BIOL-3320 Exam 4

Genomics:​

Study of genomes, or ALL the DNA of an organism

The mapping or hierarchical approach

divide the genome into segments with genetic and physical maps, then home in on the details​

The whole-genome or shotgun approach:

entire genome broken into random, overlapping segments that are then sequenced

genetic map

Genetic crosses and frequency of crossing over are used with polymorphic genetic markers to map the location of genes on chromosomes

Humans have 24 genetic maps-

22 autosomal (non sex) chromosomes, the X and Y chromosomes

Physical maps-

more detailed information about genetic markers obtained from genome sequence data

Sequence-tagged site:

unique genetic markers in genome, very helpful for genetic maps

Clone Contig Map:​

Higher resolution than restriction maps- can be used to sequence entire genome​

Get a bunch of YAC or BACs with partially overlapping clones that are continuous for genome’s chromosome​

Ask whether sequence tagged sites (STS) are present in this case (A-Z)

The Shotgun Approach: Steps​

• Take whole genome​

• Shear it, put it in 2 kb vectors & 10 kb vectors​

• Sequence it all (500-1000 bp at a time)​

• Presto genome​

• Very fast!​

• Limited by computer & repetitive DNA

Open Reading Frames (ORF’s):

Computer searches for start codons and stop codons to identify areas that are potential genes​

Only ORF’s with more than 100 codons are likely genes

Many Gene Functions Unknown:​

Over 35% of genes in ANY organism (including Human) ​have no deducible function! 

The Human genome:​

• Genome sequenced in 2003​

• Genes encode noncoding RNA or proteins​

• Approximately 21,000 protein-coding human genes​

• Approximately 22,000 other human genes​

• Repeat sequences are > 50% of genome.​

• Ethnicities have few unique alleles of genes. ​

• Greatest amount of genetic variation is in Africa

Human Genome Variation:​

• 80,000 years ago there were only 10,000 humans on the planet!​

• Human genomes vary by at least 9 million bp​

• More genetic diversity within races than between them in most cases

The Fabulous Fugu:​

• Fugu is an unusual vertebrate because its genome size is only 400 Mb​

• Very few introns, and few gene deserts, regions with little genes​

• Many genes in Fugu and humans are similar, so finding a gene in Fugu makes it easier to find in humans

Bioinformatics:​

• Bioinformatics is a marriage between biology with math and computer science​

  Can help to:​

• Find genes in a genome ​

• Align sequences ​

• Predict structure and function of genes​

• Figure out interaction between genes and gene products​

• Use genomes to figure out evolutionary relationships

GenBank:​

• Database that contains millions of DNA sequences for every organism you can imagine​

• For comparative genomics, you can sequence your critter, and then compare it to GenBank

syntenic/ linked genes

genes on the same chromosome

First Evidence of Linkage:​

Sweet pea cross by William Bateson, Edith Saunders & Reginald Punnett in 1905!​

Thomas Hunt Morgan

pioneered work with Drosophila fruit flies

Morgan worked with several X-linked mutations, and discovered that some genes are linked

Why are all males with recessive alleles?​

Because they must get Y chromosome from dad, and mom only has recessive alleles​

In F2 generation, Morgan noticed most offspring had parental phenotypes, but only 37% were recombinant

50% recombinants are expected if independent assortment is true​

Morgan Proposes Crossing Over:​

Parental phenotypes most common, recombinants more rare​

Equal numbers of parental phenotypes and recombinant phenotypes​

Proposed crossing over as an explanation because he hypothesized that:​

Alleles of some genes assort together because they lie near each other on the same chromosome​

In 1931, Harriet Creighton and Barbara McClintock found a heterozygote in corn with a translocated chromosome-

part of chromosome 8 had broken off and attached to one homolog of chromosome 9, and a darkly staining “knob” on the same homolog​

They found that recombinant chromosomes carried the unusual knob with the C allele, and the translocated chromosome 8 with the wx allele​

First evidence that recombination is associated with physical exchange of parts of homologous chromosomes

German American Curt Stern

published results within weeks of Creighton and McClintock that showed the same thing in Drosophila​

​Just like the results with corn, recombinant progeny were associated with physical exchanges of material in the chromosomes​

Together, the corn and Drosophila results provided strong evidence that genetic recombination occurs from crossing over during Meiosis​

So how can we determine if genes are linked?

We can use testcrosses

testcrosses

where we cross an individual with homozygous recessive alleles with a heterozygous individual​

Example: a+/a b+/b   X    a/a b/b​

IF the genes are not linked, we expect a 1:1:1:1 ratio:​

a+ b+   a+ b   a b+  a b​

If we don’t get this ratio, or significantly close to this ratio (using chi-square test), the genes are linked to some degree

coupling

This individual has two wild-type alleles on one homolog, and two recessive alleles on the other

repulsion

This individual has one wild-type allele and one mutant allele on each homolog,

Coupling & Repulsion:​

Crossing over can switch coupling to repulsion, or vice versa​

The recombination frequency for two linked genes is the same, regardless of whether they are involved in coupling or repulsion

In 1913, Alfred Sturtevant (a student of Morgan’s)

suggested that recombination frequencies could be used to calculate distances between genes on a genetic map​

Defined a map unit (mu) as the interval in which 1% of crossing over takes place- aka, centimorgan (cM)​

NOTE: for linked genes, crossover frequency does NOT equal recombination frequency

Crossover frequency:

frequency of physical exchange between chromosomes in between genes of interest

​

Recombination frequency:

frequency of recombination of genetic markers (alleles) in a cross- determined by offspring phenotypes​

Two-Point Testcrosses:​

The “two-points” are two genes- we want to obtain recombination frequencies​

A double heterozygote is crossed with a homozygous recessive individual to obtain recombination frequencies​

In every case, we should obtain equal numbers of parental phenotypes, and equal numbers of recombinant phenotypes​

Recombination frequency is estimated by:​

    # of recombinants    ​

# of testcross progeny​ x 100

This method doesn’t work well if linked genes are far apart from each other

Three-Point Testcross:

Similar to two-point testcross because we cross a triple heterozygote with a triple homozygous recessive​

In hypothetical example we cross:​

p+ r+ j+ p   r   j​

p   r   j p   r   j​

p allele = purple color (yellow wild type)​

r allele = round shape (elongate wild type)​

j allele = juicy fruit (dry wild type)​

Number of possible phenotypes = 23 = 8 because there are two phenotypes for each of the three genes​

Numbers of each phenotype from testcross can be used to determine gene order AND map units between genes

Results:  Classes 1 & 2 are parental phenotypes with no crossing over​

Other classes are recombinant- possibly one crossover or a double crossover​

Double crossover: two crossovers, one between each pair of linked genes​

In general, double crossovers are more rare than single crossovers, so doubles occur at the lowest frequency​

Thus p, r & j must be arranged in a way that the center gene changes from parental class to classes 7 & 8​

So the double crossovers give us the new gene order ​

For the p — j distance, we must add the progeny for Classes 3 & 4 AND 7 & 8, and divide by total progeny​

Double crossovers must be included because each includes a crossover in Region I​

For j—r distance we must add the progeny for Classes 5 & 6 AND 7 & 8, and divide by total progeny​

Based on this, estimate the recombination frequencies for Regions I & II, and then try to draw a gene map showing distances between genes p, j and r​

Recombination frequencies are as follows:​

For the p—j distance:​

(52 + 46) + (4 + 2)/500 = 0.208 or 20.8%​

For the j—r distance:​

(22 + 22) + (4 + 2)/500 = 0.1 or 10%

Which of the following women are responsible for demonstrating that recombination is associated with physical exchange of parts of homologous chromosomes?​

1. Barbara McClintock​

2. Harriet Creighton

Development:

the irreversible process organisms undergo from single-celled zygote to multicellular organism

an interaction of the genome, cell cytoplasm and environment, and involves a programmed sequence of events

Zygote starts as a totipotent cell-

has potential to be any cell in body​

Determination:

process where genetics “programs” a cell to become specialized (fate)- often done through induction, or chemical signaling

Differentiation

process in which determined cells undergo physical changes to become specific cell types- e.g., nerve cells, antibodies, etc.

Differentiation controlled by gene expression- synthesis of specific proteins guide fate of the cell

Individual cells change to actually become…

Morphogenesis:

“generation of form,” process or anatomical structure formation and cell shape and size changes

• Structures form by changes in cell #, shape, position​

Human development is an inefficient process: ​

50% of conception do not implant (implantation 8-10 dpf,​

Heart beat at 21 dpf).​

 a further ~30% die and abort after implantation.​

​3-4% of all live births possess a macroscopically​ visible congenital defect (120,000 babies/year in the USA).​

1% of all babies are born with a heart defect.​

20% of neonatal deaths are caused by congenital defects​ (the leading cause of neonatal death in the USA)

congenital disorders are the cause of 50% of pediatric ​admissions in the USA

Developmental defects seen at birth (congenital) are caused by ​defects in the cellular processes of development.Model Organisms:​

Model Organisms:​

To understand genetics of development, model organisms must have mutants that affect development, and involved genes must be mapped and cloned for study​

Many model organisms we’ve seen before:​

• Yeast (Saccharomyces cerevisiae)​

• Fruitfly (Drosophila melanogaster)​

• Soil worm (Caenorhabditis elegans)​

• Mouse-ear cress plant (Arabidopsis thaliana)​

• Mouse (Mus musculus)​

• Zebrafish (Danio rerio): especially good for development because embryos are transparent- genetics heavily studied​

In developing cells, is DNA lost to accommodate specific cell types, OR​

Are only certain genes expressed in a constant-sized genome?

Experiments with carrots in 1950’s: differentiated cells could be used to grow an entire new carrot..​

…so DNA is NOT lost during development

Clearly shows differentiation is not from loss of DNA, but from gene expression

1975: nuclei from skin cells of frogs were injected into eggs to make tadpoles, but few survived to adults, those that did were sterile​

1996: scientists in Scotland (Ian Wilmut) cloned the first animal (a sheep) from an adult cell nucleus, meant adult cell nuclei could become totipotent again

Problems with Animal Cloning:​

Cats cloned at Texas A&M- complicated relationship between genotype/phenotype and environment means clone is not the same as donor “mother”​

As seen with sheep, most clones die before or soon after birth​

Remember microarray analysis?  Scientists used this on cloned mice and found many have abnormal gene expression- explanation is differentiated nuclei must be reprogrammed ​

Long way from cloning humans or bringing back dinosaurs from extinction

Are we a long way from cloning humans or bringing back dinosaurs?

• Jack Horner and colleagues have isolated blood vessels from Tyrannosaurus rex bones- iron in animal’s body prevented decay of collagen and other proteins​

• Blood vessel structure nearly identical to modern ostrich- bolsters evidence that birds are direct descendents of dinosaurs​

• Over time, DNA degrades (half life is 521 years), and after 6.8 million years, all bonds would be broken​

• BUT, chemicals similar to DNA have been found with iron-preserved collagen & with dino sequence, it would be possible to clone with ostrich egg- Horner wants to do this!​

Oldest known DNA:​

In December 2022, scientists published environmental DNA (eDNA) data from plants and animals that are 2 million years old​

Samples were obtained from permafrost in Greenland, and include 135 different species ranging from mastodons to ants​

“The survival of such ancient eDNA probably relates to its binding to mineral surfaces.”

Gene Expression & Hemoglobin Development:​

• Humans have two α and two β chains in their Hb-A hemoglobin, controlled by separate genes​

• Two genes are similar because one was duplicated from the other during our evolutionary history​

• During development, several genes code for different hemoglobin polypeptides​

• In embryos, two ζ (zeta) and two ε (epsilon) chains are made in yolk sac​

• After 3 months, fetal hemoglobin (Hb-F) is synthesized in liver and spleen, with 2 α and 2 γ (gamma) chains​

• Before birth, synthesis shifts to bone marrow, which makes Hb-A and some δ (delta) chains​

• Arrangement of hemoglobin chains on chromosomes matches order of gene expression in development​

Antibody Production:​

• Lymphocytes are white blood cells involved in immune response​

• Small lymphocytes include B and T cells​

• B cells develop in bone marrow, when activated by an antigen (e.g., foreign protein on virus or bacteria) they form plasma cells that make antibodies after a few days​

• ​Antibodies attach to antigens, and mount the body’s defense system

We develop immunity over time from clonal selection-

cells with antibodies to an antigen are stimulated to proliferate and make more antibodies

Antibodies are Immunoglobins:​

Antibodies are proteins called immunoglobins, with 2 identical short or light (L) chains and 2 identical long or heavy (H) chains​

Disulfide bonds (–S-S–) hold chains together

The two arms of the Y-shaped antibody contain the antigen-binding sites, which attach to antigens and stimulate ________.

clonal selection​

____________ allows antibody arms to move independently, and bind to separate antigen sites to help disable infecting agents

Hinge region

Mammals have 5 classes of antibodies

gA, IgD, IgE, IgG, and IgM with 5 different H-chain polypeptides and 2 L-chain polypeptides​

IgG is the most abundant class in blood, and IgM is the class that recognizes new antigens-

Each polypeptide chain in an antibody is organized into domains of 110 amino acids each-

part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain 

H chains have 4 domains _______________________. L chains have 2 domains ________________.

(3 constant regions, 1 variable region)………… (1 constant, 1 variable)

Somatic recombination:

random DNA rearrangements during B cell development that join different gene segments and exclude others

As B cell develops, certain V, J, and C segments become associated with each other to the exclusion of others- this is somatic recombination​

Light chain: 

V (variable), J (joining) and C (constant) gene segments are widely separated on the chromosome at beginning of B cell development

Heavy Chain: 

• includes V (variable), D (diversity), J (joining) and C (constant) gene regions that can be shuffled​

In mice, 500 V regions, 12 D regions, 4 J regions, and 1 constant region​

…thus 500 X 12 X 4 = 24,000 rearrangements for heavy chains​

Combined probability of heavy (24,000) and light (1400) chains is 33,600,000 possible antibody molecules​

In placental mammals, Y chromosome makes _____.

testes

Absence of Y chromosome defaults to ______.

ovaries

Genes on the Y chromosome (maybe SRY gene) code for testis-determining factor (TDF)

causes testis formation

most important even in development for sex determination

If for the light chain there are 250 different V segments and 4 J segments, how many different light chain combinations are possible?

250 X 4 = 1,000

If for the heavy chain there are 200 V segments, 13 D segments and 4 J segments, how many different heavy and light chain combinations are possible?

200 X 13 X 4 X 1,000 = 10,400,000

Discontinuous or discrete traits:

each trait has only a few distinct phenotypes​

Continuous traits:

a wide distribution of phenotypes are possible

Phenotype of continuous traits must be quantified or measured, so…

…aka quantitative traits, and the study of such traits is quantitative genetics

Multifactorial traits

traits affected by a combination of genotype and environment​

Polygene hypothesis

for quantitative traits says that multiple genes control the traits…    …should make sense when environment’s impact is limited

Quantitative trait loci:

chromosome regions with genes that affect quantitative traits

Has human genetics changed in 60 years…

NO

Has human environment changed in 60 years?

YES

Mean (x), or average

tells us center of distribution of phenotypes = ∑xn/n

Variance:

how much individual observations spread out around the mean​

​

s2 = ∑(xi – x)2​

           n – 1

Standard deviation:

square root of variance- provides same information but in same units as measurements

Tobacco Plant Example:​

Researcher crossed tobacco plants with long flowers (mean = 93.1 mm) and short flowers (mean = 40.4 mm)​

F1 individuals had a mean of 63.5 mm​

When he crossed F1 individuals, F2 offspring had a mean of 68.8 mm​

BUT variance of F1 = 8.6 mm, whereas F2 = 42.2 mm​

In a continuous trait, we often find that F1 is intermediate to parents, but F2 has more variance

pleiotropy

where one gene affects multiple traits

Is it possible that genotype and environment can affect several measures of size in same individual? 

YES

Correlation coefficient:

measures strength of association between two variables in the same experimental unit, usually individuals

To calculate correlation, we first calculate covariance

Correlation does not imply causation

covariance:

amount of variation in two characters that is shared in an individual

Correlation coefficient ranges from –1 to 1, but what does this tell us?​

Sign indicates direction of correlation, so negative sign means decrease in one variable (x) gives an increase in other variable (y)

So what does the value tell us?​

Absolute value (not considering sign) gives strength of correlation ​

1 is very strong- so increasing x always has an effect on y​

0 is weak- increasing x has no effect on y​

Regression

tells us more precisely about relationship of two variables, and predictions from data

Regression analysis can tell us how much of a trait is genetically determined

Slope of the line

tells us how much of an increase in x corresponds to an increase in y

ANOVA:

Analysis of variance asks if two or more means are significantly different​

If there are only two groups, ANOVA is equivalent to the t-test

is a test used to determine differences between research results from three or more unrelated samples or groups

Heritability:

proportion of a population’s phenotype that is due to genetics and not environment

Phenotype can also be affected by

• Covariance:  What if genotype and environment are connected?  Example: superior genotypes live in super environments? COVG,E​

• Genotype/phenotype interactions:  What if genotype and environment interact with each other?  VGXE​

• Additive allele effects:  What if allele A contributes twice as much as allele a?  Example: haploinsufficiency.  Additive genetic variation = VA​

• Dominance/recessiveness:  Heterozygotes will have less variation than populations with homozygous recessive.  This is dominance variance, VD​

• Epistasis and other gene interactions:  This is interaction variance, VI 

• General environmental effects: Nutrition, temperature or other effects during development that affect adult phenotype. VEg​

• Special environmental effects:  Immediate effects from environment. Example: increased skin pigmentation after being in the sun.  VEs​

• Family environmental effects:  Specific habitats of closely related individuals can affect phenotype in same way- can easily be confused with genetic effects. VEcf​

• Maternal effects:  Variation in litter size, gestation period, milk production can all affect phenotype. VEm​

• So in total, VP = VA + VD + VI + VEg + VEs + VEcf + VEm + 2COVG,E + VGXE

broad-sense heritability:

Quantitative genetics are most interested to know how much VP is attributable to VG. 

the ratio of total genetic variance to total phenotypic variance

Broad-sense heritability = H2B = VG/VP​

Value can range from 0 to 1, with zero being no heritability and 1 being maximum heritability with minimal influence of environment

captures the proportion of phenotypic variation due to genetic values that may include effects due to dominance and epistasis

narrow-sense heritability:

geneticists want to know how likely parents are likely to resemble offspring, which is most affected by additive variation:​

Narrow-sense heritability: H2N = VA/VP​

Narrow-sense heritability can track phenotypes from generation to generation, and helps predict changes from selection (artificial or natural)

Limitations of Heritability Estimates:​

1. Broad-sense heritability does not define all of the genetic contributions to a trait: it only measures proportion of phenotype that is due to genetics, not the genes that affect the trait​

2. Heritability does not indicate what proportion of a phenotype is genetic: heritability is based on variance of a population, not individuals​

3. Heritability is not fixed for a trait: depends on genetic makeup and environment of a population, which can shift often​

4. High levels of heritability for a trait does not imply that trait differences among populations is genetic: environment can have a major effect on phenotype even if heritability is high, so population differences may not be genetic​

5. Traits shared by members of a family do not imply high heritability: similar family environments can lead to similar phenotypes regardless of genetics

If we keep environment constant, ___________.

closely related individuals should be similar in phenotype because they have more genes in common

BUT, if environment is important for a trait, relatedness won’t matter as much

Midparent value, or mean of mom/dad’s phenotype equals ______.

value for offspring if variation is due to additive genetic variation- gives a slope of 1​

f slope is less than 1, gene interactions (epistasis) and environment are a factor​

If slope is 0, environment is main factor

Evolution:

genetic changes in populations over time

Natural selection

individuals with certain traits leave more offspring than others

Artificial selection

only selected individuals are bred, causing genetic changes over time

When were the first dogs domesticated in Siberia?

23,000 years ago

How did dogs learn to “manipulate” you?

Dogs “hijacked” the human oxytocin “love” response, including reduction of stress, but main effect is parent/offspring bonding​

Allowed dogs and humans to form strong emotional bonds​

This oxytocin release does not occur in wolf/human interactions

Friendly Dogs & Williams Syndrome:​

Study in 2017 linked “hypersocial” dog behavior to mutations in GTF2I and GTF2IRD1 genes​

Deletion of these genes in humans leads to Williams Syndrome:​

Affects 1 in 70,000 people​

Elfin facial features​

Cognitive difficulties​

Tendency to love everyone​

If for the light chain there are 380 different V segments and 2 J segments, how many different light chain combinations are possible?

380 X 2 = 760

If for the heavy chain there are 350 V segments, 3 D segments and 8 J segments, how many different heavy and light chain combinations are possible?

350 X 3 X 8 X 760 = 6,384,000

monolayer

growth checked by contact inhibition

Cancer: 

disease where eukaryotic cells are transformed- divide uncontrollably and abnormally