week 10
What are some evolutionary trends in miRNA?
- 336/800 miRNAs are primate specific, not found in any other mammals - Spatial expression patterns vary amongst even closely related species - Timing and location of miRNA expression is not necessarily conserved
What are characteristics of miRNA families?
- Continually evolving, very plastic - Classified based on sequences - Expand by duplication, contract by deletion; observed by lineage trends - Some present in many animals/plants, early origin, often involved in development - Some present in only few animals/plants, recent origin, more variety of roles - Many models for how they originate, some derived from transposons and repeat sequences
What are characteristics of siRNAs?
- involved in gene silencing - induce DNA methylation, histone modifcations, chromatin inactivation - some originate from TE and heterochromatin regions - some prevent expression of TEs
What are some characteristics of lncRNAs?
- no open reading frame, do not code for proteins - many are intergenic, sometimes overlap into coding regions - unknown mechanism of action - some function in chromatin remodelling complex to regulate gene expression - can be similar sized to miRNA - don't appear to have structural rules like miRNA
What are the functions of miRNA?
- post transcriptional gene silencing, down-regulation - usually bind to 3' untranslated region two main: 1. transcript cleavage: get incorporated into a multi-protein complex, RNA-induced silencing complex (RISC), act as guides to target/degrade complementary mRNA by cleavage 2. blocks translation: RISC prevents binding of mRNA to ribosomes http://upload.wikimedia.org/wikipedia/commons/a/a7/MiRNA.svg
What are some features of miRNAs?
- short, 20-24 nucleotides - produced from larger precursor transcriptions of 70-100 nucleotides that have a hairpin structure - Hairpin cleaves by dicer endonuclease to create miRNA
What are the components of promoters?
1) Core promoters are the site of assembly of the transcription complex, not a site of transcription regulation 2) Transcription factor binding sites are where enhancers, repressors bind
How can gene duplication effects on cis-reg. elements be studied?
1) DNase1 seq data 2) chromatin immunoprecipitation
What are two experimental methods to detect cis-regulatory elements?
1) Deletion constructs - delete a region, determine expression based on reporter gene like GFP 2) Site directed mutagenesis to identify exact nucleotides
What are the steps in whole genome shotgun sequencing?
1) Fragmenting entire genome into 1-3 kb fragments, 2) Amplification via cloning vectors, 3) Sequence amplified regions from both ends and 4) Align sequence via computational methods to create physical maps
What are two intron loss mechanisms?
1) Genomic deletions (due to recombination) and 2) Integration on an intronless cDNA back into the genome (due to recombination with the original gene)
What are two consequences of gene duplication?
1) If the breakpoints do not include cis-reg. elements, then there will be no gene expression of the duplication gene 2) If there are some but not all cis-reg. elements are included in the duplicated region, the duplicated gene will have an altered transcription profile, this leads to an immediate expression pattern change
What are four intron gain mechanisms?
1) Intron transposition, 2) Transposition insertion, 3) Tandem duplication causing new splice site insertion and 4) Intron transfer
What are some effects of novel mutations in promoter sequences in cis-regulatory elements?
1) Most common - modulate/eliminate TFBS 2) Different transcription factor may start to bind instead 3) Can generate new binding sites de novo 4) insertions/deletions disrupts spacing between binding sites, affects transcription profiling
What are the types of selection that act on cis-reg. elements?
1) Purifying selection - most common, long term conservation of binding sites 2) Positive selection - if mutation is advantageous, ex. changes transcription binding site
What are two computational methods to detect cis-regulatory elements?
1) Using programs that use databases of known binding sites to scan sequences for potential binding sites 2) Phylogenetic footprinting - comparisons with homologous chromosomal regions from from other species
What is are examples of phenotypic change resulting from cis-regul. element change?
1) ts1 gene in maize, change within the cis-regulatory elements 2) fw2.2 gene in tomatoes, codes for a negative regulator of cell division that controls cell number
What species is an example of a project involving comparison of populations and ecotypes?
1001 ecotypes from around the natural populations around the world of Arapidopsis thaliana, comparison to a reference sequence
What is the average coding sequence length?
1300 bp.
When did the Human Genome Project start/end?
2001-2004 through whole genome shotgun sequencing
What is the composition of the human genome?
45% repetitive DNA (including TEs), 1.5% exons, 25% intron and regulatory sequences, 15% unique noncoding DNA, 15% repetitive DNA (not related to TEs), 10% SINE/Alu elements
What are tRNAs?
A Transfer RNA (abbreviated tRNA and archaically referred to as sRNA abbreviating soluble RNA) is an adaptor molecule composed of RNA, typically 73 to 94 nucleotides in length, that serves as the physical link between the nucleotide sequence of nucleic acids (DNA and RNA) and the amino acid sequence of proteins. Involved in nuclear, mitochondrial, chloroplast gene expression
What is the FOXP2 gene?
A gene that has been positively selected for in hominin evolution, that has been implicated in the capacity of language along with other genes
What are microRNAs?
A microRNA (abbr. miRNA) is a small non-coding RNA molecule found in plants and animals, which functions in transcriptional and post-transcriptional regulation of gene expression.
What is selective sweep?
A selective sweep is the reduction or elimination of variation among the nucleotides in neighboring DNA of a mutation as the result of recent and strong positive natural selection.
What is the top gene category within the molecular function of Gene ontogeny?
ATP binding, DNA binding, zinc ion binding
What are some adv/disadv to using programs that use databases of known binding sequences for potential binding sites to identify cis regulatory elements?
Adv - rapid identification of possible binding sites Disadv - many of the potential binding sites identified have no biological function and are simply matches
What are selective sweeps?
An adaptive mutation rises, and the allele rises to fixation and displaces all other alleles in the population over a few generations.
What are some plants with sequenced genomes?
Arapidopsis thaliana, rice, cottonwood (B.C. economic value), corn, cacao, potato, tomato, banana
What are BAC?
Bacterial artificial chromosomes, vectors used for cloning, amplifying and sequencing large sequences of DNA from other organisms.
What are 6 distinct adaptations during hominin evolution that were partially influenced by the environment?
Bipedalism, smaller canine teeth, enlarged cheek/jaws, more use of foot, larger brain, more vertical face without snout, sophisticated use of tools
What are some disadv to phylogenetic footprinting to identify cis regulatory elements?
Can only identify regions that are highly conserved between species, other conserved regions may have no biological/regulatory function, lots of false positives Big assumption that nucleotides within binding sties are more likely to be conserved by selection
What are some sequenced vertebrate genomes?
Chimpanzee, gorilla, bonobo, orangutan, cow, horse, pig, chicken, mouse, rat - mostly related to agricultural, economical interest.
How does conservation compare from lincRNA to protein coding genes?
Conservation significantly higher in protein coding genes than lincRNA in Arapidopsis, related genera?
Muscle cells in the mammalian heart are multinucleate, meaning that multiple nuclei are present in the cytoplasm of a large cell. Predict what is different about the cell cycle in a muscle cell. 1. M phase is inhibited. 2. The G1 and G2 phases are extended. 3. Cytokinesis does not occur. 4. S phase happens twice.
Cytokinesis does not occur.
How did some of the anatomical changes that accompanied domestication of maize from teosinte occur?
Due to changes within the cis-regulatory elements of the transcription factor gene tb1 (represses outgrowth of axillary meristems and branch elongation by effecting cell cycle)
What is the largest gene in humans?
Dystrophin, 2.4 Mb with 97 exons
Where are cis regulatory factors found?
Either 5' or 3' UTR (untranslated region) or introns - all upstream of the promoter region where transcription initiates, mostly a few hundred base pairs away
How many genes do humans have?
Estimated to be 100,000. Actually 20,000 due to alternative splicing
What is an EST?
Expressed sequence tags are single read sequences from cDNA, which are complementary to mature mRNA. They are usually 500-800 base pairs long and contain partial sequences of genes.
Cells arrested in G2 will contain the same quantity of DNA as cells arrested in G1: True False
False
Exiting early from G2 would cause the mutant cells to look this way: True False
False
The same cyclin protein will regulate the transition to S phase and the transition to M phase: True False
False
How is the genome annotated?
Find/verify known genes, repeat masking, identify intron/exon by transcriptome/EST analysis, comparison for gene homologs in other species, detect/identify non-coding RNA and TE
What are some migration patterns originating from Africa?
First Africans migrate into Middle east and interbreed with Neanderthals, then modern humans expand to Europe and some interbreed with Denisovans in SEA, and most recently migration into the New World (western hemisphere)
What are some characteristics of the chimpanzee genome?
First non-human genome sequenced from one sample, allows comparison between human and primate evolution, 1.23% single nucleotide substitution when compared to humans, 98% conserved regions (but not accounting for insertions/deletions), 30% identical gene orthologs and others differ by most of two amino acid
What is one example of a regulatory network?
Genes associated with the transition to flowering
Why is it hard to do phylogenetic analysis on hominin evolution?
Hard to find viable DNA samples in the fossils
What is the order of genome size (approximate number of base pairs) of human, Drosophila, chicken, C. elegans?
Human (3 billion) > Drosophila (180 million) > Chicken (120 million) > C. elegans (100 million)
What is the Human ENCODE project?
Human Encyclopedia of DNA elements, project from 2007-2012 to determine functional properties within the entire genome (promoter, enhancer, represser, exons, RNA transcripts, methylation sites, chromatin modification sequences, DNA replication sites)
What are the benefits of knowing the human genome?
Identification of disease genes, expansion of drug targets, know sequence variation in populations/individuals, learn vertebrate genome evolution trends, applications towards all of biology
What are some uses of comparative genomics?
Identification of new genes by homology, identify potential regulatory elements, learn how genomes expand and contract, gene family evolution insight, detection of ancient polyploidy events, medical applications
Where do introns come from?
Increased identification due to restricted phylogenetic distribution, other introns/exons/transposons and RNA intermediate step re-inserting itself into new locations
What is one model for the acquisition of a new miRNA?
Initially, new miRNA are expressed at low level and target specific spatio-temporal domains (vary in deleterious, neutral, advantageous results). Eventually, natural selection purges miRNA that targets sequences with deleterious results, whilst preserving miRNA with adv/neutral targets. These later miRNA can have increased expression without being deleterious to the genome
What is one example where phenotypic effects of a gene was due to cis-regulation elements further away from the gene of interest?
LCT gene (which allows for lactose persistence due to lactose breaking down into glucose and galactose) has SNP variations in the two introns (9, 13) in the MCMT gene (adjacent to LCT) has led to lactose persistence
What is an issue with the African Origin of Hominins?
Localization of fossils could be due to preservation, not actual trends in migration
What are long non-coding RNAs?
Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides (Perkel 2013). This somewhat arbitrary limit distinguishes long ncRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs (Ma 2013).
What are the three gene types or gene ontology in the maize genome?
Molecular function, biological process, cellular compartment
What are two examples of miRNA gene expression regulation?
Mutation in the miRNA may increase affinity to binding 1. Sheep musculature - GDFS protein represses muscle development, results in normal muscle mass. However, when targeted with a specific miRNA, there is a decrease of GDFS protein, so de-repression results in increased muscle mass 2. Tourette's syndrome - appropriate repression of SL1TRK1 protein results in normal brain development, stronger repression due to miRNA binding causes abnormal brain development
What are regulatory networks?
Networks of gene products regulation expression of other genes, either positively or negatively They evolve by changes in interactions Vary in complexity
Unrepaired single-stranded DNA breaks can lead to double-stranded DNA breaks during the next round of replication. Which repair pathway would be most appropriate for repairing double-stranded DNA breaks? 1. Nonhomologous end joining 2. Base excision repair 3. Mismatch repair 4. DNA proofreading
Nonhomologous end joining
What are some characteristics of the rice genome?
One of the top three crops in the world, both indica and japonica varieties studied by both clone-by-clone/BAC and whole genome shotgun approach, studied by both private companies and public international consortiums Has 12 chromosomes, relative small genome (390 mb), about 40,000 non TE-genes which are almost double the number of genes in humans, high percentage of homologs alignment with arapidopsis, 35% of non-centeromeric regions is TE Most genes involved in metabolic pathways and plant defence allelopathy mechanism/production
What are physical maps?
Provide a tiling path of minimally overlapping clones along a chromosome
What are cis-regulatory elements?
Regions of DNA that regulate expression of nearby gene(s), are often binding sites for regulatory proteins (like transcription factors), size is highly variable
What is the function of cis-regulatory elements?
Regulate timing and patterns of gene expression; often plays a role in cell differentiation
What are rRNAs?
Ribosomal RNA composes 60% of the ribosome, present as tandem repeats, upto 10 kb in length
When in the cell cycle would you find sister chromatids? 1. G2 2. G1 3. S 4. S and G2
S and G2
How did hitchhiking occur around the LCT gene?
SNPs associated with selection of lactose persistence trait also caused the hitchhiking of alleles from other genes closeby
What are some characteristics of the bonobo genome?
Second chimpanzee lineage to be sequenced, completed in 2012, very heterozygous alleles
What are transcription factors?
Short sequences approximately 5-12 bp. long where regulatory proteins bind
What are small interfering RNAs (siRNAs)?
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length. siRNA plays many roles, but its most notable is in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence. siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome. The complexity of these pathways is only now being elucidated.
What are snoRNAs?
Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. They basically process RNAs
What are some issues with whole genome shotgun sequencing?
Some gaps may still remain AND repeated sequences and transposable elements are challenging to align in certain regions and species (sunflower, but not arapidopsis)
Why do some mutations at cis regulatory elements not effect transcription?
Some nucleotides are not part of transcription
What would happen to the variation between organisms in a population if their DNA polymerase did NOT have a proofreading function? 1. The amount of variation would decrease. 2. The amount of variation would stay the same. 3. The amount of variation would increase.
The amount of variation would increase.
What would happen to the daughter cells if the G2 phase of the parent cell is shortened? 1. The cells would be missing chromosomes. 2. The cells would not undergo cytokinesis. 3. The cells would be larger than normal. 4. The cells would be smaller than normal.
The cells would be smaller than normal.
What is Incomplete Lineage Sorting (ILS)?
The population split between bonobo/chimpanzee and bonobo-chimp-ancestor/humans happened relatively close together, hence around 2% of genomic regions show patterns where bonobos or chimps are more closely related to humans than to to each other
What is the largest protein in humans?
Titin protein, fibrous structure, largest exon is 17 kb
Where are maize TE's mostly located?
Towards the ends of the chromosomes; different from arapidopsis where it is mostly towards the middle, scattered near the centromere
What types of repeat sequences exist in the human genome?
Transposon-derived and simple sequence repeats (3%)
Cells arrested in M will have more condensed DNA than cells arrested in G2: True False
True
How to detect proteins bound to DNA?
Use DNase1 on hypersensitive DNA (chromatin, not heterochromatin or DNA protected by protein) to cleave sites where protein is bound, then remove the protein to sequence the DNA
How are BAC used for physical mapping of the genome?
Used as landmarks in the genome to help place shotgun sequence reads
What is the conservation of promoter sequences in cis-regulatory elements?
Varied - some are highly conserved in distantly related species, some evolve rapidly
What are some characteristics of the maize genome?
Very large genome compared to rice (2.3 GB), size is why it took so long to sequence, 85% TE and ancient allelotetraploid (many gene losses from region disrupts colinearity with closely related species) explains size
What is some evidence for migration to the New World?
When reconstructing the Native American population history by a large scale SNP study, it revealed three distinct migrations from Siberia
What is one example of lincRNA?
Xist gene: - X inactive specific transcript - expressed only in the inactive X chromosome, initiated at X inactivation centre, spreads to the rest of the chromosome - noncoding RNA gene of 17 kb - induce heterochromatin formation - Xist is methylated and silenced in the active chromosomes
Which of the following types of DNA damage is MOST likely to be repaired incorrectly to produce a mutation? 1. a single-stranded DNA break 2. a double-stranded DNA break 3. a missing base 4. a single-base mismatch 5. cross-linked thymines
a double-stranded DNA break
Which one of the following would MOST likely contribute to uncontrolled cell proliferation (i.e., cancer)? 1. a mutant cyclin that cannot bind to its normal CDK binding partner 2. a mutant kinetochore protein that causes reduced microtubule attachment 3. a mutant enzyme needed for microtubule synthesis/polymerization 4. a mutant DNA replication mechanism causing extra chromosome copies 5. a mutant CDK that was active in the absence of its cyclin binding partner
a mutant CDK that was active in the absence of its cyclin binding partner
What are copy number variants?
alterations of the DNA of a genome that results in the cell having an abnormal or, for certain genes, a normal variation in the number of copies of one or more sections of the DNA.
How to identify the binding sites of a transcription factor?
chromatin immunoprecipitation followed by illumina/tiling sequencing
What is the order of gene mutation (insertion/deletions/substitutions) frequencies on different types of sequences from highest to lowest?
introns, non-coding intergenic regions > promoter regions > coding sequences
What is the top gene category within the biological process of Gene ontogeny?
metabolic process, protein amino acid phosphorylation, regulation of transcription/DNA dependent
A gene that normally has the sequence CAGAGCC is replicated as CATAGCC. Which of the following repair mechanisms would MOST likely be employed to fix this replication error? 1. non-homologous end joining 2. base excision repair 3. nucleotide excision repair 4. None of the other answer options is correct. 5. mismatch repair
mismatch repair
What is the top gene category within the cellular compartment of Gene ontogeny?
nucleus, membrane, intracellular
Colchicine is a drug that is used in plant breeding to create polyploids. It blocks the assembly of microtubules. If dividing cells are treated with colchicine, at what stage of mitosis would you predict the arrest would occur? 1. G1 of interphase 2. late anaphase 3. prophase 4. telophase 5. metaphase
prophase
What are the major non-coding RNAs?
rRNAs, tRNAs, snoRNAs, long-non coding RNAs, mmicroRNAs
What is an example of selective sweep?
tb1 gene in maize, selection due to domestication, only 2% of nucleotide diversity in 5' region, upto 90 kb upstream, caused expression change, more tb1 expression = less axillary meristem growth, less bushy growth
You are studying the cell cycle using fission yeast. A model of the fission yeast cell cycle is shown below. You have observed some mutant cells that are much longer than wild type cells. Use these data to determine whether each of the following statements is true or false. http://www.macmillanhighered.com/brainhoney/Resource/9844275,8,0,2,0/Assets/Pombe%20Cells.png Blocking the transition from G1 to S would cause the mutant cells to look this way: True False
true
Mismatch repair, base excision repair, and nucleotide excision repair are similar in that each: (Select all that apply.) 1. repairs multiple mismatched or damaged bases across a region. 2. None of the answer options is correct. 3. uses an undamaged segment of DNA as the template to repair a damaged segment of DNA. 4. repairs a single mismatched base. 5. repairs a short strand of mismatched nucleotides.
uses an undamaged segment of DNA as the template to repair a damaged segment of DNA.