Exam 3: Chapters 10-14 Flashcards
(134 cards)
1
Q
Does a wild-type human genome sequence exist? Example?
A
No
Example: Genome sequences of only 3 people reveals over 5 million DNA polymorphisms: sequence differences
2
Q
How do polymorphisms influence phenotype?
A
- Most do not influence phenotype
- Seen in reactions to drugs (no/bad/good effect)
3
Q
What is the percentage of Codons in the human genome? How do mutations affect it?
A
- < 2% human genome (most is regulatory)
- many mutations in codon don’t change amino acids
- many deleterious mutations disappear form the population through natural selection (Evolutionary) - not what we observe, rather they accumulate
4
Q
What are the four categories of genetic variation? *
A
1) Single nucleotide polymorphisms (SNP):
2) Deletion-insertion polymorphisms (DIPs or indel):
3) Simple sequence repeats (SSRs or microsatellite):
4) Copy number variants (CNVs):
Tools for looking at genomic DNA
- rare is more likely to be inherited
kb - 1,000 bases
5
Q
What is the single nucelotide polymorphism?
A
SNP: 1 base pair change - 1 per 1kb
6
Q
What is the deletion/insertion polymorphism?
A
short insertions or deletions of a single or a few base pairs (1-100pb) - 1 per 10kb
7
Q
What are simple sequence repeats?
A
1-10 base sequence repeated 15-100x in tandem (back-to-back) - 1 per 30kb
8
Q
What are copy number variants?
A
large blocks of duplication or deletion (10bp-1Mb) with population frequency of < 1% 1 per 3 Mb
9
Q
How does crossing-over interact with copy number variants?
A
Unequal crossing over produces new alleles of copy number variants (CNVs)
- Misalignment during meiosis
- Not common (most inherited not mutated)
10
Q
How is the genotype determined? What are two methods?
A
- isolating a gene and analyzing the alleles
- one method: polymerase chain reaction (PCR) or gel electrophoresis (sort by size)
11
Q
What is polymerase chain reaction? Who invented it? What are the benefits?
A
- method of making many copies of a target region of DNA
- first developed Kary Mullis
- Faster, less expensive, & more flexible way to amplify specific fragments of DNA (compared to molecular cloning)
- Extremely efficient: can amplify DNA from a single cell or from some archaeological samples
12
Q
What is the oligonucleotide and what is its function?
A
- Two primers defining target region for PCR method w/o lagging strand
- heating up makes single strands then primers bind before strands reattach
- One primer: complementary to one strand of DNA at one end of the target region
- Other primer: complementary to the other strand of DNA at the other end of the target region
– primers work together to isolate the desired region
13
Q
What are the three steps in each PCR amplification cycle?
A
1) Denature strands
2) Base pairing of primers
3) polymerization from primers along templates
* each cycle it doubles –> exponential increase of DNA
(5-6 min per cycle)
* an hour or two results in millions so can make visible band in gel electrophoresis
14
Q
How is PCR used to determine diseases? Example?
A
By sequencing PCR products
Example: sickle cell anemia: cased by SNP in HbB gene chromosome 11 (Gln GAG to Val GTG)
- PCR Genotyping can identify carriers and homozygous individuals
- Amplify the chrom 11 sequence: see one (homozygous) or two bands (heterozygous)
15
Q
PCR product size determine genotype?
A
- Target regions containing SSRs or DIPs can be amplified by PCR
- PCR products vary in size
- Size variation detected by gel electrophoresis
(Diff alleles have different sizes)
16
Q
What is the PCR analysis of Huntington disease?
A
- PCR determines number of triplet CAG repeats
- Normal allele has < 34 repeats
- Disease causing alleles have 42+ repeats (more repeats lead to earlier onset of Huntington’s disease, autosomal dominant disorder)
17
Q
What are two ways PCR is used with fetal and embryonic cells?
A
- Prenatal genetic diagnosis: genotypic fetal cells isolated by amniocentesis (fetal cells in amniotic fluid are extracted using a needle)
- Preimplantation embryo diagnosis: utilizes in Vitro fertilization and PCR, genotype embryos before placing in womb
(not always accurate)
18
Q
How does genetics influence DNA fingerprinting?
A
- Short tandem repeat (STR) loci are highly polymorphic
– many alleles exist in the population
– an individual person carries only two - Genotype is discovered through PCR at many STR loci
– 20 pairs of PCR primers are labeled with fluorescent dyes
– probability that two people have the same alleles at 20 STR loci is very remote - CODIS database is maintained by FBI
- Data from all 20 STR loci
- Data can match DNA from crime scene to a person or can establish innocence
(It is UNIQUE to the person)
19
Q
What is multiplex PCR used for?
A
DNA fingerprinting – looks at multiple loci at once
20
Q
What can short hybridization probes do?
A
Distinguish between single-base mismatches
- “short” = < 40 base oligonucleotides of sample (target) DNA
- no mismatch b/w probe & target = hybrid will be stable at high temperature
- mismatch b/w probe & target = hybrid will not be stable at high temperature
21
Q
How are hybridization probes used on microarrays?
A
Genotyping
- Allele-specific oligonucleotides (ASO) are attached to a solid support (like a silicon chip): many put on one tray
- Add DNA
- Use computer to determine binding (homozygote/heterozygote, dominant/recessive)
- Up to 4 million loci can be genotyped simultaneously
22
Q
What is positional cloning?
A
Identify disease-causing genes by genetic linkage to polymorphic loci
23
Q
What is the strategy of positional cloning?
A
- Same as linkage analysis using two phenotypes, except one gene tracked by phenotype, the other by DNA genotype
- Use microarrays to simultaneously analyze millions of two-point crosses, each one a test for linkage between a disease locus and a DNA marker
24
Q
What are the steps of positional cloning?
A
- Narrow region of interest by finding closely linked DNA markers
- Locate candidate genes in the region of interest
- Determine sequence and expression of candidate genes in normal and diseased individuals
25
How can genetic disease display heterogeneity?
*
**Allelic heterogeneity**: disease caused by different mutations in the same gene
- **Compound heterozygote:** individual with different mutate alleles of the same gene
- Individuals with certain alleles may respond to drug treatment while others do not
**Locus heterogeneity** disease caused by mutation in one of two or more different genes
26
How has genome sequencing changed today? What i the name of the sequencing?
- become routine
- cost of entire genome: $400-$500
- Whole-exome sequencing: less expensive
- High-throughput or massively parallel sequencing is like Sanger sequencing with a few modifications
27
What are the modifications of the high-throughput or massively parrallel sequencing?
- Anchoring of individual DNA molecules (in place)
- Identifying each base before the next one is added (additions are controlled & known)
- Increased sensitivity eliminates need for cloning or PCR
28
What was Sanger sequencing?
Colored probe that dead ends nucleotide chain
(*Fluorescent tags on nucleotides,
adds blocking group (by random chance) to terminate DNA sequence creates random fragments that can be analyzed
- sort by size*)
29
What does genome sequencing reveal?
Variation
- each individual differs at > 3 million locations from the RefSeq human genome
30
How can we tell which polymorphism causes a disease?
- Transmission pattern
- Predicted effect on protein function
- Clues from other genome sequences
31
What are the points of cloning cats? (Copy Cat & Rainbow)
- All cells have identical DNA
- Each cat has many types of cells
- The cats are dissimilar in many phenotypes: not affected by same **environmental** factors in development
- Take cell from host: new cells have age of host so clones do not live as long
32
What is chromatin? Their composition? Purpose?
- Definition: any complex of DNA & protein found in a nucleus of a cell
- chromones = separate pieces of chromatin that behave as a unit during cell division
- Composition: 1/3 DNA, 1/3 histones, 1/3 non-histone proteins
- Purpose: DNA interaction w/ histones & non-histone proteins produces sufficient level of compaction to fit into a cell nucleus
(Chromosomes support the packaging, replication, segregation, and expression of genetic information)
33
Why compact chromosomes?
- All DNA in the cell stretched out together would be 6 ft long
- Compaction allows the DNA to fit in a cell nucleus
34
What are the different levels of chromosome compaction?
- Nucleosome: (confirmed) Naked DNA 7-fold to 100 A fiber
- Supercoiling: (hypothetical) additional 6-fold compaction --> 40 to 50-fold condensation relative to naked DNA
- Radial loop-scaffold: (hypothetical) 10,000x more compact than naked DNA
35
When can we see DNA? *
When it is wound up in prophase
- unraveled, one cell would be **6 ft long**
36
What are histone proteins?
- Small, positively-charged, and highly conserved
- Bind to and neutralize negatively charged DNA
- Five types - H1 (outside- holding DNA from unwrapping), H2A, H2B, H3, and H4
- Core histones 2x (H2A, H2B, H3, and H4) make up the nucleosome (8 subunits, 4 types)
- DNA loops around twice
37
What are the nonhistone proteins?
- hundreds of other proteins that make up chromatin and are not histones
- 200 - 200,000 molecules of each kind of non-histone protein
38
What are the functions of nonhistone proteins?
- Structure - chromosome scaffold
- chromosome replication (DNA polymerase)
- chromosome segregation (kinetochore proteins)
- transcription - largest group
39
What are aspects of nucleosomes?
- resemble beads on a string
- spacing & structure affect genetic function
-- accessibility for proteins that initiate transcription, replication, and further compaction
-- arrangement along chromatid highly defined and varies in (1) different cell types and (2) under different conditions
(level 1 condensation)
40
What are the facts about the DNA wrapping around the histones?
- 160 bp of DNA wraps twice around a nucleosome core
- 40 bp of linker DNA connects adjacent nucleosomes (# = eukaryotic average)
- Histone H1 associates with linker DNA as it enters and leaves the nucleosome core
- DNA bends sharply at several places as it wraps around the core histone octamer
- Base sequence dictates preferred nucleosome positions along the DNA
41
What is the level 2 condensation?
- nucelosome supercoiling?
- 100 A nucleosomal chromatin compacted into 300 A fiber
42
What is level 3 condensation?
- radial loop-scaffold model
- several nonhistone proteins (NHPs) bind to chromatin every 60-100kb and tether the 300 A fiber into structural loops
- several loops father into daisylike rosettes
- condense with other daisies into a compact bundle
43
What is the G-banding of chromosomes? What phase,
comprises the bands, and pattern?
- metaphase chromosomes stained with Giemsa stain have alternative bands of light and dark staining
- each band contains many DNA loops and ranges from 1 - 10 Mb in length
- Banding patterns on each chromosome are highly reproducible (always condenses the same way) - in species
44
What are the terms for locations of genes in relation to chromosomal bands? *
- Short arm = **p arm**
- Long arm = **q arm**
- within each arm, light and dark bands are numbered consecutively
- arms may have subdivisions
- ter: terminus
45
What is FISH? What does it do? What in the phase? What is the process?
Fluorescent in situ hybridization
- used to characterize genomes
- depends on hybridization between metaphase chromosomes and a labeled DNA sequence
- Chromosomes are spread on a glass slide and denatured to make them single strand
- DNA sequence is labeled with a fluorescent tag to make a probe
- probe hybridizes to chromosomes at complementary regions
46
What is SKY? what is it used for?
Spectral karyotyping (a variation of FISH)
- probes specific for each chromosome are labeled with a different fluorescent dye
47
What is heterochromatin? * What are the two types?
highly condensed, usually inactive transcriptionally
- dark stained regions of chromosomes
- constitutive: condensed in all cells (most of Y and all pericentromeric regions)
- facultative: condensed in only some cells and relaxed in other cells (position affect variegation, X chromosome in female mammals)
48
What is euchromatin? *
relaxed, usually active transcriptionally
- lightly stained regions of chromosomes
49
What does transcription require from chromosomes?
requires changes in chromatin structure
- promoters of inactive genes are hidden in nucleosomes
- activate: transcription factors behind to enhancers and recruit chromatin remodeling proteins (chromatin remodeling complexes)
- promoters: exposed by removing or repositioning nucleosomes
50
What are the aspects of the 8 subunits of the histone?
- Each subunit have tails that extend outward from nucleosomes
- enzymes can add chemical groups (methyl, phosphate, ubiquitin)
- Modifications can alter nucleosomes and bind chromatin modifier proteins
51
What is methylation on a histone tail?
- Histon methyltransferases add methyl group to histone tails
- adding methyl group (to H3 lysine 9) favors heterochromatin formation
- process reversed by methyltransferases
52
What is acetylation of histone tail modification?
- Added by histone acetyltransferases
- Prevents close packing
- Favors euchromatin expression of genes
- Reversed by histone deacetylases
53
What is X-chromosomes inactivation Why? What is it called?
- heterochromatin formation (facultative)
- **dosage compensation**: in mammals so X-linked genes in XX & XY expressed at same level (all only need one X)
- random inactivation of all except one X in each cell
- **Barr body**: darkly stained heterochromatin masses observed in somatic cells at interphase
54
What is the process of X chromosome mosaicism?
- very early embryo both X active
- humans: random X-inactivation ~ 2 weeks after fertilization (500-1000 cells)
- all those cell descendants have same inactive X
- adult females are **mosaic** in X-linked genes
55
What does Xist gene do? What is Xist RNA?
(Xist: X inactivation specific transcript)
- expressed on the inactive X
- Xist RNA: large, non-coding, cis-acting regulatory RNA
-- binds to expressing X
-- initiates histone modifications (methylation, deacetylation), --> heterochromatin formation inactivating X
56
What is important about the origins of replication in eukaryotes?
- there are multiple
- DNA synthesis rate humans ~ 50 nt/sec
- Would take 800 hours to replicate human genome w/ one origin
- Mammalian cells have ~ 10,000 origin sites many being active at the same time (genome-wide)
- **Accessible regions**: devoid of nucleosomes
- Replication unit (replicon): DNA being replicated in both directions one origin
57
What happens to nucleosomes during DNA replication?
Disassembly and reformation
- DNA packed in nucleosomes w/i minutes of synthesis
- Tetramer of H3 and H4 associate with DNA then two dimers of H2A and H2B
- New nucleosomes: recycled & new histones
- Chromatin is open to histone modification just after replication
58
What occurs in replication at the ends of chromosomes?
- RNA primers removed leaves unreplicated DNA at 5' end
- Need special mechanism to not loose DNA information
59
What protects the ends of eukaryotic chromosomes? What are they and what does it prevent?
Telomers: specific repetitive sequences that do not contain genes
- Species-specific (TTAGGG humans, TTGGGG Tetrahymena)
- 250-1500 repeats: varied between cell types
- Prevents: chromosome fusions & maintain integrity of chromosomal ends
- Does shorten with replication: limits cell replication, part of aging process
60
What is telomerase?
Ribonucleoprotein that extends telomers
- telomerase RNA is complementary to telomere repeat sequence
- template to add new DNA repeat sequences to telomere
-- additional round occur after telomeres translocate to newly-synthesized end
(not rate to prevent loss)
61
What is telomerase activity in different cells? *
- Level of telomerase and cellular life-span varies between different types of cells
- Most somatic cells have low expression of telomerase
-- shorten at each division
-- **senescence** after <50 generations in culture
- **Germ, stem, tumor cells** have high expression of telomerase
-- length is maintained
62
What are the two main themes underlying the observations on chromosomal changes?
1. Karyotypes generally remain constant within a species (
- most genetic imbalances result in a selective disadvantage - may not reach maturity
2. Related species usually have different karyotypes
- closely-related differ only few rearrangements
- distantly-related differ by many
- correlation between karyotypic rearrangements and speciation
63
What are the types of **chromosomal** rearrangement?
Deletion, duplication, inversion (different order), and translocation (transfer sections between nonhomologous chromosomes)
64
How does FISH detect large chromosomal rearrangement?
SKY with probes specific for two different chromosomes shows a chromosomal translocation
- only passed on if occur in germ cells
65
What are the effects of deletions in chromosomes?
homozygosity:
- often lethal or harmful depending on size of deletions and affected genes
heterozygous: potential
- mutant phenotype due to gene dosage effects (haploinsufficiency) *(snapdragon flower color, red/white/shades of pink)*
- altered phenotypes due to mutation in the other copy of the gene
- uncover existing recessive mutant allele (star player gets sick)
66
What is an example of a reciprocal chromosomal translocation?
Chronic myelogenous leukemia:
- Translocation of chromosomes 9 & 22 create an altered protein causing cancer
67
What are transposable elements?
(TEs): movable genetic elements
- any DNA segment with the ability to move from place to place within a genome
68
Who discovered transposable elements?
- Marcus Rhoades (1930s) and **Barbara McClintock** (1950s) inferred existence of **TEs** from genetic studies of **corn**
-- Nobel Prise 1983
69
What is now discovered about transposable elements? (size, present, function)
Now found in all organisms
- Previously: considered selfish DNA - not useful
- Now: beneficial function
- Size: range 50bp - 10kb
- Present: hundreds of thousands of copies (can)
- Function: disrupt a gene (middle of gene malformed protein) and/or cause gene to be unstable (affect length --> mismatch during crossing over) (can)
(probably hoping within chromosome but many present in genome)
70
How are transposable elements seen in corn?
- Mottling of kernels caused by movement of TE into and out of a pigment gene (disrupt red color gene)
- nonautonomous (cannot move around on own - lacks activator) TE inserts into a gene and disrupts function
- autonomous: shows instability by TE hoping out restoring gene function - Mottling result
- activators are important for proper hopping
71
What about transposable elements in humans?
May not be able to see effect
72
What are DNA transposons?
- inverted repeats of 10-200 bp long at each end of a transposase gene
- gene encoding transposase enzyme which recognizes the IRs and cuts a border between the IR and genomic DNA
73
What are three ways
transposable elements disrupt genes and alter genomes?
1) Alter phenotypes
- insert within coding region of a gene
- insert near a gene and affect its expression (promoter
- TE-associated alleles can be unstable
2) trigger spontaneous chromosomal rearrangements
- unequal crossing over between TEs
3) Gene relocation due to transposition
- formation of composite TE
74
What is aneuploidy? * The types?
The loss or gain of one or more chromosomes
Euploidy - normal chromosomes (2n)
Nullisomy - loss of chromosome set (2n-2)
Monosomy - loss of single chromosome (2n-1)
- usually lethal
Trisomy - addition of one chromosome (2n+1)
- most are lethal
Sex chromosome aneuploidy tolerated due to X-chromosome inactivation
75
What are aneuploids?
Individuals who chromosome number is not an exact multiple of the diploid number for that species
76
What causes aneuploidy?
Nondisjunction: the failure of chromosomes to segregate normally and can occur during meiosis I or meiosis II
77
Where do we see that some euploid species not diploid?
Monoploidy and polyploidy rarely observed in animals
- exceptions: ants, bees, hermaphroditic worms, some fish
- polyploidy in humans lethal
78
How is a polyploid organism formed?
- tetraploid: diploid gametes
- from tetraploid parent or defects in meiosis (spindle or cytokinesis)
- artificially: can cross diploid gamete and monoploid gametes
- odd chromosome numbers are typically sterile
79
What occurs in meiosis in a triploid organism?
- no way for gametes to contain balanced set of chromosomes
- must have even number of chromosomes
- sterile (used to advantage in crops - seedless fruit)
80
How might tetraploid cells be produced?
can occur during mitosis in diploid when chromosomes fail to separate into two daughter cells
- tetraploid in gamete precursors produces diploid gametes
- union 2 diploid gametes --> tetraploid organisms
81
What is autoployploid?
All chromosome sets are derived from the same species
82
What happens in meiosis of tetraploids?
- 4 copies of each group of homologs pair 2-by-2 to form 2 bivalents
- Successful tetraploid produce balanced gametes and are fertile
83
How are polyploids seen in agriculture?
- 1/3 all known flower plant species are polyploid
- Polyploidy often results increased size and vigor
- Often selected for agricultural cultivation
-- tetraploids: alfalfa, coffee, peanuts, Macintosh apples, Bartlett pear
-- octaploids: strawberries,
84
What is a euploid? *
Complete set of chromosomes (usually diploid)
85
What is polyploid? *
A euploid species that carries 3 (or more) complete sets of chromosomes
86
What is allopolyploid? *
Hybrids in which chromosome set come from distinct, but related, species
- usually infertile b/c different chromosome sets cannot easily pair and segregate properly
87
What is an amphidiploid? *
Has two diploid genomes, each from a different (not closely related) parental species
- Raphanobrassica: sterile F1 from crossing cabbages and radishes, has 18 chromosomes (9 from each parent)
88
What happened in the creation of the alloployploid Triticale?
- F1 hybrid of wheat (tetraploid) and rye (diploid)
- Both have 7 chromosomes but wheat x4, rye x2
- **sterile** because there are no paring partners for the rye chromosomes
89
How is fertile triticale made? What are some combinations of triticale?
- Treatment with **colchicine** causes chromosome doubling in germ cells
- some combine high yield of wheat w/ ability of rye to grow in unfavorable environments
- some combine high level wheat protein with high level rye lysine
90
How does colchicine treatment work?
Prevent spindle formation resulting in doubling of chromosome numbers
91
What happened on the island of Madeira?
- Mice separated geographically
- Genomic instability led to changes some populations no longer able to interbreed (diff species)
- What likely happen as species come off ark and isolate --> new species
92
Why are bacteria important for humans?
- Nine bacterial cells for every human cell (9 x 30-40 trillion)
- Most in intestines then skin/mouth/respiratory tract
- Most bacteria aid human health, small number cause disease
93
What are the characteristics of bacteria?
- variety shapes & sizes
- lack nucleus & membrane-bound organelles
- single **genophore** (prokaryotic chromosome) contained within nucleoid
- most have cell wall
94
What are pathogenic bacteria? What do they do
bacterial strain that cause diseases
- invade tissues
- may produce toxins, proteins that interfere with cell function or destroy cells
- tetanus toxin (*Clostridium tetani*) - results in paralysis by interfering with communication between nerves and muscles
95
What composes the typical bacterial genome? What is its form and how it is passed along?
- one circular chromosome
- 4-5 MB of DNA most commonly studied bacterial species
- DNA molecule condenses by supercoiling and looping
- replicate and divide by binary fission into two daughter cells
96
What is the core genome?
About 1000 genes found in all strains
(genomes of 100s E. coli strains have been sequenced)
97
What is the pangenome?
core genome plus all genes found in any other strains (about 15,000 genes)
(individual E. coli strains (~4,700 genes) contain a subset of the E. coli pangenome)
98
What are plasmids?
- small circles of double stranded (additional) DNA
- may contain genes that benefit host bacterium or contribute to bacterial pathogenicity
- use them in molecular work as cloning vectors
99
What does it mean for bacteria to be monoploid? (Five examples)
All mutations express their phenotype
1) Colony morphology altered: large/small, shiny/dull, round/irregular
2) Bactericide resistance: antibiotics/bacteriophages
3) **Auxotrophs**: unable to reproduce in minimal media: defective in enzymes required to synthesize complex compounds (amino acids, nucleotides)
4) Defective in using complex chemicals from the environment (breaking down lactose into glucose and galactose)
5) Defective in proteins essential for growth: conditional lethal mutations (temperature-sensitive)
100
How are the ways genes are transferred in bacteria? *
- Transformation: take up DNA laying around (not great at it but many cells to try)
- Conjugation: direct transfer through connecting tube (F+ build bridge to F-)
- Transduction: bacteriophage invades, destroys, passed along
(ways share/pass along genetic information)
101
What is transformation and the forms?
Competent cells can take up DNA fragments from surrounding environment
- Natural: spontaneously from surroundings
- Artificial: in lab making cell membrane compromised (calcium make permeable or use electroporation)
102
What are the indications of the demonstration of gene transfer by conjugation?
- use pilus
- can make indicated by + cannot make indicated by -
(A could grow on some, B could grow on others, together some could grow in minimal media - only see ones that could survive, powerful screening method)
103
What are the aspects of F plasmids in bacteria?
- F plasmids: contains genes for synthesizing connections between donor and recipient cells
- Donors for conjugation are F+ (carry F plasmid)
- Recipients for conjugation are F- (do not carry F plasmid)
(F+ cannot receive information)
104
What is the process of conjugation?
1. F pilus binds to F- cell wall
2. Pilus retracts and cells are drawn together
3. Gene transfer
4. Two F+ cells don't conjugate
105
What is an Hfr?
High frequency recombinant: cells formed when F plasmid integrates into bacterial chromosome through recombination between IS elements
- F plasmid has three insertion sequences (IS) elements identical to IS elements at various positions on bacterial chromosome
106
What is an episome?
- F plasmid that can integrate into bacterial genome
- Location and orientation of episome differentiate Hfr strains
- Hfr strains: retain all F plasmid functions & can be a donor for conjugation with F- strain
107
What is the gene transfer between Hfr donors and F- recipients?
- Starts in F plasmid at origin of transfer
- Chromosomal genes located next to F plasmid sequences are transferred to the recipient
- Transferred chromosomal DNA recombines into homologous DNA in recipient
- F- stays F- even after Hfr transfer
108
What is F' plasmid how is it formed? What else can they do?
- Formed: excision from Hfr chromosome plus some adjacent bacterial chromosomal DNA
Can:
1) replicate independently in bacterial cells
2) transferred to F- cells by conjugation
109
What results from the passing of an F' plasmid? What does this allow?* What is the trp example?
- F- partial diploid, called merodiploid
*allow for studies to be done on bacteria that otherwise could not happen
- If merodiploid can grow without tryptophan, 2 trp- mutations are in different genes
- 5 genes trp biosynthesis (A, B, C, D, E, )
- each of five must be present to make trp
110
What are the two types of bacteriophages?
- Virulent phages: always enter lytic cycle after infecting cell, multiply rapidly, and kill cell
- Temperate phages: can enter either lytic cycle or enter an alternative lysogenic cycle
110
What are bacteriophages?
Viruses that infect, multiply within, and kill various species of bacteria
- widely distributed in nature
- most bacteria are susceptible to one or more phages
110
What is transduction?
The process by which a phage transfers DNA from one host cell to another host cell
111
What are the lytic and lysogenic cycles?
Lytic: leads to cell lysis
Lysogenic: circular phage chromosome enters bacteria genome and is now called prophage
- prophages: do not produce viral proteins for virus particles
- Lysogens are induced into lytic cycle
- Prophages excises from chromosome, undergo replication, form new virus particles
112
What is generalized transduction?
- Incorporation of random fragments of bacterial DNA from donor into bacteriophage particles
- DNA from donor cell injected into infected recipient cell
- **transduced** chromosomal DNA recombines into homologous DNA in recipient
113
What is the effect of penicillin on bacteria?
- interferes with synthesis of the bacterial cell wall
- binds to transpeptidase inhibiting enzymatic activity (attaching peptide cross-links, NAG & NAM) preventing cell wall cross-linking
- Entry, action (at peptidoglycan), exit
114
What are methods of penicillin resistance?
1) pen^r deactivates penicillin
2) penA encodes transpeptidase
- mutation **decrease affinity** for penicillin
3) penB encodes a porin, a protein in outer cell wall regulating entry into periplasm
- mutation **decreases entry** of penicillin to cell
4) mtr encodes repressor of efflux pump
- mutation results in **increased removal** of penicillin from cell
(increased resistance leads to decreased efficiency in cellular processes - requires more energy)
115
What causes the green or variegated leaves in Four-o'clocks?
Maternal inheritance due to genes on chloroplast genome
*Mitochondria and chloroplasts are nonnuclear organelles with their own small genomes*
116
What is the structure and function of the mitochondria?
- membrane bound cytoplasmic organelles: outer membrane surrounds wrinkled inner membrane
- many in each eukaryotic cell
- Produces energy packets (ATP) through Krebs cycle and oxidative phosphorylation
116
What are the aspects of the human mitochondrial genome?
Compact gene arrangement
- **16.5 kb circular genome**
- **37 genes**: encoding tRNAs, rRNAs, and proteins for oxidative phosphorylation
- **No introns**
117
What is the variation in mitochondrial genomes across creation?
- Size: 6-2400 kb
Some...
- have introns and space between genes
- are circular (people, animals)
- are linear (plants, fungi)
- different (protozoan) have a single mitochondrion (kinetoplast) with interlocking circles of mtDNA
118
What are the mitochondrial exceptions to the universal genetic code?
- its genetic code varies in different organisms
- humans: five differences
UGA - Stop - Trp (mtDNA)
AGG/AGA- Arg - Stop (mtDNA)
AUA - Ile - Met (mtDNA)
AUU - Ile - Ile - elongation, Met-initiation (mtDNA)
119
What is the structure and function of chloroplasts?
- Membrane bound cytoplasmic organelles in plants: Outer membrane surrounds wrinkled inner membrane: inside- stroma, granum stacks of thylakoids
- Corn: cells have 40-50 chloroplasts
- Captures solar energy and stores in chemical bonds of carbohydrates
120
What are the aspects of chloroplast genomes?
- Most **120-160 kb** long
- More than one copy in each chloroplast
- Compact gene arrangement: **do have introns**
- Forms: **circular, linear & branched**
- More genes than mitochondria
121
What is the endosymbiont theory? The evidence?
Mitochondria and chloroplasts are theorized to have descended from bacteria that fused with nucleated cells
- have own DNA
- mt/cpDNA not in nucleosomes
- mt use N-formyl methionine and tRNA^fMet in translation
- bacterial translation inhibitors block mt and cp translation not eukaryotic translation
- Comparisons rRNA gene sequences suggest mt & cp genomes form common ancestor of nonsulfur & cyanobacteria respectively
122
What are the aspects of the cooperation between nuclear ang organellular genomes?
- mt & cp require nuclear gene products to assemble and function
- Cytochrome oxidase c:
-- functions in mt electron transport
-- 7 subunits, 3 encoded by mt genome, 4 by nuclear genes
-- *how would bacterial DNA be inserted into the host? no lost information...*
- nuclear genes encode majority of protein required for gene expression in mitochondria and chloroplasts
123
What results from mutations in organelle genes?
- often whole-organism phenotypes
- mtDNA: may slow cell growth leading to small cell colonies or weak tissues
- cpDNA: may decrease chlorophyll production leading to a change in leaf color
- DNA polymorphisms can be followed by DNA sequencing
124
What are the mechanisms leading to maternal inheritance?
1) Gamete size:
- female may be much larger
- zygote receives many female organelles and few paternal organelles (may be seen as foreign and harmful)
2) Paternal organelles may be...
- actively excluded or destroyed
- segregated into non-embryonic cells
- prevented from entering egg by fertilization
125
What causes plant variegation?
1) Two types of chloroplasts
- Wild-type cpDNA genes: make chlorophyll
- Mutant cpDNA genes: mutation prevents chlorophyll production
2) Two types of cells
- Heteroplasmic cells: mixture of organelle genomes
- Homoplasmic cells: only one type of organelle genome
*non-beneficial for plants, cell cannot photosynthesize*
126
What is the mitosis of the cells that cause plant variegation?
Mitotic progeny of
- homoplasmic: homoplasmic
- heteroplasmic: either hteroplasmic, homoplamic wild-type or mutant
Uneven distribution of organellular genomes has distinct phenotypic consequences
(cytokinesis: random division of cell contents)
127
What is the relationship between cytoplasmic segregation and variegation?
Threshold effect: certain fraction of wild-type organelles sufficient for normal phenotypes
(four-o'clocks, heteroplasmic make enough chlorophyll to be green)
128
What are the characteristics for pedigrees for mitochondrial disease?
Several human nervous system diseases by mutations in mitochondrial genome
- Mutations from mothers to **all children**
- Symptoms vary by **heteroplasmy**
(squares- males, circle-female)
129
How might mitochondrial mutations impact aging?
- Oxidative phosphorylation system in the mitochondria generates free radicals, which can damage DNA
- accumulation mtDNA mutations over time may result in age-related decline in oxidative phosphorylation
130
What is the evidence in support of role of mtDNA and aging?
- Percentage of heart tissue with a mt deletion increases with age
- Alzheimer's disease brain cells have abnormally low energy metabolism
- 20-35% mt in AD brain cells have mutations in cytochrome c oxidase genes possibly explaining the low energy metabolism
131
What is the purpose of oocyte nuclear transfer?
To sidestep transmission of mitochondrial disease
- insert mother's nucleus in donor's egg
