Exon shuffling can lead to a common domain being found in a variety of proteins.

Via Non-Viral Retrotransposon

If a LINE has a weak poly(A) signal, then sometimes transcription will continue and include an adjacent 3′ gene (eventually terminating at that gene’s strong poly(A) signal). ORF2 then reverse-transcribes the RNA transcript of the LINE and gene, eventually inserting the gene at a new location along with the SINE in a phenomenon known as exon shuffling.

Via Interspersed Element Crossover

Exon shuffling can occur double crossover between interspersed repeats.

Via DNA Transposons

The most common type of repetitive DNA are interspersed repeats or moderately repeated sequences. These are present as a single copy at very many different loci and can move or jump to new locations.Interspersed repeats account for almost half of human DNA. These do not occur in tandem arrays. Individual copies of the same, or nearly the same sequence, ~100 bp to ~10 kb long, are found at tens of thousands to more than 1 million different positions dispersed all over the genome. This dispersion is the result of repeated insertions of transposons into new sites during the evolution. The interspersed elements are either transposons themselves or are derived from other genomic sequences acted on by transposon enzymes.

Class			Length	Copy #	Genome	Overview
DNA Transposons			2 – 3 kbp	~300,000	3%	Transposons either move by: direct excision and reinsertion of one DNA element; or insertion of a reverse-transcribed RNA product. DNA transposons can jump during S phase from a daughter strand into unreplicated DNA, thus increasing its copy number. A DNA transposon integrates with a staggered cleavage of the target DNA followed by ligation of the target 5’ ends to the transposon and filling the gaps.
LTR Retrotransposons			6 – 11 kbp	~440,000	8%	Retrotransposons are transcribed normally, then reverse-transcribed to form a DNA copy that is inserted into a new site. LTR retrotransposons are retroviruses that have lost the ability to exit and reinfect a cell. The upstream LTR acts as a promoter and the downstream LTR contains a poly-A site to produce transcripts from the integrated element. These both encode the proteins needed for transposition and serve as a template for making the DNA copy. Between them is a coding region that encodes proteins for transposition and also acts as a copy template. The retroviral genomic RNA is copied into DNA via priming, extension, jumping and repriming steps: A tRNA binds to the primer binding site and copies the 5′ end. Another tRNA at the 3′ end copies the body of the virus. This 3′ end copy is replicated. This 3′ end copy replica reprimes at the 5′ end, forming a virus with both LTR’s. The double-stranded DNA of an LTR retrotransposon integrates by the same mechanism as a DNA transposon, with a staggered cleavage of the target DNA followed by ligation of the target 5’ ends to the transposon and filling the gaps. revise_new_w.png
Non-LTR Retrotr’sons
	LINEs		6 – 8 kbp	~860,000	21%	Non-LTR retrotransposons also encode a reverse transcriptase, but use a different mechanism for insertion. The two proteins encoded by LINEs are: ORF1, an RNA binding protein; and ORF2, a reverse transcriptase and endonuclease. ORF2 protein makes a nick in an A/T rich region of the target DNA to allow priming on the Poly-A tail of the LINE RNA. Reverse Transcriptase uses the 3’ end of the nicked target to extend into the LINE RNA, making a DNA copy. RT reaches the end of the LINE RNA and continues into the target at the staggered cleavage. Insertion is completed by cellular enzymes that copy the second strand, degrade the RNA and ligate the fragments together. Most LINE elements are truncated due to incomplete copying of element during insertion. This makes them inactive for transposition, but they can still be mutagenic upon insertion and can still induce aberrant recombination events. LINEs can result in exon shuffling.
	SINEs		100 – 300 bp	~1,600,000	13%	Short Interspersed Elements (SINE’s) do not encode proteins but transpose by the same mechanism as LINE’s, presumably using the LINE proteins. SINEs carry within them a promoter for RNA Pol III allowing new RNA copies to be made. The most common SINE is the Alu Element named for an Alu restriction site it contains. Alu elements originally derived from the 7SL RNA, a cytoplasmic small RNA involved in protein secretion.Single Alu Elements can evolve into new exons. Alu elements can result in exon shuffling.
Processed Pseudogenes			Variable	1 – ~100	~0.4%

Also, there is unclassified spacer DNA that accounts for ~25% of the genome.

Also, there is unclassified spacer DNA that accounts for ~25% of the genome.

Class			Length	Copy #	Genome	Overview
Tandem Repeats			Variable	20 – 300	0.3%	Encode rRNAs, tRNAs, snRNAs and histones. In some cases, the multiple copies allow increased production of identical gene products – rRNA and histones. In these cases, the genes usually exist as tandem arrays. Allows production of millions of copies of the gene product per cell division – needed for ribosome, snRNPs and histones.
Simple-Sequence DNA			1 – 500 bp	Variable	3%	Commonly called “satellite DNA” — Sheared DNA has buoyant density dependent on base content. Total DNA gives rise to a main band of average base content. Certain overrepresented simple sequence repeats give rise to satellite bands due to skewed base content. Microsatellite DNA is defined as having very short repeat units of 1-15 nt, such as CAGCAGCAG etc, repeated 50 or more times. Many human diseases are caused by triplet (especially CAG) repeat expansion mutations. These are thought to accumulate during rare mistakes in DNA synthesis when the nascent daughter strand slips backward along the template strand to insert additional bases into the daughter strand.
						Minisatellite DNA has a longer repeat unit length of 15-100 nt or so With tandem array lengths of 500 bp to 20 kb. Differences between individuals in the number of repeats of a minisatellite sequence arise through unequal crossing over between chromosomes during meiosis. Some minisatellite sequences are highly variable in repeat number between individuals. This is the basis of the DNA fingerprinting.
						Most simple sequence DNA is comprised of 14-500 bp units tandemly repeated in long stretches of 20-100 kb. Most of these very long simple sequence DNAs are either at the centromeres of chromosomes where they may affect chromosome segregation, or serve as telomeres at the chromosome ends.

Also, there is unclassified spacer DNA that accounts for ~25% of the genome.

Intron Definition

Introns have sequences that directs the splicing apparatus during RNA splicing, part of RNA processing:

Transposable elements were first discovered by Barbara McClintock in kernels of corn, where certain mutations caused loss and reinstatement of purple pigment (due to gain and loss of an insertion element that activated pigment genes). The human genome contains ~300,000 DNA transposons, which have extensively accelerated evolution due to the modularity of exons and regulatory regions. Transposition is one avenue for exon shuffling to occur, whereby an exon and two flanking transposons are all excised and reinserted elsewhere as a single element (potentially adding a new exon to a gene). There are conservative (cut + paste) and replicative (copy + paste) mechanisms for transposition. However, transposition is potentially mutagenic and over-transposition is very deleterious; thus, it remains a rare event that occurs in about 1:10⁵ or 1:10⁷ cells per generation. Transposable elements occur in both eukaryotes (transposons; retrotransposons) and prokaryotes (insertion elements).

A gene is the nucleic acid sequence needed to synthesize a particular gene product. A gene includes more than just the coding region that encodes an RNA transcript; there are also control regions controlling synthesis, processing and translation of the RNA transcript. In prokaryotes, the entire coding region encodes a continuous polypeptide sequence. In eukaryotes, coding regions contain exons (50-250 nucleotides) that encode polypeptide sequences and introns (500-50,000 nucletides, removed during RNA processing) that do not. Higher eukaryotes not only have introns within genes, but large intergenic regions. For example, a ~80 kb region in Saccharomyces cerevisiae (baker’s yeast) contains 40 genes; the ~80 kb region encompassing the human β-globin cluster contains only 5 genes. This extra DNA comes from multiple repeats described here. Exons often encode modular units that are included or excluded via RNA processing. Exons are usually highly conserved while introns are barely conserved. For example, SUR2 exons are 90% identical between mice and humans while SUR2 introns are less than 10% identical between mice and humans. A lack of inter-species sequence conservation indicates a lack of function.

A transcription unit is a region of DNA that is transcribed under the control of a particular promoter. While a gene and a transcription unit (like the LAC operon) are distinguishable in prokaryotes, the two terms are used interchangeable in eukaryotes. There are simple and complex eukaryotic transcription units. A simple transcription unit RNA transcript is processed to yield a single mRNA encoding a single protein. Complex transcription units, which are more common, encode an RNA transcript that is processed to form different monocistronic mRNAs each encoding a different protein. A single transcript can undergo different mRNA pathways via:

The total mass of histones associated with DNA in chromatin is about equal to that of the DNA. Interphase chromatin and metaphase chromsomes also contain small amounts of a complex set of other proteins. For instance, a growing list of DNA-binding transcription factors have been identified associated with interphase chromatin. The structure and function of hese critical nonhistone proteins, which help regulate transcritpion, are examined in Chapter 11. Other low-abundance nonhistone proteins associated with chromatin regulate DNA replication during the eukaryotic cell cycle.

A few other nonhistone DNA-binding proteins are present in much larger amounts than the transcription or replication factors. Some exhbit high mobility during electrophoretic separation and have thus been designated high-mobility group (HMG) proteins.W hen genes encoding the most abundant HMG proteins are deleted from yeast cells, normal transcription is disturbed in most other genes examined. Some HMG proteins have been found to bind DNA cooperatively with transcription facors binding to specific DNA sequences to stabilize multiproteins complexes regulating transcription of a neighboring gene.

A mammalian chromosome is massive, with tens to hundreds of megabases of DNA. The entire haploid human genome contains about 3 billion base pairs of DNA. Only about 5% of this encodes functional RNAs or Proteins or controls their production. Eukaryotic chromosomes are bound to structural proteins to form chromatin. During metaphase, chromatin is highly condensed into the recognizable structure seen at left. During interphase, chromatin is highly decondensed so that regulatory proteins can access the DNA.

During evolution large rearrangements can occur in the size and number of chromosomes. A syntenic region contains genes that are found in the same order in different species, although not always on the same chromosome. For example, the Indian Muntjac has three large chromosomes and a tiny X chromosome; the very similar Reeves Muntjac has just as much DNA — and often in the same sequence — but divided among 23 chromosomes. These chromosomal rearrangements are rare, but are extremely important for speciation because they make productive mating impossible. The number, sizes and shapes of metaphase chromosomes constitute the karyotype (distinctive for each species). During metaphase, chromosomes are distringuished by banding patterns and chromosome painting.

The region of the chromosome where the sister chromatids are held together is called the centromere. This assembles a structure called the kinetochore that is required for attachment to microtubules during alignment at the metaphase plate, splitting of the sister chromatids, and movement to the spindle poles. Because of the nature of DNA replication, a linear chromosome requires special sequences at the ends called Telomeres. DNA replication requires an RNA primer to initiate synthesis, which is degraded after priming. The loss of these primers on the lagging strand of the chromosome ends will result in a loss of information with each round of replication. Telomerase is a special enzyme that uses its own RNA template to add telomeric repetitive DNA to chromosome ends. Chromosomes require replication origins (ARS), centromeres, and telomeres for proper replication, mitotic segregation, and maintenance. These sequences were first identified in yeast. Adding telomeric DNA to a DNA containing an ARS and Centromere allows its maintenance as a linear chromosome. Yeast artificial chromosomes containing ARS, Cen, and Tel elements allowed the cloning of large fragments of human chromosomes.

Some specialized eukaryotic cells increase cell volume via endomitosis, where DNA synthesis is repeated without cell division and a normal chromosome develops into a giant polytene chromosome results (first observed in Drosophila melanogaster larval salivary glands). Centromeres and telomeres endoreplicate poorly, leading to a bundle of duplicate chromatids (as many as 1,000) stuck at the chromocenter; thus, the ploidy of the cell remains constant.

Chromosome puffs are diffuse uncoiled regions of the polytene chromosomes where RNA transcription occurs; a large chromosome puff is a Balbiani ring. In addition to increased nucleic and cellular volume, polytene cells have metabolic advantages since multiple gene copies facilitate high levels of gene expression (which would be particularly useful in larval cells). Polytene chromosomes have very distinctive banding patterns that are useful for mapping the location of genes and observing their transcriptional activation.

To pack DNA into the tiny nucleus (the DNA packing problem), DNA is tightly wound around special proteins to form a nucleoprotein complex called chromatin. Chromatin proteins are predominantly histones, a family (H1, H2A, H2B, H3 and H4) of small proteins that are conserved in eukaryotes and contain many positively-charged basic amino acids that interact with negatively-charged DNA phosphate groups.

Condensed vs Decondensed

Actively transcribed chromatin is in 10 nm form (beads-on-a-string). This can revert to 30 nm when genes are repressed. Highly inactive chromatin, such as that containing repetitive DNA, is still further condensed around the chromosome scaffold. Histones have unstructured tails (not seen in crystal structure) that are specifically modified (including acetylation, methylation, and phosphorylation) to mediate the regulated condensation and decondensation of the chromatin.

Nonhistone proteins provide a structural scaffold for long chromatin loops. Although histones are the predominant proteins in chromosomes, nonhistone proteins are also involved in organizing chromosome structure. Electron micrographs of histone-depleted metaphase chromosomes from HeLa cells reveal long loops of DNA anchored to a chromosome scaffold composed of nonhistone proteins. This scaffold is shaped like the metaphase chromosome and persists evn when DNA is digested by nucleases. Loops of 30-nm chromatin fiber a few megabases long associate with a flexible chromsome scaffold, yielding an extended form characteristic of chromosomes during interphase. Folding of scaffold produces highly condensed structure characteristic of metaphase chromosomes. But the geometry of scaffold folding in metaphase chromosomes has not yet been determined.

In situ hybridization with different fluorescent-labeled probes to DNA in human interphase cells support loop model shown. Some probe sequences mllion of base pairs apart in linear DNA appeared reproducibly very clse in interphase nuclei from different cells. hese are postulated to lie close to specific sequences in DNA called scaffold-associated regions (SARs) or matrix-attachment regions (MARs) bound to chromosome scaffold. SARs have been mapped by digesting histone-depleted chromsoemes with restriction enzymes and recovering fragment bound to scaffold proteins. In general, SARs are found between transcription units. Genes are located primarily within chromatin loops attached at bases to chromosome scaffold. Experiments with transgenic mice indicate that in some cases, SARs are required for transcription of neigboring genes.

Individual interphase chromosmes are less condensed than metaphase chromosomes and cannot be resolved by standard microscopy or electron microscopy. Nonetheless, it is associate with extended scaffolds and is further organized into specific domains. This can be demonstrated by in site hybridization of interphase nuclei with a large mixture of fluorescent-labeled probes specific for sequences along the length of a particular chromosome. Little overlap between chromosomes in interphase nuclei. Precicse positions are not reproducible between cells.

The pre-mRNA binds many proteins during transcription. Although collectively called hnRNP proteins, they in fact belong to many different structural and functional families. Like transcription factors, RNA binding proteins can be modular and contain and RNA-binding domain and a protein-protein interacting domain.

RNA-Binding Domain

RNA-binding domains often recognize short sequences (like a base triplet) and thus have less sequence specificity than DNA-binding domains. To increase specificity, many proteins have multiple RNA-binding domains; ssRNA is flexible enough that different domains can bind the same strand. Common RNA-binding domains are shown below, in order of decreasing frequency.

III. Biosynthetic Pathways

In the 40’s Beadle and Tatum suggested the “one-gene-one-enzyme” hypothesis which explained how genes can code for a biosynthetic pathway ultimately producing a useful compound.

Rule of Thumb

• The compound that rescues most number of mutants is the last one in the pathway.
• The mutant that is rescued by most supplements is the first one in the pathway.

A B C D
Mut 1 - + + +
Mut 2 - - + +
Mut 3 - - - +

Click Χ² for information on how to use the Χ² table (Chi-Squared Table).

A mutation is any change in the nucleotide sequence or arrangement of DNA; mutations are one of only four evolutionary agents. There are three categories of mutations: genome mutations, which arise from chromosome missegregation and change how many intact chromosomes are in a cell, leading to aneuploidy; chromosome mutations, which arise from chromosome rearrangement and restructure an individual chromosome; and gene mutations, which are base pair mutations affecting an individual gene. An inherited mutation is a mutation passed on from ancestors; a de novo mutation is a new and non-inherited mutation. Disease mutations interfere with protein synthesis at one of the steps in the list to the bottom right.

Somatic mutations are not passed on to progeny and only affect some cells of the body; a tumor is a common example of a somatic mutation. Germline mutation affect germ cells (cells which differentiate into eggs or sperm) and are passed onto progeny. Many germline mutations occur either in the fertilized egg (the zygote), leading to both germ and somatic cells containing the mutation. Dynamic mutations worsen during gametogenesis, leading to a worsened phenotype in each generation. Examples of dynamic mutations include Huntington Disease and Fragile X Syndrome, which involve repeat sequences extending during gametogenesis.

Some mutations are more likely to be maternal and other mutations are more likely to be paternal. Primary oocytes develop fetally and ovulate years and decades later; the older an oocyte is, the more likely it is to undergo nondisjunction. This nondisjunction leads to trisomy (which can be viable) and monosomy (which is almost never viable). For this reason, aneuploidy is maternal in at least 80% of all cases and occurs more often with maternal age. On the other hand, spermatogenesis occurs throughout a man’s entire life. With mutations accumulating during each round of replication, point mutations are almost always paternal and increase with paternal age.

Point Mutations

Point mutations are substitutions of one base pair for another. A transition is when one pyrimidine is swapped for another — such as C for T, or T for C — or one purine is swapped for another — such as A for G, or G for A. A transversion is when a pyrimidine and a purine swap. Transitions are more frequent because when cytosines methylate to form 5-methylcytosine, they can spontaneously deaminate to thymidine.

Deletions, Insertions, Inversions & Translocations

There are deletions (removal of DNA), insertions (addition of DNA), inversions (reversal of the orientation of a DNA segment), translocations (moving of a DNA segment) and combinations thereof. Some deletions and insertions are small changes which are detectable only via PCR or genome sequencing; these usually shift the reading frame (a frameshift mutation) and lead to truncation of the mRNA by an early stop codon in the new reading frame. Some larger mutations can be detected via Southern blotting. For a mutation to be detectable by chromosome banding, it must involve at least 2 million base pairs.

Deletions, insertions, inversions and translocations often arise via faulty recombination. For example, unequal crossing over is crossing over without proper exchange of genetic information, leading to insertions in one chromosomes and deletions in another. Another form of faulty recombination is when mispaired chromosomes or sister chromatids exchange genetic information.

Mutation Nomenclature

Effect on Protein Function

Probability can be sued to predict the types fo progeny that will result from a monohybrid or dihybrid cross. The Punnett square is a graphical representation of these possible outcome. Phenotypes are the result of the genotype of an organism, more than one genotype may result in the same phenotype. Distinct segregation patterns result from monohybrid, dihybrid, and test-crosses.

Probability (expected frequency): probability of an outcome…# of times event is expected to happen/# of opportunities (trials)

The sum of all the probabilties of all possible events = 1

General rules:
Step 1: For outcome “A,” what events must happen? Multiply the probability of each event.
Step 2: Are there different ways to get outcome “A”? Calculate the probabilities of each different way and add them up.

Multiplication Rule (Product Rule). The probability of two independent events, A and B, being realized simutaneously is given by the product of their separate probability
Prob{A & B} = Prob{A}â‹…Prob{B}
The probability of indepdent events occuring together is the product of the possibilties of the individual events: p(A and B) = p(A)p(B)

If you roll two dice, what is the chance of getting two 5′s? A 5 on the 1st die and a 5 on the 2nd die? 1/6*1/6

Addition Rule (Sum Rule). The probability of the realization of one or the other of two mutually exclusive events, A and B, is the sum of their separate probability. The probability of either of two mutually exclusive events occurring is the sum of their individual probabilites.
Prob{A or B} = Prob{A}+Prob{B}

If you roll two dice, what is the probabiltiy of two 5′s or two 6′s? Probability of a 5 on 1st die and a 5 on 2nd die or a 6 on 1st die and a 6 on 2nd die.

1/36+1/36=1/18

The Punnett Square is a way of depicting the product rule. Using Mendel’s Law of segregation, we know that both alleles are equaly likely to occur. So for a cross:

1/2R 1/2 r
1/2R 1/4RR 1/4 Rr
1/2r 1/4 rr 1/4 Rr

1/4 RR 1/4 Rr 1/2 Rr

t T
t tt tT
T Tt TT

These are extension to the 3:1 Ratio, the ratio of monohybrid crosses (1 gene):

Incomplete dominance

Definition: The phenotype of the heterzygote is different from the phenotype of either of the homozygote, and the heterzygote’s phenotype lies somewhere in the range between the phenotypes of the homozygotes.

Example: Four O’Clock flower

Red x White ⇒ Pink
Pink x Pink ⇒ 1:2:1 Red:Pink:White

Instead of seeing 3:1 ratio, we observe a 1:2:1 phenotypic ratio.

(Also note that the phenotypic ratio is the same as the genotypic ratio)

Codominance

Definition: Codominance is the condition under which the heterozygote manifests phenotypically the features of two different alleles, rather than an intermediate phenotype between the homozygous dominant and homozygous recessive phenotypes.

Although there is 1:2:1 phenotypic ratio, as in incomplete dominance, the heterozygote does not represent an intermediate phenotype, but rather a phenotype in which the traits from the two different alleles coexist.

Example 1: ABO blood types. IA, IB, and IO are the alleles for a gene that produces blood antigen:

IA and IB alleles encode for antigen A and B, respectively.
IO alleles does not encode for any antigen.

An AB individual has both A and B antigen. So allele IA and IB are codominant.

Example 2: The L and M antigens are similar to the ABO system. The ability to produce M and N antigens is determined by alleles of single gene.

Homozygotes for M allele LMLM produce only M antigens.
Homozygotes for N allele LNLN produce only N antigens.
Heterozygotes LMLN produce both M and N antigens.

Question: If two AB blood type individuals mate, what type of blood will the offspring have?

Answer: There will be a 1 :2: 1 ratio, in which 1/4 of the offspring will be type A, Y2 will be type AB, and 1/4 will be type B.

AB x AB ⇒ 1A : 2AB : 1B

Multiple Alleles

They are often multiple forms of the same gene present in a population. As a diploid organism, only two of the alleles are expressed in the organism. Many genes have more than two alleles: for example: blood groups (discussed above) have 3 (A, B, O).

Example: Multiple alleles determine hair color in rabbits.

Genotype Phenotype
cc
Albino -white hair allover the body
Ch Ch Himalayan- Black hairs on the extremities and white hair on the body

Cch Cch Chinchilla- White hair with black tips over the entire body CC colored hair(wild type)

Cch and Ch show codominance: the genotype Cch Ch has white hair with black tips on the body but black hair at the extremities.

Cch and c show incomplete dominance: Crossing a chinchilla with an albino produces a rabbit with white hair with gray tips (light chinchilla). It’s genotype is Cch c.

Lethal Alleles

Some alleles turn out to be lethal if carried by individuals. Most of these lethal alleles are only lethal when homozygous. Such alleles may distort the expected 3:1 ratio a 2:1 ratio may be observed because the individuals that are homozygous for the lethal allele die.

Example: coat color in mice.
Yellow coat color (Y) is dominant over brown coat color (y). When two heterozygous (Yy) mice mate, 2/3 of the offspring are yellow (Yy) and 1/3 are brown (yy). The 2:1 ratio results because the homozygous yellow (YY) mice die.

Yy x Yy ⇒ 1 YY (dead!) : 2 Yy (yellow) : 1 yy (brown)

Question: What happens if the lethal allele is sex-linked?

Answer: The offspring will skew toward having more females than males.

The 3:1 ratio deals with one gene. The 9:3:3:1 ratio deals with two genes. However, there are many extensions to this ratio. The classical 9:3:3:1 ratio is produced when we cross two double heterozygote individuals exhibiting traits with complete dominance. The table below explains patterns which result via extensions of the 9:3:3:1 ratio.

The following questions refer to offspring ratios after the mating of individuals heterozygous for the suppressor (gene A) and heterozygous for the gene it suppresses (gene B), e.g. AaBb x AaBb

Question 1: What ratio of offspring phenotypes would you expect if suppression occurs when “gene A” possesses a dominant allele and the target gene produces a mutant phenotype when “gene B” is homozygous recessive?

A = suppressor gene: suppresses mutant pigment when genotype is AA or Aa
B = pigment gene: produces mutant pigment when genotype is bb
9 A_B_ normal
3 A_bb normal (mutant pigment is suppressed)
3 aaB_ normal
1 aabb mutant (not suppressed)

Ratio is 15 : 1 (normal : mutant).

Question 2: What ratio of offspring phenotypes would you expect if suppression occurs when “gene A” is homozygous recessive and the target gene produces a mutant phenotype when “gene B” possesses a dominant allele?

A= suppressor gene: suppresses the mutant pigment when genotype is aa
B= pigment gene: produces a mutant pigment when genotype is BB or Bb
9 A_B_ Mutant (not suppressed)
3 A_bb normal
3 aaB_ normal (mutant pigment is suppressed)
1 aabb normal
Ratio is 7 : 9 (normal : mutant).

Linkage is the tendency of genes, which are closely located on the same chromosome, to segregate together. The physical distance two genes on the chromosome is proportional to how frequently they cross over together during homologous recombination. Therefore, frequency of recombination is a tool for mapping chromosomes. Linkage analysis follows DNA regions, allowing not only mapping of a chromosome but also identification of disease genes. Association analysis is a similar approach for identifying disease genes, but instead just analyzes which alleles are present in diseased individuals throughout a population.

Loci cross over together in proportion to how near each other they are. Adjacent loci almost always cross over together; far-apart loci rarely cross over together. The proportion of recombinants to nonrecombinants is the recombination frequency, measured in Morgans and labeled θ (theta). The smaller θ gets, the closer the two loci. Recombination frequency can be used to draft a chromosome map.

A low recombination frequency (θ≈0) means two loci are tightly linked (close together and almost always assort together), while a high recombination frequency (θ≈0.5) means two loci are unlinked and could even be on different chromosomes. θ can be used to calculate the LOD score (determining whether two loci are linked) and the map distance (how far apart are two loci).

To determine recombination frequency, recombination events must be deduced. This requires that: at least one parent be heterozygous (informative); and the phase of the alleles must be known. The phase of an allele is simply whether it is on the maternal or paternal homologue. Two alleles on the same homologue are in coupling (they are cis) and two alleles on opposite homologues are in repulsion (they are trans). In a DdMm individual, D could have the same phase as either M or m; more information is needed to deduce which homologue bears which allele.

Linkage Equilibrium

Imagine that genes HAIR and BRAIN are two genes present on Chromosome 13, and that HAIR and BRAIN each have two alleles. Alleles HAIR^brown and HAIR^blonde are equally prevalent throughout the population; thus, any Chromosome 13 is equally likely to bear HAIR^brown or HAIR^blonde. However, alleles BRAIN^smart and BRAIN^dumb are not equally prevalent throughout the population. In fact, BRAIN^dumb is four times as prevalent as BRAIN^smart; thus, any given Chromosome 13 has a 20% chance of bearing BRAIN^smart and a 80% chance of bearing BRAIN^dumb.

When two alleles are in linkage equilibrium, then 20% of Chromosome 13′s bearing HAIR^blonde or HAIR^brown would also bear HAIR^smart and 80% of Chromosome 13′s bearing HAIR^blonde or HAIR^brown would also bear HAIR^dumb. When an allele is in linkage disequilibrium, then it is more likely to associate with a certain allele of a different gene. For example, although BRAIN^smart and BRAIN^dumb are present on 20% and 80% of Chromosome 13′s, a Chromosome 13 with HAIR^blonde might also bear only BRAIN^dumb alleles while HAIR^brown bears the remaining 30% BRAIN^dumb and all the 20% BRAIN^smart alleles. In this case, HAIR^blonde and BRAIN^dumb are in strong linkage disequilibrium.

For most diploid species, sex is determined by one of the homologous pair of sex chromosomes. In humans and fruit flies, the sex chromosomes are X and Y (XX=female ; XY=male). In birds, female is WZ and male is ZZ. There are many ways of determining sex:

• Sex chromosome: humans, silkworms, drosophila, nematodes.
• Ploidy: bees, wasps, and ants.
• Environment: bonellia (sea worm larvae are undifferentiated; if lands on ocean floor, becomes female; if lands on female, becomes male).

Since in humans, Y chromosome carries very little genetic information, sex-linked genes usually refer to X-linked genes (genes on X chromosome). To determine if a gene is sex-linked, you perform pedigree analysis, use Drosophila, or perform a reciprocal cross.

Also known as the testes-determining gene or sex-determining region on the Y chromosome, SRY is a gene located on the short arm of the Y chromosome and encodes a DNA-binding transcription factor known as testes-determining factor (TDF) early (and briefly) in development. Although SRY does not determine sex in all cases — it is not present in 10% of unambiguous XX males — it is a key instrument in the concert of sex determination.

The LOD score (aka Z) gives an estimation of how closely two loci are linked (for example, a marker locus and a disease locus) and quantitates sample size (data reliability). A LOD score less than 2.0 means the two loci are not linked; a LOD score between 2.0 and 3.0 is inconclusive; and a LOD score greater than 3.0 strongly indicates linkage. Lod scores are always reported in association with the recombination frequency θ (theta), measured in Morgans, which describes linkage without taking into account sample size.

Step	Description
Step 1	Look at nothing more than affected/unaffected individuals and determine the mode of inheritance.
Step 2	Cross out individuals who cannot be identified as recombinants or nonrecombinants (uninformative individuals). Persons whose parents are not shown. Individuals without at least one heterozygous parent. Children are uninformative when the child and both parents have the same haplotype. If the allele of interest is dominant, then children with a homozygous recessive parent.
Step 3	Determine whether each informative individual is recombinant or nonrecombinant. Considering an informative individual’s haplotype, is it consistent with their parent’s haplotype and the mode of inheritance? Yes, consistent with parental haplotype -> Not Recombinant. No, inconsistent with parental haplotype -> Recombinant. Remember that even unaffected individuals can be recombinant!
Step 4	Count how many recombinants and non-recombinants there are.
Step 5	If you are provided a recombination frequency (θ, aka theta) then go straight to the equation below. If you are provided the gene-marker distance (measured in centimorgans or cM) then divide that value by 100 to calculate θ. For example, a marker 10cM from a gene yields a Θ of 10/100 = .10. If you are supposed to calculate the recombination frequency (θ), then use the equation below with θ values of .001, .10, .15, .20, .25, .30, .35, .40., .45 and .50. The correct θ value will yield the highest LOD Score.

A genetically normal human has a maternal copy and a paternal copy of 23 different chromosomes. With 23 pairs of each chromosome, a genetically normal human has 46 chromosomes total. There are exceptions — for example, sperm cells are haploid and contain only one copy of each chromosome (23 chromosomes total). The number of chromosomes within a haploid cell is referred to as n. Thus, a cell with 2 copies of each chromosome (a diploid cell) has 2n chromosomes; a cell with 3 copies of each chromosome (a triploid cell) has 3n chromosomes; and a cell with 4 copies of each chromosome (a tetraploid cell) has 4n chromosomes.

Not including reproductive cells, if an individual has any other than 2n chromosomes then they are heteroploid. If a heteroploid has an exact multiple of n chromosomes (such as 69 or 92, in humans) then that individual is euploid. If a heteroploid does not have an exact multiple of n chromosomes (such as 45, 47 or any value ≠ 46) then that individual is aneuploid. Although rare, triploidy and tetraploidy are forms of euploidy. Paternal chromosome triploids (individuals with an extra copy of every paternal chromosome) have abnormal placentas and are known as partial hydatiform moles; however, maternal chromosome triploids are spontaneously aborted early during gestation.

Aneuploidy is more common than euploidy. Most aneuploidy is trisomy (three copies of an individual chromosome, instead of a pair) or monosomy (one copy of an individual chromosome). The most common trisomy is trisomy 21, which occurs in 95% of Down Syndrome patients. Chromosomes 21, 13 and 18 contain the fewest genes of any other chromosome — trisomy of any chromosome other than these three deadly. Monosomy of any chromosome except Chromosome X (a condition called Turner Syndrome) is lethal. Aneuploidy is usually caused by meiotic nondisjunction, meaning that (usually during meiosis I) chromosomes fail to disjoin properly and daughter cells either have too many or too few chromosomes.

Chromosome structure abnormalities can be either unbalanced rearrangements or balanced rearrangements. The various kinds of unbalanced rearrangements are detailed below. Unbalanced rearrangements involve deletion, duplication or both. Deletion of a chromosome segment leads to partial monosomy of that segment. Duplication of a chromosome segments leads to partial trisomy of that segment. Any mutation leading to an imbalance like monosomy, trisomy or other can result in an abnormal phenotype.

High-resolution banding can detect deletions and duplications on the magnitude of several thousand base-pairs, and FISH can detect very small or uncertain deletions.

Chromosome structure abnormalities can be either unbalanced rearrangements or balanced rearrangements. Balanced rearrangements usually do not lead to phenotypic abnormalities; all genetic information is present in the right amount, just in a different order or in the wrong location.

There are numerous diseases which arise from expansions in (CAG)_n repeats, which encode glutamine. In a coding region, a (CAG)_n repeat expansion can either inactivate a protein or exhibit a dominant phenotype due to poly-glutamine’s toxic effects; examples include Huntington Disease and Spinocerebellar Ataxia (SCA) type I. In a non-coding region, a (CAG)_n repeat expansion can interfere with chromatin structure and disrupt transcriptional regulation, and/or exhibit a dominant phenotype by distracting splicing factors with the triplet repeat; examples include Myotonic Dystrophy, Fragile X Syndrome and other SCA forms.

Patients with Chronic Myelogenous Leukemia have undergone a translocation between chromosomes 9 and 22 to form a Philadelphia chromosome (so named due to where it was discovered) containing the tip of chromosome 9 and a portion of chromosome 22, and with an activated oncogene.

Thalassemias are a heterogeneous group of hemoglobinopathies whereby a reduction in synthesis/stability of α or β globin chain causes α- or β-thalassemia, respectively. The clinical problem is excess β peptides (in α-thalassemia) or excess α peptides (in β-thalassemia); this imbalanced α:β ratio leads to early cell destruction and iron overload. The chain produced at a normal rate becomes relatively overabundant, eventually precipitating in the cell, damaging the membrane and causing premature hemolysis.

The heterozygote advantage is that thalassemias weaken red blood cells and confer some resistance to malaria. Upon infection, the cell just dies and prevents spread. Individuals that are α^-α^-/α^-α^- die in utero, although this does not include heterozygous mutations in different globin genes. Expression of the globin gene locus is managed by a locus control region (LCR).

β-thalassemia

β-globin deficiency. Mostly due to single base-pair substitutions in the β-globin genes. Much more heterogenous than α thalassemias, even in high frequency populations. Complex β thalassemias involve β globin plus one or more of the genes from the β globin cluster LCR. β globin cluster is totally dependent on its LCR for expression.

α thalassemia

Due to α-globin deficiency. Excess γ-globin chain form g4 tetramer (Hb Bart’s). Excess β-globin chains form b4 tetramer ( Hb H). These are ineffective oxygen carriers.

Nearly all disease genes have mutant alleles which affect splicing of the mRNA, rather than protein coding. Exon skipping can arise via mutations in the 5′ splice site, the 3′ splice site, and/or exonic or intronic splicing enhancers. Activation of cryptic splice sites (thus shortening the exon) can arise via mutated exonic splicing enhancers. Examples of diseases due to mRNA splicing errors include Familial Isolated Growth Hormone Deficiency (mutations in the GH-1 gene), Cystic Fibrosis (mutations in the CFTR gene), Spinal Muscular Atrophy and some thalassemias (mutations in the β-globin gene that activate a cryptic 3′ splice site).

Spinal Muscular Atrophy is the most common genetic cause of infant death, occurring in 1 in 6,000-10,000 births. This is an autosomal recessive disorder caused by mutations in the SMN1 gene, encoding a protein involved in snRNP maturation. Humans have undergone an SMN gene duplication generating the SMN2 gene. Even though it encodes an identical protein, the presence of SMN2 cannot compensate for the loss of SMN1 in spinal muscular atrophy, because its splicing is abnormal and it does not produce functional protein.

Splicing targeted therapies are being tested for Spinal Muscular Atrophy. A single C to T mutation in an exonic splicing enhancer of SMN2 exon 7 causes exon skipping and production of a truncated protein from 80% of the SMN2 mRNA. If drugs or other therapies could be found that increase exon 7 splicing of SMN2, one could potentially treat the disease even without correcting the SMN1 defect.

A hemoglobinopathy is any disorder of hemoglobin, the red blood cell protein which binds oxygen. Oxyhemoglobin has a reduced orion (does not carry oxygen); methemoglobin has an oxidized iron (carries oxygen). Methemoglobin reductase reverses the oxidation, thus unload the oxygen. Some mutant hemoglobin is resistant to methemoglobin reductase, and the heme iron thus remains permanently oxidized. Heterozygotes accumulate methemoglobin, leading to asymptomatic cyanosis (blue skin). Homozygotes are completely unable to unload oxygen and have thus not been found. Separate from reductase-resistant methemoglobin are diseases where hemoglobin has altered oxygen affinity. For example, Hb Kempsey binds oxygen very strongly and cannot unload oxygen to tissues. Encoded by an autosomal dominant, Hb Kempsey heterozygotes over-proliferate RBCs in an attempt to resolve low oxygen levels in tissues; HB Kempsey is lethal for homozygotes.

Clinically significant structural variants of normal hemoglobin are synthesized in the proper amount, but have structural defects which can cause or interfere any of the mechanisms described below. Structural variants are typically caused by point mutations, and are responsible for Sickle Cell Disease. Recall that Amino1#Amino2 means ‘Amino1′ replaced ‘Amino2′ at position ‘#’ on the polypeptide.

A hydatid cyst (also known as a mole) is a mass of placenta-derived cells resembling a grape cluster. Complete hydatidiform moles contain only paternal chromosomes but have a 46,XX karyotype. Complete hydatidiform moles (or complete moles, for short) develop into a hydatid cyst. Complete moles contain no normal placenta and no fetus. Ovarian tetranomas are cases with only maternal chromosomes which exist only as benign tumors.

Partial hydatidiform moles (or partial moles, for short) are triploids containing either an extra maternal chromosome set or an extra paternal chromosome set. Cases with an extra set of maternal chromosomes are severely retarded with small, fribrotic placentas. Cases with an extra set of paternal chromosomes have abundant trophoblast (“pre-placental”) growth and poor embryonic development.

X-Linked Severe Combined Immunodeficiency (SCID) stems from a mutation in either common γ chain gene or IL-7 receptor-α gene. A mutation in the common γ chain gene leads to an inability to bind (and hence respond) to cytokines IL-2,4,7,9,15,21. Without activity of those cytokines, the individual cannot produce T cells, B cells nor NK cells; the patient has bubble boy disease and almost no ability to fend off infection. A mutation in the IL-7 receptor-α gene leads to a lack of T cells but present B cells and NK cells. Based on this — and the paragraph above — it is clear that IL-7 is critical for T cell formation, but not B cell nor NK cell formation.

It is worth noting that mice require IL-7 for both T cell and B cell formation. SCID mice are extremely useful research tools. Lacking B and T cells, they are unable to mount an adaptive immune response. Thus, they do not reject transplanted tissues and are also useful for studying how to restore hematopoiesis. Also, SCID has been successfully treated via gene therapy techniques to introduce a functioning common γ chain gene into hematopoietic stem cells.

Down Syndrome (aka trisomy 21) is one of only three thoroughly-studied viable non-mosaic autosomal disorders (the others being trisomies 13 and 18), all of which result in stunted growth, mental retardation and multiple congenital deformities. The phenotype of Down Syndrome involves: hypotonia (low muscle tone) at birth; dysmorphic facial features; folded, low-set ears; flat nasal bridge; mental retardation, with an average adult IQ or 30-60; and, in about a third of all cases, congenital heart disease.

Down Syndrome is the most frequent and studied chromosomal disorder, and can arise in several different ways. All of these avenues somehow involve trisomy of Chromosome 21, and are detailed below.

Trisomy 18 is one of only three thoroughly-studied viable non-mosaic autosomal disorders (the others being trisomies 18 and 21), all of which result in stunted growth, mental retardation and multiple congenital deformities. Trisomy 18 frequently arises via translocation of all or most of Chromosome 18. Trisomy 18′s phenotype is mental retardation in all cases, with most cases also involving: severe heart malformation; a receded jaw (overbite); low-set malformed ears; rocker-like feet; and hypertonia, usually leading to unique clenched fists characteristic of trisomy 18.

Trisomy 13 — occurring in 1 in ∼20,000 births — is one of only three thoroughly-studied viable non-mosaic autosomal disorders (the others being trisomies 18 and 21), all of which result in stunted growth, mental retardation and multiple congenital deformities. Trisomy 13 (aka Patau Syndrome) leads to severe growth and mental retardation, usually accompanied by: a sloped forehead; malformed ears; clenched fists and rocker-bottom feet (as in trisomy 18); congenital heart defects; urogenital defects; and polycystic kidneys. Most trisomy 13 individuals gain their extra chromosome via maternal nondisjunction in meiosis I.

Many thanks to ThereseAnn, the commenter who provided incredible links to learn more about trisomy 13. I urge StudentReader.com visitors to peruse LivingWithTrisomy13.org, a site about fellow human beings living with Trisomy 13.

Sickle cell hemoglobin (HbS) results from a single nucleotide substitution which changes the codon of the sixth β-globin amino acid from glutamic acid to valine (GAG→GTG: Glu6Val) and is the causative agent of sickle cell disease when homozygous. Homozygotes have α₂^Aβ₂^S hemoglobin. Heterozygotes (said to have sickle cell trait) have α₂^Aβ₂^S (HbS), α₂^Aβ₂^A (HbA) and α₂^Aβ₂^Sβ₂^A (HbS/HbA hybrid) hemoglobin, and are clinically normal despite mild symptoms under low oxygen pressure. In Sickle Cell Disease, the HbS β chains form chains, aggregating into long fibers which deform the cell, impair its function and result in hemolysis for homozygotes.

Hyperphenylalaninemias are enzymopathies (enzyme inactivity) and aminoacidopathies (inappropriate amino acid processing) that lead to an increase in phenylalanine blood levels due to improper pheylalanine catabolism. They are all autosomal recessive and exemplified by phenylketonuria and phenylalanine hydroxylase deficiency. All hyperphenylalaninemias stem from loss of function in phenylalanine hydroxylase (PAH or PheH, a liver enzyme which catabolizes phenylalanine to tryosine) and/or genes required to synthesize its cofactor, tetrahydrobiopterin (BH₄).

Classic phenylketonuria (PKU) stems from an autosomal recessive PAH mutation that renders it inactive. Unable to degrade phenylalanine, PKU patients accumulate phenylalanine in their body fluids and the ensuing hyperphenylalaninemia damages the developing nervous system and mature brain. It is treated by complete diet restriction of phenylalanine; if no phenylalanine is ingested, then it cannot accumulate in the body. Phenylalanine hydroxylase variants are frequently benign, with many people being heterozygous at the gene encoding PAH. The few clinically significant PAH alleles can lead to near-complete PAH activity (classic PKU) or variant PKU (aka non-PKU hyperphenylalaninemia). Variant PKU occurs when PAH carries enough residual activity to lessen the disease phenotype, or prevent the disease despite elevated phenylalanine blood levels.

Rarely, a patient with hyperphenylalaninemia will have normal PAH but be unable to properly synthesize or recycle its cofactor, tetrahydrobiopterin (BH₄). Since BH₄ is necessary for the activity of other enzymes, phenylalanine restriction alone will not fully treat the patient. Fortunately, normal BH₄ can be administered in large oral doses that allow normal development and even unrestricted consumption of phenylalanine. In addition, BH₄ administration can help lower phenylalanine blood levels in patients whose PAH has a weak BH₄ affinity.

Hyperphenylalaninemias lead to mental retardation due to an imbalance of amino acids crossing the blood brain barrier. With too much phenylalanine in the bloodstream, other amino acids are outcompeted and their levels in the brain significantly decrease. This decrease in brain levels of other amino acids leads to disruption of brain development and, to a lesser extent, mature brain function. Patients with PKU are allowed slight flexibility in their diet during adulthood, as elevated phenylalanine blood levels will not have as detrimental an effect after development. However, elevated phenylalanine levels are detrimental for fetal life. Thus, females with hyperphenylalaninemia expecting to conceive must vigorously restrict their phenylalanine consumption to avoid severe developmental retardation in their offspring.

Lysosomes are membrane-bound organelles which contain hydrolases that degrade unneeded proteins and infectious bacteria. When these substrates accumulate without degradation inside the lysosome, the cells die lysosomal storage diseases (a subset of enzymopathies ensue. Affected tissues grow noticeably large, and mental dysfunction occurs in diseased brains. Tay-Sachs disease is a group of disorders where hexosaminidase A (hexA) cannot degrade the sphingolipid GM₂ ganglioside. Since GM₂ ganglioside synthesis is most prevalent in the brain, it is most severely affected by Tay-Sachs.

hexA

Normal hexA consists of α and β subunits (encoded by HEXA and HEXB) that associates with an activator protein to cleave GM₂ ganglioside. Disruption of the α subunit leads to Tay-Sachs disease; disruption of the β subunit o the activator protein leads to, respectively, Sandhoff disease and activator protein deficiency. Tay-Sachs homozygotes are clinically normal at first, but mentally deteriorate by 3-6 months of age and die by 2-4 years. HEXA alleles with some residual activity can lead to late onset, motor dysfunction, psychosis or sometimes no disease at all.

Lysosomes are membrane-bound organelles which contain hydrolases that degrade unneeded proteins and infectious bacteria. When these substrates accumulate without degradation inside the lysosome, the cells die lysosomal storage diseases (a subset of enzymopathies ensue. Hurler Syndrome is a mucopolysaccharidose, a kind of disease involving accumulation of mucopolysaccharides (or glcyosaminoglycans) within lysosomes due to deficiencies in degradative enzymes. An autosomal recessive disease, Hurler Syndrome is caused by extreme impairment of the enzyme α-L-iduronidase, leading to facial dysmorphia, developmental retardation and death by ∼10 years. In Scheie Syndrome, also autosomal recessive, α-L-iduronidase has residual activity and patients sometimes have normal life spans and normal intelligence (despite facial dysmorphia).

Hunter Syndrome is a phenotypically similar mycopolysaccharidose, but is X-linked recessive and impairs iduronate sulfatase (not α-L-iduronidase). Hurler Syndrome fibroblasts synthesize normal iduronate sulfatase, and Hunter Sydrome fibroblasts synthesize normal α-L-iduronidase. When grown together in culture, they uptake secreted proteins and thereby complement each other to produce non-diseased cells. Complementation analysis is the study of genetic complementation, where mutant gene products can complement each other to neutralize disease.

Duchenne Muscular Dystrophy (DMD) is untreatable, severe, deteriorative and relatively common X-linked recessive disorder caused by a mutation of the structural protein dystrophin. DMD causes worsening muscle weakness, with wheelchair confinement by ∼12 years and death by ∼20 years. Females very rarely have DMD, since it is lethal in males and thus cannot be passed on to XX progeny; however, females with Turner Syndrome or X chromosome translocations have been known to have DMD. About 1/3 of DMD patients have new mutations, while 2/3 have maternal inheritance of the DMD allele.

At ∼2,300 kilobases (1.5% of the X chromosome), the gene encoding dystrophin is among the largest in the animal kingdom. It contains 79 exons, and its 7 tissue-specific promoters allow differential splicing into tissue-specific and developmentally regulated isoforms. Dystrophin is a massive complex with a myriad of functions that maintain muscle integrity. Most DMD patients have deletions in either the 5′ or central regions, while most other patients have randomly distributed point mutations. Due to its sheer size, the gene for dystrophin has one of the highest frequencies of mutation.

PCR analysis of carriers and fetuses can identify deletions in dystrophin, and molecular sequencing can identify point mutations. Also, linked markers are useful for prenatal diagnosis when direct analysis does not detect a mutation. If a DMD patient is the first affected family member and a mutant dystrophin allele is not found in his mother, then most of the time it is due to a new mutation. However, ∼10% of such cases are in fact due to maternal germline mosaicism, meaning only some of her cells carry the mutant allele; this is significant due to an increased risk of recurrence.

Cystic Fibrosis (CF) is caused by an autosomal recessive mutation of the cystic fibrosis transmembrane regulator gene (CFTR), which was identified via positional cloning. Cystic fibrosis is due to abnormal fluid and electrolyte transport across epithelial apical (pointed) membranes. This leads to disease of the lung, pancrea, intestine, hepatobiliary tree and male genital tract. Noticeable when kissing a baby with cystic fibrosis, loss of CFTR activity leads to chloride and sodium build-up in sweat glands. Also, the mucous layer of the lung becomes ‘sticky’ and traps pathogens, leading to chronic pulmonary infections..

The first sign of cystic fibrosis is usually high concentration of sodium and chloride in sweat. The average patient survives to ∼33 years, eventually succumbing to chronic lung and heart infections, and maldigestion due to a deficiency of pancreatic enzymes. Many males are infertile due to absence of a vas deferens; most females have near-normal fertility. Postnatal lower intestinal tract obstruction also may occur, treatable by surgery. Some individuals with mutations in the CFTR may have just one or all of these phenotypes.

CFTR

Present on Chromosome 7 (7q31, to be precise), the CFTR gene is ∼190kB and encodes the large 170kD cystic fibrosis transmembrane conductance regulator (CFTR) protein. Likely an ABC transporter, it contains a chloride channel with five domains: two membrane-spanning domains, each of which contain six transmembrane sequences; two nucleotide-binding domains which bind and hydrolyze ATP to power opening and closing the channel; and a regulatory domain, mediated via phosphorylation.

Approximately 2% of white (European) populations carry a cystic fibrosis disease allele, although carriers are almost nonexistent in other populations. The most frequent CFTR defect is δF508, a missense mutation that reduces ATP binding and accounts for 70% of all CF alleles in white populations. Patients homozygous for δF508, or with premature stop codons in CFTR all have insufficient pancreatic activity, but differing pulmonary (lung) disease. Two alleles of a modifier gene encoding transforming growth factor β1 (TGFβ1) are frequent in cystic fibrosis patients with severe pulmonary disease. Also, a cystic fibrosis genocopy (aka phenocopy — an identical phenotype for different genetic reasons) is a mutation in SCNN1, which encodes an epithelial sodium channel. With ∼85% of CFTR disease alleles identified, DNA diagnosis is useful prenatally and to identify carriers amongst relatives of a patient with no familial history of cystic fibrosis. However, the only current treatments for cystic fibrosis are antibiotics, exercise and a nutritious diet.

Fragile X Syndrome is an X-linked mutation in FMR1, which encodes an abundant neuron protein named FMRP. Almost every FMR1 mutation is an expansion of the CGG-repeat sequence in the 5′ untranslated region of the gene. Normal individuals between 6 and 50 CGG repeats, while diseased patients have over 200 repeats. So many repeats does not happen within one generation; it takes several expansions (premutations) over several generations to acquire hundreds of CGG repeats (full mutation). Having over 200 CGG repeats leads to hypermethylation of the repeat region and the adjacent FMR1 promoter, leading to obliteration of FMRP expression. Mental retardation ensues, with a more severe phenotype in males than females. AGG triplets nestled between CGG repeats inhibit AGG expansion; thus, individuals with reduced AGG triplets are predisposed to become carriers of a diseased FMR1 allele.

Almost all males and ∼50% of females with a disease allele will have the Fragile X phenotype. Some patients have a mixture of premutant and full mutant cells (repeat length mosaicism) due to a worsening genotype with every round of mitosis. Patients with over 200 CGG repeats but lacking methylation in some of their cells (repeat methylation mosaicism) or none of their cells range from normal to fully affected. When analyzing a pedigree, increasing prevalence of the disease in successive generations and inheritance from unaffected mothers are both signs of fragile X syndrome.

Huntington Disease (HD) is an autosomal dominant, late-onset, progressively neurodegenerative disorder caused by mutation of the ubiquitously expressed HD gene. Normal HD alleles have 10 to 26 CAG repeats, while disease HD alleles typically contain more than 36 CAG repeats; 97% of disease alleles are inherited, while 3% of disease alleles are new mutations arising from inherited premutations of 20 to 35 repeats. The function of huntingtin — the gene product of HD — is unclear, and it is also unknown how huntingtin encoded by a disease allele leads to the Huntington Disease phenotype.

Age of onset is inversely proportional to the number of CAG repeats in the HD allele. Juvenile onset ensues from over 60 CAG repeats, adult onset ensues from 40-55 CAG repeats and reduced penetrance (possibly no disease phenotype) ensues from 36-41 CAG repeats. Alleles with more than 36 repeats expand during paternal transmission, with somewhat less expansion during maternal transmission. The phenotype includes cognitive loss, behavioral instability and even schizophrenia. As death approaches, motor skills fail such that incontinence and mutism develops; however, behavioral disturbances lessen. The only current treatment for Huntington Disease is pharmacologic treatment of behavioral instability, and lots of support.

A homeodomain is a highly conserved protein domain (first identified in Antp and Ubx proteins) containing 60 amino acids that fold into three conserved DNA-binding helics (indicating a role in transcriptional reglation). Many homeodomain proteins have been shown in vitro to bind to regulatory regions of genes known to regulate by genetic criteria. The DNA sequence encoding a homeodomain is a homeobox.

Specific and highly conserved residues in the homeodomain’s third helix (helix 3) bind the major groove of DNA, meaning all homeodomain proteins bind the same DNA sequence in vitro. Specific homeodomain proteins have different regulatory properties via interaction with different co-factors; the co-factor and the homeodomain protein bind a specific DNA sequence together, but are unable to bind it individually.

Patients with two defective retinoblastoma (Rb) alleles invariably develop retinoblatomas, leading to its discovery as a tumor suppressor. E2F is a key host cell transcription factor in activating cellular genes required for synthesis of dNTPs, DNA polymerases, and other proteins required for cells to begin S-phase. Also, E2F binds and activates transcription from the adenovirus E2 promoter. The E2 region encodes viral proteins required for adenovirus DNA replication. In uninfected cells, retinoblastoma (aka Rb) is bound to E2F and inactivates it; in infected cells, Rb is not bound to E2F — the adenovirus protein E1A binds to retinoblastoma and removes it from E2F. Since E1A deactivates retinoblastoma by binding it and displacing it from E2F, it accomplishes the equivalent of mutations in both retinoblastoma alleles: there is no functional cellular retinoblastoma. SV40 and polyomavirus Large T-antigen also binds Rb; mutations of SV40 and polyomavirus Large-T that did not bind retinoblastoma did not transform cells. p110 is another tumor suppressor that was found to be absolutely identical to retinoblastoma.

The most important message of the Hardy–Weinberg equilibrium is that allele frequencies remain the same from generation to generation unless some agent acts to change them. With that in mind, the Hardy–Weinberg equilibrium allows scientists to determine whether evolutionary agents are operating and their identity (as evidenced by the pattern of deviation from the equilibrium). The equilibrium also shows the distribution of genotypes that would be expected for a population at genetic equilibrium.

No real-life population is ever at Hardy-Weinberg equilibrium, but fortunately aberrations are usually rare enough that it can be assumed the population is at Hardy-Weinberg equilibrium. The five requirements for Hard-Weinberg are:

1. Population size is very large.
2. There is no migration between populations.
3. There is no mutation.
4. Mating is random.
5. Natural selection does not affect the alleles under consideration.

If the conditions of the Hardy–Weinberg equilibrium are met, then the frequencies of alleles at a locus remain constant from generation to generation, and after one the genotype frequencies will not change after one generation of random mating. If p and q represent the frequencies of the dominant and recessive alleles at a locus, then p² and q² are the frequencies of the homozygous genotypes and 2pq (or pq+qp) is the the frequency of the heterozygous genotype. These can be related by the Hardy-Weinberg equation:

p² + 2pq + q² = 1

Below are examples of different types of DNA-binding proteins. The most common and best studied DNA-binding proteins are the Zinc finger proteins, the Helix-turn-helix proteins, and the Leucine zipper proteins.