1/2 is 3' to 5' & other 1/2 is 5' to 3'
Topoisomerase
enzymes responsible for supercoiling
Helicase
enzyme responsible for flattening out the helix
DNA Polymerase III
enzyme responsible for replication
Continuous synthesis
located on the leading strand; one direction & a nice long strand
Discontinuous synthesis
located on the lagging strand
Primase
responsible for adding an RNA primer (aids DNA polymerase III)
DNA Polymerase I
repairs DNA; alot in cell. looks for errors takes out RNA & replaces with DNA
establishes the replication fork
DNA ligase
responsible for forming phosphodiester bonds
Initiation
origin of replication
Okazaki fragment
before replicator there are fragments called
transcription
making an RNA message from DNA
translation
making proteins from the message
Prokaryotic cells
replication, transcription, & translation occur in cytoplasm
transcription stages
initiation, elongation, & termination
Transcription initiation
RNA polymerase finds "recognition sites" on DNA
RNA polymerase
makes the RNA polymere
Transcription elongation
RNA polymerase polmerizes RNA copies
Transcription termination
A "stop site" is recognized
Products of transcription
mRNA, tRNA, rRNA, & catalytic RNA
mRNA
working blue print
rRNA
building block used in protein synthesis factories (ribosomes)
tRNA
transports RNA brings monomers for protein synthesis from cytoplasm to ribosome
Products in transcription are used in...
translation
Genetic code is...
universal (almost)
punctuation
degeneracy
wobble
Punctuation
there is a start and stop
degeneracy
multiple codons for the same amino acids
wobble
for a group of codons some errors can occur & not mess up the protein
Translation
aka protein synthesis
tRNA
transfers amino acids to ribosome
Amino acids attach to tRNA
at 3'
Anticodon
triplet of complementary nucleotides; form hydrogen bonds & match up with 3 other codons
AUG
start
UAA
stop
UAG
stop
UGA
stop
codon
located on messenger RNA
lines up with anticodon
AUG in Eukaryotes
Met.
AUG in Prokaryotes
FMet.
Initiation
initiation complex
Elongation
tRNAs bring amino acid building blocks to the mRNA/ribosome site where peptide bonds are formed
Termination
stop site is recognized
Missense mutation
produces a full protein with one nucleic acid changing the entire amino acid
Aminoacyl-tRNA syntheases
the enzyme responsible for adding AA to tRNAs
Point mutation
change in one nucleotide - an A instead of a C
Frameshift mutation
insertion or deletion of nucleotides ; shifts the entire reading frame
Missense point mutation
change in one nucleotide & will have full length proteins but will have different proteins & a different function
Nonsense point mutation
changing in one nucleotide on DNA or RNA that results in placing a premature stop codon producing an incomplete protein
mutation
change in the genes
phenotype
biochemical make-up & produces physical characteristics
silent point mutation
change in one nucleotide that results in a change in a codon that is for the same amino acid as in the original message so the same protein is made
Ionizing radiation
x-rays & gamma rays
Non-ionizing rays
ultraviolet light
Lateral gene transfer
exchange of genetic material & creates diversity
transformation
naked DNA is transferred through the environment and taken up by the recipient cell & then incorporated
uptake of "naked" DNA
transduction
through a bacterial virus genetic material is transferred ; a mid range of DNA transferred
transfer by temperate bacteriophage
conjugation
largest amount of DNA is transferred; chromosomal & plasmids are transferred; donor & recipient cells are connected by a bridge (pili)
Frederick Griffiths
1928
DNA transferred by transformation
Plasmids
way of delivering the pay load (DNA)
Integrated plasmid
high frequency recombinant F' cells
Ti plasmid
can be used to transfer genetic material in plants
Recombinant DNA
double stranded DNA from 2 different sources that get spliced together
Agrobacterium tumefaciens
contains Ti plasmid
Resistant plasmids transfer _______ through conjugation
antibiotic resistance
Transfection
introducing new genetic material (nucleic acid) of a virus into a mammalian cell
Chargaff's rule
looking at how much (percentage) of nucleotides in an organisms cell
Genetically engineered prokaryotes & eukaryotes
insulin production
hemoglobin
growth hormone
interferon
albumin
oil degraders
alcohol producers
pesticide control
vaccines
frost resistance
DNA hybridization
heat to denature & 2 complementary sides are reanneal regardless of the source
2 pieces of single stranded DNA from 2 different organisms combine to make a new DNA
cDNA
complementary DNA (copies) of DNA from plasmids to carry or transfer DNA
Sticky ends
form a staggered cut & want to stick back to (reanneal) single stranded DNA tail
Restriction endonucleases
facilitate cleavage of phospodiester bond; will do it on both sides of the DNA; but is looking for a specific sequence
DNA probe
a tiny piece of single stranded DNA that has the same complementary nucleotides as what is being looked for & it is tagged
small ssDNA with tagged reporter gene
DNA probes are used to...
find a unique gene
How to form a DNA probe
streak DNA to form colones => transfer colonies to a plastic membrane => soak membrane in an alkaline solution => you then have lysed cells with naked DNA => probe is then added => membrane is washed & tag is looked for
"tag" are..
radioisotopes
chemiluminescent or
colorimetric
radioisotope
long half lives & have dangerous disposal & dangers for workers using it
chemiluminescent
glows in the dark
colorimetric
look for a color change
Blue colonies
normal intact functional lac z' protein
white colonies
have recombinant DNA & nonfunctional lac z' proteins
DNA electrophoresis
technique that separates biological material by masses & sizes onto a gel
Practical uses for DNA electrophoresis
forensics
crime solving
hair
skin
semen
saliva
agarose
pure agar
Gel electrophoresis
very fragil
electric field separate DNA fragments by size in an agars gel matrix
Southern blot method
DNA transfer from gel to nylon or nitrocellulose pieces of membrane
RFLP
Restriction fragment length polymorphism
"restriction nucleases used and many shapes produced"
Dr. Southern
first lab to come out with a gel electrophoresis method
Benefits in addition to forensic application of RFLP analysis
medicine
sickle cell anemia
cystic fibrosis
paternity issues
Northern blot
separates RNA in an electrical field by gel electrophoresis in agarose, stained with ethydium bromide & then onto a membrane & stains it
Western blot
proteins transfer, poly acrylamide separation (PAGE) of proteins separated in an electrical field by gel electrophoresis in agarose, stained with ethydium bromide, & then are separated onto a membrane
Use of western blot
diagnose lyme disease & HIV
Southern western blot
Finds DNA binding proteins (DNBP)
technique used to isolate fragments of DNA containing genes or segments of genes. DNA fragments are seperated by an electrical field by gel electrophoresis in agarose. stained with ethydium bromide fragments are then transferred from the gel to nitrocellulose or nylon
DNA polymerase
example of DNA binding protein
PCR
multiple copies of the same small sample of DNA are produced
the procedure by which billions of copies of a sequence of DNA can be made in a few hours
Microarrays
a molecular microscope slide with 100's of samples form DNA & they use the DNA probe to find where diseases originate & look for distinct differences from person to person
Customized medicine
medicine made for an individual
PCR needs:
RNA primer
Nucleotides
TAQ DNA polymerase
Thermus aquaticus
all enzymes are heat tolerant so often used in PCR
Targeting Cytochrome P450
genotypes (phenotypes) of over 99% of the world's population
& is around 3.5 billion years old
biotechnology
using an organism to produce a commercial or industrial product
Products of ancient biotechnology
beer & bread
genetic engineering
artificially manipulating the genetic make-up of an organism
in vivo
in an organism
in vitro
in the labratory
recombinant DNA (rDNA)
double stranded DNA made from 2 different sources or species
Gene library
term used to describe a collection of different DNA fragments
colonies
identical copies of organisms or cells
bioreclamation
use of genetically engineered organisms to reclaim garbage or oil spills
DNA denaturing
heating DNA to separate double stranded DNA into single stranded DNA ;
phosphodiester are not broken
DNA probes
short segments of single stranded DNA which have been labeled that recognizes or binds to the DNA sequence of a gene from another source
Reporter gene
gene that has been tagged with a radioisotope usually by a DNA probe
DNA hybridization
complementary binding of two sources of ssDNA to form one piece of double stranded DNA - complete, incomplete, or partial
Restriction endonucleases
enzyme used to nick dsDNA and make sticky ends
complementary DNA (cDNA)
copies of DNA made from the mRNA derived from the specific cells producing the desired gene product needed - rHGH, rInsulin
reverse transcrpitase
A VERY IMPORTANT enzyme used to produce cDNA from mRNA
transgenic animals
animals which have been genetically engineered in vitro to have specific genes from a different species
plasmids
one of the most common vectors used in genetic engineering
Ti plasmid
the most elegant way of introducing recombinant DNA into a plant cell
viruses
vectors that are more commonly used in human gene therapy
RFLP
the DNA fingerprint. DNA is cut with restriction endonucleases producing different sized fragments. the different sized fragments are separated by electrophoresis generally DNA from some people will produce different mixtures of fragments. used in criminal investigations & paternity testing
water pollution can occur in 3 ways...
physically (stuff in it)
chemically
biologically (massive growth of algae)
Polluted water supports...
a greater than normal microbial load
Waterborne illnesses
viruses are the most common cause
diarrheal diseases occur world wide
water borne diseases are ________ in the US
rare
Water treatment removes most
waterborne pathogens
Potable water
water that is considered safe enough to drink but is not completely sterile but it is expected to have no pathogens and levels must be low enough to not be considered a health concern
Coliform bacteria
gram-negative rods that are lactose users & are found coming from mammalian digestive tracts
Coliform bacteria example
E. coli
E. Coli may indicate that other pathogens may be present as well such as ...
salmonella & shingella
these are gram-negative bacteria but are lactose negative
Presence of coliforms in water indicates...
fecal contamination & an increased likelihood that disease-causing agents are present
Water treatment stages:
1. sediment
2. flocculation
3. filtration
4. disinfection
sediment stage
take out solids
flocculation
add an agent to make the little particles settle
filtration
uses sand, activated charcoal, or membrane to filter water
disinfection
chlorine gas, ozone, or UV light are used to clean the water