hmb265 2nd half

Lecture 16: Transposable Elements

do genes exist in fixed places in the chromosome?

no. one woman revolutionalized…. with only classical genetics and a light microscope. what a woman

Barbara McClintock

• late 1940s

• discovered movement of small segments of DNA transposable elements

• used maize 

• one train of maize showed chromosome 9 broke often at a particular site

  • involve Ds element and Ac element

• molecular structure of DNA was unknown

• using classical genetics and a light microscope

transposable elements

movement of small segments of DNA

Ds element

dissociation factor

site of the break

non autonomous element - cant mobilize itself

incomplete mutated form of Ac element

Ac element

• unlinked genetic factor necessary to be present to activate the breakage at the Ds locus

• autonomous element - can moblilize itself and other mobile elements in the same family

• can become a Ds element (when it cant jump back out again)

• inverted repeats at the end, encodes a single gene which encodes a transposase protein

chromosome 9

controls genes that control several phenotypes of maize kernels;

pt traits: pigmented/colorless, plump/shrunken, nonshiny/waxy

transpoable/unstable alleles can lead to novel/odd phenotypes

spotted phenotype: independent exicsion of Ds alleles

unstable phenotype

characteristic of mobile/transposable elements

*add more detail/definiton

mechanism of transposition in corn

transposase

insertion of element

cuts with staggered ends (like restriction enzymes), inserts transposable elements between staggered ends→cell repairs ends

net result: flanking sequences, direct repeats reflecting a repeat of the target site dna generated by processes of dna repair

T or F? Transposable elements are common.

True.

Found in all kinds of organisms; bacteria, Drosophila (12.5%), humans (34% of the genome)

2 main classes of transposable elements in eukaryotes

retrotransposons & DNA transposons

retrotransposons

transposable elements going through an RNA intermediate

different types; some have LTRs long terminal repeat sequences (100s of bases long; direct repeats)

similar to retroviruses; go through RNA intermediate which then gets translated into a reverse transciptase → cDNA→reinserted into genome in a different place. however they do not get packaged and leave the cell; stay moving around the genome.

DNA transposonas

• smaller than retrotransposons

• gene encodes a transposase in the gene

• eg P element DRosophila,

• recognizes inverted repeats, excies element, transposes to new genomic lovcation

  • original location has a gap and either a)homologous chromosome without P elemnt is repaired with no transposon or b) p element reconsitiuted in original position if sister chromatid

transposable elements in humans

LINEs, SINEs, DNA transposons

LINEs

req reverse transcriptase to mobilize

autonomous

SINEs

req reverse transcriptase to mobilize

non autonomous

how do organisms survive with so many mobile elements?

so much of our DNA is transposable elements- but we can control this mobilization

• they are found in introns

• often defective unable to transpose again due to mutations (lack of repeats or active transposase)

• epigenetic changes in heterochromatin vs euchromatin “silencing” parts of genome make “safe havens”

but mutation can occur

chromosomal rearrangments

Transposable elements can result in unqueal lining of homologous chromosomes; alter dna 

can relocate dna sections

lead to inapporpriate expression of gene product

laboratory uses of TrElems

allows for the transfer of any gene of interest to make transgenic flies

replace transposase gene with marker gene and inject into drosophila embryo

subject will have rosy- phenotype, but with a successful transposition some of progeny crossed with WT will be rosy wildtype

Lecture 13: Quantitative trait loci

How to define heritability?

What is heritable in a gene?

P=G+E

phenotype is the sum of genes + environment

variation in phenotype = Sum of variation of genetics and variation of environment

Broad Sense Heritability (H²)

the extent of phenotype variation attributable to genetic variation

H² = 1 if…

all phenotypic variation because of genetics

H² = 0 if…

all phenotypic variation because of environment

(the minimum)

H² limitation

H² does not predict how progeny will perform on the basis of the phenotype of their parents

progeny=/= look like their parents

why? most variation is not transmissible from parent to offspring bc of context. offspring inherit alleles but not genomic context. interactions b/w loci are not inherited. all effects not transmitted; only additive effects

experiments for H², Vg, and Vp

measure effect of genotypes in many environment

• model species, genetically identical strains

  • hold Vg as close to 0 

• twin studies

  • show environmental effects, where genes are “held constant”

If H² is high…

the phenotype of an individual is probably because of genotype IN THAT FAMILY (in the same population where env and stuff are relatively constant)

What H² DOESN’T tell us

• phenotype of individual based on parents phenotypes, even if H² is high

• what is going on in other families/populations

What H² DOES tell us

the proportion of phenotypic variability attributed to genetics for the entire population.

3 components of genetic variation

additive, dominance, epistatic effects

Vd

phenotype not predictable - may be homozygous or heterozygous

Vi

cant predict phenotype

context of another locus matters

Va

predictable

narrow sense heritability (h²)

• extent of phenotypic variation attributable to additive genetic variation

• what is commonly thought of as ”heritability”

if h² = 1…

• all the phenotypic variation is attributable to additive variation

• the maximum

if h² = 0…

• if phenotypic variation is attributable to other genetic & environmental effects 

• the minimum

h² does tell us

the phenotype is predictable based on parent phenotype for that specific place, time people, family, population etc

eg. selective/artificial breeding if h² is high (eg. pick a large pig to make more large pigs)

h² doesnt tell uss

• whats happening in other families

• what the genes are

Lecture 14: Genetic Mapping & Complex Traits

quantitative effect

• usually two or more important genes

Quantitative Trait Loci (QTL)

genes controlling the trait in a quantitative loci

regions of genome associated with variation in a complex trait

• identified using genetic markers (eg SNPs), association studies, statistical methods

Step 1 

cross b/w inbreds which differ at trait(s) of interest

because limited by genetic diversity present in inbreds (less recombination) → variation in phenotype due to env. causes

Step 2: F2 frequency distribution

does gene distribution match phenotype distribution?

Step 3: molecular markers; genotyping

molecular markers spread out across genome at regular intervals to find markers that co-segregate with trait ()

sequence all F2

Step 4: statistics ; are markers co-segregating?

statistically significant →

associating with the trait or not

step 5: LOD score

plot the degree of association (logsrithm of oddds LOD) on a linkage map

statistical association between a marker and a trait of interest?

Odds Ratio (OR)

probability of linkage →in favor of QTL

LOD = log OR

100 → 100 odds in favor of there being a QTL

LOD score example

LOD > 3 → linked

Lecture 17: Chromosome Packing & Number

chromosome packing : nucleosome

• double stranded helix wrapped around histone core, 2 sets of 4 subunits

• organizational purposes

• can be open or closed

  • accessible or inaccessible for transcription & regulation

• can protect from cleaving/digestion

heterochromatin

• transctriptionally inactive

• extremely closed conformation

• dna silenced

euchromatin

transcriptionally active

chromatin remodelling

• histone tail modifications (100+ diff ways)

  • acetylation (by HATs)

  • methylation (usually of K or R) by HMT

• alter chromatin structure

• huge flexibility in complexity

x-chromosome inactivation

• heterochromatin formation inactivates an entire x chromosome (into a Barr body)

  • random which one gets inactivated

• early during development

  • 2nd x is still essential until 100 cell count

• inactivation is hereditary through cell division i.e. clonal

  (except reactivated in germ cells)

• reason—dosage compensation

• still a small number of genes on barr body expressed

mechanism of x inactivation

• XIST RNA coats a chromosome creates Barr body/ heterochromatin

• hypoacetylation of a Lys in two histones & histone methylation

euploidy

complete sets of chromosomes 

eg. diploid (2 sets of chromosomes, humans), monoploid (one set), triploidy

aneuploidy

loss or gain of one or more chromosomes

eg. monosomy (missing a chromosome in a pair of homologous chromosomes in chrom 3 

usually caused by meiotic nondisjunction; diff distributions of chromosomes in gametes (extra or lacking chromosome)

monoploidy

• common in male social insects

• male bees, wasps and ants

• develop gametes vua parthenogenesis

• unfertilized egg into an embryo, males produce gametes via modified meiosis

• can create monoploid plants uncover recessive mutations

  • take meisois product in anther and cause haploid cell in lab to be induced to grow as an embryo and eventually formed monoploid plantlet and observe phenotype visualizing traits directly and study mutations

polyploidy

• associated with origin of new species

• + correlation with size and vigor

• reason fruits are getting bigger

  • more polyploid (more sets of chromosomes) , bigger fruits

autopolyploids

form spontaeneously or in lab

autotriploid (or other odd set)

• tend to be sterile

• issue pairing chromosomes in meiosis

• may form unbalanced gametes

  • chance of complete set of balanced chromosomes is very low but possible

• commercial bananas and watermelons are sterile; propogated asexually

autotetraploids

• can be spontaneous doubling (2n→4n) or drug induced (colchicine)

• usually source of new species because new plant self fertilizes is not distinctly tetra ploid and cant be crossed back to the diploid parent

tetraploid meiosis

• in tetraploidy you dont have 2 homolog chromosomes you have 4 homologous chromosmes for each different type of chromosome, when pairing up, only homologues can pair up, they have a choice of three partners to pair up with (1+2 new)

• once paired with 3 possible partners, different ways of assorting”left door or right door” for one partner and the other

• pairing and alignment varies

• 4 alleles instead of 2

• all possibilities are equally likely

• choices of who they pair with and which way they assort

• probability of aa aa gametes for example are 2/12 or 1/6 ² because 2 parents → 1/36

  • unique dihybrid ratio→ 1AA:4Aa:1aa

allopolypoloids

hybrids of two closely related species where chromosomes are partially but not fully homologous

F1 sterile

homeologous

• chromosomes or genes that are similar but have originated from different parental species

• Homoeologs are pairs of genes that originated by speciation and were brought back together in the same genome by allopolyploidization

(fertile) amphidiploid

doubled diploid: doubling in germ cells

type of alloploid

monosomy

usually lethal in humans in utero

missing a whole chromosome

eg monosomy 21

turner syndrome (XO)

trisomy

2n+1

trisomy 21 down syndrome

smaller size →less bad result (eg down syndrome chromosome 21 is smaller than other maybe more lethal disorders from other chroomosomes)

down syndrome

chromosome 21 has 3 chromosomes instead of 2 like all the other ones

bivalent chromosomes

i think just homologous chromosomes

trisomies XXX

less bad

XYY: Usually fertile – X pairs with one Y; other Y does not

pair and is not transmitted to gametes

i.e. X or Y gametes, not XY or YY

XXX: Usually fertile - Two X chromosomes pair; third does

not pair and is not transmitted

i.e. only X gametes, not XX

Thus, conditions are not passed on to progeny

genomic hybridization: microarrays

stained two different colors based on type of mutation to determine where large scale insertions and deletions occurpre

prenatal testing

• associated with fetal abnormalilties not a diagnostic test just risk assessment

  • ultrasound, blood test for hormone levels, plasma protein levels

chorionic villi sampling

take placenta cells associated with fetus to test

less invasive

earlierin pregnancy

amniocentesis

requires needle

directly samples fetal cells in amniotic fluid to test for chromosomal abnormalities

preimplantation embryo diagnosis

in vitro fertilization

extracting eggs and fertilizing in vitro grow them in lab then extract a cell from embryo and use it to sequence genome and determine what alleles that embryo has

can screen for diseases, see which embryos to reimplant based on what alleles they have

use info to select for traits you want * controversial

eg. select for eye color

leads to many regulation, ethical, and societal questions

Lec 19: Epigenetics

epigenetics

heritable modifications in gene function not due to changes in the base sequence of DNA

DNA methylation

generally repressive

replication fork- dedicated methyl transferases that can copy pattern to newly synthesized dna

molecular mechanisms of epigenetics

1) dna methylation

2) covalent modification of histones (acetylation, methylation, etc)

3) non-covalent mods of histones (chromatin remodeling, histone variants)

4) non coding RNAs - transcriptional silencing

mice coat color variance due to epigenetics

agouti gene responsible for coat color

retrotransposon element insertion upstrream of agouti 

IAP (cryptic promotor) can affect expression

IAP methylation → silencing of gene → brown

unmethylated IAP →yellow

varying phenotype gradient

environental effects (epigenetic)

feed methyl donors, modify mothers diet → epigenetic changes in offspring

can persist in F2

epigenetics is not required for normal development. T or F?

False.

Epigenetics is required for normal development.

A very tightly controlled process of gene regulation and expression that must occur.

PRE element

with PRE: Hox gene repressed

allows cell divisions with no attrition (?) of methylation marks

no recruitment of methylation

Hox genes are still repressed

stable thru cell divisions

parental imprinting

one type of epigenetic control

occurs in placental mammals and controls development in utero

receive maternal and paternal imprints (egg and sperm)

old imprints erased during meiosis (for zygote)

new imprints

example of maternal vs paternal imprint differences

methylation on ICR region, no H19 transcription in paternal

deletions in the same part of a chromosome can lead to different effects based on paternal or maternal allele

effects of mutations in imprinted genes

effect of mutation only seen in genes that are actually expressed

eg mutation in gene A is imprinted in the mom, only express the dad’s copy

impringint of Igf2

only expressed from fathers allele

no mutation in mothers allele will have an effect

deletion inherited from dad → effect

deletion inherited from mom → no effect

Prader-Willi syndrome PWS

paternal SNRPN gene inactivated by mutation, maternal SNRPN gene inactivated by imprinting

Angelman syndrom

same part of chromosome 15

maternal copy has deletion which inactivates AS gene, paternal copy inactive via imprinting