Genetics

Which sex is disproportionately affected with X-linked dominant inheritance?

Females, no male-to-male transmission but daughters can get mutant from mom or dad

Proband

First affected family member who seeks medical attention for a genetic disorder.

Why are only a small fraction of the hundreds of thousands of potential binding sites used?

First, the binding site must be accessible (in a relaxed open chromatin conformation and away from direct contact with nucleosomes). Secondly, binding is combinatorial—different transcription factors work in concert by binding to adjacent recognition sequences.

Fluorescence in situ hybridization

Fluorescent DNA or RNA probe binds specific gene. Localize genes and direct visualization of anomalies at molecular level. Fluorescence = gene present. Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections. Identifies chromosomal abnormalities. Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination.

How to observe transcriptome?

Focussed Experimental Approaches: -Northern Blotting Analysis -Real time PCR (quantitative or semi-quantitative) Highthroughput Approaches: -Closed System Profiling: Microarray expression profiling -Open System Profiling: Serial analysis of gene expression (SAGE) Massively Parallel Signature Sequencing(MPSS)

miRNA

(microRNA) about 20 nucleotides long, a small, single-stranded RNA molecule, generated from a hairpin structure on a precursor RNA transcribed from a particular gene, it associates with one or more proteins in a complex that can degrade or prevent translation of an mRNA with a complementary sequence, up to 1/3 of all human genes may be regulated by miRNAs

Transposon

(or transposable element) is a small piece of DNA that inserts itself into another place in the genome.

What do most hybridization reactions require?

(synthetic) Oligonucleotide probe

Satellite DNA

(variable in length, located at centromeres and heterochromatic regions). occur about every 30kb of DNA, usually in euchromatin, and are highly variable in # of repeats make good genetic mapping markers.

Non-penetrance in an autosomal dominant disorder

* are asymptomatic disease gene carriers. Note that if the mutant allele came from their father (*) vs from their mother the pattern is different. This is due to epigenetics (which is covered in the next lecture) in a process called imprinting that may result in a different phenotype based on parent of origin

What are uses of FISH?

-Locate chromosomes throughout the cell cycle -Identify relative sizes of all chromsomes -Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections -Identifies chromosomal abnormalities -Standard FISH (homogenous DNA probe = fragment) or chromsomal painting (heterogenous DNA probe = different types of DNA fragment) -Isolation and labeling of whole-genomic control DNA (rhodamine) and whole-genomic cancer DNA (FITC), hybridization of differentially labeled whole-genomic probes to normal metaphase chrosomomes, loss of DNA shifts color region to red and addition of DNA shifts color region to green -Detection of high concentrations of base pairs -Special telomeric probes specific to individual chromosomes have been designed (Probe is based on the TTAGGG repeat present on all human telomeres) -Detect gene fusion

What percent of genes is coding?

1.5%

Minisatellite

100 bp to 20 Kb, found in telomeres and subtelomeric regions

Alu repeats

100-300 bp sequences that are dispersed in over 10^6 copies in human DNA, and are inserted into other genomic sites, but have an internal Pol III RNA promoter (similar to tRNAs) and is transcriptionally active to produce further tRNAs to undergo reverse transcription, allowing exponential expansion; sometimes called "selfish DNA".

Solenoid

146 bp of DNA are wrapped approximately 1.65 times around a histone octamer (each histone consists of 2 H2A, H2B dimers, and H3, H4 tetramer) which together are called a nucleosome.

How many genes do we have?

20,000-22,000

How many genes are on mitochondria?

37

Sequence studies reveal how many SNPs?

38 million. Most genetic variation has neutral effect on phenotype

Tertiary structure

3D shape of single polypeptide using hydrogen, hydrophobic forces, ionic, covalent, disulfide bonding The third level of protein structure; the overall, three-dimensional shape of a polypeptide due to interactions of the R groups of the amino acids making up the chain. Globular, rod-like, tube, coil, sheet

Loss of a chromosome is lethal except in which case?

45X, Turner syndrome

What is the max cloning capacity of a plasmid?

5 - 10 kb

What percent of genome is subject to evolution?

5%

Oligonucleotide hybridization analysis of short sequences

50 nt can be set up so that only perfectly matched hybrids form, a single mismatch will not hybridize and therefore reveal an SNP.

What percent of the genome is repeats?

50%

What is the max cloning capacity of a YAC?

>1 Mb

Consequences of repair failure?

>170 human genes are involved in repair; mutations in those genes correlate with a number of genetic disorders. With time & age, we acquire more mutations some of which will generate greater susceptibility to a variety of diseases including cancer. Repair defects lead to more chromosome aberrations e.g. ataxia telangiectasia, Fanconi anemia (any 1 of 13 different genes involved in cross link repair)

Zinc finger

Frequently found in regulatory proteins in eukaryotes, binds a zinc ion. Part of the finger of aminos acids that is created forms alpha helix. And this recognition helix interats with DNA in the major group. Usually atleast 2 or 3 zinc fingers on proteins that use them for DNA binding.

Enhancer

A DNA sequence that recognizes certain transcription factors that can stimulate transcription of nearby genes. A DNA sequence that controls the efficiency and rate of transcription of a specific promoter. Can be upstream or downstream from promoter. Enhancer may be hundreds or thousands of bases distant from the promoter, and come into contact with the transcription factors by DNA looping.

Single Nucleotide Polymorphism

A DNA sequence variation occurring commonly within a population (e.g. 1%) in which a single nucleotide — A, T, C or G — in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes. Definition: "SNPs" are the variation at one base pair within a coding or noncoding sequence.

Signal Recognition Particle

A RNA protein complex that binds to the ER signal sequence in polypeptide as it emerges from a ribosome and transports the ribosome-polypeptide complex to the ER membrane where synthesis of the polypeptide is completed. The ribosome + signal sequence + SRP complex then attaches to an SRP receptor in the ER membrane itself.

Genetic Drift

A change in the allele frequency of a population as a result of chance events rather than natural selection. Bottleneck and Founder Effect

Mutation

A change in the order of the bases in an organism's DNA; deletion, insertion, or substitution. DNA replication errors of mismatch or replication slippage Chromosome segregation and recombination errors.

Microsatellites (Simple Sequence Repeats)

A class of genetic polymorphism commonly used for mapping, linkage analysis and to trace inheritance patterns. Microsatellites are tandemly repeated sequences, where the repeating unit is 1 to 4 nucleotides long. The majority of microsatellites occur in gene introns or other noncoding regions of the genome.

NimbleGen sequence capture system

A commercial system using DNA microarrays to permit hybridization mediated enrichment of desired DNA sequences within a complex starting DNA such as human genomic DNA. Linker oligos attached at the ends of the fragments allow the fragments to be eluted from the microarray and amplified and sequenced. Genome wide capture of all exomes is possible (~180,000)

Pedigree

A diagram that shows the occurrence of a genetic trait in several generations of a family

Gene

A discrete unit of hereditary information consisting of a specific nucleotide sequence in DNA (or RNA, in some viruses). Most of the genes of a eukaryote are located in its chromosomal DNA; a few are carried by the DNA of mitochondria and chloroplasts.

Huntington's Disease

A fatal hereditary disorder caused by accumulation of the protein huntingtin that leads to degeneration of the basal nuclei and cerebral cortex. The allele for this disease contains a long string of bases in which the codon CAG—coding for the amino acid glutamine—repeats over and over again, more than 40 times. The greater the number of codon repeats, the earlier the disease appears, and the more severe are its symptoms.

Manifesting heterozygote

A female carrier of x-linked recessive gene who expresses the phenotype b/c the normal allele is inactivated in some tissues

Turner's syndrome

A genetic defect in which affected women have only one X chromosome, causing developmental abnormalities and infertility.

Haplotype

A group of alleles of different genes on a single chromosome that are closely enough linked to be inherited usually as a unit

Epimutation

A heritable change in gene expression that is not accompanied by a change in DNA sequence.

Sickle Cell Disease

A human genetic disease caused by a recessive allele that results in the substitution of a single amino acid in the hemoglobin protein; characterized by deformed red blood cells that can lead to numerous symptoms.This disorder is caused by a defective allele for beta-globin, one of two polypeptides in hemoglobin, the oxygen-carrying protein in red blood cells.

Cystic Fibrosis

A human genetic disorder caused by a recessive allele for a chloride channel protein; characterized by an excessive secretion of mucus and consequent vulnerability to infection; fatal if untreated. Most cases result from the deletion of just three bases in the gene for a protein called cystic fibrosis transmembrane conductance regulator (CFTR). As a result, the amino acid phenylalanine is missing from the protein

Run-off transcription

A linear template (e.g. plasmid with inserted gene of interest, blue) at a restriction site about 200 bp downstream from transcription start is added to a nuclear extract along with radioactive nucleoside triphosphates. RNA polymerase is able to activate at the start site (red arrow). The length of the radioactive RNA product (green) is determined by gel electrophoresis. Knowing the position of the restriction site at the 3' end of the modified gene insert allows the run-off transcript size to locate the transcription start site.

Variable Number Tandem Repeat (or VNTR)

A location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals.

Whole genome shotgun

A method for determining the DNA sequence of an entire genome. After a genome is cut into small fragments, each fragment is sequenced and then placed in the proper order.

What is the difference between thymine and uracil?

A methyl group on 5' carbon

Nonsense mutation

A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.

Synonymous mutation

A mutation that does not result in a different amino acid

Penetrance

A mutation that fails to manifest the phenotype shows a lack of penetrance. Seen in late-onset diseases

RNA polymerase I

A nucleolar RNA polymerase, RNA polymerase I, is dedicated to making three of the four different ribosomal RNAs (rRNAs) in our cyto-plasmic ribosomes (the 28S, 18S, and 5.8S rRNAs).

Nucleoside

A nucleoside is a nucleobase linked to a sugar, a nucleotide is composed of a nucleoside and one or more phosphate groups. Thus, nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH) to produce nucleotides.

Nucleosome

A nucleosome is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores.

Genetic anticipation

A phenomenon where the onset of symptoms of a hereditary disease appears to occur at progressively earlier ages in successive generations within a particular lineage

DNA Vector

A plasmid or viral genome where a desired gene is inserted. The vector inserts the DNA into a new cell so that a clone may be grown. Large amounts of gene product can be harvested from the clones.

Missense mutation

A point mutation in which a codon that specifies an amino acid is mutated into a codon that specifies a different amino acid.

Human Genome Project

A project coordinated by the National Institutes of Health (NIH) and the Department of Energy (DOE) to determine the entire nucleotide sequence of the human chromosomes

TATA box

A promoter DNA sequence crucial in forming the transcription initiation complex.

Telomere

A region of repetitive nucleotide sequences at each end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes.

Transcription factor

A regulatory protein that binds to DNA and affects transcription of specific genes.

Dicer

A ribonuclease that cleaves double-stranded RNA molecules into fragments approximately 20 to 25 nucleotides long that ultimately interfere with the expression of the corresponding gene.

Recombinant DNA

A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from two different sources. Recombinant DNA represents two sources of DNA each cut with a restriction enzyme or two and recombined at the cut sites. Some product is reannealed DNA, the recombined is the desired product...to be selected and amplified.

Enhancer

A segment of eukaryotic DNA containing multiple control elements, usually located far from the gene whose transcription it regulates. A DNA sequence that recognizes certain transcription factors that can stimulate transcription of nearby genes

Centromere

A specialized condensed region of each chromosome that appears during mitosis where the chromatids are held together to form an X shape.

Five-prime cap

A specially altered nucleotide on the 5′ end of some eukaryotic primary transcripts such as precursor messenger RNA. This process, known as mRNA capping, is highly regulated and vital in the creation of stable and mature messenger RNA able to undergo translation during protein synthesis. Mitochondrial[1] and chloroplast mRNA[2] are not capped.

Promoter

A specific nucleotide sequence in DNA that binds RNA polymerase, positioning it to start transcribing RNA at the appropriate place.

Locus

A specific place along the length of a chromosome where a given gene is located.

Southern blot

A technique that enables specific nucleotide sequences to be detected in a sample of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe. Looking for medium sized changes (hundreds to several kilobases) in genes/DNA in test sample.

childhood B-cell acute lymphocytic leukemia

A translocation between chromosomes 12 and 21 with breakpoints at bands 12p13 (TEL)[telomere] and 21q22 (AML1) occurs in at least 25 percent

Hybrid gene

A translocation such as the Philadelphia chromosome results in a ________ which causes leukemia.

Epistasis

A type of gene interaction in which one gene alters the phenotypic effects of another gene that is independently inherited.

Amplification

A usually massive replication of genetic material and especially of a gene or DNA sequence.

Wobble base pair

A wobble base pair is a pairing between two nucleotides in RNA molecules, that does not follow Watson-Crick base pair rules.[1] The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C)

Male lethality in a X linked dominant trait

Absence of a normal allele is lethal before birth and therefore none of the males with the gene are born. The trait affects females and they pass it on to half their daughters but "none of their sons."

Why are transposons important?

Accounts for interspersed highly repetitive DNA sequences that increase gene novelty in a variety of ways including forming novel exons and regulatory sequences and mediating exon shuffling between genes.

Mismatch repair

After the new DNA has been replicated, this mechanism scans over the DNA to ind any mismatches of bases. It mismatches are found, it repairs the mismatch by removing the incorrect base and replacing it with the proper one. Certain forms of cancer arise because of failure in mismatch repair, because if a mutation is able to pass through the cracks of this system, the DNA sequence from then on is altered.

Allele Frequency

All alleles possess a frequency that is between 0.0 and 1.0

What is the stringency?

Amount of fit between two sequences in a hybridization reaction

Branch site

An additional control element, the branch site, is located very close to the splice acceptor; it contains an invariant A nucleotide and is responsible for initiating the splicing reaction.

Allele

An allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous. Though the term "allele" was originally used to describe variation among genes, it now also refers to variation among non-coding DNA sequences.

Nondisjunction

An error in meiosis or mitosis in which members of a pair of homologous chromosomes or a pair of sister chromatids fail to separate properly from each other.

Kindred

An extended family covering many generations

Compound heterozygote

An individual bearing two different pathogenic mutations in the same gene that together are sufficient to manifest a recessive phenotype.

Compound heterozygotes

An individual bearing two different pathogenic mutations in the same gene that together are sufficient to manifest a recessive phenotype.

Genetic mosaic

An individual with somatic regions that are genetically different from each other

What happens to TF during elongation?

As elongation continues, many of the transcription factors are detached from the promoters and elongation factors are added. Speed is about 20 nt/sec.

Variable Expression

As with non-penetrance, variable expression is seen in dominant conditions (Waardenburg syndrome) where different family members have different symptoms

Which amino acids have amide groups?

Asparagine and Glutamine

What are the acidic amino acids?

Aspartic acid and Glutamic acid

What is hybridization?

Association between two complementary NA strands = annealing or hybridization

Imprinting

Autosomal dominant characters affect both sexes but may manifest only when inherited from a parent of one particular sex. Glomus body tumors occur in men or women who inherit the trait from their father. Beckwith-Wiedemann syndrome has the opposite legacy: this congenital abdominal wall defect, oversized tongue, and excessive birth weight is expressed only in babies who inherit it from their mother. At a single locus, only 1 allele is active. The other is inactive (via methylation). Deletion of the active allele = disease

Huntington disease

Autosomal dominant single gene disorder with mutant alleles producing an abnormal protein especially toxic to neurons. Neuron loss leads to dementia, mid to late adult life onset is more common but onset can occur in teens. In the case of Huntington disease age of onset is linked with number of trinucleotdie repeats within the gene.

U2 snRNA

Binds to U1 and recognizes the branch point

Aberrant methylation

C in CpG when methylated silence transcription; A + CH3 distorts DNA blocking protein:DNA interactions

Eukaryotic mRNA degradation

Cap prevents 5'-3' exonuclease attack and poly A prevents 3'-5' exonuclease attack. Destabilizing elements within sequence often near 3' end cause cleavage, loss of 3' poly A and turn over of mRNA. Within coding region, mutations may create termination codons and trigger surveillance system that degrades mRNA. AUUUA (ARE) upstream of poly A tail attracts a protein which when bound to the mRNA generates cleavage and turnover of mRNA.Deadenylation allows decapping to occur and then endonucleolytic turnover.

Facultative heterochromatin

Changes from condensed and inactive to open and active. X inactivation and transition of globin during development are examples.

Chimeras

Chimeras are the result of fusion of two zygotes to form a single embryo (reverse of twinning)

Chromatin

Chromatin is a complex of macromolecules found in cells, consisting of DNA, protein and RNA. The primary functions of chromatin are 1) to package DNA into a smaller volume to fit in the cell, 2) to reinforce the DNA macromolecule to allow mitosis, 3) to prevent DNA damage, and 4) to control gene expression and DNA replication. The primary protein components of chromatin are histones that compact the DNA. Chromatin is only found in eukaryotic cells (cells with defined nuclei).

Homolog

Chromosomes that have the same sequence of genes, that have the same structure, and that pair during meiosis. Humans.

Constitutive heterochromatin

Consists of highly repetitive DNA, structural areas, centromere and telomeres and constricted regions

Which amino acid has a sulfhydryl group?

Cysteine

HR and Fanconi anemia DNA repair pathway

DNA interstrand cross-links, some uncertainty about mechanism. Cross linking between bases on complementary strands of DNA occur when chemicals of exogenous or endogenous origin. Cisplatin, causes interstrand cross linking & exposure to UV radiation causes intrastrand crosslinks (T-T)

How does DNA methylation work?

DNA methylation involves adding a methyl group to certain cytosine residues, forming 5-methylcytosine (5-meC). The cytosines that are methylated occur within the context of a palindromic sequence, the CG dinucleotide (also called a CpG dinucleotide, where p represents phosphate). The 5-meC base pairs normally with guanine (the methyl group is located on the outside of the DNA double helix, minimizing any effect on base pairing). It is recognized by specific 5-meCG-binding proteins that regu-late chromatin structure and gene expression.

What dictates changes in chromatin structure?

DNA methylation, histone modification, positioning of nucelosomes, noncoding RNAs

What do genetic controls depend on?

DNA sequence

Next Generation Sequencing

DNA sequence is recorded while being synthesized. Massive parallel DNA sequencing is indiscriminate: all the different fragments in a complex sample can be simultaneously sequenced without need for gel electrophoresis, therefore it is faster and the output is much larger.

Allele

DNA sequence variant, all DNA sequences, not just genes, can have more than one allele.

Analytical methods to discover novel SNPs and detect known SNPs?

DNA sequencing capillary electrophoresis mass spectrometry single-strand conformation polymorphism (SSCP) electrochemical analysis denaturating HPLC and gel electrophoresis restriction fragment length polymorphism hybridization analysis

Leucine zipper

DNA-binding motif found in many transcription factors; formed by an interaction between a helices in two polypeptide chains that are "zippered" together by hydrophobic interactions between leucine residues.

Why C-T mutations are common

Deamination of C produces U seen as foreign base in DNA, cut out by uracil DNA glycosylase. BUT---in 5meC deamination, C is converted to T.

Hydrolytic damage

Depurination, depyrimidation, deamination, hydrolysis between sugar and base (depurination), loss of NH group (deamination) e.g. C->U

Detecting hybridization by dye-quenching

End labeling with fluorescent label on end and quenching compound on the other will "block the signal" until the hybrid forms and releases the hairpin and the fluorescent signal is restored.

Why is gain/loss usually lethal?

Gene dosage, too little or too much protein expressed

Gene duplication

Gene duplication offers possibility of different forms of a protein.

What are the nonpolar amino acids?

Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Phenylalanine, Tryptophan

What genes are on the Y chromosome?

Have male-specific functions or have equivalent gene copy on X (pseudoautosomal region)

Disease related phenotypes in carriers

Heterozygotes for sickle cell disease have some sickle shaped cells (mutated beta globin co-dominantly expressed) and experience anemia

Autosomal Recessive

Heterozygotes often unaffected or asymptomatic carriers If both parents are carriers, chance of future child being affected is 25% or ¼ (1/2 x ½ = ¼)

PAX6

Heterozygous loss-of-function mutations in PAX6 (ch11-15) cause aniridia (absence of iris of the eye). Patients with aniridia may not have a mutation within the coding sequence of PAX6, but have translocations with breakpoints up to 125 kb downstream of the last PAX6 exon. Experiments with transgenic mice reveal a cluster of regulatory sequences in the downstream region.

What are the basic amino acids?

Histidine, Arginine and Lysine

Which mRNAs are not polyadenylated?

Histone. One alternative is a stem loop structure with 6 bp stem and 4 nt loop with a conserved 9 nt sequence CAAGAAAGA about 12 nt downstream of the hairpin. The conserved sequence is complementary to part of U7-snRNA. How and what enzyme cuts the RNA is under study.

Ortholog

Homologous genes that are found in different species because of speciation.

Y RNA

Human Y RNAs are functionally required for DNA replication.

Chromosome conformation capture

Identify DNA sequences that may be separated in the genome but are physically close within the nucleus due to folding of chromosomes and positions of neighboring chromosomes. Crosslinking captures these interacting sequences containing a DNA fragment from each of the parts. Restriction digestion of the two will have sticky ends and ligase produces the ends of two different regions of DNA.

Pyrosequencing

Immobilized DNA template strand with detectable pyrophosphate groups that get released as polymerase adds nucleotides. Each pyrophosphate group indicates which nucleotide was just added, and by the intensity, you can see whether one or more bases were just added. It's a real-time method.

Structural Variation

In addition to the SNP and VNTR variations, there are rearrangements, translocations, balanced and unbalanced that occur at chromosomal level.

RNA editing?

In some RNA transcripts, certain nucleotides naturally undergo deamination or transamination. When this happens in the coding sequences of mRNAs, the amino acid sequence of the protein will differ from that predicted by the genomic DNA sequence. eg adenine to inosine (acts like guanosine by base pairing with cytosine) occurs in CAG to give CIG which acts like CGG to make glutamine to arginine = Q/R editing, mRNAs that make neurotransmitter receptors or ion channels. Some other types of RNA editing are known, including C → U editing (used in making apolipoprotein B mRNA, for example) and U → C editing (used in making mRNA from the WT1 Wilms tumor gene).

Chromosome

In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure.

What is the most important cause of deviation between gene frequencies and genotype frequencies?

Inbreeding

How does alternative splicing work?

Include intron, exon skipping, intron-exon junction changes (changing length of exon). Produces alternative forms (isoforms) from individual genes

Nucleotide excision repair

Includes the removal and re-synthesis of several nucleotides spanning the altered site. Helicase unwinds 30nt stretch, excision nuclease cuts them out, excised oligonucleotide degraded, DNA polymerase and ligase repair DNA. Helix-distorting DNA lesions (large DNA adducts, DNA intrastrand crosslinks) repair of bulky, helix distorting DNA lesions, once detected the site is opened, DNA cut on both sides of the lesion, and gap resynthesized. Transcription-coupled repair is triggered by transcription complex being stalled at a damaged site. Genome wide repair depends on another pathway: global genome NER pathway.

Epigenetic mechanisms

Independent of DNA sequence. Can bring about differential changes in chromatin structure DNA methylation, histone modifications, microRNAs, genomic imprinting.

Why is CF allele still in population?

Individuals heterozygous for CF would have had an advantage when living in cities with poor sanitation and polluted water (typhoid), and—because they also carried a normal allele—these individuals would not have suffered from cystic fibrosis.

Why is Sickle Cell allele still in the population?

Individuals with just one copy of the sickle cell allele are generally healthy, and are also highly resistant to the parasite, giving them a great advantage against malaria.

What are the 3 stages of translation?

Initiation, elongation, termination

What are the three stages of transcription?

Initiation, elongation, termination

Hierarchical shotgun

Isolate the chromosomal DNA, clone large fragments into BACs or YACs and create a contig, shear into smaller pieces and clone into vectors, and based on overlapping regions create one contiguous sequence.

What is hybridization used for?

It is used to find sequences in test DNA. Using a defined probe of NA sequence and a test sample of DNA hybridization allows complementary fragments to bind (re-anneal or form a hybrid with a test sequence). Use of solid supports to capture labeled probe-test sample duplexes. The probe is bound to the matrix and denatured and then exposed to test sample also denatured.

What are the 3 classes of transposons?

LINES, SINES, retrovirus-like elements

Primer extension

Label DNA primer ~ 18 nt with sequence id to region about 50-150 nt downstream of transcription start is synthesized and annealed to specific mRNA in a mixture of cellular mRNAs. The primer is extended to the RNA template's 5' end by RT and the DNA/RNA hybrid is denatured. The size of the DNA product is determined by gel electrophoresis. The location of the initiation site can be determined by comparing the mobility of the extended primer with that of products produced by dideoxynucleotide DNA sequencing method using the same primer.

S1 mapping

Labeled DNA probe is added to mixture of RNA extracted from cells and allows probe to anneal with complementary RNA to produce hybrid with 3' poly nt overhang at one end and 3' polydeoxy nt overhang at the other. Then S1 nuclease is added to remove the SS overhangs. The length of the SS DNA is determined by gel electrophorsis & represents the distance from the 5' probe to the transcription start site.

Elongation

Large subunit joining the initiation complex in which the P (peptidyl site) has tRNA-met in position, the second site, A or aminoacyl site covers the second codon in the ORF. eEF-1 has 4 subunits, a, b, d, and g. The subunits within the rRNA at the A site ensure correct tRNA acceptance. Once both tRNAs are in place with their linked amino acid, the peptidyl transferase (a ribozyme) forms the peptide bond, and requires hydrolysis of GTP. Translocation allows advancement of the mRNA another codon forward moving the dipeptide - tRNA to P and opening A for the incoming tRNA-aa. This too requires a GTP.

How do TFs bind DNA?

Like other DNA-binding proteins, a transcription factor typically recognizes a specific short sequence (often four to nine nucleotides long) using a DNA-binding domain that contains some motifs that physically bind DNA, such as zinc fingers or leucine zippers

mRNA surveillance

Locates incorrect termination codons

What are the three levels of heterogeneity?

Locus heterogeneity (eg deaf) Allelic and phenotypic heterogeneity

LINES

Long interspersed nuclear elements, Repeated DNA sequences of 6000 to 8000 base pairs in length that alternate with lengths of DNA sequences found in the genomes of higher organisms

How are males/females both hemizygous for certain genes on X chromosome only?

Males constitutionally hemizygous, females functionally hemizygous

X-linked recessive disorder affects which sex more?

Males, mother is a carrier and no male-to-male transmission because father only contributes Y

Allelic heterogeneity

Many different mutations within a given gene are found in different patients with a common clinical condition. Some phenotypes/traits/ diseases have both locus and allelic heterogeneity. Ex: Beta-thalassemia is a deficiency of beta-globin which can be caused by many different mutations in the globin gene.

VNTR: variable number of tandem repeat polymorphism

Microsatellites, minisatellites, satellite DNA

What happens if child has 2 copies of mutant allele?

More severe phenotypic effects or earlier onset

How does a mutation affect cis and trans-acting gene regulation?

Mutation of a cis-acting regulatory sequence often has consequences for just one allele of one or a few neighboring genes; mutation of genes specifying a trans -acting gene regulator may have consequences for the expression of multiple genes distributed across the genome.

Locus heterogeneity

Mutations in one of several different loci may produce the same clinical phenotype. One mechanism is epistasis: gene A controls the action of gene B, or is upstream of B in a pathway. A second explanation is when genes A and B function in separate pathways but affect the same phenotype.

Clinical Heterogeneity

Mutations/variations in one gene produce more than one disease or phenotype. HPRT gene can produce gout or Lesch-Nyhan syndrome (severe mental retardation with behavioral problems (OMIM 300322).

How does PCR work?

Need to design a forward and reverse primer, have DNA sequence of interest, add extra nucleotides heating and annealing. Polymerase chain reaction amplifies DNA between primers. Primers identify start and stop of PCR Key foundation of reaction is DNA replication using a heat tolerant polymerase. Cycles determine amount of amplified DNA. qPCR types vary, use machines to give relative quantitation of the sequence of interest. Real-time PCR is a qPCR with a special machine to give absolute quantitation of target amplified.

Are all of the genes in a cell expressed?

No, only a subset of genes are expressed and it varies by cell type

Competing endogenous RNA

Non coding mRNA transcripts can have 3'-UTR sites for miRNA binding, which competes out the miRNA from binding to its target site on a coding mRNA. e.g., Regulation of PTEN gene

Oxidative Damage

O2 -> O2- -> H2O2 superoxides, hydroxyl radicals are reactive oxygen species (ROS) are generated in mitochondria

Founder effect

Occurs when a new colony is started by a few members from the original population (e.g. migration or survivor being founders). Genetic drift that occurs when a few individuals become isolated from a larger population and form a new population whose gene pool composition is not reflective of that of the original population.

Chromatid

One copy of a duplicated chromosome, which is generally joined to the other copy by a single centromere.

What does TRIIH do?

Opens the helix at the start site, and phosphorylates C terminal domain of polymerase which activates polymerase binding to DNA. [promoter strength is influenced by presence or absence of DNA binding promoters, and presence or absence of a nearby enhancer sequence]

Nucleotides

Organic molecules that serve as the monomers, or subunits, of nucleic acids like DNA and RNA. The building blocks of nucleic acids, nucleotides are composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

Autonomous replicating sequence

Origin of replication of DNA in yeast. Consists of a 11bp "consensus" sequence surrounded by a more variable 50bp AT-rich sequence (AT because weaker than GC).

Guide mRNA

Pairs with pre-edited RNA to provide a template for editing

piRNA

Piwi-interacting RNA: RNA involved in silencing transposable elements in the germ line and in stem cell populations.

Mosaics

Post zygotic mutations produce mosaics with two or more genetically distinct cell lines. Mosaics can be the product of chromosomal aberrations or gene mutation. Somatic and/or germ lines. If mutation occurs in early embryo, it may affect a tissue or organ. If the mutation is in a germ cell, the person may be germinal mosaic.

RNA editing

Process whereby the genetic information is changed at the level of mRNA. It is revealed by situations in which the coding sequence in the RNA differs from the sequences of DNA from which is was transcribed. ADARs = adenosine deaminases acting on RNA are enzymes that carry out RNA editing by deamination of adenosine to inosine. (AI editing) CU editing happens in apolipoprotein gene APOB (example next slide). UC editing has been documented in Wilms tumor transcript of exon 6 Some target mRNAs are selectively edited within introns before splicing which may affect the splicing pattern in alternative splicing. (Hyperediting is more common in viral infections than in human genes)

Post-translational processing of proteins

Processing of proteins after translation includes folding of the protein into its correct tertiary structure; cleavage by proteases, modification of side chains of amino acids, e.g. phosphorylation, and intein splicing. Inteins are intervening sequences in some proteins similar to mRNA introns. Inteins must be removed and the peptide ligated for the protein to be active.

Termination

Protein synthesis ends when the A site has the stop codon and fails to recruit a tRNA/aa. Eukaryotes have two release factors: eRF-1 that recognizes the termination codons, and a ribosome recycling factor (RRF in bacteria and hypothetically eRF3 in eukaryotes) which unlocks the ribosome.

Proteomics

Protein-protein AND protein-DNA interactions. Proteomics attempts to account for all proteins expressed in a cell and organism. The expression analysis profiles subcellular localization of proteins. Large scale assays of phenotypes generated by gene knockout or RNAi induced gene knockdowns can reveal gene function. Protein microarrays (protein chips) are similar to DNA arrays, with proteins on glass slides, proteins may be antibodies, or enzyme activity e.g. kinase rx. Protein-protein -> functional protein networks

7SL RNA

RNA that is required for the secretion of newly synthesized polypeptides through the endoplasmic reticulum. Involved in signal recognition particle (SRP) that delivers protein molecules to the ER after synthesis.

RISC

RNA-induced silencing complex

Pedigree limitations

Rarely can a mode of inheritance be defined unambiguously in a single pedigree Family size is limited Patterns overlap Rare conditions Recessive condition can mimic a dominant pedigree if the gene is common enough in the population that unrelated persons marrying into the family often carry it. Example: Blood Group O

Pseudoautosomal inheritance and X-Y recombination

Recombination in male meiosis occurs between pairs within the pseudoautosomal regions, effectively the genes in these regions are alleles...since such a gene can move between the X and Y chromosomes, it is neither X linked nor Y linked and so the pattern of inheritance resembles autosomal inheritance.

How does new population from founder effect differ?

Reduced genetic variation from original population Non-random sample of the genes in the original population (often called sampling error) Most often use "founder" in deleterious alleles (disease phenotypes)

Pseudoautosomal

Regions of X and Y that have short gene-containing regions in common, called pseudoautosomal regions, may recombine in male meiosis. Recombination between X and Y is very limited. The pseudoautosomal regions are located just before the telomere associated repeats at the ends of the short and long arms

What are boundary elements?

Regulatory elements that ensure that signals from regulatory elements do not act on genes other than their intended targets. Act as barriers between neighboring euchromatin and heterochromatin regions. Insulator can block inappropriate interactions between enhancers and promoters by binding regulatory proteins. (eg CFTC regulator)

What causes base mismatch?

Replication errors, small inserts/deletions due to replication slippage

Techniques to find start site?

S1 mapping, run off assay and primer extension

serial analysis of gene expression

SAGE allows short sequences of cDNAs (RT product of mRNA) to be analyzed. Generally 12bp tags are enough to identify a mRNA. PolyT captures the polyA mRNA tail, RT converts RNA to DNA, then digestion with Alu (5'AGCT3') reduces the size of the fragment because of its frequency in the genome. BsmF1 cuts 10-14 bp downstream of the recognition sequence generating the small fragment representative of the mRNA.

What makes up the Signal Recognition Particle?

SRP9 SRP14 SRP19 SRP54 SRP68 SRP72 7SL RNA

DNA nick repair

SS break repair or DNA nick repair can be repaired by ligase unless gap is made and then it is filled by polymerase and sealed with ligase

Sequence tagged site

STS is a short DNA sequences 100-500bp easily recognized and is singular that is it occurs only once in the chromosome or genome being studied. To map a series of STS markers we need overlapping segments each of which has a unique STS.

What else contributes?

Selective migration and mortality also contribute (bottleneck). Founder effect (population derived from a small number of founders) and heterozygote advantage may account for autosomal recessive conditions being very uneven in ethnic distribution (CF).

Which amino acids have hydroxyl groups?

Serine, Threonine, Tyrosine

Hearing loss

Several different genes contribute to hearing loss. A man and woman with hearing loss may marry and produce normal hearing children if each of them carries a recessive mutation in a different gene. The parents are each double recessive at that allele, but the children are heterozygous at each allele and therefore normal of hearing.

SINES

Short interspersed nuclear elements--type of transposon, left over from evolution but doesn't code for anything, is inactive but can be moved by LINE1 enzymes. Can be replicated and moved easily. 13% of human genome, also known as Alu repeats. 70% belong to Alu repeat family with 1.5 million copies in genome. Often transcribed by adjacent promoters but cannot make proteins but can use the RT from LINES

T-loop

Single-stranded overhang causes the DNA at 3' terminus to loop back and displace one of the strands in the double-stranded molecule (forms the single stranded d-loop) in an upstream telomeric repeat, then pairs with the complementary strand; DNA in these t-loops is protected from degradation and/or modification by DNA repair processes; telomere-specific protein complex (shelterin) bind/coat DNA preserving t-loop structure

X-linked recessive

Skips generations, males cannot be carriers of x-linked conditions. Sex linked inheritance always expressed in the male offspring

snoRNA

Small nucleolar RNAs: RNA molecules that guide chemical modifications of other RNAs. Base modification of rRNA.

Anticipation

Some disorders show increase in severity generationally: fragile X mental retardation, myotonic dystrophy, Huntington Disease because each is generated by unstable repeats often called dynamic mutations. This increased severity, earlier onset, is called anticipation.

How does the transcription initiation complex come together?

Some of the protein subunits of the transcription initiation complexes recognize and bind specific short DNA sequence elements of a promoter; others are recruited by binding to previously bound proteins. For a protein- coding gene, most core promoter elements are upstream of the start site, and the spacing of the elements is important.

Which assays use a labeled probe and unlabeled test sample?

Southern blot, tissue in situ, chromosome in situ

Alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins. An intron is optional Alternative donor site Alternative acceptor site Optional exon Alternative exons

Where does transcription start?

Start of transcription occurs in the initiator sequence (Inr) 25-30 bases 3' to the TATA box or equivalent. Downstream of start is another core promoter (DPE) which is functional only when it is 28-32 bases 3' to A in the Inr.

What are the 4 classes of chemical damage to DNA?

Strand breakage, Base deletion, Base modification, Base cross-linking

TBP

TATA binding protein, binds directly to TATA box in RNA polymerase II

What are core promoter elements found in genes transcribed by RNA polymerase II?

TATA box, bound by TFIID, initiator (Inr) element, downstream core promoter (DPE) +28 to +32 to A of Inr, TFIIB binds to TFIIB recognition element (BRE) which posiions polymerase at start site *not all necessary and some promoters lack all of them*

What are the are generic transcription factors that assemble prior to the polymerase?

TRIID, TRIIB, TRIIE, TRIIF, TRIIH

TERC

Telomerase RNA component, also known as TERC, is an RNA gene found in eukaryotes, that is a component of telomerase used to extend telomeres. TERC serves as a template for telomere replication (reverse transcription) by telomerase.

TERRA

Telomeric repeat-containing RNA (TERRA)is a long non-coding RNA that forms integral part of telomeric heterochromatin along with the telomeric binding proteins. It plays a key role in maintaining the telomeric structure and plays an important role during the processes like cell differentiation and development. TERRAs are transcribed from the telomeric end of the DNA and almost all the DNA ends have been shown to transcribe the molecule.

Chiasmata

The X-shaped, microscopically visible region representing homologous chromatids that have exchanged genetic material through crossing over during meiosis.

Duchenne muscular dystrophy

The dystrophin gene stretches over 2.4 Mb of DNA with 79 exons, 7 alternative promoters (3 upstream and 4 within the gene) each of which results in a different mRNA and protein. The three upstream promoters are active in cortical, muscle, and cerebellum respectively. The four promoters within the gene are produced in a tissue-specific manner and produce a shorter transcript and truncated protein.

Base excision repair

The excisional repair of DNA may involve the removal of a single altered base (oxidation, deamination, methylation, etc); Single base deletion by hydrolysis. Glycosylase removes base (creating abasic site) and then sugar/phosphate removed (endonuclease and phosphodiesterase) to insert single new nucleotide (DNA polymerase/ligase).

Primary structure

The first level of protein structure; the specific sequence of amino acids making up a polypeptide chain.

The global gene expression of a cell dictates what 3 things?

The form of a cell, how it behaves, and its identity

Quaternary structure

The fourth level of protein structure; the shape resulting from the association of two or more polypeptide subunits. The fourth level of protein structure; the shape resulting from the association of two or more polypeptide subunits. Can be stabilized by disulfide bridges and ligand bonding.

Barr bodies

The inactive X in each cell of a female condenses into a compact object, lies along inside of nuclear envelope. Genes on X in Barr Body are not expressed

How do remote cis elements work?

The intervening DNA is looped out to allow the remote elements to come into contact (proteins of the enhancer can interact with proteins of the promoter)

Germ line

The lineage of reproductive cells that contributes to the formation of a new generation of organisms, as distinct from somatic cells, which form the body and leave no descendants in the next generation.

Secondary structure

The localized, repetitive coiling or folding of the backbone of a protein due to hydrogen bond formation between amino acids. Coils (alpha helix) & folds (beta pleated sheets)

Retropseudogene

The most common retrosequences. Dead genes derived from RNA.

Acetylation

The process where an acetyl functional group is transferred from one molecule (in this case, Acetyl-Coenzyme A) to another. Deacetylation is simply the reverse reaction where an acetyl group is removed from a molecule. Acetylation of histones neutralizes its positive charge, thus making it less attracted to the DNA --> allows for increased transcriptional activity

Transcription unit

The segment of DNA that is transcribed into mRNA.

short interfering RNA siRNA

The siRNA duplexes are bound by different complexes that contain an Argonaute-type endoribonuclease (Ago) and some other proteins. Thereafter, the two RNA strands are unwound and one of the RNA strands is degraded by the Ago ribonuclease, leaving a single-stranded RNA, the guide strand, bound to the Argonaute complex. The complex is now activated, and the single-stranded RNA will guide it to its target by base pairing with complementary RNA sequences in the cells.

Transcriptomics

The study of characteristics and regulation of the functional RNA transcript population of a cell/s or organism at a specific time. -the population of functional RNA transcripts. -the mechanisms that regulate the production of RNA transcripts -dynamics of the trancriptome (time, cell type, genotype, external stimuli)

What is the variation in hybrization?

The variation is whether the probe or the test sample is labeled

What happens to isoforms?

They may be retained, mayb e secreted or sent to different cellular components

What do splicing enhancers/repressors bind?

To help keep the spliceosome in place splicing enhancers bind SR proteins (so called because they have a domain based on repeats of the serine-arginine dipeptide). Splicing suppressors bind hnRNP proteins that are active in removing bound spliceosomes.

Paralogous genes

Two mRNAs which have similar sequences and cross-hybridize to each others probe on the array. The transcriptome with related mRNAs reflects the gene family. Likewise a gene that is differentially spliced requires a design that detects unique features of each alternative.

snRNA

Type of RNA found only in the eukaryotic nucleus that is involved in processing of initial mRNA transcription products to a mature form suitable for export from the nucleus to the cytoplasm for translation.

Where are cis-acting regulatory elements in mRNA located?

UTRs

Protein degradation

Ubiquitin added to a specific sequence in the N terminus of a protein will mark the protein for degradation. The sequence contains lysine to which the ubiquitin peptide is linked. The sequence is called the N-degron.

Microarray hybridization

Uses parallel hybridization to immobilized probes and allows numerous assays to be done simultaneously on a common sample on a grid format. Within the grid are millions of identical copies of one probe (each grid square is called a feature). A test sample of a complex population of fluorescently labeled denatured DNA or RNA is added to different probe populations on the microarray, washed. Detection of hybridization signal using a laser scanner, intensity is a quantitate measure within the population.

Consanguinity

Where two identical mutant alleles are present because parents are close relatives

Polygenic theory of quantitative traits

With multiple loci contributing to a trait, the one to one relationship between genotype and phenotype disappears. As the number of loci increases the distribution looks like a Gaussian curve

Patterns in pedigrees

X inactivation Mosaicism due to X inactivation Few Genes on the Y Genes in Pseudoautosomal region Mitochondrial DNA mutations

Y linked holandric traits

Y specific region of Y chromosome is non-recombining male-specific region Population genetics says that non-recombining regions are eventually lost because harmful mutations in these regions cannot be removed by recombination. Over millions of years of evolution, Y ch has contracted so that now Y has 38% of X DNA. Y has fewer genes making only about 31 different proteins. X and Y are thought to have originated as a homologous pair of autosomes that began to diverge in sequence after one of them acquired a sex-determining region.

How do we test for protein:protein interaction?

Yeast two hybrid screening (Y2H) Affinity purification FRET: fluorescence resonance energy transfer Co-immunopreciptation (Co-IP)

Retrogene

a DNA gene copied back from RNA by reverse transcriptase.

cis-acting regulatory DNA element

a DNA sequence whose function in gene regulation is limited to the single DNA molecule on which it resides (eg promoter). A cis-acting RNA sequence regulates the expression of the single RNA transcript that it is located on (located in untranslated regions).

Klinefelter's syndrome

a chromosomal trisomy in which males have an extra X chromosome resulting in an XXy condition; affected individuals typically have reduced fertility

Clone library

a collection of cloned DNA fragments. Used for mapping and sequencing.

Clone contig

a contiguous DNA sequence of fragments ordered along a whole chromosome or subchromosomal region. Clones containing overlapping fragments provide base material for a larger continuous DNA sequence, selection of unique sequences in each of the clones provides a STS source, and if the clones derive from a restriction digest with accompanying map, the physical location of each overlapping clone is positioned for generating a genetic map.

Population

a group of interbreeding individuals

Leucine zipper

a helical stretch of amino acids rich in hydrophobic leucine residues, aligned on one side of the helix. These hydrophobic patches allow two individual α -helical monomers to join together over a short distance to form a coiled coil. Beyond this region, the two α -helices separate, so that the overall dimer is a Y-shaped structure. The dimer is thought to grip the double helix much like a clothes peg grips a clothes line. Leucine zipper proteins normally form homodimers but can occasionally form heterodimers (the latter provide an important combinatorial control mechanism in gene regulation).

dideoxy sequencing

a method for determining the sequence of a DNA fragment using dideoxynucleotides, which cause termination of DNA synthesis during the procedure. Principle of dideoxy method is based on DNA synthesis and the inability to elongate a strand when the base is connected to a dideoxyribose.

When do you use a BAC?

a modified plasmid vector with tight constraint on copy number that allows large fragments to be stably propagated (E. Coli)

X-inactivation

a process by which one of the two copies of the X chromosome present in female mammals is inactivated

Genetic abnormality

a rare, uncommon version of a trait

trans-acting gene regulator

a regulatory protein or RNA molecule that is free to migrate by diffusion so as to recognize and bind specific short target nucleic acid sequences, thereby regulating the expression of both alleles on distantly located genes (Figure 1C) or the RNAs they produce (Figure 1D). Often regulates multiple different genes or mRNA transcripts

Telomerase

a ribonucleoprotein enzyme whose polymerase function depends on an RNA subunit (TERC = telomerase RNA component) and a protein subunit (TERT telomerase reverse transcriptase). An enzyme that catalyzes the lengthening of telomeres. The enzyme includes a molecule of RNA that serves as a template for new telomere segments.

Transcription factor

a sequence-specific DNA-binding pro-tein that binds at or close to cis- acting DNA sequences that are important in gene expression. In addition to ubiquitous general transcription factors that bind to core promoter elements, many other transcription factors bind to additional, often remote, cis- acting sequences, such as enhanc-ers, as described below.

Gene family

a set of closely related genes with slightly different functions that most likely arose from succession of gene duplication events.

Antisense RNA

a single-stranded RNA molecule complementary to, and thus targeted against, an mRNA of interest to block its translation

retrovirus-like elements

a type of interspersed repetitive segment that moves by reverse transcription. They closely resemble retroviruses. human endogenous retroviruses (HERVs) are 6-11 kb contain LTRs and sometimes pol gene from ancient retroviruses (pol contains RT). Little evidence for transposing human HERVs.

Real time PCR

absolute quantitation (# copies) of product

Positive Selection

advantageous variant has increased survival and reproductive success rates (FOXP2 OMIM 602081 may confer language ability)

X-Linked Dominant

affects males and females equally, tends to be in each generation, 50% chance of being affected if mother is affected but an affected father will have unaffected sons, while his daughters are at risk. In some cases daughters will have a milder phenotype as a result of X inactivation.

Gene pool

all alleles at all loci in all individuals in a population

Gene pool

all of the alleles at a specific gene locus within the population

The Hardy Weinberg Law

allele and genotype frequencies remain constant in succeeding generations in a population at equilibrium. No mutations Large population No migration No selection No selective mating

Gel retardation assay

allows the fragment of DNA bound by protein to be "retarded" that is move more slowly in the gel compared to the non-protein bound counterpart. Once the fragment is found, it can be separated from the protein and sequenced. If the fragment is large, the precise binding sequence will not be found by this assay and a protection assay will need to be done.

Genetic disorder

an inherited disease or disorder that is caused by a mutation in a gene or by a chromosomal defect. Each genetic disorder is characterized by a specific set of symptoms—a syndrome.

Rhodamine

an organid cye that absorbs dye in the yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). it can be used in two-color immunogluorescence assays. it can be used with fluroscein because it is easly distinguishable from it.

restriction fragment length polymorphism

any DNA polymorphism that causes a change in the recognition sequence for a restriction enzyme.

microRNAs

are single-stranded regulatory RNAs that down-regulate the expression of target genes by base pairing to complementary sequences in their transcripts. Typically about 20-22 nucleotides long, they are formed by multiple processing events, including cleavages in the cytoplasm that are performed by the same endoribonucleases used in RNA interference.

Functional variants

are those effecting gene function, changing structure of gene product and its function.

What are the five basic Mendelian inheritance patterns?

autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, and Y-linked (not Y-linked dominant or Y-linked recessive because males are never heterozygous for Y-linked sequences

U2 snRNA

base pairs with the branch site sequence

U1 snRNA

base pairs with the splice donor sequence

TFIID

binds TATA box, helps assembly of IIB, A and TAFs.

TFIIB

binds TBP, Pol II and promoter DNA helping fix the transcription initiation site.

TFIIE

binds promoter may help stabilize transcription bubble in the open complex

Insulator

can block inappropriate interactions between enhancers and pro-moters by binding regulatory proteins, notably the CTCF regulator

cDNA library

capture mRNA, reverse transcribe to cDNA, clone into a vector, amplify, map. The cDNA corresponds to the mRNA expressed in the cell. Since most of the mRNAs are polyadenylated at the 3' end, they can be captured with an oligoDT column. Reverse transcription converts the mRNA into ds DNA. The cDNA is cloned into a vector, amplied in the host cell or by PCR, and represents the exons of the gene from which it was transcribed. REMEMBER: cDNA lacks the regulatory sequences in the genome which control transcription.

Phenotype

characteristics or traits of a person, or cell

Promoters

cis -acting regulatory DNA sequences that are important in establishing which segments of a DNA strand will be transcribed. Each promoter is a collection of very short sequence elements that are usually clustered within a few hundred nucleotides from the transcription start site. For each DNA strand, transcription begins at fixed points on the DNA where the chromatin has been induced to adopt an 'open' structure

Regulatory nucleotide sequences and proteins can be divided into what 2 classes?

cis-acting and trans-acting

RNase MRP

cleavage of pre-rRNA

How does consanguinity impact recessive autosomal inheritance?

compound heterozygote v. true homozygote

Trisomy 21

condition in which an individual has three number 21 chromosomes, resulting in Down syndrome

Splice acceptor site

contains an invariant AG dinucleotide that defines the 3' end of an intron at the RNA level and is embedded within a larger sequence that includes a preceding polypyrimidine tract.

Splice donor site

contains an invariant GU dinucleotide that defines the 5' end of an intron at the RNA level

Unprocessed Pseudogenes

copy of a functional gene that has arisen as a result of a gene duplication event and subsequently acquired mutations that caused it to become nonfunctional. They usually have all the same characteristics of genes, including an intact exon-intron structure and promoter sequences.

FITC (fluorescein isothiocyanate)

detected by using laser scanners/fluorescence microscopy

Digoxigenin (a steroid found in digitalis plants)

detected via a digoxigenin-specific antibody that is coupled to a fluorophore or suitable enzyme

biotin (vitamin B7)

detected via streptavidin (a bacterial protein with an extraordinarily high affinity for biotin) that has been conjugated to a fluorophore or enzyme

Homologous recombination (HR)-mediated DNA repair

dsDNA breaks, accurate DNA repair but requires an intact homologous DNA strand as a template (post replication in S phase or occassionally G2). Trimming back of ds break at 5' ends, strand invasion and DNA synthesis, finish DNA synthesis and ligate ends, resolution. If the sister chromatid is present it can provide a template for the repair by crossing over and gap synthesis.

Nonhomologous end joining (NHEJ)

dsDNA breaks, doesn't rely on a template strand and so is available throughout cell cycle. Less accurate than HR-mediated DNA repair. no template strand is present so the broken ends are fused together by special proteins that bind the broken ends and recruit ligase to rejoin the ends. Most important in G1 before DNA replication.

Dosage effect

duplication can allow more gene product

FITC

excited by blue, emits green

What are other regulatory elements during splicing?

exonic splice enhancer (ESE), exonic splice suppressor (ESS), intronic splice suppressor (ISS), intronic splice enhancer (ISE)

Chromosome painting

extension of F.I.S.H. Instead of using one or two random small probes you are using a combination or set of overlapping probes and you lit the entire chromosome.

What is an example of translational regulation?

ferritin (an iron-binding protein used to store iron in cells) and the transferrin receptor (which helps us absorb iron from the diet). When iron levels are low, the priority is to maximize the amount of iron that can be absorbed from the diet: transferrin receptor mRNA is protected from degradation so that it can make a protein product. Conversely, when iron levels are high, ferritin production is activated to store iron in cells. This happens without any change in the production of ferritin or transferrin receptor mRNAs. Instead, both these mRNAs have iron-response elements (IREs), which can be bound by a specific IRE-binding protein that regulates the production of protein from these mRNAs; the availability of this binding protein is also regulated by iron concentrations.

What are 3 labeling systems?

fluorescence, antibody detection, specific protein interaction

Genomic library

fragment genome, capture all fragments, clone into vector, amplify, map

Genes

functional units of DNA that make some product needed by cells, either the polypeptide chain of a protein or a functional noncoding RNA.

TFIIH

functions in transcription and DNA repair, has kinase and helicase activity.

2 techniques to find DNA binding sites?

gel retardation and DNase footprint

alternative promoters

genes have multiple promoters which results in different primary transcripts that contain the same protein-coding regions. drive transcription from alternative versions of a first exon, (UDP glycosyltransferase gene UGT1A1 has 13 alternative first exons). different regulatory elements (tissue specific), use of alternative first exons may produce protein isoforms with different properties, e.g. soluble or membrane bound isoforms

Housekeeping genes

genes that are switched on all the time because they are needed for life functions vital to an organism

microarray comparative genome hybridization

genomewide search for large (megabase sized) changes in the DNA

Massively parallel sequencing of amplified DNA

genomic DNA is fragmented, ligated to oligo adaptors, separated into SS. Fragments are bound to beads favoring one fragment per bead, each of which is caught in a droplet of a microreactor that does clonal PCR amplication and sequencing.

Consanguineous

having the same lineage or ancestry; related by blood

Co-immunopreciptation (Co-IP)

helps validate two proteins interaction. Antibody specific for one of the two (or more) proteins binds capturing the complex

Mutation

heritable change in DNA sequence

Which DNA is poorly conserved?

heterochromatin, transposon repeats, other (mostly unique sequences

What is a heteroduplex?

hybrid between two sources of DNA and or RNA/DNA

RNase P

in tRNA modifications, the enzyme that cleaves the tRNA to remove the extra bases at the five prime end; the RNA is the enzyme, not the protein-->ribozyme

Mendelian Pedigrees

include autosomal dominant, autosomal recessive, X linked dominant, X linked recessive and Y linked holandric. Pedigrees are graphical representation of a family tree.

Silencer

inhibit or prevent transcription when the appropriate trans-acting elements (repressors) are bound to them.

U1 snRNA

internal U1 in spliceosome base pairs with the target sequence in the intron to locate the 5' end of the intron, selects the donor splicing junction

Zinc finger motif

involves the binding of a Zn 2+ ion by four conserved amino acids (normally either histidine or cysteine) so as to form a loop (finger). They usually occur in clusters of sequential zinc fingers. The so-called C2H2 (Cys 2 /His 2 ) zinc finger typically comprises about 23 amino acids, with neighboring fingers separated by a stretch of about seven or eight amino acids. The structure of a zinc finger may consist of an α -helix and a β -sheet held together by coordination with the Zn 2+ ion, or of two α -helices, as shown here. In either case, the primary contact with the DNA is made by an α -helix binding to the major groove.

cap analysis of gene expression

is a high throughput method that sequences the first 18 nt at the 5' end of the mRNA. The 5' capture method works by biotinylating the cap and selecting with streptavidin magnetic beads. MmeI (type II restriction enzyme) recognizes sequence and cuts away from it, producing 18 nt fragment of cDNA with 2 nt sticky end which is ligated to form concatemers for sequencing.

rapid amplication cDNA ends

is an application of RT-PCR that allows whole mRNA amplification between an internal primer and the 5' end of the mRNA. It reveals different promoter and 5' exon use in different tissues, a variant on differential gene expression (unrelated to splicing)

FRET: fluorescence resonance energy transfer

is used to test protein interaction. Two proteins are expressed as hybrid proteins, each with different fluorescent tag (cyan-CFP) or (yellow-YFP). Exposure to light that excites only one of the two fluorophores, yields signal if the two do not interact (A) or a signal at a different wavelength if the two proteins interact (B)

What are the 3 phases of PCR?

lag phase, exponential phase, saturation phase (can be viewed on a amount of product v. number of cycles graph)

Copy number variations (CNV)

large scale indels that generate increase or decrease of copy number of genes or sequences

Microsatellites

less than 100 bp long, widely distributed in euchromatin

Class I gene

make rRNA

Class III gene

makes tRNA, snRNA, miRNA

Affinity purification

mass spectrometry used to screen for protein partners of a test protein. (AP-MS) uses a bait protein immobilized by being linked to insoluble supporting matrix (on beads or in a column). Cell lysate added the proteins attach to the "bait" and nonbinding proteins are separated from the matrix by washing or with magnet. Increased salt concentration allows proteins that stick to be eluted. They are then analyzed by mass spectrometry. TAP (tandem affinity purification) tagging uses a fusion protein to link to the target protein, captured with antibody, purified, then separated by specific protease.

Mitochondrial mutation

matrilineal inheritance. Mitochondrial genome is 16.5 kb ~ 37 genes more prone to mutation and these mutations tend to show up in muscle and brain where the tissues have high energy requirements. Affects males and females equally but only the mother's mitochondria are stably inherited.

DNA libraries

may contain complete sets of plamid-containing cell clones in bacterial cells or clones of phages; may contain artificial chromosomes; may contain cDNA

Quantitative PCR

measures amount of sequence of interest

Which assays use an unlabeled probe and a labeled test sample?

microarray comparative genome hybridization, microarray-based expression profiling

Variant

mutated site, irrespective of frequency

Negative selection

mutations that lower reproductive success gradually become more rare and eventually should be eliminated from the genome.

Processed Pseudogenes

non-functional copies of normal genes that originate when processed mRNAs are accidentally reverse transcribed to DNA then inserted back into the genome at a new location

What controls rate of transcription?

non-ubiquitous, transcription factors bind to cis-acting regulatory elements other than those of the core promoter (repressors or silencers). Co-activator or co-repressor proteins recruited by bound TFs (do not bind DNA directly).

Protection Assay

nuclease digestion cuts all the backbone links in the DNA unless protected by a protein bound to the sequence. Label one end of fragments; allow protein binding; treat with DNaseI, Using limiting amounts of enzyme, the assay reveals a protected domain that differs by one nucleotide in the ladder resolved on the gel.

Tandem gene duplication

occurs after sister chromatids or non-sister chromatids of homologous chromosomes mispair -dosage effect -Insure against mutation when there are multiple copies, e.g. rRNA genes, histones -Exon duplication may give advantage to a structural motif in a protein -Novel variants produce divergent genes that have related proteins e.g. globin family

Penetrance

of a single-gene disorder is the probability that a person who has a mutant allele will express the disease phenotype. Dominantly inherited disorders, by definition, are manifested in heterozygotes and might be expected to show 100% penetrance. That might be true for cer-tain dominant disorders. For many others, however, penetrance is more variable and the disorder can sometimes appear to skip a generation so that a person who must have inherited the disease allele is unaffected ( non-penetrance

TSIX

overlaps Xist and is expressed in the opposite direction,, expressed only during early embryonic development.-encodes RNA coplementary to Xist RNA, believed to bind to Xist RNA and inhibit its function...

Why are some genes only expressed in certain tissues or at certain times in development?

partly because the activity of the promoters relies on tissue-specific or developmentally regulated transcription factors that bind to non-core elements.

TFIIF

prevents nonspecific DNA binding, binds Pol II

Hardy-Weinberg

principle that allele frequencies in a population will remain constant unless one or more factors cause the frequencies to change 1). No mutations. 2). Random mating. 3). No natural selection. 4). The population size must be extremely large, no genetic drift. 5). No gene flow. i.e. transfer of pollen.

When do you use a YAC?

recombinants are effectively small linear chromosomes that consist mostly of human or other foreign DNA (S. cerevisiae) A library of YACs can accommodate much more DNA.

STRPs: short tandem repeat polymorphisms

refer to microsatellites specifically. Generated by replication slippage

What are additional types of cis regulation?

regulatory noncoding RNA acts on DNA or on nascent transcripts that remain attached to DNA. These types of gene regulation are partic-ularly important in certain epigenetic phenomena such as X-chromosome inactivation or imprinting

When do you use a plasmid?

replicate independently of host chromosome and can reach high copy numbers; widely used for cloning small DNA fragments (E. Coli)

Recombinant DNA

represents two sources of DNA each cut with a restriction enzyme or two and recombined at the cut sites. Some product is reannealed DNA, the recombined is the desired product...to be selected and amplified.

Initiation

requires formation of the preinitiation complex, binding of the tRNA for Met, phosphorylation of eIF-2, addition of other eIF factors, cap binding complex, and other eIF cofactor proteins that aid in scanning the mRNA for the AUG-start codon. (Rarer starts include 5' GUG or 5'UUG). The initiation complex scans from the 5' end of the mRNA to the first AUG 30 plus bases inside the mRNA. The leader can be hundreds of nucleotides in length and may contain hairpins and other base-paired structures. The usual start codon, AUG is often within a short consensus sequence 5'ACCAUGG3' - the Kozak consensus. Once in place the large subunit joins the complex and translation begins.

Yeast two hybrid screening (Y2H)

reveals protein-protein interactions. This illustration is similar to the one in last week's lecture: identifying the DNA binding domain (BD) of a TF and the second protein with a AD: Transcription activation domain. Since these two proteins may be from genes from unlinked alleles, one can be used to trap the other.

microarray-based expression profiling

simultaneously tracking expression of very many genes; cancer profiling

SMRT Chip

single molecule real time sequencing proceeds at speeds of 20,000 times faster than second generation sequencers. It tracks the DNA polymerases as they synthesize DNA in real time. It records which nucleotides get incorporated by using nucleotides tagged with one of four different fluorophores. SMRT chip is a thin metal film over a glass substrate. The metal film has thousands of tiny sub-wavelength holes known as nanophotonic zero-mode waveguides (ZMWs). ZMW allow naophotonic visualization for direct recording of a DNA polymerase as it generates the sequence

scaRNA

small cajal RNA, used to modify snoRNA and snRNA

What do you get when you denature (heat) and anneal (cooling) homologous DNA molecules?

some original homoduplexes and artificial heteroduplexes

What are the 3 fundamental cis-acting RNA regulatory sequences?

splice donor site, branch site, splice acceptor site

What other short cis-acting regulatory RNA elements regulate splicing?

splice enhancer sequences (which stimulate splicing) and splice suppressor sequences (which inhibit splicing). They are located close to splice junctions and can lie within exons or introns.

Variant of BE repair

ssDNA break, initiated by poly(ADP ribose) polymerase binding to cleavage site

TFIIA

stabilizes TBP and TFIID binding

Chromosome banding

straining of chromosomes in such a way that light and dark areas occur along the length of the chromosome in a repeatable pattern; each chromosome can be identified by banding patterns (each light/dark strip=band)

Allele-specific oligonucleotides

stretch of DNA capable of either base pairing with a specific allele or not. can be used in hybridization to detect minor differences in DNA sequences. Very short probes (<21 bp) that specifically hybridize to one allele or the other = probes are allele-specific oligonucleotides (ASOs).

Chromosome in situ

studying large scale changes using chromosomes fixed on a slide as a test sample

RNA interference

technique to silence the expression of selected genes in nonmammalian organisms; uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA

Exome

the fraction of the genome that contains only exons

Allele frequency

the number of times an allele occurs in a gene pool, compared to the total number of alleles in that pool for the same gene

Transcriptome

the population of mRNAs expressed by a genome at any given time. The complete collection of transcribed elements of the genome. mRNAs: 35, 913 transcripts (including alternative spliced variants)

Are there differences surrounding the invariant GU and AG sites?

the sequence surrounding the invariant GU and AG signals is vari-able—some splice sites are strong and readily used, whereas others are weak and used only occasionally.

Genotype

the underlying corresponding genetic factors responsible for the trait(s). Genotypes are homozygous or heterozygous at a given locus (as long as the allele is diploid) thus males at a Y or X locus are hemizygous.

Tissue in situ

tracking RNA transcripts in tissues and embryos

RNA polymerase II

transcribes all protein-coding genes, genes that make long noncoding RNAs and some short RNA genes including many miRNA genes.

RNA polymerase III

transcribes tRNA genes, the 5S rRNA gene, and some other genes that make short RNAs.

Helix-loop-helix

transcription factors have three helices with two of them separated by a loop. Provides a surface for protein-protein interactions, therefore the proteins are active as dimmers. The third helix has positive charges and is thought to be the DNA binding domain.

What are the different levels of gene regulation?

transcription, post-transcriptional processing (to make mRNA or a mature noncoding RNA), translation of mRNA, post-translational modification, folding of gene products, incorporation into a multisubunit functional molecule, and degradation of gene products.

Class II gene

use polymerase II, the transcript is processed into mRNA (capping, splicing, polyadenylation), transported to cytoplasm for translation into protein

Bottleneck

when population size is reduced (e.g. natural disaster), allele frequencies of survivors are not same as allele frequency of larger prior generation. Genetic drift acts more quickly to reduce genetic variation in small populations, undergoing a bottleneck and this reduces genetic variation in the population which may last several generations.

What is the max cloning capacity of a BAC?

~200 kb