How do DNA microarrays work?

DNA microarrays work on the principal of base-pairing . Base-pairing allows probes to hybridize to targets on the microarray.

At a basic level microarrays are implemented as follows: a cell's RNA is extracted. This RNA (targets) is then multiplied, labeled with fluorescence and hybridized to existing DNA (probes) on the microarray. After hybridization, the probes that were hybridized with targets are fluorescent and a computer scanner is able to detect this fluorescence. Those probes that are fluorescent correspond to the genes that were expressed in the cell.

The Cell Membrane

• All cells are enclosed by a thin, film-like membrane called the plasmalemma or more popularly as the plasma membrane
• Danielli and Davson (1935) proposed a “trilaminar model” according to which, the plasma membrane is formed of a bimolecular layer of phospholipids (35 Å thick) sandwitched between two layers of proteins (each 20 Å thick). The model was proposed even before the plasma membrane was seen under the electron microscope
• J.D. Robertson (1959) proposed a “unit membrane concept” according to which, all biological membranes shared the same basic structure:
  • thickness of about 75 Å
  • a characteristic trilaminar appearance when viewed with electron microscope
  • the three layers are a result of the same arrangement of proteins and lipids as proposed by Danielli and Davson
• S. J. Singer and G. Nicolson (1972) put forward the “fluid mosaic model” of membrane structure which is presently the most widely accepted model.

Components of the Plasma Membrane

The Plasma membrane structure

• According to the fluid mosaic model, the cell membrane consists of a highly viscous fluid matrix of two layers of phospholipid molecules which serve as a relatively impermeable barrier to the passage of most water soluble molecules
• The plasma membrane contains lipids (32%), proteins (42%), carbohydrates (6%) and water (20%) although variations are always there
• Protein molecules or their complexes occur in the membrane, but not in continuous layer; instead, these occur as separate particles asymmetrically arranged in a mosaic pattern

  

  Illustration of a transmembrane protein

  • some of these proteins (peripheral or extrinsic proteins) are loosely bound at the polar surfaces of lipid layers
  • some proteins (integral or intrinsic proteins) penetrate deeply into the lipid layer
  • other proteins (transmembrane or tunnel proteins) penetrate through the phospholipid layers and project on both the surfaces
• The carbohydrates occur only at the outer surface of the membrane and their molecules are covalently linked to other components of the plasma membrane thus constituting the glycocalyx of the cell surface:
  • the polar heads of some lipid molecules (forming glycolipids) 
  • most of the proteins exposed at outer surface (forming glycoproteins)
• Cholesterol, found particularly in animal cell membranes, is an amphipathic lipid that is found in lipid bilayers that serves as a temperature-stability buffer

Nature of the Plasma Membrane

Membrane proteins

• Integral membrane proteins
  • membrane proteins differ in the degree to which they span lipid bilayers and most of the integral membrane proteins completely span the lipid bilayer
  • they are typically hydrophobic where they interact with the hydrophobic portion of the membrane and are typically hydrophilic where they interact with the hydrophilic portion of the membrane and overlying (and underlying) H₂O
• Peripheral membrane proteins
  • contrasting with integral membrane proteins, peripheral membrane proteins do not enter the lipid bilayer but are instead attached to the outside of the membrane via attachment to portions of integral membrane proteins jutting out of the membrane interior
• Functions of membrane proteins
  • transport of substances across membranes
  • enzymatic activity (e.g., smooth endoplasmic reticulum)
  • signal transduction (e.g., cell communication)
  • intracellular joining (e.g., intercellular junctions in animals)
  • cell-cell recognition (e.g., cell communication)
  • attachment to the cytoskeleton and extracellular matrix
• Fluidity of membrane proteins
  • membrane proteins are capable of diffusing within the membrane, a diffusion that is similar to that of phospholipids within membranes, though not as rapid. Other membrane proteins are tied in place by attachment to the cytoskeleton or the extracellular matrix

Oligosaccharides (glycoproteins)

• many eukaryotic membrane proteins are glycoproteins, proteins to which carbohydrate molecules of intermediate length (oligosaccharides) have been covalently attached. The attached oligosaccharides are always found on the extracellular side of the plasma membrane. The extracellular placement of oligosaccharides on membrane proteins makes intuitive sense since the oligosaccharides are added to these proteins within the lumen of the endomembrane system
• oligosaccharides play important roles in cell-cell recognition (i.e., oligosacherides of specific monomer sequence and branching pattern are recognized by other cells)

Membrane Asymmetry

• a typical cell membrane tends to have a different composition on one leaflet (monolayer) than on the other. Differences between leaflets tend to include different ratios or types of amphipathic lipid-based molecules found in each leaflet, different kinds of proteins facing in or facing out, or fixed orientations of proteins spanning the membrane.
• this asymmetry allows the cell to automatically differ its intracellular environment from that existing extracellularly. Thus, asymmetries tend to be rigidly maintained via minimal flip-flopping

Membrane Fluidity

• There are typically 3 types of phospholipid mobility in a lipid bilayer which are required for the membrane to function properly:
  • lateral diffusion → lipid molecules within a monolayer constantly exchange places with their neighbors
  • rotation → lipid molecules within a monolayer rotate very rapidly around their long axis
  • “flip-flop” → movement lipid molecules very rarely flip from one monolayer to the other
• The fluidity of a lipid bilayer depends on the nature of the hydrocarbon tails → the closer and more regular the packing of the tails, the less fluid the bilayer will be
• Membrane fluidity depends on:
  • length of the hydrocarbon tails → a shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another and therefore increases the fluidity of the bilayer
  • level of saturation of the hydrocarbon tails with respect to hydrogen → lipid bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower proportions
  • presence of cholesterol → at higher temperatures, cholesterol serves to impede phospholipid fluidity whereas at at lower temperatures, cholesterol interferes with solidification of membranes (i.e. functions similarly to the effect of unsaturated fatty acids)

Genomic Imprinting V: DNA methylation and gene silencing

The fact that genomic imprinting is mediated through epigenetic differences between the maternally and paternally inherited gene copies. That is, at an imprinted locus, the maternally inherited allele will have one pattern of epigenetic modifications, while the paternally inherited allele has a different pattern. These differences are first established in the male and female germ lines, when the alleles that will eventually become maternally and paternally derived are in physically different locations. It is not hard to imagine, then, how these differences could be established. One pattern of gene expression in spermatogenesis results in the paternal-specific epigenetic modifications. A different pattern of gene expression in oogenesis results in maternal-specific epigenetic modifications.

But what are these epigenetic modifications, and how do they change the expression pattern of the gene?

There are a number of modifications involved in imprinting, but for the moment, we're going to focus specifically on the simplest and best-understood mechanism: DNA methylation.

The two horizontal lines in this picture represent the two copies of a gene.  The big, solid box is the part of the gene that actually codes for the protein. The open box is the promoter region, which is the part of the DNA sequence responsible for regulating expression of the gene. The lollipop things indicate DNA methylation on cytosine residues (the "C" of the A, C, G, T alphabet that makes up DNA).

In this simplest type of scenario, the DNA sequence in the promoter region binds to a variety of proteins that recruit the molecular machinery that will transcribe the gene, leading eventually to production of the corresponding protein. The addition of methyl groups to the DNA changes its binding properties, so that it no longer binds to this machinery, and that copy of the gene is not transcribed.

If you're not a molecular biologist, you can think of it like this. The transcription machinery is a bit like a Xerox machine, and the gene is like the master copy of some document. The promoter region is like a lock that has to be unlocked before you can copy this particular document. There are a number of proteins called "transcription factors" that function like a key to this lock. These transcription factors fit nicely on the promoter region, unlocking the gene and resulting in the production of many copies of the gene product.

Adding methylation to the promoter region is a bit like squirting epoxy into the lock. The presence of the methyl groups actually changes the physical shape and chemical properties of the DNA. So, when you try to put the key in, it no longer fits right, and the gene can not be copied.

In the top part, we see the red transcription factor binding to the black promoter region, which will activate transcription from the gene. In the bottom part, methyl (CH3) groups have been chemically added to the promoter region, preventing binding, and thereby preventing transcription.

So, these relatively subtle chemical changes are able to completely alter the functional properties of the gene.

Next time, we'll talk about how these methylation patterns are maintained through development, and how the two gene copies are able to maintain distinct epigenetic states across multiple rounds of cell division and DNA replication. Make sure to tune in, because it's really slick!

The two references represent the first proposals that DNA methylation might be the thing that permits the stable transmission of patterns of gene expression across cell divisions.

Allele-Specific DNA Methylation and the World of Epigenetics

The ability of species to adapt to changing and challenging conditions is remarkable and due to a wide variety of molecular mechanisms. Many of these mechanisms fall into the broad category of epigenetics of which we are still learning the details.

One of the best known epigenetic mechanisms is DNA methylation in which a small molecule (a methyl group) is added to the DNA macromolecule at particular locations. Like a barcode or marker, the methyl group indicates, for instance, which genes in the DNA are to be turned on. This DNA methylation is accomplished via the action of a protein machine that adds the methyl group at precisely the right location in the DNA strand.

The methylation occurs at certain target sites along the DNA sequence where specific short DNA sequences appear. These sequences are found by protein machines as they move along the DNA. The protein machines search for these sequences and add a methyl group to the appropriate DNA base.

The protein machine binds to the DNA, twists the helix so the DNA base rotates into a precisely shaped pocket in the protein, and the protein then facilitates the transfer of the methyl group from a short donor molecule to the DNA base.

In bacterial studies it has been found that the short donor molecule does more, however, than just supply a methyl group. It also helps to control the protein. First, the short donor molecule binds to the pocket of the protein so the methyl group is ready for transfer. But the donor molecule also binds to another site on the protein. This binding serves to alter the structure of the protein, enhancing its function. So the protein is designed to do its job when it is charged with a donor molecule.

But not all of the DNA target sequences are methylated. This complex DNA methylation function doesn’t occur if the target sequence is protected by another protein that binds to the sequence. This protein binds to some of these DNA target sequences but not all. The result is a particular DNA methylation pattern which influences which genes are expressed.

Mark the marker

Furthermore, the methyl group marker can, itself, be modified. That is, the mark can be marked, thus adding another layer of information to the epigenetic mechanism. For instance, the methyl group can be hydroxylated. And of course a different molecular machine is required for the task, and the information of when and where to go to work is needed.

All of this makes for a complex DNA methylation pattern which is superimposed on the DNA macromolecule. In fact, this encoding of epigenetic information varies substantially across different regions of the DNA and it varies between the two alleles of a given gene and this allele-specific methylation can be tissue-specific.
DNA methylation is also transmitted across generations, but in the embryonic stages of development can be erased by yet another protein machine.

Histones

In addition to the DNA macromolecule, methyl groups are also used to tag the histone proteins about which the DNA is wrapped. The histones have a hub, around which the DNA wraps, and a tail that sticks out on which chemical markers are attached. As with DNA, these markers are signals for the protein machinery. And like DNA, these tags are removed as well. Such modifications and removal of these chemical tags means that these codes are dynamic, and there are protein inspectors that double-check these complex encodings.

These subtle codes are also context dependent. In one type of cell a histone modification may turn off a gene whereas in another type of cell the same histone modification may turn on the gene.

In addition to methylation, histones can also vary by tiny differences in their amino acid sequence. This histone sequence variation serves as yet another type of tag used for gene regulation.

Furthermore, histone variants are not merely static sign posts that influence gene expression. These variants are moved, by other proteins, between different locations in the genome, resulting in migration patterns that occur in the embryonic development phases.

Did epigenetics evolve?

Evolutionists do not think twice about the question of whether the epigenetic world evolved. Of course it did. Evolution is a fact, and so all of biology is its handiwork. This despite the “hint at an unimagined complexity of the genome” as one science writer admitted.

With evolution we must be believe that levels of complexity we could never have dreamed of, and which contradict evolution’s predictions, arose from random mutations (no, natural selection doesn’t change that fact, the mutations are still random). And as those complex machines and mechanisms arose, we must either believe there would be no use for them, or just luckily there would be some intermediate use for them that they happened to fulfill, while waiting for a later time for their epigenetic functions to be realized.

In fact, beyond sheer speculation, there is no explanation for how the epigenetic world evolved. The conviction that it did evolve is not a scientific conclusion—it comes from the belief that evolution is a fact.

Of course none of this means that the epigenetic world absolutely could not have evolved. But there certainly is no justification for taking up positions at the other end of the spectrum. There is no scientific justification for proclaiming that evolution, including the evolution of the epigenetic world, is an undeniable fact, as evolutionists insist.

It is unfortunate that we stake out such hard-edged, dogmatic positions that can be defended only by shouting down and blackballing dissent. In spite of the science, evolution must be true and all who disagree must be rejected.

Evolutionists are not the first, and undoubtedly won’t be the last, to engage in religious narrow-mindedness and parochial intolerance, in defiance of the facts. Religion drives science, and it matters.

DNA Package

How DNA is Packaged (Basic) 

 

How DNA is Packaged (Advanced)

 

The Perfect Package to Protect DNA : SASP-DNA Complex