ant wrangle 3
Hartl, section 10.4 and go back to CRISPR section from last exam module: (P 342-345) more than on this quizlet. the players
-CRISPR repeat in the bacterial genome, which for concreteness we suppose is part of the genome of the bacterial virus. the repeat is transcribed into a guide RNA (gRNA). the guide RNA is one key component of the system -the other two components are a trans-acting CRISPR RNA (tracrRNA) and the CRISPR-associated protein 9 nuclease (Cas9)
microarray chips
-depend on hybridization of cDNAs to pre-selected oligos bound to a surface, each representing a unique region of genes DNA microarrays are widely used tools to simultaneously measure the expression of many different genes. They consist of thousands of probes—each representing a different gene—immobilized on "chips" or slides, and rely on complementary hybridization to evaluate gene expression in different biological conditions.
advantages of RNA-seq over microarray hybridization
-differences in efficiency of hybridization among transcripts can affect results from microarrays but not those from RNA-seq, bc in RNA-seq, each transcript is identified according to sequence -differences in the level of transcription of alternative alleles in heterozygous genotypes can be detected -unlike microarray which involves probing for known RNA sequences, it can profile gene expression from organisms with unsequenced genomes -can accurately measure a larger range of transcript expression levels than microarrays, especially at very low/high levels
limitations to RNA-seq
-difficulty in detecting or accurately estimating the abundance of rare transcripts present in just a few molecules per cell
steps of CHIP
-first step is to cross-link the protein to the DNA with the help of a cross-linking agent, such as formaldehyde. this immobilizes the protein on the DNA, marking the binding site of the protein -after cross-linking, the chromatin is mechanically sheared into short fragments ranging from 100-200 bp (X-CHIP) -N-CHIP is the alternative method -> nucleases directly digest DNA from the chromatin into short fragments without any prior crosslinking -immunoprecipitation is carried out by introducing antibodies that target the regulatory proteins in the solution containing sheared/digested DNA -these antibodies are linked to agents that help in selective isolation -one common method is linking the antibodies to magnetic beads -a magnetic is used to isolate the antibodies along with any bound molecules -the complex is rinsed to wash off any loosely associated contaminants -the target molecules are detached with the help of a detergent, such as SDS -in the case of X-CHIP, cross linking is reversed with the help of elevated temps, and the associated regulators of histones are degraded with the help of proteases, leaving behind the DNA -sequencing of the associated DNA can help identify the cis-regulatory sequence that the protein of interest was bound to or the gene whose expression is modulated by a particular histone modification
prader-willi syndrome
-if the spontaneous deletion that include chr region 15q11 takes place in the father
angelman syndrome
-if the spontaneous deletion that include chr region 15q11 takes place in the mother -more about these page 305
genomic imprinting
-in mammals, some genes are imprinted by methylation in the germ line mammals feature an unusual type of epigenetic silencing. a process with the following characteristics -imprinting occurs in the germ line -it affects at most a few hundred genes (many of them located in clusters) -it is accompanied by heavy methylation (though the primary signal for imprinting is unknown) -imprinted genes are differentially methylated in the female and male germ lines -once imprinted and methylated, a silenced gene remains transcriptionally inactive during embryogenesis -imprints are erased early in germ-line development, then later reestablished according to sex-specific patterns more info page 304
regulation of gene expression
-many mechanisms that have evolved to allow eukaryotic and prokaryotic cells to turn different genes on and off, or express them at higher or lower levels, in response to the environment -in addition to transcription factors and microRNAs that work in eukaryotic cells to regulate expression, mechanisms exist that can be passed down to daughter cells. such heritable changes to gene expression that do not involve DNA mutations are known as epigenetic changes.
transcription profiling
-methods used to measure transcription -measuring which genes are being expressed and at what level under some given condition -usually involves comparing expression levels of genes between at least two samples: for example, cells under a specific condition, such as infection with a virus, or exposure to a toxin or hormone, compared with cells under "normal" conditions
Reporter Genes video
-researchers use reporter genes to dertermine when and where a gene of interest is expressed -a reporter gene codes for a protein that can be tracked, such as a protein with a known enzymatic activity or one that is fluorescent -gene expression is controlled by cis-regulatory sequences located upstream or downstream of the gene's coding region in DNA -the pattern and timing of gene expression can be determined by creating recombinant DNA with a reporter gene under the control of a cis-regulatory sequence of interest and introducing it into cells of an organism -this recombinant gene is introduced into diff cell types, and both the gene of interest and the reporter gene are allowed to express -since the gene of interest and the reporter gene have the same cis-regulatory sequence, they are expressed in the same cells and at the same time -so by monitoring the expression of the reporter gene, a scientist can track where the gene of interest is being expressed -one of the well-known reporter genes is one that encodes for a protein called GFP (green fluorescent protein) -GFP= discovered in the jellyfish aequoreavictoria. this protein produces a green fluorescence under UV light, which enables researchers to track its location within a cell -for example, to study the expression of beta-tubulin in C elegans, the coding sequence of the beta-tubulin gene is replaced by the GFP gene -the GFP gene expression is now under the control of the promoter for the beta-tubulin gene. this recombinant DNA was introduced into the worm using a microinjection and the gene was expressed similarly to beta-tubulin -the location of GFP was monitored using fluorescence microscopy as GFP fluorescence was shown in the touch receptor neurons. this is likely a site where beta-tubulin is expressed
CHIP
-technique for studying the protein-DNA interactions that regulate gene expression -chromatin is broken down and antibodies that bind to histone modifications or regulatory proteins are used to isolate the target molecules with the associated DNA During ChIP, chromatin—which consists of DNA and its associated proteins—is "immunoprecipitated," meaning that it is isolated through the use of antibodies. With this method, researchers can assess which proteins associate with which DNA sequences. With ChIP, specific histone modifications can be targeted by antibodies, which can be "pulled down" along with the surrounding DNA. Researchers can then employ PCR, microarrays, or sequencing to identify DNA regions associate with histone modifications of interest. By varying the antibodies used during ChIP, this technique can also help pinpoint DNA regions bound by transcription factors and other regulatory proteins.
10.4 steps
-the gRNA initiates a series of events that ultimately results in destruction of the genome of any invading virus that contains a complementary sequence -in the first step, the gRNA invades the viral target DNA by base pairing with its complementary DNA strand -at this point, the tracrRNA joins the complex and recruits the Cas9 protein -Cas9 protein is a nuclease, and it cleaves both strands of the target viral DNA -the cleaved ends of the target DNA are then attacked by exonucleases in the cell, and the target viral DNA is degraded
RNA-seq
-uses next-gen sequencing to sequence cDNAs from transcripts -makes use of the techniques of massively parallel sequencing -the poly-A tail of mRNA is targeted by a poly-T oligonucleotide primer, and reverse transcriptase is used to produce a ssDNA complementary to each of the mRNA molecules -these DNA strands are then replicated to produce a ds cDNA corresponding to the population of mRNA molecules present in the cells at the time of extraction -the collection of cDNAs is analyzed with massively parallel sequencing, and each cDNA sequence is compared with the reference genome of the organism to identify the gene to which the transcript corresponds -technique that can provide info on the sequence and quantity of every RNA expressed, known as the transcriptome in a cell population
in mammals, cytosines are modified preferentially in
5'-CG-3' dinucleotides. many mammalian genes have CG-rich regions upstream of the coding region that provide multiple sites for methylation; these are called CpG islands, where the "p" represents the phosphate group in the polynucleotide backbone
an overview of epigenetics video some questions being asked by epigeneticists
At the most basic level, scientists are still actively studying the mechanisms by which epigenetic marks, such as histone modifications and DNA methylation, are created, removed, and interpreted. Researchers continue to characterize the enzymes that carry out these functions, as well as how the marks interact with the transcription machinery to activate or repress gene expression. A deeper question that arises is whether there exists an "epigenetic code," analogous to the well-defined "genetic code," that dictates how information in DNA is translated into protein sequence. Researchers are trying to determine if the combination of epigenetic marks form a similarly predictive code that will one day make it possible to deduce the expression pattern of every gene. Recently, scientists have been interested in the biological roles of lncRNAs. While a prevailing model is that lncRNAs help recruit epigenetic factors to specific genomic locations, their exact mechanisms, and whether all lncRNAs function similarly, are still being studied. Finally, because epigenetic marks are chemical "add-ons" that are not simply replicated along with DNA, scientists are still trying to learn how the marks continue through cellular generations. Even more controversial is the potential trans-generational inheritance of certain epigenetic processes. Because it is observed that epigenetic marks are dramatically erased or "reprogrammed" early in embryogenesis, and again during gamete formation, how and whether these trans-generational phenomena actually occur remains hotly debated.
gene-editing technique
CRISPR-Cas
histone modifications
Current evidence suggests that changes in chromatin structure that regulate gene expression are mediated by chemical modifications made to histones, usually in their freely moving "tails." The most common of these are acetyl, methyl, and phosphate groups that are added to, or removed from, specific amino acids, and these different histone modifications are observed to be associated with different levels or modes of gene expression. For example, the addition of three methyl groups to the 27th lysine residue in histone subunit H3—a modification termed H3K27me3—has been linked to gene silencing. Alternatively, the H3K9ac modification, where an acetyl group is added to the 9th lysine residue on histone H3, has been associated with gene activation. Histone modifications are hypothesized to play a role in the epigenetic regulation of gene expression by marking regions of chromatin as "active" or "silent." One mechanism by which histone modifications are believed to exert their effects is to recruit transcription factors or chromatin "remodeling" enzymes, the latter of which physically move the positions of nucleosomes.
CRISPR-Cas9 system
DNA editing tool that stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein 9 -simple method for precise manipulation of the genome by altering specific sequences of DNA -first observed in bacteria, it is a means of defense against viruses. refers specifically to the immune system of streptococcus pyogenes, the first to be described in detail. used generally to refer to any such system -as foreign, viral DNA enters a bacterium, it is processed into smaller fragments which may be inserted into a region of the bacterial genome called a CRISPR locus -when the region is transcribed, the product associates with smaller RNAs called tracrRNAs, which may help to orient both the Cas9 protein and RNAase to the molecule, the latter of which cleaves the transcript -the end result is several complexes, each consisting of a Cas9 protein, tracrRNA, and a CRISPR RNA, derived from DNA in the locus -the CRISPR RNA in these structures recognizes and guides Cas9 to viral DNA, which is then cleaved and destroyed -scientists harness CRISPR-Cas9 by synthesizing individual RNA molecules that mimics tracrRNA and CRISPR RNA, which can target a gene of interest. for ex, when 2 such guide RNAs are introduced into cells with Cas9 and both target the same gene, a sequence can be excised -once this target region is removed, the cut ends are reconnected, and the effects on the cell are observed -thus, the CRISPR-Cas9 system is modified from a bacterial mechanism, and can be employed for an array of gene editing techniques
DNA methylation analysis video: DNA methylation
DNA methylation is a chemical modification of DNA that affects gene expression under different cellular contexts. Many researchers are interested in the mechanism and functions of this process, as aberrant DNA methylation has been associated with diseases such as cancer.
DNA methylation analysis video:
During this biochemical process, a cell adds a chemical tag known as a methyl group to cytosine bases in its DNA. Most methylated cytosines occur next to guanine bases in the same DNA strand, and these adjacent nucleotides—and the phosphodiester bond that links them—are referred to as "CpGs." Although most vertebrate CpGs are methylated, those that are unmethylated tend to occur close together in CpG "islands" near the promoters of actively expressed genes, and various other regulatory sequence elements. How changes in DNA methylation contribute to gene regulation is still a subject of intense investigation. Methylation of CpG islands seems to be important for the stable, long-term gene silencing seen in epigenetic processes such as genomic imprinting, which is the parent-of-origin specific expression of certain genes, as well as X-chromosome inactivation, the silencing of one of the two X-chromosomes in each cell of female mammals. Repression of critical genes due to aberrant methylation of CpG islands has also been shown to contribute to uncontrolled cell growth, which can lead to cancer. Mechanistically, DNA methylation may contribute to gene silencing by either preventing transcription factors from associating with promoters, or by recruiting proteins that modify histones and remodel chromatin into a transcriptionally non-permissive state.
CHIP fuller version video gene expression is regulated by:
Gene expression is regulated by "nucleosome occupancy," or whether a stretch of DNA is packed into nucleosomes. Transcribed DNA tends to be located in "nucleosome-free" regions, which allow proteins to associate with a gene's regulatory sites, and enable RNA polymerase to carry out transcription.
An Overview of Epigenetics key moments in history of epigenetics
In the 1930s, Hermann J. Muller observed a phenomenon known as position-effect variegation in Drosophila. He found mutant flies with mottled eyes, and linked this phenotype to the variable spread of condensed "heterochromatin" that silenced the gene responsible for eye color. This would be the first identified "epigenetic" phenomenon where a phenotypic change was observed without a corresponding change to the genetic sequence. In 1959, Susumu Ohno observed in female rat liver cells that one of the two X-chromosomes was condensed. Two years later, Mary Lyon hypothesized that this condensed X-chromosome is genetically inactivated, that the choice of which X chromosome to be inactivated is random, and that this inactivation is stably inherited by the cell's offspring. This process, now called X-chromosome inactivation or XCI, causes females to be biological mosaics. In 1964, Alfred Mirsky published the earliest work on the role of histone modifications in gene regulation. Histones form the core of nucleosomes, which are the basic repeating unit of chromatin in eukaryotic cells. Mirsky studied how methylation and acetylation of histones affected RNA synthesis, and it is now known that numerous modifications alter the "activity state" of nearby chromosomal regions. In 1975, Robin Holliday and his student John Pugh, and independently Arthur Riggs, proposed that methylation of CpG dinucleotides in DNA might be involved in stable epigenetic silencing, for example during XCI. Adrian Bird and colleagues lent further credence to this idea in 1985 by identifying clusters of unmethylated CpG sites throughout the genome that were later associated with transcriptionally active promoters. He would later also discover regulatory proteins that bind methylated DNA, eventually repressing transcription. In 1984, Davor Solter, Azim Surani, and others observed that mouse embryos containing only maternal or paternal genetic material—created via nuclear transplantation experiments—did not develop normally. This marked the discovery of genomic imprinting, or parent-of-origin specific gene expression. The first imprinted genes were discovered in 1991, where only the copy inherited from either the father or mother is ever expressed. One of these genes, H19, turns out to be rather unusual—its final product is a 2.3 kilobase RNA that does not get translated into proteins. More of these "long noncoding RNAs" or lncRNAs were soon discovered, including Xist, which is required for shutting down the X-chromosome during XCI. Current evidence suggests that these RNAs may function as scaffolds to recruit regulatory factors. Today, researchers continue to work out how interactions among lncRNAs, DNA methylation, and histone modifications regulate epigenetic processes.
microarray principles video
One of the earliest methods developed to assess gene expression in biological samples is Northern blotting, which involves "probing" for specific RNA molecules immobilized on membranes. "Free-floating" probes recognize complementary RNA sequences in the sample, and are typically labeled with radioactive or fluorescent molecules so that they can be visualized. Advances in microfabrication, genome sequencing, and other technologies have led to the development of the microarray biochip. Like Northern blots, microarrays are based on the principle of complementary binding between probe and sample nucleic acid sequences. Unlike Northerns, however, in microarrays it is the oligonucleotide probes that are immobilized on a glass slide or chip. The "free-floating" samples are generated from RNA isolated from cells or organisms of interest, which is reverse transcribed to complementary or "c"-DNA. This can either be directly labeled with fluorescent molecules, or their amounts may be further amplified by in vitro transcription into cRNA. The sample is then hybridized to the chip. Because probes on microarrays designed for different applications may either be "sense," which means their sequences are in the same direction as an organism's expressed RNA, or "antisense," researchers must ensure that the strand directionality of the sample is complementary to that of the probes. The "raw" fluorescence intensity data for each gene-specific dot on the chip can then be quantified and processed. The data can be subjected to further statistical tests, like the Student's t test, to determine if the fluorescence signals—and thus expression levels—for a gene of interest are significantly different between two cell types or experimental conditions. Researchers can also use this data to "cluster" or group genes based on similar patterns of expression. For example, when comparing expression patterns between two cell populations, certain genes may be found to demonstrate expression changes by roughly equivalent amounts in the same direction, and would thus be grouped together. Researchers can depict these relationships in a type of tree diagram or "dendrogram" where heights and arrangements of "branches" indicate how similar—or dissimilar—gene expression patterns are. This type of analysis can provide insight into gene networks, as "clustered" genes may participate in the same biological pathways.
DNA Methylation Analysis video: There are several methods for detecting the methylation state of DNA.
One technique, the HELP assay, involves two restriction endonucleases called HpaII and MspI, which respectively cleave only unmethylated, or both methylated and unmethylated, CCGG sequences. By comparing the digestion patterns produced by these two enzymes, the methylation status of DNA can be deduced. Another method, called methylated DNA immunoprecipitation or "MeDIP," uses antibodies that bind to methylated cytosines to enrich for methylated DNA sequences. Finally, bisulfite analysis is used to distinguish methylated from unmethylated cytosine in DNA, by carrying out a chemical reaction that converts unmethylated cytosine to uracil. Following this conversion, bisulfite-treated DNA can be subjected to PCR, sequenced, and compared to a reference genome. Unmethylated cytosines are those that are present in the reference, but replaced with thymines following bisulfite analysis and PCR. By subjecting bisulfite-treated DNA to mass spectrometry, researchers can also create "methylation epigrams," which linearly represent different CpGs in the genome and depict the degree of methylation at each of them. Such epigrams are particularly useful if researchers wish to compare methylation patterns between different cell types.
protein factors and gene regulation
Protein factors play important roles in the gene regulation; not only do they organize DNA in chromosomes, but they also bind to specific DNA sequences—called regulatory sites—to activate or repress expression.
two methods commonly used for purpose of comparing expression levels of genes between at least two samples in transcription profiling
RNA-seq and microarray chips -both used to estimate the relative level of gene expression of each gene in the genome
Testing the strength of promoters or enhancers
Recombinant DNA techniques allow researchers to piece together regions of DNA from different organisms in whatever way they want in order to study aspects of gene expression. Reporter constructs, for example, allow us to test the strength and specificity of a promoter region by inserting it upstream of a gene with a clear readout, such as luciferase or green fluorescent protein (GFP). The light generated can be quantified under different conditions, acting as a readout of the strength of the promoter under those conditions.
gene-editing for research purposes
Researchers have learned a lot about the functions of various genes by examining phenotypes associated with mutations in those genes. In the past, researchers could cause random mutations by exposing cells or organisms to mutagens such as radiation or certain chemicals. These days, however, they can alter the DNA sequence of a specific gene, in a sense "editing" the gene. One of the most powerful means of editing genes is through the CRISPR-Cas system.
CHIP fuller version video: generalized procedure for CHIP technique
To begin, cells of interest are collected and treated with chemicals like formaldehyde, which act as "cross-linking" reagents and help affix proteins to the DNA sequences they associate with by facilitating the formation of covalent bonds between them. Care must be taken to not "over treat" cells with formaldehyde, as this can impact the ability of antibodies to recognize their target histone modifications at later ChIP stages. To stop the cross-linking process, glycine is added to the formaldehyde solution with which cells are being treated. The cells are then collected and lysed to release the chromatin. To solubilize chromatin and precisely define the DNA regions that associate with modified histones, chromatin is mechanically "sheared" into smaller pieces using sound waves—a process called sonication. Typically, scientists aim to create chromatin fragments 200 to 1000 base pairs in length. Once chromatin fragments of the desired size are generated, an antibody is added to the solution, and the mixture is incubated to give the antibody time to recognize its target histone modification. Magnetic beads to which the antibodies can bind are then introduced into the mixture, immobilizing the antibody-associated chromatin complexes. The beads are collected through the use of magnetic racks, and washed several times to rinse off any unbound chromatin or antibodies. To release the chromatin from them, beads are placed in a solution containing the detergent SDS, and after collecting the beads with a magnet, the supernatant is kept. The enzyme proteinase K is then added to this solution to degrade all proteins, including histones, so that the DNA component of the chromatin can be isolated. The resulting DNA is then purified and analyzed.
principles of RNA seq (video)
Transcriptome sequencing requires isolating a population of transcripts whose levels are to be measured. Most RNA in cells is ribosomal RNA, or rRNA, the central component of the cell's protein-production machinery. To facilitate recovery of other types of transcripts, rRNA is typically removed prior to sequencing by hybridizing the sample to complementary oligonucleotides attached to magnetic beads, and using a magnet to separate the rRNA from the rest of the sample. Alternatively, a specific population of RNA can be selected for sequencing. For example, protein-coding messenger RNAs, or mRNAs, can be captured with "oligo-dT"—short stretches of deoxy-T nucleotides that bind to the sequence of A bases known as a poly-A tail at the end of these transcripts. The contaminating rRNA is then removed. MicroRNAs, which are 22-nucleotide regulatory RNAs, can be selectively isolated for sequencing based on their size. Because RNA is inherently prone to degradation, it is first reverse transcribed to double-stranded DNA. Oligonucleotide sequences known as adaptors are then ligated onto the DNA fragments. The adaptors contain constant regions that serve as primer-binding sites for subsequent PCR amplification, and these are usually asymmetric so that the "strandedness" of the template is preserved. The adaptors also contain unique sequences, known as "barcodes," that identify all fragments originating from a single sample. The library is then amplified by PCR. A sequencing chip, on which there are oligonucleotides complementary to the adaptors, is used to immobilize the library sample, which is diluted such that the DNA molecules anneal onto the chip at low density. The DNA is amplified on the chip via a process called "bridge amplification" to form "clonal clusters." Short fragments, each 30-150 bases in length, are then synthesized from one or both ends of these DNA templates, generating hundreds of millions of products known as sequencing reads. The sequencing results are then analyzed for quality and the data are processed. Analysis of the sequences can reveal a wide variety of information, including differences in expression levels of RNAs between samples and previously unknown transcripts or forms of transcripts.
epigenetics
broadly refers to the study of heritable differences in gene function that cannot be explained by DNA sequence changes. The term "epigenetics" was first introduced by Conrad Waddington in the 1950s, to explain how diverse cell types in the body could arise from one set of genetic material. Researchers have identified many processes thought to have an epigenetic basis, but there is still significant debate about many fundamental principles of the field. -heritable changes in gene expression that are due not to changes in the DNA sequence itself, but to something "In addition to" the DNA sequence, usually either chemical modification of the bases, or protein factors bound with the DNA. gene expression can be affected by heritable chemical modifications in the DNA
transcriptional inactivation is associated with
heavy methylation -a number of observations suggest that heavy methylation is assoc with genes for which the rate of transcription is low ie the inactive X chr in mammalian cells which is extensively methylated -more on page 303
Changes in gene expression can come about through
modifications of histones (that uncoil or tighten these structures) or methylation of CpGs -gene expression regulated also by proteins that associate with cis-regulatory regions on the DNA
functional genomics
new approach to genetics made possible by genomic sequencing. focuses on genome-wide patterns of gene expression and the mechanisms by which gene expression is coordinated -as changes occur in the cellular environment the patterns of gene expression also change. but genes are usually deployed in sets, not individually. as the level of expression of one coordinated set is decreased, the level of expression of a diff coordinated set may be increased
why is there imprinting
page 305 -one suggestion is that it evolved in early mammals with polyandry (each female mating with a series of males). in such a situation, it is to a male's benefit to silence ggenes that conserve maternal resources at the expense of the fetus, bc this strategy maximizes the father's immediate reproduction. but it is to a female's benefit to silence genes that allocate resources to the fetus at the expense of the mother, bc this strategy maximizes the female's long-term reproduction -this hypothesis is supported by the fact that some imprinted genes do affect the allocation of resources between mother and fetus in the direction that would be predicted. on the other hand, many genes that are imprinted have no obvious connection to maternal-fetal conflict
Microarray procedure
page 327 -mRNA is first extracted from both the experimental and control samples -this material is then subjected to one or more rounds of amplification into DNA copies using the enzyme reverse transcriptase -in the experimental material, the primer for reverse transcripton includes a red fluorescent label. in the control material, the primer includes a green fluorescent label. when a sufficient quantity of labeled DNA strands has accumulated, the fluorescent samples are mixed and hybridized with the DNA chip -genes overexperssed in experimental over control will have more red strands hybridized to the spot and those underexpressed in exp relative to control will have more green strands hybridized to the spot -after hybridization, the DNA chip is placed in a confocal fluorescence scanner that scans each pixel (the smallest discrete element in a visual image) first to record the intensity of one fluorescent label and then again to record the intensity of the other label. these signals are synthesized to produce the signal value for each spot in the microarray -the signals indicate the relative levels of gene expression by volor -a red/orange spot indicates high/moderate overexpression of the gene in the experimental sample, a spot that is green/yellow indicates high/moderate underexpression of the gene in the experimental sample, and a spot that is perfectly yellow indicates equal levels of gene expression in the samples -in this manner, DNA chips can assay the relative levels of any mRNA species that has an abundance in the sample of more than one molecule per 10^5, and differences in expression as small as about 2 fold can be detected
in most higher eukaryotes, a proportion of the cytosine bases are modified by
the addition of a methyl (CH3) group to the 5 carbon. the cytosines are incorporated in their normal, unmodified form in the course of DNA replication, and then the methyl group is added by an enzyme called DNA methylase
prader-willi syndrome and angelman syndrome
the epigenetic, sex-specific gene silencing associate with imprinting is dramatically evident in this pair of syndromes characterized by neuromuscular defects, mental retardation, and other abnormalities -both conditions are associated with rare, spontaneous deletions that include chr region 15q11
10.4 most straightforward application of CRISPR-Cas9
to create targeted deletions, often called knockout mutations bc a deletion results in loss of function -first step is to synthesize a sgRNA (synthetic guide RNA) that is complementary to the genomic sequence to be deleted, and the second is to introduce the sgRNA and Cas9 into the nucleus of cells to be engineered -the sgRNA invades the target DNA by base pairing with its complementary strand, and then the tracrRNA component of sgRNA recruits Cas9, which cleaves both strands of the target DNA -exonucleases then begin to degrade the target DNA from the cleaved ends, enlarging the gap -the DNA in eukaryotic chr is much longer than in a bacterial virus, and euke cells have mechanisms to detect broken DNA and rejoin the ends -one such mechanism is known as nonhomologous end joining. alternative is template-directed gap repair (both on p 343)
in eukes, DNA is
wrapped around histone proteins and forms a complex called a nucleosome, which further groups together into a structure known as chromatin to aid in the tight packaging of DNA in the cells
