genetics 3
Barbara McClintock
discovered transposable elements -•PhD in Botany from Cornell •Professor at University of Missouri-Columbia •Strange leaf patterns in maize •Albino patches on the leaves ****Compared chromosomes and found that some portions were missing and others had gained portions**
reverse genetics
discovering gene function from a genetic sequence -have a genotype and want to know gene function and what is its phenotype. -have gene and what does it do •Understanding the phenotype associated with a gene •Gene silencing •Targeted mutagenesis •RNAi •CRISPR
Barr Bodies
inactivated X chromosomes found only in females (silenced) -•Genetic mechanism in MAMMALS compensates for X chromosome dosage disparities. •Lies against nuclear envelope of interphase cells. •Provide mechanism for dosage compensation.
XX males
incorrect crossover of SRY gene to the X chromosome; sterile (hypogonadism, azoospermia) -place SRY gene on female egg and she will develop as male. ***happens in fathers sperm** his XY chromosome synapse -recombination in pArs SRY gene goes to X chromosome -genetically female but SRY gene makes male traits.
Haplodiploid Sex Determination
insects, bees, wasps males develop from unfertilized eggs (haploid) females develop from fertilized eggs (diploid)
EMS mutagen
is a mutagen given to model organism
LTR retrotransposons
retrovirus-like elements; all are flanked by long terminal repeats and encode gag and pol Long terminal repeat regions moved around in the DNA -These repeats range from ~100 bp to over 5 kb! -Share many features with retroviruses -A retrovirus can turn into an LTR retrotransposon if the gene for the envelope protein is damaged
Genetic Balance Theory
It is the ratio of the genes in the X chromosome(s) (which are female determining) to the genes in the autosomes (which are male determining) that determines sex in Drosophila. •Threshold of maleness is reached when X:A ratio is 1:2 (X:2 A). •Presence of additional X (X X:2 A) alters balance and results in female differentiation.
Case 3 Patterns of Temperature Sex Determination
Low and high temperatures yield 100% females •Intermediate temperatures yield various proportions of males
Case I Patterns of Temperature Sex Determination
Low temperatures yield 100% females; high
MSY
Male-specific region of the Y -the remainder of the Y chromosome about (95%) does not synapse or recombine with the X chromosome and is referred to as MSY.
Morpholinos
!!!short pieces of modified DNA that block translation!! -have synthetic nucleotides -no phosphodiaster bond (-Bound to methylenemorpholine rings linked through phosphoramidate groups instead of phosphastes.) -25 bps. - Target mRNAs to block their transcription/ translation ***************** (mRNA is processed and leaves nucleus where the morpholinos binds and prevents translation) -Do not destroy the mRNAs -Their bases are the same as RNA
Case 2 Patterns of Temperature Sex Determination
high temperatures yield 100% females; high
ZW Sex Determination System
reptiles and birds homogametic here is male (ZZ) -oocyte determines sex!!
Class 2: DNA transposons, steps for transposition
"cut and paste" 1. Excision transposase needs to be transcribed and translated first then these enzymes will bind to ITR, and cleave out the TE (encoded by the TE (autonomous) or from another TE (non-autonomous) -(two DS breaks) -(cell will repair break where TE was cut from) -(transposase recognizes ITR, and the ITR tells enzyme where to grab) -"makes sticky ends" (staggered SS breaks where DR will fill in) ( careful what sequences and what strand you are adding for DR) **look at picture
horizontal gene transfer (HGT)
(also, lateral gene transfer) transfer of genes between unrelated species
Stem Cells Often Need a Niche
(an organism that fits in the ecosystem.) •is an area of a tissue that provides a specific microenvironment, in which stem cells are present in an undifferentiated and self-renewable state. (keeping stem cells protected in a tissue, from differentiated. -screw up a niche you could get cancer •Cells of the stem-cell niche interact with the stem cells to maintain them or promote their differentiation. -all these other helper cells to maintain the healthy stem cells.
TALENS
(transcription activator-like effector nucleases) -second gene editing tool to come available after ZINC finger -This one is more specific -DOES NOT NEED TO BE IN PAIRS (like Zinc fingers do) -IS a Transcription factor -•Restriction enzymes that can be engineered to cut specific sequences(fok 1) •Made by the fusion of the DNA binding domain of a bacterial transcription factor (TAL) to a DNA cleavage domain********** •DNA cleavage domain is not sequence-specific •The researcher can engineer the TAL domain to be sequence specific (binding TAL to a specific domain) -again just like Zinc Fingers , you can introduce donar DNA into the DNA break!!
Cryptons
** subclass of DNA TE** -also have a circular DNA intermediate -Discovered in pathogenic fungi -Long boys - one has been shown to be 4 kb in length! -Have a long CDS with introns!
What is the Goal of Cleavage?
**** to kick start development.*** *** to sort out maternal determinants**** (cells differentiate) (cleavage is a subtype of mitosis, 1 big cells are cleaved into many small ones, but in mitosis you get 2 daughter cells, cleavage does not do that) -•Polarized Oocytes differentially localize mRNAs and proteins (different molecules types need to subdivide, so when cleavage happens those cells will then begin to differentiate.) **** -but these polarized eggs have different molecules in different parts of the egg •Maternal Determinants (aka cytoplasmic determinants) -are special molecules which play a very important role during oocyte maturation, in the female's ovary. During this period of time, some regions of the cytoplasm accumulate some of these cytoplasmic determinants, whose distribution is thus very heterogenic. They play a major role in the development of the embryo's organs. Each type of cell is determined by a particular determinant or group of determinants. Thus, all the organs of the future embryo are distributed and operating well thanks to the right position of the cytoplasmic determinants. The action of the determinants on the blastomeres is one of the most important ones.
How Can We Make Cells Different During Development?
****Differential Gene Expression (DGE)*** -early on in embryonic development different cells express different genes, through out time. (cis regulatory and trans regulatory elements and how they interact and how they are turned on in specific cell types. ) Same genome but gene expression changes. -methylation, epigenetic
siRNAs in RNAi
***1 pathway to RNAi*** have a foreign long DSRNA molecule, cell signals dicer to initiate interference. 1. Dicer- protein that cleaves the original dsRNA into siRNAs***23 bps 2. siRNAs are then loaded into the RISC 3. then argonaute will take either strand -Guide strand- antisense to the mRNA -Passenger strand - the other strand; degraded by the Argonaute The guide strand acts as a template to "recognize" the target mRNA (directs RISC and argonaute to something that will base pair with it. mRNA) -now you have a double stranded RNA molecule, because you got rid of passenger strand and left with guide strand that is ssRNA and once it base pairs with the mRNA of choice it will be DS. •Once found, Argonaute cleaves the mRNA
Genomic (Nuclear) Equivalence
***dolly the sheep -this demonstrated genomic equivalence, (two different species of lamb. ) 1.non nucleated egg cell from mom (removed nucleus) 2. took udder cells from another sheep 3. sheep breed is the same from udder cells, -- use just need one genome, the donor tissue genomic equivalent to the donor sheep
Dissociation (Ds)
**nonautonomous transposable element*** (DOES NOT HAVE OWN TRANSPOSASE) named by Barbara McClintock for its ability to break chromosome 9 of maize but only in the presence of another element called Activator (Ac). ***Ds moved ONLY if Ac was present**
Zinc-finger Nucleases (ZFNs)
**type of transcription factor** part of a TF -•Artificial restriction enzymes (restriction enzymes are made by bacteria to target viral genome by cutting it) zinc-finger transcription factor domain + DNA-cleavage domain (restriction enzyme) = ZFN •DNA-cleavage domain is non-specific (Specificity attained by engineering the zinc-finger transcription factor domain) TF= binds DNA -Recognizes triplets -can tell what sequence to bind to this is the zinc finger) zinc finger domain recognizes a sequence in the DNA, that researchers are interested in a DS break, and the ZFN encompasses the ZF plus the DNA cleavage domain (restriction enzyme), binds to it and the cleavage domain cleaves it. but they come in pairs of two to complete the DS cut. (the cut will mutate that sequence or you can add a sequence in to that) •******Can ONLY use ZFNs in pairs!!!! •The nuclease domain needs to dimerize *** after ZFN makes its cut, the cell has two ways to repair that. homologous Recombination or non-homologous end joining. ** was the first DNA editor used
Polinton AKA Mavericks
- are large DNA transposons which contain genes with homology to viral proteins and which are often found in eukaryotic genomes. - the largest and most complex known DNA transposons. - encode up to 10 individual proteins and derive their name from two key proteins, 1. DNA polymerase and 2. a retroviral-like integrase •Also encode a capsid-like protein --Virus turned into TE __have similar DNA to virus!! (retrovirus use reverse transcriptase)
Sexual differentiation in humans
- in early embryo development, every human undergoes a period when it is potentially hermaphroditic. -by the fifth week of gestation, gonadal primordia (the tissues that will form the gonad) arise as a pair of gonadal (genital ) ridges associated with each embryonic kidney. (hermaphroditic because at this stage its gonadal phenotype is sexually indifferent, male or female reproductive structures can't be distinguished, and the gonadal ridge tissue can develop to form male or female gonads. as development progresses, primordial germ cells migrate to these ridges, where an outer cortex and inner medulla form (cortex and medulla are the outer and inner tissues of an organ the cortex is capable of developing into an ovary, while the medulla may develop into testis. (in early development , two sets of undifferentiated ducts exist in each embryo)******* -wolffian ducts= male -mullerian = female -the presensce or absence of a Y chromosome is the key, -if cells of the ridge have an XY , development of the medulla into a testis is initiated around the 7th week. -however if , no Y chromosome is present, then no maleness is present, the cortex of the ridge forms ovarian tissue, and the mullein ducts forms oviducts and other portions of the vagina. -depending on which pathway is initiated, parallel development of the appropriate male or female duct system occurs, and the other duct system degenerates. -if testes differentiation occurs, the embryonic testicular tissue secretes hormones that are essential for continued male sexual differentiation -Y chromosome and development of testes inhibit formation of female reproductive system. -in females oocytes become arrested in meiosis and remain dormant until puberty. -males don't produce primary cells until puberty.
LTR transposition (more like how does reverse transcription work for LTR to encorporate into genome)
--this step involves reverse transcribing mRNA, occurs in cytoplasm, forms cDNA which is also the LTR retrotransposon that will then be inserted somewhere in the genome -integrase then binds LTR to the cDNA -then the cDNA is complete and is transported back into nucleus, ultimately inserting itself into the genome **** very first thing, your cells machinery is going to be picked up by this LTR transposon and transcribe a mRNA, reading 3' to 5' making a mRNA 5' to 3'****** 1.A host tRNA (blue) base-pairs to a sequence, the primer binding site, near its 5 ́ end •tRNA serves as the primer for DNA synthesis for reverse transcriptase (Rtase) (tRNA is RNA so it can bind to itself, in this case tRNA binds to mRNA, actually base pairs with LTR (** to synethize DNA, you need a primer(starting sequence) as seen in Okazaki fragments so then you can begin to build off primer) -the tRNA binds to the u5 site!!!!!!! 2.Rtase copies the R and U5 sequences into DNA (is a RU5 tRNA molecule now) and falls off the mRNA transcript at the 5' side to go base pair at the 3' LTR portion of mRNA) 3.This new DNA sequence (tRNA primer) can then base-pair to the R of the 3' LTR (tRNA base pairs with R site of mRNA) 4.The first strand of DNA is then reverse transcribed (these first few steps, build primer of tRNA and RU5 molecule bu synthesizing on 5' of LRT which is still the mRNA, and then once its got its primer, normal synthesis of DNA can occur, by going over to 3' of mRNA and reverse transcribing into DNA in normal fashion of reading to mRNA 3' to 5' and building DNA in the proper direction. ) 5. The RNA template is degraded by RNAse leaving only a fragment at the 3' site (RNAse chomps up RNA sequence, and leaves a little behind to act as a primer so synthesize the other strand of cDNA •Reverse transcriptase has to switch templates again using complementarity between the two copies of R and U5. •Synthesis then proceeds in both directions to give double-stranded DNA with and LTR, made up of U5, R and U3, at each end.
two types of retrotransposons
-LTR (long terminal repeat) -and non-LTR
splicing enhancers
-are cis-regulatory sequences found in exons!!! -sequence that tell spliceosome where to sit (spliceosome are protein and rna molecules that cut out introns and tie together exons. How does the cell know how to make a different mRNA and thus different protein? =splicing enhancers -depending on what proteins are around the mRNA, the spliceosome knows where to splice. (proteins are floating around cytoplasm and be attracted to splicing enhancers and thus promote different gene expression through splicing some genes have multiple exons, and their are many variants of each exon, depending on how they are spliced , making 1000's of different proteins.
Non-LTR Retrotransposon mechanism
-do not have LTR sequences at their ends ; 1. SINEs- short interspersed elements; 2. LINEs- long interspersed elements; (together makeup 34% human genome) **Contain 1 or 2 open reading frames (ORFs) -Contain genes for reverse transcriptase, RNA-binding protein, nuclease, and sometimes ribonuclease H domain -•Following transcription and nuclear export, the ORF(s) are translated and assemble to form a ********ribonucleoprotein particle (RNP)****** •These particles are then placed back into the nucleus and then integrated into the chromosomes
Potency defines stem stem cells
-drives from potential to become specific things -the potential of that stem cell to become different cell types. totipotent- can make any cell (not limited, the first 4 to 8 cells are toti -pluripotent, can't become everything but almost everything. -mulitpotent- only can become certain types of cells, the c (gut stem cell can become any cells of the gut but can't become neuron. only that specific cell in that tissue its in.
LTR retrotransposon genes
-gag - Group-specific antigen •Makes up the inner shell of the viral coat --the retro transposon still thinks its a virus , codes for inner coat of viral shell, it won't be functional but still evolved from virus pol - One giant gene; encodes... •Reverse transcriptase •Integrase •Protease - cleaves the products of these genes into separate units ****** this pol gene codes for one long protein, but protease cleaves this protein to make two separate proteins****
steps in retrotransposon transposition
-involve the actions of both retrotransposon-encoded proteins and those that are part of the cells normal transcriptional and translational machinery. -first step the cells RNA polymerases transcribe the retrotransposon DNA into one or more copies -second step the RNA copies are translates into the two enzymes required for transposition (reverse transcriptase and integrase) -third step the retrotransposon RNAs are then converted to DS DNA copies through the actions of reverse transcriptase. The ends of DS DNAs are recognized by integrate, which then inserts the retrotransposons into the genome. ---because many RNA copies can be converted to DNA and transposed in this way, retrotransposons can accumulate rapidly and may create mutations at many sites in the genome.
mRNAi
-maternal RNA interfernce (all off spring will have this interference and that gene will be turned off)
miRNA
-micro (a little bigger then siRNA) -ALSO DOUBLE STRANDED a class of functional RNA that regulates the amount of protein produced by a eukaryotic gene (actually encoded in our genes) -come from internal or inside our genes and cells -miRNAs are abundant in many mammalian cell types[8][9] and appear to target about 60% of the genes of humans and other mammals -a way for your cell to regulate gene expression
structure of GRN
-nodes represent genes/proteins/mRNA -Edges between two nodes= are lines that show the interaction of the nodes -G1 interacts with G2 to turn on g3 (an example)
siRNA
-small interfering -made in response to a virus!!!! (external of our cells) (-are derived from longer double stranded RNA (dsRNA) molecules and chops them up into smaller or siRNA molecules . these long dsRNA may appear with cells as a result of virus injections or the expression of TE, both of which may synthesize dsRNA as part of their cell cycle. dsRNA gets into cell and knows it is bad and once to get rid of it by initiating RNAi now these siRNA bind complementary to the target mRNA or the virus. •Targets the mRNA for degradation •Each strand has a 2-3 bp overhang (has sticky ends on both sides)
Developmental Genetics
-studies how genes are expressed during development (broad name) •Development: Slow processes of progressive change in an organism (interface of genotype and phenotype) in the middle of genotype and phenotype is development.**** look it as, making different cell types from 1 cell (zygote) to make 1000's of different cell types •Embryology: The study of development between fertilization and birth •NOT ALL DEVELOPMENTAL BIOLOGY IS EMBRYOLOGY!!! (small part of developmental study) •You have never stopped developing! •Regeneration, metamorphosis, etc! -anything that develops
How do you make all differentiated cell types? How do you translate the genome?
-three inter related processes. 1.cell division 2. cell differentiation 3. morphogenesis -The process by which an organism takes shape and the differentiated cells occupy their appropriate locations. (what different cell types you need to make an organ) -different cell types come together to make a specific organ.
ribonucleoprotein particle (RNP)
-translated into particles that aid in the transposition of non-LTR!!! NON-LTR •Contain 1 or 2 open reading frames (ORFs) •Contain genes for reverse transcriptase, RNA-binding protein, nuclease, and sometimes ribonuclease H domain -•Following transcription and nuclear export, the ORF(s) are translated and assemble to form a ribonucleoprotein particle (RNP) •These particles are then placed back into the nucleus and then integrated into the chromosomes
The Genetics of Maintaining Potency
-we can identify where a stem cell is based on where its at in the cell cycle. -differes from somatic cell cycle •Embryonic Pluripotent Stem Cells •Modified cell cycle •Shortened G1( a lot of differentiated goes on ) not specific cell type so won't spend a lot of time here. •G0 Absent •Generally, as a cell differentiates, the G1 and G2 phases lengthen •At the G1 phase, the cells are more sensitive to differentiation •No oscillatory expression of cyclins or CDKs (not in stem cells of cell cycle) ******Cyclin E is always "on" or active (g1 and S) right to m to s, not differentiating, just want to replicate and grow tissue. •Allows for the direct transition from M to Late G1, which is shortened •ESCs are also characterized by a non-functioning G1 checkpoint synthesizing gene products in g1 and g2 needed for that cell type. turning on genes. starting to differentiate
U3 region
-•Contains the Enhancer and promoter sequences necessary for transcription found in LTR (remember a promotor binds RNA polymerase to allow for transcription) (enhancer bind transcription factors
Primary Sex Determination in Mammals (XX)
-•the "pre-ovaries" produce estrogen •Enables the Mullerian duct to form the uterus, oviducts, cervix and the upper portion of the vagina
the processing steps of bacterial crispr system?
1. Foreign DNA acquistion (viral bacteriophage inserts its dna into bacteria cell) 2. crispr locus transcription (this locus is where it stores spacers and repeated sequences) -now called pre-crRNA 3. Crispr RNA processing -pre-crRNA is processed into a repeated segment and spacer. now this segment is sequence specific to attack a sequence of viral element (the crRNA has stem and loop that can interact with cas-9) 4. RNA guided targeting of viral element. (interferes with viral genome and chops it up) -cas genes cut up genes of viral genome
Mutation by Double Strand Breaks (DSBs) -the two methods of recombining breaks?
1. Non-homologous end joining •"non-homologous" because the break ends are directly ligated 2. homologous recombination ( holiday junction)
miRNAs in RNAi
2. pathway of RNAi Encoded by endogenous genes (endogenous= in our genome) -we use it to Regulate our own gene expression, acting as a dimmer switch •Require processing before activation •Then processed to a 70 bp pre-miRNA by the enzymes Drosha and Pasha(DGCR8) 1. we have miRNA gene , and expressed by RNA poly 2, 2. will form a primary RNA (Initially expressed as a long pri-miRNA - may have up to 5 miRNA precursors) 3. then DRosha and pasha turn the pri-miRNA into pre-miRNA -then the miRNA is shuttled out of nucleus and same process repeats itself. -dicer -small rna -then risc and guide strand and binds to target.
the target site of crispr plus 3bp pam is ?
23 bp -20 bp of target sequence. picture
androgen insensitivity syndrome
A condition caused by a congenital lack of functioning androgen receptors; in a person with XY sex chromosomes, causes the development of a female with testes but no internal sex organs -•In humans, some XY individuals... •Do have SRY •Thus, make testes and make anti-Mullerian hormone •However, they have a mutation in the testosterone receptor •They CAN respond to the estrogen made by the adrenal glands
RISC (RNA-induced silencing complex)
A protein complex that is targeted to specific mRNA molecules by base pairing with short regions on the target mRNA, inhibiting translation or degrading the RNA. -contains argonaute family protein that binds RNA and has endoribonuclease or "slicer" activity. -RISC cleaves and evicts one of the two strands of the DS siRNA and retains the other strand as a single-stranded siRNA "guide" to recruit RISC to a complementary mRNA. RISC then cleaves the mRNA in the middle of the region of siRNA-mRNA complimentary. (its going to grab a siRNA/miRNA and find anything that base pairs with it and destroy it)
restriction enzyme
A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes.
Ac-Ds system in maize
Ac (Activator) - transposable element with functional transposase Ds (Dissociation) - does not contain a functional transposase gene (deletion) so it requires the AC transposase to move. -•Mobile controlling elements in corn plants found using A c-D s system. •Two mutations of transposable elements. ***picture from book***
Hemophilia
An X-linked recessive disorder in which blood fails to clot properly, leading to excessive bleeding if injured. -LInes (long interspersed elements) -can transposition into this gene and cause a mutation
reverse transcriptase
An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis. -also seen in retrotransposons
CRISPR/Cas9 in humans
Cas 9= nuclease (cleave nucleic acids) Cas 9 needs to know where to cut -this is where crRNA comes in crRNA = spacer = viral DNA, guide the nuclease (cas 9) (to base pair to specific sequence) tracr RNA= needed to help crRNA integrate into cas 9, this is repeated segment stem and loop (transactivating crRNA) partially complimenary base pairs with crRNA or spacer -together form gRNA.
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats -An Adaptive Immunity System in Bacteria -•These are DNA sequences •Derived from bacteriophages that had previously infected the bacteria •Used to detect and destroy DNA from similar viruses during subsequent infections. (this is the adaptive immunity part) (bacteria learn by incorporating the viral DNA into its own genome, and now has memory to attack it if it is introduced again into bacteria cell) -this has evolved from evolution, not something scientists developed -repeated segments of DNA over and over -
transposase
Cuts DNA backbone, leaving single-stranded "sticky ends" -binds to the end of a transposon and catalyses its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. -picture** gene encoding transposase consists of 1 ORF , 4 exons 1.binds to ITR 2. cut to make sticky ends -if gene encoding transposase is mutated you could get a non functional enzyme ** transposase gene hacks the machinery of cell to make enzyme. (does not have own machinery= parasite)
How are TALENS MADE? WHAT FUSED together?
DNA binding domain of a bacterial transcription factor (TAL) to a DNA cleavage domain
trans regulatory elements
DNA sequences that modify the expression or activity of genes that are not nearby on the chromosome, often by coding for transcription factors*** -•Genes that modify the function of distant genes •DNA sequences that encode trans-acting factors (sequence that codes for TF) •These are usually transcription factors •These are also HIGHLY pleiotropic ( hox gene, for example, that regulate 100's of genes.
Even-skipped gene (eve)
DNA that is needed to segment embryo (different enhancers that drive the expression of these genes in different stripes of embryo) -seen in drosophila and other insects can drive gene expression by regulating what enhancers you turn on
Wolffian ducts
Early embryonic ducts that can develop into male internal genitalia under the proper stimulation (testosterone). -medullla formed by germ cells and gonadal ridges.
GRN's as control system
GRN's control animal development -genes often evolve together as one unit!!! -regulate the expression of 1000's of genes -can be homologous (GRN can be homologous, because they are so important for a specific function, they are conserved through many organisms. -example eye development has many of the same genes (GRN) amongst different organisms.
transposable elements
Genetic element that has the ability to move (transpose) from one site on a chromosome to another. -jumping genes - present in genomes of all organisms (bacteria, human) -45% of human genes -the movement of TE from one place to another has the capacity to disrupt genes and cause mutations, as well as to create chromosomal damage such as DS breaks -•Contribute to... • spontaneous mutation(framshieft= indel/ nonsense and missense) • genetic rearrangements •horizontal transfer of genetic material •Aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) •Cells must depress transposition to insure genetic stability __ telomerase is a TE, The fruit fly Drosophila melanogaster lacks telomerase, but instead uses retrotransposons to maintain telomeres
DNA transposons
Mobile genetic elements that move without making an RNA intermediate. *****"CUT AND PASTE"***** (transposon is physically cut out of the genome and then inserted into a new position in the same or a different chromosome. the site in which it was cut from is usually repaired with no trace of the original DNA transposon -inverted terminal repeats (ITRs) are located on each end of the TE, and an open reading frame (ORF) codes for the enzyme transposase. (both are required for movement) -ITR is essential for transposition and are recognized and bound by the transposase enzyme -direct repeats (DRs) are short DNA sequences that flank transposable elements in which the DNA sequence is repeated in the same direction (these flanking DRs are creates as a consequence of the TE insertion process) classifies as either 1. autonomous 2. nonautonomous
The Ratio of X Chromosomes to Sets of Autosomes Can Determine Sex (C. elegans)
Nematode (worm); used as a model organism in studies of animal development -- have no Y chromosomes •Two sexual phenotypes: •Males with one testes have only one X chromosome, no Y. •Hermaphrodites (have both testes and ovaries): (Have two X chromosomes, no Y.) •Eggs are fertilized by stored sperm-self-fertilization. •Majority of offspring are hermaphrodites; less than 1% are males. hermaphrodites fertilize each others eggs!!!! have same number of body cells as each other. interesting fact
Which of the following elements allows for homologous pairing in meiosis?
PARS -The pseudoautosomal regions refer to regions of homology between the X and Y chromosomes in humans that undergo recombination during meiosis
Tsix gene
Prevents X chromosome inactivation -This gene product is ncRNA from the inactivated X chromosome. -is expressed on the inactive chromosome and not on the active one. -• - antagonizes the Xist RNA (blocks Xist ncRNA) -used by good copy X chromosome
Endogenous genes
Produced within the body
what is viral dna called in bacteria crispr?
Spacer!! -specific to the virus that it attacks (viral DNA that have been integrated into crispr locus of bacteria)
pluripotent stem cells
Stem cells that can become almost all types of tissues and cells in the body. -germ cells, endoderm,
passenger strand
Strand that is degraded and decomposed after cut by dicer in RNA -other strand the guide strand is antisense to the mRNA
forward genetics
The classical approach to genetic analysis, in which genes are first identified by mutant alleles and mutant phenotypes and later cloned and subjected to molecular analysis. -you have a phenotype, and now you want to find what gene is broken and what is its genotype. 1. give MALE Drosophila EMS mutagen and now their sperm and other cells have mutations. 2. now cross that wildtype females 3. each offspring will carry a variety of mutations 4.
Secondary Sex Determination
The determination of the male or female phenotype by the hormones produced by the gonads. •Male and female duct systems and external genitalia
Morphogenesis
The process by which an organism takes shape and the differentiated cells occupy their appropriate locations. -what different cell types you need to make an organ) -different cell types come together to make a specific organ. -ex. to make the eye, you need multiple cell types to make up the eye.
SINEs (Short Interspersed Nuclear Elements)
The second most abundant class of transposable elements in human genomes; can create copies of itself and insert them elsewhere in the genome. -•Non-LTR Retrotransposons •100-700 bps long •All are non-autonomous •Rely on LINEs •SINE RNAs form a complex with LINE proteins and are inserted into the genome by target primed reverse transcription rely on Lines*****
Retrotransposons
Transposable elements that move within a genome by means of an **RNA intermediate** -amplify and move within genome by way of RNA intermediate (CLASS 1) (transposon is first transcribed, then reverse transcribed by reverse transcriptase )****** -"copy and paste" (viruses replicate like this) -contrast to class 2 (DNA transposons) ***42% of human genome, other 3% is DNA transposons -2 types of retrotransposons ---- long terminal repeat (LTR) ---- and non LTR (lines and sines) -- can be either 1. autonomous 2. non autonomous -encode proteins that are required for their transposition and are flanked by direct repeats act their insertion sites.
Drosha
cuts up primary miRNA (pri-miRNA) to generate pre-miRNA (occurs in nucelus!!) -POSHA binds to primary miRNA so that drosha can cut
Primary Sex determination
determination of gonads
XIC
X inactivation center -4 genes -2 that matter -these gene products come from "inactivated X" XIst- coats X chromsome to signal for Barr body Tsix- counters Xist in good X chromosome
How do we get about inactivating an X chromosome to make Barr body?
Xic: Mechanism of Inactivation (X inactivation center) -a region on the X chromosome -in placental mammals -has 4 genes 2 important ones -Xist- activated by the chromosome that will soon be Barr body! -Tsix- protects from Xist on good X chromosome (these names are mirror images of each other) -these two genes expression only occurs on inactivated chromosome!!
Studying Gene Function
how genes work? -you break them, or you mutate them and see what happens. •Forward Genetics •Finding the genetic basis for a particular phenotype •Human Genetic Disease Screens; linkage mapping •Example: Drosophila segmentation genetic screen
what is a stem cell
a cell that can differentiate and become any type of human cell -the stem, from where all the branches that all the cells come from -Gives rise to differentiated cells through cell division (a cell that is 100% cell type that it will be the rest of its life. neuron stays neuron will never go back. -when it divides , one daughter cell becomes differentiated cell and the other stays stem cell (to maintain the population or homeostasis of stem cells) most adult stem cells are quiescent. stay dormant until needed for tissue regeneration
long terminal repeat (LTR)
a direct repeat of DNA sequence at the 5' and 3' ends of retroviruses and retrotransposons ** LTR is the control center** (allows to jump around) *** NOT MIRROR IMAGE** (same order upstream and downstream U3RU5) -U3 region- Contains the enhancer and promoter sequences necessary for transcription -R domain encodes the 5′ capping sequences (5′ cap) and the polyA (pA) signal -U5- purpose unclear; may aid in polyadenylation
Autonomous transposons
able to transpose by themselves, as they encode a functional ****transposase enzyme**** and have intact ITRs ****class 2 DNA transposons
Methylation
another form of cell differentiation -epigenetics-modification of ACTG's (only C and A, mostly C) -turn genes on and off (depends on the gene) •Methyl groups can be added to DNA sequences to modify expression •Only cytosine and adenine can be methylated •Cytosine methylation is more common •CpG Islands where most methylation occurs**** (Methylated regions of the vertebrate genome (CG repeats) •Often occur in promoters( on and off) (if one methyl then promotor can still bind TF, if heavily methylated then steric hinderance and TF can't bind. depending on cell development, a promotor can be active and turn on gene, once gene expression is no longer needed, the promotor will become methylated and gene expression is turned off.
guide strand is sense or antisense?
antisense to the mRNA -the guide strand will guide argonaute to find something to base pair with it. ---In double-stranded DNA, only one strand codes for the RNA that is translated into protein. This DNA strand is referred to as the antisense strand. The strand that does not code for RNA is called the sense strand.
fate maps
diagrams showing organs and other structures that arise from each region of an embryo -shows how cleavage can shuttle maternal determinants (mRNA that are moved around) -one nucleus, egg cell, as it matures, it will move around the mRNA around before cleavage, then those cells will inherit the proper proteins and mRNA needed for that cells function or differentiation
what cleaves pre-miRNA?
dicer
cis regulatory elements
are DNA sequences*** that modify the expression of other genes that are nearby on the chromosome, often by acting as binding sites for transcription factors cis= same side -regions of ncDNA, that are close to the DNA or gene that they regulate -regulating genes by binding to TF. **which in turn control morphogenesis, the development of anatomy, and other aspects of embryonic development. -TF are DNA binding proteins (A single transcription factor may bind to many CREs, and hence control the expression of many genes (pleiotropy).)********* -•Cis-regulatory Elements •Regions of non-coding DNA •Found in the vicinity of the genes that they regulate •Typically regulate gene expression by binding to transcription factors •Include... •Promoters- bind polymerase •Enhancers- TF can bind to enhance gene expression •Silencers- TF can bind to repress gene expression •Insulators- keep genes protected from other TF
stem cells
are needed for the formation of organs - zygote is a stem cell, will give rise to every cell in your body -adult stem cells, important for maintaining tissue and homeostasis. (cheek cells that are constantly dividing, intestinal stem cells that give rise to new cells cancer stems, from misregulation of GRN , to make stem cells, stem cell that keeps dividing and not regulated. you want it to be regulated and divide when necessary.
Maternal Determinants (aka cytoplasmic determinants)
are special molecules which play a very important role during oocyte maturation, in the female's ovary. (everything else other then DNA = ribosomes and mRNA that is moved around that will then get cleaved to begin development of organs. -anchor mRNA appropriately in the egg, so then can express genes needed for organ development. During this period of time, some regions of the cytoplasm accumulate some of these cytoplasmic determinants, whose distribution is thus very heterogenic. They play a major role in the development of the embryo's organs. Each type of cell is determined by a particular determinant or group of determinants. *****Thus, all the organs of the future embryo are distributed and operating well thanks to the right position of the cytoplasmic determinants. **** The action of the determinants on the blastomeres is one of the most important ones. ************************************************************* those cells, once cleavage happens, will inherit those maternal determinants. The determinants are all sorted before cleavage. then after cleavage , those cells have the proper proteins and mRNA needed to differentiate -also goes along with maternal inheritance (if mutations in these determinants, does not matter what the genotype of fetus, will inherit these defects, because these determinants are needed for cell differentiation.
Activator (Ac)
autonomous TE (HAS OWN TRANSPOSASE)
Alternative RNA Splicing
can be used for cell differentiation -a single gene can encode multiple proteins (1 gene doesn't necessarily code for one protein) -Eukaryote genes have introns (prokaryotes do not) -have different isoforms (can splice exons differently, can combine them differently) -depending how you splice and arrange exons, you can produce different proteins. a way to regulate genes, (depending on what gene products the cell needs, it can splice introns differently and produce those products -take gene (pre-mRNA) = Introns and exons 1. spliceosome will cut out introns and tie together exons to make spliced mRNA that can now leave nucleus -(introns can be identified by the sequence 5' GU and ends with the sequence 3' AG) -A branch site spliceosome (snRNA= RNA and protein components)
Non-autonomous transposons
can't move on their own because they DO NOT encode their own functional ***transposase enzyme*** **require the presence of an autonomous transposon elsewhere in the genome, so that the transposase synthesized by the autonomous element can be used by the non autonomous element for transposition.
multipotent
cell with limited potential to develop into many types of differentiated cells -(gut stem cell can become any cells of the gut but can't become neuron. only that specific cell in that tissue its in. -most stem cells in adult stem cells ( certain range of cells from which tissue it came from)
Class 1 vs Class 2 transposons
class 2 = DNA transposons (no rna intermediate) "cut and paste" class 1= retrotransposon (rna intermediate) "copy and paste" transcription to rna and then reverse transcription by reverse transcriptase.
quiscent
dormant, not proliferating ***not dividing until needed*** (but still has cell function) adult stem cells -reversible and stable (a kind of epigenetic) organs need regenerated (lungs intestines, skin) -cell has two competing choices, 1. it can transcribe its DNA into proteins for cell function or 2. it replicates its DNA and goes into s phase (competition inside the cell whether DNA poly will be active or RNA poly)
RNA Interference (RNAi)
double stranded RNA is usually an intermediate for retro virus and the cell recognizes as foreign and bad -the dsRNA virus will then encode mRNA and soon protein products. the cell freaks out and use RNAi to target all of the mRNA from the dsRNA (virus) -2 subtypes of ncRNA involved in this process 1. siRNA (small interfering) 2. miRNA (micro) -these are the two pathways in which interference can be done. **same pathway but what is your starting RNA molecule •Utilizes dsRNA (dsRNA is rare inside eukaryotes.) Rare in the cell, except those that are made by viruses **a mechanism by which noncoding RNA or (ncRNA) (miRNA or siRNA) molcules guide the postranscriptional silencing of mRNA's in a sequence specific manner. ** ***!!!!!!!!most important thing to remember of RNAi is that since ncRNA can associate with mRNA through complimentary base pairing, ncRNA are able to TARGET SPECIFIC mRNA and, via associated proteins, destroy the mRNA or block their translation. RNAi may have evolved as a mechanism to recognize these dsRNA and inactivate them, protecting the cell from external (viral) or internal (TE) assaults. is the silencing of gene expression triggered by the presence of ds RNA homologous to portions of the gene. The gene silencing generally results from the cleavage and degradation of a target gene's mRNA, but it can also result from blocking translation of intact mRNA. --•An ancient immunity pathway against transposable elements and/or viral elements (used to get rid of virus's) -•Also used for gene-regulation •Likely evolved at the base of the eukaryotes
RNAi as a Molecular Tool
dsRNA can be synethesized from scientists (Just need the sequence of the target mRNA) •Can be made via PCR •eRNAi - embryonic RNAi •mRNAi-maternal RNA **** RNAi has off target effects!!!!!
what can you do with crispr?
edit DNA -cut it -NHEJ -introduce another piece of DNA, HDR -piece of a gene is inserted with homologous remodeling with Holliday junctions. how do you validate it? PCR amplify gene that had crispr run on gel -- you can also sequence it to be sure!!
The Weird Monotreme System
egg laying mammals -no nipples -more reptile like sex chromosomes -10 sex chromosomes (if 10X then female -if 5X and 5Y then male) during meiosis , all the x's go together and all the y's go together.
eRNAi
embryonic RNAi
XIST gene
encodes an RNA that binds to and inactivates the X chromosome -codes for ncRNA (does not code for protein) -The inactivated X chromosome is coated by XIst RNA's -turns on this gene in inactive X chromosome!!!!
R domain
encodes the 5′ capping sequences (5′ cap) and the polyA (pA) signal on LTR -5' cap comes out first from RNA polymerase guanine cap -3' adds poly A tail
Protease
enzyme that breaks down proteins
Dicer
enzyme that cleaves and processes double stranded RNA (foreign) to produce siRNAs or miRNAs that are 21-25 nucleotides in length -different protein from RISC is initiated by the cell in response to invading dsRNA -is a ribonuclease -targets the long DSRNA, and dice it into shorter siRNA
Aromatase
enzyme that converts testosterone to estradiol -•Temperature effects on enzymes: •Aromatase converts androgens (male hormones such as testosterone) to estrogens (female hormones such as estradiol). •Case II
Drosha and Pasha
enzyme that take primary miRNA and turns into pre miRNA picture
Pluripotent Stem Cell Research
ethical concerns about embryos can use mice to study same thing in humans Once harvested, the pluripotency of the stem cells are maintained via niche transcription factors •Oct4, Sox2 and Nanog take early embryo , 8 cell state, more likely pluripotent -suck up with needed -place in tube and maintain niche, -we can figure out how potency works in stem cells -constantly changing -are all stem cells equal in potency _nope there not all equal -2 different type -1 naive -(greatest potential for pluripotentcy.
Totopotent Stem Cells
first 4-8 cells, can become any cell type
vertical gene transfer
flow of genetic information from one generation to the next **picture**
gonadal ridge
future gonads in 5-6 week old fetus -usually refered to as bipotential gonads. -the pretensce or absence of a Y chromosome is the key, -if cells of the ridge have an XY , development of the medulla into a testis is initiated around the 7th week. -however if , no Y chromosome is present, then no maleness is present, the cortex of the ridge forms ovarian tissue, and the mullein ducts forms oviducts and other portions of the vagina.
transposon (class 2) anatomy
gene that encodes transposase (1 ORF and 4 exons) -flanking the transposase gene is 2 ITRs (ITRs to understand their sequence just flip twice of each other and you will get the inverted sequence) -direct repeats, these flanking DRs are created as a consequence of the TE insertion process (fill in the gaps of the sticky ends after insertion) ****** these are not part of the transposon, and stay behind when the TE leaves********
How is Self-Renewal Achieved?
mitosis (two identical cells) -•Symmetric Cell Division •Results in either the production of two identical stem cells, or two cells that are committed to differentiate (make one stem cell will divide to two new stem cells) -two new cells, two identical cells, either two tissue cells or two stem cells -two of the same •Asymmetric Cell Division •Results in a stem cell, and one that is ready to differentiate •Stabilizes the stem cell pool •This is called "Single Stem Cell Asymmetry" (stem cell, divides into differentiated cell and the other daughter cell will stay stem cell maintain population •Population Asymmetry •Some stem cells are more prone to make cells to differentiate, (spermagenesis, one becomes sperm, one becomes sperm stem cell) •While others are prone to make more stem cells. •Some stem cells are more "potent" than others due to asymmetry
Are All ESCs Equal in Potency?
nope -•We now recognize two different pluripotent states of ESCs 1 Naïve: has the greatest potential for pluripotency (can still become lots of other things 2. Primed: an ICM cell with some maturation towards the epiblast lineage (starting to differentiate to more multipotent. ) not multipotent yet, but pluripotent primed stem cells are starting to express genes needed for differentiation ***Primed for differentiation*** all development is, totally totipotent -turning on different genes to kick start - computer program, turn on a gene in one cell, to make specific cells needed for development.
ribosomal selectivity
not all ribosomes are the same!! -different ribosomes, through evolution, have been selected to translate only a few or certain mRNA. -will translate one mRNA over another
maternal effect
nuclear genotype of mom affects phenotype of progeny through substances present in egg -offsprings phenotype for a specific trait, are under the control of mothers nuclear gene products present in the egg -determined by mothers genotype -maternal determinants are positioned in the egg, and the way they are arranged the offspring will express a phenotype.
Temperature-Dependent Sex Determination
occurs when the sex of an individual is determined by the temperature at which eggs develop -•Environment, specifically temperature, has profound influence on sex determination. •Enzymes involved in steroids, androgens, and estrogen synthesis are affected (inhibited) by temperature. (certain enzymes are only activated at the correct temp **Phenotype is ALWAYS the combination of genes interacting with the environment.
what codes for transposase?
open reading frame
the Pam sequence is found on what?
opposite strand to which guide RNA on chispr binds too -crispr with guide rna binds to the target sequence and on the opposite strand of that the pam sequence is found.
exogenous genes
outside source of DNA
GRN (gene regulatory network)
part of cell differentiation (gene regulation) -a network of genes that interact, or gene products that interact to make a specific thing. -a set of -proteins -mRNA -genes (all interact to control a specific function) - a specific function and all the regulatory network that interact and talk to each other. -visualize interactions to see a specific function.
TSP
period of incubation that temperature is important to determine sex. -•Critical period of incubation is known as the thermosensitive period (TSP)
Stem Cells Can Differentiate Via Many Factors
physical mechanisms maintain this way or instruct to differentiate depending on signal depending on what you grow stem cell, it will differentiate different (what they grow on) -elasticity, Mesenchymal Stem Cells (MSCs) can be influenced by their cell matrices •Laminin keeps MSCs in a state of stemness •If grown on soft collagen, they differentiate into neurons •If grown on moderately elastic collagen, they become muscle cells •If grown on harder matrices, they can become bone cells
Mullerian ducts
precursors to female internal structures -cortex forms by germ cells and and gonadal ridges
Argonaute
protein that is part of RISC thatr binds to one strand of the miRNA or siRNA molecule -most important part of RISC -is an RNA nuclease enzyme that cuts RNA -binds RNA and has endoribonuclease or "slicer" activity.
the PAM
protospacer adjacent motif -DNA sequence that is upstream of target of cas 9 -part of the virus -a way to distinguish viral DNA, part of invading virus but not part of crispr locus. -NGG -cas 9 cuts 3/5 bp upstream of PAM. -great picture
LINEs (long interspersed nuclear elements)
repetitive DNA sequences, interspersed throughout the genome **Non-LTR Retrotransposons** •Roughly 21% of the human genome •Each LINE is roughly 7,000 bps long -possibility to jump into a coding region of your DNA and cause a mutation. (frame shift= indel/ missense or nonsense) -nonautonomous** -endonuclease cuts DNA, RNA binds one of the DNA strands, now reverse transcriptase builds the rest of the DNA strand using the RNA as a template, then ligates the newly synthesized to the break in DNA. then reverse transcription builds the second strand using the first strand (that strand it just made) as a template to make compliment strand that will now be inserted into genome. (non autonomous so it needs help)!!!
The Ratio of X Chromosomes to Sets of Autosomes Can Determine Sex (drosophila)
same sex chromosomes as mammals -homogametes- XX -heterogametes- XY •Drosophila have same sex chromosomes as humans: •Female XX and male XY. •However, Y chromosome does not determine sex in Drosophila (no SRY gene) -if you have two X chromosomes you are female, if not you are male -•The Y chromosome is NOT involved in determining sex (Has a collection of genes involves in making sperm) •If three sets of autosomes and two X chromosomes, the fly is a mosaic (phenotype is half female , half male) sterile!! 1. chromosome is sex 2,3,4 are autosomal haploid =4 diploid =8 ****sex is determined by Ratio!!!!!!!!
repeat sequences in crispr?
sections of repeated sequences that are between spacer/viral dna that has been inserted into bacterial genome. -RNA sections that are foldable into stem and loop structures
Sex determination and sexual differentiation is controlled by
sex chromosomes
homogametic
sex chromosomes are of one type Females (XX) vs heterogametic that is XY
Direct repeats (DRs)
short DNA sequences that flank transposable elements ( dna transposons) in which the DNA sequence is repeated in the same direction -these flanking DRs are created as a consequence of the TE insertion process (will stay behind if TE leaves, these repeats are necessary to ligate the gaps after transposase cleaved at the ITR) ***foot print it leaves after transposition occurs***
inverted terminal repeats (ITRs)
short segments of DNA that have the same nucleotide sequence as each other but are inverted relative to each other. -essential for transposition and are recognized and bound by the transposase enzyme --- very important for transposase to recognize this sequence, if it does not, then it can't bind
Cell Differentiation
taking one genome and making many different cell types. Genomic Equivalence (All cells in the zygote have the same genome!) (except gametes) and SNP's *****Differentiation results from differential gene expression***** (different cell types turn on different genes at different times during development. )
no presence of Y chromosome
the cortex of the ridge forms ovarian tissue, and the mullein ducts forms oviducts ( fallopian tubes) and other portions of the vagina.
Integrase
the enzyme responsible for integrating viral DNA into the host cell's DNA -seen in retrotransposons -grabs DNA and inserts into genome
bipotential gonad
the gonad of the developing embryo that is capable of differentiating along two developmental pathways toward the development of either a testis or the ovary -•Both male and female gonads are formed by the same precursor -aka gonadal ridges...
stem cells
trans regulatory elements, that code for TF, and these cells will differentiate . only giving them TF, give them different fates.
blue section of X chromosomes
translocated from marsupials
how do we hack into crispr cas 9
use to edit DNA -cas 9 protein -crRNA -tracr RNA (need all 3) but you can make a single gRNA from fusing crRNA and tracrRNA) -can modify the target, target sequence must be by PAM (don't want off target effects) look a lot like your sequence of interest. *** target site must be around 20 bp!!!!!
PAR inherited autosomal fashion
when far apart they crossover like not linked
Common Mechanisms to Maintain Stem Cells in a Niche
will know what it is touching -chemical regulation 1.Physical Mechanisms - structural and adhesion factors 2.Chemical Regulation - proteins and hormones secreted from other cells 3.Intracellular Mechanisms (gene expression) 1.Cytoplasmic determinants - movement of these as the cell divides 2.Transcriptional Regulation - the internal network of transcription factors that silence or maintain specific gene expression 3.Epigenetic regulation - chromatin accessibility (heterochromatin , want stem cells tightly bound keep them quite to keep undifferentaited.
Often a stem cell can generate (give rise to) many different cell types
zygote stem cell will give rise to trillions of different cell types in your body -depending on what chemical signals they get, they will give rise to needed cells chemical cues during development
SRY gene
• Y Chromosome carries the SRY gene-encodes a transcription factor •Organizes the bipotential gonad into testes •The product of the SRY gene has to function at a specific window of time to suppress ovary formation and promote testis formation •If not, ovary-specific genes begin to function IS Transcription factor for signaling male***
Embryonic Development
•All organisms arise from a single cell •This single cell gives rise to many different cell types, each with a different structure and corresponding function How do you make all differentiated cell types? How do you translate the genome? - you do it by three interrelated processes •Cell division •Cell differentiation •Morphogenesis
Embryonic Cell Division
•Cleavage - subtype of mitosis (The goal of mitosis is to make identical daughter cells) •Cleavage doesn't necessarily produce identical cells •Cleavage is mitosis without growth •Sub-divides the fertilized zygote (not increasing the size of embryo, -is subdividing, not growing, 1 big cells cleaves into many small cells ****goal of cleavage is to kick start development.*** (cells differentiate)
Dosage Compensation Prevents Excessive Expression of X-Linked Genes in Mammals
•Dosage compensation: •Females have potential to produce twice as much product for X-linked genes: •May create a "genetic dosage" difference between genders. •Balances dose of X chromosome gene expression in males and females Barr bodies!! Need to compensate for this!!
Y chromosome houses genetic information for "maleness"
•Early embryo is hermaphroditic •By 5th week, gonadal ridges form either ovaries or testes (bipotential gonads). -**** it is genes rather than chromosomes that determine sex. (The Y , in some cases, play no role in sex determination, the presence of two X chromosomes could could femaleness; or maleness could occur from the lack of a second X chromosome) -but found that the Y chromosome does indeed determine maleness in humans.
Methods of Differential Gene Expression (DGE) Enhancer modularity?
•Enhancer Modularity -Enhancers or repressors for a gene "code" for expression in different tissues (different cis regulatory and trans regulatory elements that interact and drive the expression of the same gene but in different tissues. ) - for example ***look at picture** take a gene that has 3 different enhancers, these enhancers turn on in different tissues. 1. for brain, one enhancer might turn on to drive brain development 2. one for limp development, one enhancer turns on to drive limp growth. (enhancers that only turn on in specific cell tissues)*** The principle of enhancer modularity dictates that many separate enhancers allow a particular proteins to be expressed in some tissue while not expressed in others. The bottom line is that although there is just one genome, the infinite combination of enhancers and transcription factors working in collaboration is really the driving factor for gene expression
Transposons, Mutation, and Evolution
•Human diseases •Hemophilia A: Disease caused by LINEs inserted at two points within gene. •Study showed L I N E was not on mother's X chromosome but on both parents' chromosome 22. -•Insertions of T Es in various locations and their effects: •Coding region: Translation disruptions. •Intron: Termination signal in intron can terminate transcription. •Intron: Splicing of RNA transcribed from a gene. •Gene's transcription regulator region: Effects gene expression. •Identical T Es in genome: Potential for transposon recombination. -•T Es contribute to evolution by altering genes and chromosomes. •Drosophila telomeres •L I N E-like elements evolved to act as telomeres. •Maintain length of Drosophila chromosomes.
Pseudoautosomal regions (PARs):
•Present on both ends of Y chromosome—share homology with regions on X chromosome. •Synapse and recombine with X chromosome during meiosis. •Pairing region critical for segregation of X and Y chromosomes during male gametogenesis. *** this and SRY gene are only sections that recombine in meiosis.
Y chromosome and male development
•Pseudoautosomal regions (PARs): •Present on both ends of Y chromosome—share homology with regions on X chromosome. •Synapse and recombine with X chromosome during meiosis. •Pairing region critical for segregation of X and Y chromosomes during male gametogenesis. SRY gene -sex determining region -MSY-male specific region of the Y ***SRY is very close to Par 1 which can lead to problems.
RNAi - The Components
•RNA •siRNA (small interfering) - 21-22 bps •miRNA (micro) - 19-25 bps; encoded by the genome •RISC - RNA Induced Silencing Complex - cleaves the mRNA •Complex of proteins; precise structure not yet resolved •Main component is Argonaute 1. when long dsRNA (micro or siRNA) are present in the eukaryotic cell, they are cleaved into approx. 22-nucleotide long siRNA by an enzyme called Dicer. 2. These siRNAs then associate with the RISC complex. RISC contains argonaute family protein that binds RNA and has endoribonuclease or "slicer" activity. 3. RISC cleaves and evicts one of the two strands of the DS siRNA and retains the other strand as a single-stranded siRNA "guide" to recruit RISC to a complementary mRNA. RISC then cleaves the mRNA in the middle of the region of siRNA-mRNA complimentary. 4. cleaved mRNA fragments lacking cap or a poly-A tail are then quickly degraded in the cell.
Designing TALENs
•TALs recognize DNA through 34 AA repeat sequence, but the 12th and 13th position are hypervariable (you can change that position to get the TAL to bind to a specific sequence of researchers choice) ************************* •There is a "code": •NI = A, HD = C, NG = T, and NN = G or A. •You can use one TALEN to induce a single break, or two to make a double break -picture shows that hyper variable position!!!
Reverse Genetics: Editing the Genome
•The "gold standard" for reverse genetics •All methods involve cutting DNA •Main methods: •TALEN •Zinc finger nucleases •CRISPR
Patterns of Temperature Sex Determination
•Three different patterns of temperature sex determination in reptiles •Case I: Low temperatures yield 100% females; high •Case II: Exact opposite occurs. (high temps = female) •Case III: Low and high temperatures yield 100% females •Intermediate temperatures yield various proportions of males •Seen in various species of crocodiles, turtles, and lizards. •Critical period of incubation is known as the thermosensitive period (TSP)
Mechanism- LTR Retrotransposons
•Transcription start site -starts in one LTR and ends in the other •Carried out by the host's enzymes! •Remember, the LTR's are not mirror images. •Transcription starts at the 5' LTR at the 5' R sequence •Terminates through the 3' LTR ***** transcription reads 3' to 5' builds the transcript (mRNA) 5' to 3' (unlike virus's this does not move from one cell and infect another!!)
CRISPR-Cas
•Upon viral infection, bacteria use CRISPR-associated (Cas) proteins to cut off a piece of viral DNA •This sequence is integrated into the bacterial genome in between specific spacer regions •By storing it, the bacteria remember this virus and will be able to counter-attack it if it invades again •Repeat sequences- separate the spacer/viral sequences Spacer- viral DNA, that is inserted between repeats!! -the spacer and repeat sections are then going to guide the Cas 9 protein to the viral DNA.
Primary Sex Determination in Mammals (XY)
•testes form via SRY and secrete... •Mullerian-inhibiting factor (or AMH) •This destroys the Mullerian duct •These also start to secrete testosterone •Activates the formation of the penis, male duct system, and inhibits the development of breast primordia
