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11.2: DNA Sequencing - Biology

11.2: DNA Sequencing - Biology


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DNA sequencing determines the order of nucleotide bases within a given fragment of DNA. This information can be used to infer the RNA or protein sequence encoded by the gene, from which further inferences may be made about the gene’s function and its relationship to other genes and gene products. DNA sequence information is also useful in studying the regulation of gene expression. If DNA sequencing is applied to the study of many genes, or even a whole genome, it is considered an example of genomics.

Dideoxy sequencing

Recall that DNA polymerases incorporate nucleotides (dNTPs) into a growing strand of DNA, based on the sequence of a template strand. DNA polymerases add a new base only to the 3’-OH group of an existing strand of DNA; this is why primers are required in natural DNA synthesis and in techniques such as PCR. Most of the currently used DNA sequencing techniques rely on the random incorporation of modified nucleotides called terminators. Examples of terminators are the dideoxy nucleotides (ddNTPs), which lack a 3’-OH group and therefore cannot serve as an attachment site for the addition of new bases to a growing strand of DNA (Figure (PageIndex{1})). After a ddNTP is incorporated into a strand of DNA, no further elongation can occur. Terminators are labeled with one of four fluorescent dyes, each specific for one the four nucleotide bases.

To sequence a DNA fragment, you need many copies of that fragment (Figure (PageIndex{2})). Unlike PCR, DNA sequencing does not amplify the target sequence and only one primer is used. This primer is hybridized to the denatured template DNA, and determines where on the template strand the sequencing reaction will begin. A mixture of dNTPs, fluorescently labeled terminators, and DNA polymerase is added to a tube containing the primer-template hybrid. The DNA polymerase will then synthesize a new strand of DNA until a fluorescently labeled nucleotide is incorporated, at which point extension is terminated. Because the reaction contains millions of template molecules, a corresponding number of shorter molecules is synthesized, each ending in a fluorescent label that corresponds to the last base incorporated.

The newly synthesized strands can be denatured from the template, and then separated electrophoretically based on their length (Figure (PageIndex{3})). Since each band differs in length by one nucleotide, and the identity of that nucleotide is known from its fluorescence, the DNA sequence can be read simply from the order of the colors in successive bands. In practice, the maximum length of sequence that can be read from a single sequencing reaction is about 700 bp.

A particularly sensitive electrophoresis method used in the analysis of DNA sequencing reactions is called capillary electrophoresis (Figure (PageIndex{6})). In this method, a current pulls the sequencing products through a gel-like matrix that is encased in a fine tube of clear plastic. As in conventional electrophoresis, the smallest fragments move through the capillary the fastest. As they pass through a point near the end of the capillary, the fluorescent intensity of each dye is read. This produces a graph called a chromatogram. The sequence is determined by identifying the highest peak (i.e. the dye with the most intense fluorescent signal) at each position.

Next-generation sequencing

Advances in technology over the past two decades have increased the speed and quality of sequencing, while decreasing the cost. This has become especially true with the most recently developed methods called next-generation sequencing. Not all of these new methods rely on terminators, but one that does is a method used in instruments sold by a company called Illumina. Illumina sequencers use a special variant of PCR called bridge PCR to make many thousands of copies of a short (45bp) template fragment. Each of these short template fragments is attached in a cluster in a small spot on a reaction surface. Millions of other clusters, each made by different template fragment, are located at other positions on the reaction surface. DNA synthesis at each template strand then proceeds using dye-labeled terminators that are used are reversible. Synthesis is therefore terminated (temporarily) after the incorporation of each nucleotide. Thus, after the first nucleotide is incorporated in each strand, a camera records the color of fluorescence emitted from each cluster. The terminators are then modified, and a second nucleotide is incorporated in each strand, and again the reaction surface is photographed. This cycle is repeated a total of 45 times. Because millions of 45 bp templates are sequenced in parallel in a single process, Illumina sequencing is very efficient compared to other sequencing techniques. However, the short length of the templates currently limits the application of this technology.


11 Molecular Biology of The Gene

Molecular biology is the branch of biology that concerns the molecular basis of biological activity in and between cells, including molecular synthesis, modification, mechanisms and interactions. Molecular biology arose as an attempt to answer the questions regarding the mechanisms of genetic inheritance and the structure of a gene. In 1953, James Watson and Francis Crick published the double helical structure of DNA courtesy of the X-ray crystallography work done by Rosalind Franklin and Maurice Wilkins. Watson and Crick described the structure of DNA and the interactions within the molecule. This publication jump-started research into molecular biology and increased interest in the subject.

Nucleic acids are biopolymers that are essential to all known forms of life. The term nucleic acid is the overall name for DNA and RNA. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. If the sugar is a compound ribose, the polymer is RNA (ribonucleic acid) if the sugar is derived from ribose as deoxyribose, the polymer is DNA (deoxyribonucleic acid).

In an influential published in 1941 paper, George Beadle and Edward Tatum proposed the idea that genes act through the production of enzymes, with each gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway. The concept arose from work on genetic mutations in the mold Neurospora crassa, and subsequently was dubbed the “one gene–one enzyme hypothesis” by their collaborator Norman Horowitz. In 2004 Norman Horowitz reminisced that “these experiments founded the science of what Beadle and Tatum called ‘biochemical genetics.’ These experiments are by some considered to constitute the begining of what became molecular genetics and the development of the one gene–one enzyme hypothesis is often considered the first significant result in what came to be called molecular biology. Although it has been extremely influential, the hypothesis was recognized soon after its proposal to be an oversimplification. Even the subsequent reformulation of the”one gene–one polypeptide" hypothesis is now considered too simple to describe the relationship between genes and proteins. In attributing an instructional role to genes, Beadle and Tatum implicitly accorded genes an informational capability. This insight provided the foundation for the concept of a genetic code. However, it was not until the experiments were performed showing that DNA was the genetic material, that proteins consist of a defined linear sequence of amino acids, and that DNA structure contained a linear sequence of base pairs, was there a clear basis for solving the genetic code.

In attributing an instructional role to genes, Beadle and Tatum implicitly accorded genes an informational capability. This insight provided the foundation for the concept of a genetic code. However, it was not until the experiments were performed showing that DNA was the genetic material, that proteins consist of a defined linear sequence of amino acids, and that DNA structure contained a linear sequence of base pairs, was there a clear basis for solving the genetic code.

Although genes were known to exist on chromosomes, chromosomes are composed of both protein and DNA, and scientists did not know which of the two is responsible for inheritance. In 1928, Frederick Griffith discovered the phenomenon of transformation: dead bacteria could transfer genetic material to “transform” other still-living bacteria. Sixteen years later, in 1944, the Avery–MacLeod–McCarty experiment identified DNA as the molecule responsible for transformation. The role of the nucleus as the repository of genetic information in eukaryotes had been established by Hämmerling in 1943 in his work on the single celled alga Acetabularia. The Hershey–Chase experiment in 1952 confirmed that DNA (rather than protein) is the genetic material of the viruses that infect bacteria, providing further evidence that DNA is the molecule responsible for inheritance.

James Watson and Francis Crick determined the structure of DNA in 1953, using the X-ray crystallography work of Rosalind Franklin and Maurice Wilkins that indicated DNA has a helical structure (i.e., shaped like a corkscrew). Their double-helix model had two strands of DNA with the nucleotides pointing inward, each matching a complementary nucleotide on the other strand to form what look like rungs on a twisted ladder. This structure showed that genetic information exists in the sequence of nucleotides on each strand of DNA. The structure also suggested a simple method for replication: if the strands are separated, new partner strands can be reconstructed for each based on the sequence of the old strand. This property is what gives DNA its semi-conservative nature where one strand of new DNA is from an original parent strand.

Figure 11.1: A cartoon representation of DNA based on atomic coordinates of PDB 1BNA, rendered with open source molecular visualization tool PyMol.

Although the structure of DNA showed how inheritance works, it was still not known how DNA influences the behavior of cells. In the following years, scientists tried to understand how DNA controls the process of protein production. It was discovered that the cell uses DNA as a template to create matching messenger RNA, molecules with nucleotides very similar to DNA. The nucleotide sequence of a messenger RNA is used as a template by ribosomes to create an amino acid sequence in protein this correspondence between nucleotide sequences and amino acid sequences is known as the genetic code.

With the newfound molecular understanding of inheritance came an explosion of research. One important development was chain-termination DNA sequencing in 1977 by Frederick Sanger. This technology allows scientists to read the nucleotide sequence of a DNA molecule. In 1983, Kary Banks Mullis developed the polymerase chain reaction, providing a quick way to isolate and amplify a specific section of DNA from a mixture. The efforts of the Human Genome Project, Department of Energy, NIH, and parallel private efforts by Celera Genomics led to the sequencing of the human genome in 2003.


14.2 DNA Structure and Sequencing

In this section, you will explore the following questions:

  • What is the molecular structure of DNA?
  • What is the Sanger method of DNA sequencing? What is an application of DNA sequencing?
  • What are the similarities and differences between eukaryotic and prokaryotic DNA?

Connection for AP ® Courses

The currently accepted model of the structure of DNA was proposed in 1953 by Watson and Crick, who made their model after seeing a photograph of DNA that Franklin had taken using X-ray crystallography. The photo showed the molecule’s double-helix shape and dimensions. The two strands that make up the double helix are complementary and anti-parallel in nature. That is, one strand runs in the 5' to 3' direction, whereas the complementary strand runs in the 3' to 5' direction. (The significance of directionality will be important when we explore how DNA copies itself.) DNA is a polymer of nucleotides that consists of deoxyribose sugar, a phosphate group, and one of four nitrogenous bases—A, T, C, and G—with a purine always pairing with a pyrimidine (as Chargaff found). The genetic “language” of DNA is found in sequences of the nucleotides. During cell division each daughter cell receives a copy of DNA in a process called replication. In the years since the discovery of the structure of DNA, many technologies, including DNA sequencing, have been developed that enable us to better understand DNA and its role in our genomes.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A Learning Objective merges required content with one or more of the seven science practices.

Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A Heritable information provides for continuity of life.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.5 The student can evaluate alternative scientific explanations.
Learning Objective 3.1 The student is able to construct scientific explanations that use the structures and mechanisms of DNA to support the claim that DNA is the primary source of heritable information.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 4.1 The student can justify the selection of the kind of data needed to answer a particular scientific question.
Learning Objective 3.2 The student is able to justify the selection of data from historical investigations that support the claim that DNA is the source of heritable information.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
Learning Objective 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies.

Teacher Support

Franklin’s X-ray diffraction pictures helped lead to the discovery of the structure of DNA, but Watson and Crick did not mention Franklin in their seminal 1953 paper, which can be found here. This paper includes annotations that help place the work in historical context. Students might be interested to learn how Watson and Crick discovered the structure of DNA. Details can be found at this PBS website. If possible, find a copy of the announcement of the discovery as it appeared in The New York Times. The wording is interesting and the significance of the discovery is understated.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.3][APLO 3.5][APLO 3.13]

The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group (Figure 14.5). The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).

The nucleotides combine with each other by covalent bonds known as phosphodiester bonds or linkages. The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller in size they have a single six-membered ring structure. The carbon atoms of the five-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime”). The phosphate residue is attached to the hydroxyl group of the 5' carbon of one sugar of one nucleotide and the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond.

In the 1950s, Francis Crick and James Watson worked together to determine the structure of DNA at the University of Cambridge, England. Other scientists like Linus Pauling and Maurice Wilkins were also actively exploring this field. Pauling had discovered the secondary structure of proteins using X-ray crystallography. In Wilkins’ lab, researcher Rosalind Franklin was using X-ray diffraction methods to understand the structure of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of Franklin's data because Crick had also studied X-ray diffraction (Figure 14.6). In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine. Unfortunately, by then Franklin had died, and Nobel prizes are not awarded posthumously.

Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. Base pairing takes place between a purine and pyrimidine namely, A pairs with T and G pairs with C. Adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. The base pairs are stabilized by hydrogen bonds adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature that is, the 3' end of one strand faces the 5' end of the other strand. The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside. Each base pair is separated from the other base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, ten base pairs are present per turn of the helix. The diameter of the DNA double helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine can explain the uniform diameter. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves (Figure 14.7).

Science Practice Connection for AP® Courses

Activity

Read Watson and Crick’s original Nature article, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid,” How did Watson and Crick’s model build on the findings of Rosalind Franklin? How did their model of DNA build on the findings of Hershey and Chase, and others, showing that DNA can encode and pass information on to the next generation?

Think About It

Watson and Crick’s work determined the structure of DNA. However, it was still relatively unknown how DNA encoded information into genes. Select one modern form of biotechnology and research its basic methods online. Examples include gene sequencing, DNA fingerprinting, PCR (polymerase chain reaction), genetically-modified food, etc. Briefly describe your chosen technology, and what benefits it provides us. Then describe how Watson and Crick’s findings were vital to the development of your chosen technology.

Teacher Support

The activity is an application of Learning Objective 3.1 and Science Practice 6.5 because students are analyzing Watson and Crick’s model of DNA relative to the findings of other DNA researchers who determined that DNA is the molecule of heredity. The activity is also an application of Learning Objective 3.2 and Science Practice 4.1 because students are analyzing the historic published results of Watson and Crick and selecting evidence that Watson and Crick used to create their model of DNA and further show that DNA is the molecule of heredity.

Possible answer:

The Think About It question is an application of Learning Objective 3.5 and Science Practice 6.4 because students are researching the methods by which humans can manipulate heritable information and describing how those methods were based on the scientific theories and models of Watson and Crick.

Possible answer:

DNA Sequencing Techniques

Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process. Using radiolabeled nucleotides also compounded the problem through safety concerns. With currently available technology and automated machines, the process is cheap, safer, and can be completed in a matter of hours. Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today (Figure 14.8).

Link to Learning

Visit this site to watch a video explaining the DNA sequence reading technique that resulted from Sanger’s work.

  1. Sanger’s method can be used to sequence more than one strand at a time which is less time consuming. Challenges of Sanger’s method includes its decreased accuracy to sequence DNA strands.
  2. Sanger’s method is a reliable and accurate way of sequencing DNA strands. However, only one strand at a time can be sequenced at a time. Also, it can look for one base only at a time which can be time consuming.
  3. Sanger’s method is highly inexpensive and less accurate. However, it is not readily adaptable to commercial kits.
  4. Sanger’s method is less time consuming and highly accurate. However, it is more expensive than other methods available for sequencing.

The method is known as the dideoxy chain termination method. The sequencing method is based on the use of chain terminators, the dideoxynucleotides (ddNTPs). The dideoxynucleotides, or ddNTPSs, differ from the deoxynucleotides by the lack of a free 3' OH group on the five-carbon sugar. If a ddNTP is added to a growing a DNA strand, the chain is not extended any further because the free 3' OH group needed to add another nucleotide is not available. By using a predetermined ratio of deoxynucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes.

The DNA sample to be sequenced is denatured or separated into two strands by heating it to high temperatures. The DNA is divided into four tubes in which a primer, DNA polymerase, and all four nucleotides (A, T, G, and C) are added. In addition to each of the four tubes, limited quantities of one of the four dideoxynucleotides are added to each tube respectively. The tubes are labeled as A, T, G, and C according to the ddNTP added. For detection purposes, each of the four dideoxynucleotides carries a different fluorescent label. Chain elongation continues until a fluorescent dideoxy nucleotide is incorporated, after which no further elongation takes place. After the reaction is over, electrophoresis is performed. Even a difference in length of a single base can be detected. The sequence is read from a laser scanner. For his work on DNA sequencing, Sanger received a Nobel Prize in chemistry in 1980.

Link to Learning

Sanger’s genome sequencing has led to a race to sequence human genomes at a rapid speed and low cost, often referred to as the $1000 in one day sequence. Learn more by selecting the Sequencing at Speed animation here.

  1. Faster genetic sequencing will help in quick analysis of the genetic makeup of bacteria that can cause diseases in humans for better and more efficient treatments. Also, sequencing of a cancerous cell’s DNA can provide better ways to treat or prevent cancer.
  2. Fast DNA sequencing can help us quickly analyze the genetic information of existing only bacteria (not new strains) only that cause disease in humans, which may lead to more efficient treatments.
  3. Fast DNA sequencing can help doctors to treat and diagnose diseases which are not rare in populations.
  4. Faster genetic sequencing can be used to treat and prevent a few types of cancers and thus increase the life expectancy of patients suffering from the diseases.

Gel electrophoresis is a technique used to separate DNA fragments of different sizes. Usually the gel is made of a chemical called agarose. Agarose powder is added to a buffer and heated. After cooling, the gel solution is poured into a casting tray. Once the gel has solidified, the DNA is loaded on the gel and electric current is applied. The DNA has a net negative charge and moves from the negative electrode toward the positive electrode. The electric current is applied for sufficient time to let the DNA separate according to size the smallest fragments will be farthest from the well (where the DNA was loaded), and the heavier molecular weight fragments will be closest to the well. Once the DNA is separated, the gel is stained with a DNA-specific dye for viewing it (Figure 14.9).

Evolution Connection

Neanderthal Genome: How Are We Related?

The first draft sequence of the Neanderthal genome was recently published by Richard E. Green et al. in 2010. 1 Neanderthals are the closest ancestors of present-day humans. They were known to have lived in Europe and Western Asia before they disappeared from fossil records approximately 30,000 years ago. Green’s team studied almost 40,000-year-old fossil remains that were selected from sites across the world. Extremely sophisticated means of sample preparation and DNA sequencing were employed because of the fragile nature of the bones and heavy microbial contamination. In their study, the scientists were able to sequence some four billion base pairs. The Neanderthal sequence was compared with that of present-day humans from across the world. After comparing the sequences, the researchers found that the Neanderthal genome had 2 to 3 percent greater similarity to people living outside Africa than to people in Africa. While current theories have suggested that all present-day humans can be traced to a small ancestral population in Africa, the data from the Neanderthal genome may contradict this view. Green and his colleagues also discovered DNA segments among people in Europe and Asia that are more similar to Neanderthal sequences than to other contemporary human sequences. Another interesting observation was that Neanderthals are as closely related to people from Papua New Guinea as to those from China or France. This is surprising because Neanderthal fossil remains have been located only in Europe and West Asia. Most likely, genetic exchange took place between Neanderthals and modern humans as modern humans emerged out of Africa, before the divergence of Europeans, East Asians, and Papua New Guineans.

Several genes seem to have undergone changes from Neanderthals during the evolution of present-day humans. These genes are involved in cranial structure, metabolism, skin morphology, and cognitive development. One of the genes that is of particular interest is RUNX2, which is different in modern day humans and Neanderthals. This gene is responsible for the prominent frontal bone, bell-shaped rib cage, and dental differences seen in Neanderthals. It is speculated that an evolutionary change in RUNX2 was important in the origin of modern-day humans, and this affected the cranium and the upper body.

  1. Early humans emerged from Africa, then spread out to populate different parts of the globe. An isolated population of these early humans interbred with Neanderthals.
  2. Early humans interbred with Neanderthals, emerged from Africa, then spread out to populate different parts of the globe.
  3. Early humans emerged from Africa, interbred with Neanderthals, then spread out to populate different parts of the globe.
  4. Early humans did not interbreed with Neanderthals, but we have many genetic similarities because we share a common ancestor.

Link to Learning

Watch Svante Pääbo’s talk explaining the Neanderthal genome research at the 2011 annual TED (Technology, Entertainment, Design) conference.

  1. It has been suggested that all humans most likely descended from Africa. This is supported by the research that genetic variance in Africa was also found in the rest of the world.
  2. The theory that humans descended from Africa was supported by the research that most of the human genomes tested outside of Africa had close ties to the genomes of people in Africa but a genetic variance in Africa was not found in the rest of the world.
  3. Humans have most likely descended from Africa. This research is supported by the fact that all the human genomes tested outside of Africa had close ties to the genomes of people in Africa. Also, there is a genetic variance in Africa that was not found in the rest of the world.
  4. The transition to modern humans occurred within Africa which was sudden. Thus, human genomes tested outside of Africa had close ties to the genomes of people in Africa.

DNA Packaging in Cells

When comparing prokaryotic cells to eukaryotic cells, prokaryotes are much simpler than eukaryotes in many of their features (Figure 14.10). Most prokaryotes contain a single, circular chromosome that is found in an area of the cytoplasm called the nucleoid.

Visual Connection

  1. Compartmentalization in eukaryotic cells enables the building of more complex proteins and RNA products. In prokaryotes, the advantage is that RNA and protein synthesis occurs much more quickly because it occurs in a single compartment.
  2. Compartmentalization in prokaryotic cells enables the building of more complex proteins and RNA products. In eukaryotes, the advantage is that RNA and protein synthesis occurs much more quickly because they occur in a single compartment.
  3. Compartmentalization in eukaryotic cells enables the building of simpler proteins and RNA products. In prokaryotes, the advantage is only simpler proteins and RNA products because complex ones are not needed.
  4. Compartmentalization in eukaryotic cells enables the building of more complex proteins and RNA products. In prokaryotes, the advantage is that RNA and protein synthesis takes more time because it occurs in a single compartment.

The size of the genome in one of the most well-studied prokaryotes, E.coli, is 4.6 million base pairs (approximately 1.1 mm, if cut and stretched out). So how does this fit inside a small bacterial cell? The DNA is twisted by what is known as supercoiling. Supercoiling means that DNA is either under-wound (less than one turn of the helix per 10 base pairs) or over-wound (more than 1 turn per 10 base pairs) from its normal relaxed state. Some proteins are known to be involved in the supercoiling other proteins and enzymes such as DNA gyrase help in maintaining the supercoiled structure.

Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a different type of packing strategy to fit their DNA inside the nucleus (Figure 14.11). At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that are rich in basic amino acids and form an octamer. The DNA (which is negatively charged because of the phosphate groups) is wrapped tightly around the histone core. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the “beads on a string” structure. This is further compacted into a 30 nm fiber, which is the diameter of the structure. At the metaphase stage, the chromosomes are at their most compact, are approximately 700 nm in width, and are found in association with scaffold proteins.

In interphase, eukaryotic chromosomes have two distinct regions that can be distinguished by staining. The tightly packaged region is known as heterochromatin, and the less dense region is known as euchromatin. Heterochromatin usually contains genes that are not expressed, and is found in the regions of the centromere and telomeres. The euchromatin usually contains genes that are transcribed, with DNA packaged around nucleosomes but not further compacted.

Footnotes

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    ​Initiation

    ​The initiation of replication occurs at specific nucleotide sequence called the origin of replication, where various proteins bind to begin the replication process. E. colihas a single origin of replication (as do most prokaryotes), called oriC, on its one chromosome. The origin of replication is approximately 245 base pairs long and is rich in adenine-thymine (AT) sequences.

    Some of the proteins that bind to the origin of replication are important in making single-stranded regions of DNA accessible for replication. Chromosomal DNA is typically wrapped around histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is supercoiled, or extensively wrapped and twisted on itself. This packaging makes the information in the DNA molecule inaccessible. However, enzymes called topoisomerases change the shape and supercoiling of the chromosome. For bacterial DNA replication to begin, the supercoiled chromosome is relaxed by topoisomerase II, also called DNA gyrase. An enzyme called helicase then separates the DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs. Recall that AT sequences have fewer hydrogen bonds and, hence, havDNA gyrasee weaker interactions than guanine-cytosine (GC) sequences. These enzymes require ATP hydrolysis. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication, allowing for bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope as a result, this structure is called a replication bubble. The DNA near each replication fork is coated with single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.

    Once single-stranded DNA is accessible at the origin of replication, DNA replication can begin. However, DNA pol III is able to add nucleotides only in the 5’ to 3’ direction (a new DNA strand can be only extended in this direction). This is because DNA polymerase requires a free 3’-OH group to which it can add nucleotides by forming a covalent phosphodiester bond between the 3’-OH end and the 5’ phosphate of the next nucleotide. This also means that it cannot add nucleotides if a free 3’-OH group is not available, which is the case for a single strand of DNA. The problem is solved with the help of an RNA sequence that provides the free 3’-OH end. Because this sequence allows the start of DNA synthesis, it is appropriately called the primer. The primer is five to 10 nucleotides long and complementary to the parental or template DNA. It is synthesized by RNA primase, which is an RNA polymerase. Unlike DNA polymerases, RNA polymerases do not need a free 3’-OH group to synthesize an RNA molecule. Now that the primer provides the free 3’-OH group, DNA polymerase III can now extend this RNA primer, adding DNA nucleotides one by one that are complementary to the template strand (Figure 1).


    Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder

    We are performing whole-genome sequencing of families with autism spectrum disorder (ASD) to build a resource (MSSNG) for subcategorizing the phenotypes and underlying genetic factors involved. Here we report sequencing of 5,205 samples from families with ASD, accompanied by clinical information, creating a database accessible on a cloud platform and through a controlled-access internet portal. We found an average of 73.8 de novo single nucleotide variants and 12.6 de novo insertions and deletions or copy number variations per ASD subject. We identified 18 new candidate ASD-risk genes and found that participants bearing mutations in susceptibility genes had significantly lower adaptive ability (P = 6 × 10 -4 ). In 294 of 2,620 (11.2%) of ASD cases, a molecular basis could be determined and 7.2% of these carried copy number variations and/or chromosomal abnormalities, emphasizing the importance of detecting all forms of genetic variation as diagnostic and therapeutic targets in ASD.

    Conflict of interest statement

    Competing Financial Interests: The authors declare no competing financial interests.

    Figures

    Figure 1. Schematic of sample and data…

    Figure 1. Schematic of sample and data processing in MSSNG

    An executive committee oversees the…

    Figure 2. Characteristics and quality of WGS…

    Figure 2. Characteristics and quality of WGS from different sequencing platforms

    Figure 3. ASD-susceptibility genes/loci

    Figure 3. ASD-susceptibility genes/loci

    (a) ASD-risk genes with higher than expected mutation rate from MSSNG…

    Figure 4. CNV characterization via WGS reads…

    Figure 4. CNV characterization via WGS reads in the MSSNG Portal

    Figure 5. Phenotype comparison for the samples…

    Figure 5. Phenotype comparison for the samples with and without identified mutations


    Results

    We retained 175,326 total reads after stringent quality filtering and trimming, ranging from 3,965 to 50,063 reads across the seven sampling sites. Of those, 59,705 were identified through BLAST as 16S rRNA gene sequences. BC07 retained the lowest number of reads with 506, while 27,629 reads remained for BC02. Qiime2 analysis using closed vsearch with an identity cutoff of 80% identified a total of 53 medusozoan OTUs (Supplementary Figure 4). Despite the low read count for several samples, alpha-diversity rarefaction analysis suggested our method were sufficient to assess progress in recovering representative medusozoan eDNA by sample location, given our sequencing depth (Supplementary Figures 5,6). A BLASTN search of our recovered 16S rRNA gene filtered dataset yielded 59 unique medusozoan taxa, suggesting minor discrepancies can occur with variations in database and algorithm (clustering versus homology) used for read classification. Furthermore, although sequences corresponding to eDNA for the COI gene were largely a secondary target in this study, we were successful in detecting three medusozoan OTUs comprising two hydrozoans and C. frondosa (details provided in Supplementary Figures 5A, B) these data were not incorporated into our medusozoan biodiversity analyses.

    Medusozoan Fauna in the Florida Keys

    Overall, medusozoan OTU richness was lowest at Rock Harbor-BC02 (24 OTUs), followed by the Aquarium control-BC04 (15 OTUs) (Figures 6C,D). Buttonwood Sound (BC03) and Finger Pier (BC05), both pelagic habitats, had the greatest number of OTUs (42) (Figures 6B,E). On average, we detected 30 OTUs from sheltered sites (BC01 and BC02) and 36 OTUs from pelagic sites (BC03, BC05, BC06, and BC07) (Figure 6). Although Hydrozoa represented the class with the highest number of OTUs detected, on average, 65.2% of reads were assigned to Scyphozoa, 26.7% to Hydrozoa, followed by 18.6% to Cubozoa, with Staurozoa represented by the fewest reads (0.9%). With the exception of Buttonwood Sound, Cassiopea was consistently the most represented taxon (based on total corresponding reads) at each site.

    Figure 6. Map depicting distribution of jellyfish species at Florida Keys sampling sites based on 16S rRNA gene. Upper Keys (Key Largo and Marathon Key), water samples (May 14�, 2018). (a) Buttonwood Sound (BC03). 25.10143, �.43861. (b) Quarry (BC01). 24.74975, �.97812. (c) Rock Harbor (BC02). 25.07924, �.45245. (d) Aquarium (BC04). 25.10135, �.43861. Lower Keys (Fleming Key), water samples (May 17, 2018). (e) Finger Pier (BC05). 24.57581, �.79922. (f) SFUWOS FAA Tower (BC06). 24.59015, �.79704. (g) SFUWOS Drop (BC07). 24.59181, �.79487 Negative control (BC08) not depicted. Red symbols correspond to sampling locations on the map. Large, multicolored pie charts show medusozoan diversity detected at each site and total number of 16S rRNA gene reads detected per location based on Qiime2 analysis. Percentages above smaller monochromatic pie charts highlight the percent of total reads corresponding to Cassiopea and cubozoan species, respectively. Results from the Qiime2 barplot function were modified and visualized here using Krona (Ondov et al., 2011). Percentages reflect proportions prior to rarefaction analysis (subsample normalization), which resulted in loss of three minimally represented taxa.

    Cassiopea reads were recovered from all sampling sites, but most of the reads were from sheltered sites, with C. xamachana reads encompassing 96.3% of Cassiopea reads generated for Rock Harbor (Figure 6 and Supplementary Figure 6). Although Cassiopea medusae were not visually confirmed at Fleming Key, the detection of eDNA for this taxon was expected given that the upside-down jellyfish is the most common jellyfish in the Florida Keys and has been documented in mangroves of neighboring Key West, several kilometers away (NOAA, 2020). Phylogenetic analysis of consensus reads, as determined by BLASTN revealed that three putative Cassiopea species are likely present in the Florida Keys. C. xamachana dominates throughout the Florida Keys with respect to detected eDNA (Figure 7) and visual confirmation (Ohdera et al., 2018), whereas C. frondosa occurs less frequently (Table 1).

    Figure 7. Medusozoa fauna identified from 16S rRNA gene using FeDS. The table highlights morphological characteristics and residency in the Florida Keys sampling areas or closest geographic location. Line drawings to the left depict a representative taxon for each class, drawn in comparative scale. Arrows indicate species for which partially annotated genomes have been published (Ohdera et al., 2019). BH, bell height BW, bell width L, length of hydroid W, width ND, no data available FLMNH, Florida Museum of Natural History NMNH, National Museum of Natural History, Smithsonian Institution.

    In addition to Cassiopea, we detected a second scyphozoan genus Aurelia, for which the consensus of reads at the 80% identity threshold (BLASTN) shared �% similarity to Aurelia aurita (GenBank DQ787873) – a broadly distributed species complex of jellyfish known as moon jellyfish (Dawson, 2003), with at least one species known in Florida waters (Figure 7). Using Qiime2, this match was only recovered using percent identity cutoffs below 75%. Interestingly, the consensus of reads at the 80% identity threshold in our BLASTN search showed 㺘% identity to unpublished sequences of Aurelia sp. samples from southern Brazil (J. Lawley, pers. comm.). Therefore, it is likely that the DNA sequences of the species we detected is yet lacking from GenBank, highlighting the importance of building robust reference databases of genetic barcodes.

    Venomous species of box jellyfish (Cubozoa) detected by eDNA metabarcoding include taxa of the cubozoan families Alatinidae and Tamoyidae (Figures 4𠄷). Though previously reported from the Caribbean and Florida Keys region (Figure 7), no medusa of either family was visually confirmed during this study. As these box jellyfish medusae are rather large and conspicuous (Figure 7), it is conceivable that our sequences corresponded to eDNA from microscopic life stages (planulae or polyps) present at collection sites, in line with recent findings of eDNA signal detected for benthic cnidarians (Sawaya et al., 2019 Bolte et al., 2021). A. alata is the best documented species of the family Alatinidae, but at least one other has been reported in the Gulf of Mexico (Graham, 1998 Lewis et al., 2013 Lasley et al., 2016 Lawley et al., 2016). Our initial results indicated that two species of Tamoyidae had been detected in this study: Tamoya cf. haplonema (matching a New Jersey sample GenBank KR093033) and a very similar sequence of Tamoya ohboya (matching a Caribbean sample GenBank GQ150263). Tamoya species have been previously reported in this geographical region (Collins et al., 2011 Figure 7), although further studies are needed to properly delineate species within this genus. While two different exemplar sequences were identified by maximal BLASTN scores, consensus sequences of these reads both had nearly 100% identity to the T. cf. haplonema rRNA gene from New Jersey. Furthermore, the tiny cubozoan, Tripedalia cystophora, was detected via eDNA as well (as a singleton). First described from Jamaica, T. cystophora has only recently been documented in Florida waters (Orellana and Collins, 2011 Lasley et al., 2016). Finally, a set of 17 reads corresponding to an unidentified species of Carybdea (closely matching sequences of the Caribbean species Carybdea xaymacana), suggest the presence of another yet-to-be described species of box jelly in the Florida Keys (Figure 7).

    Although no species of the benthic stalked jellyfishes in the class Staurozoa were reported previously from Florida (Miranda et al., 2018), in this study we identified eDNA of the staurozoans Calvadosia cruxmelitensis and Haliclystus cf. tenuis (Figures 4𠄷). The former is of particular interest as its genome was recently published together with that of C. xamachana and A. alata (Ohdera et al., 2019). Staurozoans are relatively small medusozoans (Figure 7), and often cryptic and difficult to find in the field because they live on and blend in well with macroalgae. These species are exclusively benthic, so the presence of sequences corresponding to these taxa (C. cruxmelitensis being represented at every sampling location in this study) suggests that FeDS was able to detect eDNA of benthic species in addition to that of pelagic jellyfishes. H. tenuis was originally described from Japan (Kishinouye, 1910), but has recently been considered an introduced species in the north Atlantic (Holst and Laakmann, 2019) it has never been reported from the western Atlantic (Figure 7). C. cruxmelitensis is distributed throughout the British Isles, while congeners are the only known staurozoans to be distributed in warm tropical and subtropical waters, including reports of C. hawaiiensis from Hawaii (Edmondson, 1930), an undetermined species from India (Panikkar, 1944), and C. corbini from Brazil (Grohmann et al., 1999), Puerto Rico (Capriles and Martinez, 1970 Larson, 1980), and the western Gulf of Mexico (Lechuga and Fernández-Álamo, 2005). While known geographic distributions of staurozoans (Miranda et al., 2018) would suggest that C. corbini is the most likely species to be encountered in Florida waters, our data unequivocally indicate that C. cruxmelitensis inhabits the Florida Keys (Figure 7). Nevertheless, this assertion should be confirmed through visual inspection of suitable coastal Florida habitats to assess a hypothetical introduction. Overall, these results suggest that extensive eDNA analysis could rapidly advance our understanding of the distribution of cryptic organisms.

    We identified 36 Hydrozoa OTUs, despite only 15% of total reads mapping to hydrozoan sequences. Several of these taxa (e.g., species of Aglaophenia, Eudendrium, and Halopteris) lack a medusa stage, providing more evidence that our eDNA sampling captured benthic taxa (Figures 4, 7). Given the extensive hydrozoan diversity of over 3,500 known species, including hundreds described from the Caribbean, the more than 50% representation of all OTUs by hydrozoan taxa was not unexpected (Figures 4, 5).


    Contents

    Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilised point is Transcription Initiation:

    In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin structure, which can be altered as a result of histone modifications directed by DNA methylation, ncRNA, or DNA-binding protein. Hence these modifications may up or down regulate the expression of a gene. Some of these modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.

    Structural Edit

    Transcription of DNA is dictated by its structure. In general, the density of its packing is indicative of the frequency of transcription. Octameric protein complexes called histones together with a segment of DNA wound around the eight histone proteins (together referred to as a nucleosome) are responsible for the amount of supercoiling of DNA, and these complexes can be temporarily modified by processes such as phosphorylation or more permanently modified by processes such as methylation. Such modifications are considered to be responsible for more or less permanent changes in gene expression levels. [2]

    Chemical Edit

    Methylation of DNA is a common method of gene silencing. DNA is typically methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG islands" when densely clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment, whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods developed to quantify SNPs, such as Pyrosequencing (Biotage) or MassArray (Sequenom), measuring the relative amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis. [3]

    Histone acetylation is also an important process in transcription. Histone acetyltransferase enzymes (HATs) such as CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often, DNA methylation and histone deacetylation work together in gene silencing. The combination of the two seems to be a signal for DNA to be packed more densely, lowering gene expression. [ citation needed ]

    Regulation of transcription thus controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by several mechanisms. Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e., sigma factors used in prokaryotic transcription). Repressors bind to the Operator, coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene. The image to the right demonstrates regulation by a repressor in the lac operon. General transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA. Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA. Enhancers are sites on the DNA helix that are bound by activators in order to loop the DNA bringing a specific promoter to the initiation complex. Enhancers are much more common in eukaryotes than prokaryotes, where only a few examples exist (to date). [4] Silencers are regions of DNA sequences that, when bound by particular transcription factors, can silence expression of the gene.

    In vertebrates, the majority of gene promoters contain a CpG island with numerous CpG sites. [5] When many of a gene's promoter CpG sites are methylated the gene becomes silenced. [6] Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. [7] However, transcriptional silencing may be of more importance than mutation in causing progression to cancer. For example, in colorectal cancers about 600 to 800 genes are transcriptionally silenced by CpG island methylation (see regulation of transcription in cancer). Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs. [8] In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-expressed microRNA-182 than by hypermethylation of the BRCA1 promoter (see Low expression of BRCA1 in breast and ovarian cancers).

    One of the cardinal features of addiction is its persistence. The persistent behavioral changes appear to be due to long-lasting changes, resulting from epigenetic alterations affecting gene expression, within particular regions of the brain. [9] Drugs of abuse cause three types of epigenetic alteration in the brain. These are (1) histone acetylations and histone methylations, (2) DNA methylation at CpG sites, and (3) epigenetic downregulation or upregulation of microRNAs. [9] [10] (See Epigenetics of cocaine addiction for some details.)

    Chronic nicotine intake in mice alters brain cell epigenetic control of gene expression through acetylation of histones. This increases expression in the brain of the protein FosB, important in addiction. [11] Cigarette addiction was also studied in about 16,000 humans, including never smokers, current smokers, and those who had quit smoking for up to 30 years. [12] In blood cells, more than 18,000 CpG sites (of the roughly 450,000 analyzed CpG sites in the genome) had frequently altered methylation among current smokers. These CpG sites occurred in over 7,000 genes, or roughly a third of known human genes. The majority of the differentially methylated CpG sites returned to the level of never-smokers within five years of smoking cessation. However, 2,568 CpGs among 942 genes remained differentially methylated in former versus never smokers. Such remaining epigenetic changes can be viewed as “molecular scars” [10] that may affect gene expression.

    In rodent models, drugs of abuse, including cocaine, [13] methampheamine, [14] [15] alcohol [16] and tobacco smoke products, [17] all cause DNA damage in the brain. During repair of DNA damages some individual repair events can alter the methylation of DNA and/or the acetylations or methylations of histones at the sites of damage, and thus can contribute to leaving an epigenetic scar on chromatin. [18]

    Such epigenetic scars likely contribute to the persistent epigenetic changes found in addiction.

    In mammals, methylation of cytosine (see Figure) in DNA is a major regulatory mediator. Methylated cytosines primarily occur in dinucleotide sequences where cytosine is followed by a guanine, a CpG site. The total number of CpG sites in the human genome is approximately 28 million. [19] and generally about 70% of all CpG sites have a methylated cytosine. [20]

    In a rat, a painful learning experience, contextual fear conditioning, can result in a life-long fearful memory after a single training event. [21] Cytosine methylation is altered in the promoter regions of about 9.17% of all genes in the hippocampus neuron DNA of a rat that has been subjected to a brief fear conditioning experience. [22] The hippocampus is where new memories are initially stored.

    Methylation of CpGs in a promoter region of a gene represses transcription [23] while methylation of CpGs in the body of a gene increases expression. [24] TET enzymes play a central role in demethylation of methylated cytosines. Demethylation of CpGs in a gene promoter by TET enzyme activity increases transcription of the gene. [25]

    When contextual fear conditioning is applied to a rat, more than 5,000 differentially methylated regions (DMRs) (of 500 nucleotides each) occur in the rat hippocampus neural genome both one hour and 24 hours after the conditioning in the hippocampus. [22] This causes about 500 genes to be up-regulated (often due to demethylation of CpG sites in a promoter region) and about 1,000 genes to be down-regulated (often due to newly formed 5-methylcytosine at CpG sites in a promoter region). The pattern of induced and repressed genes within neurons appears to provide a molecular basis for forming the first transient memory of this training event in the hippocampus of the rat brain. [22]

    After the DNA is transcribed and mRNA is formed, there must be some sort of regulation on how much the mRNA is translated into proteins. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript. These processes occur in eukaryotes but not in prokaryotes. This modulation is a result of a protein or transcript that, in turn, is regulated and may have an affinity for certain sequences.

    Three prime untranslated regions (3'-UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally influence gene expression. [26] Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

    The 3'-UTR often contains miRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

    As of 2014, the miRBase web site, [27] an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). [28] Freidman et al. [28] estimate that >45,000 miRNA target sites within human mRNA 3'-UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

    Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. [29] Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold). [30] [31]

    The effects of miRNA dysregulation of gene expression seem to be important in cancer. [32] For instance, in gastrointestinal cancers, a 2015 paper identified nine miRNAs as epigenetically altered and effective in down-regulating DNA repair enzymes. [33]

    The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depressive disorder, Parkinson's disease, Alzheimer's disease and autism spectrum disorders. [34] [35] [36]

    The translation of mRNA can also be controlled by a number of mechanisms, mostly at the level of initiation. Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist, which often are directed to their target sequence by the secondary structure of the transcript, which may change depending on certain conditions, such as temperature or presence of a ligand (aptamer). Some transcripts act as ribozymes and self-regulate their expression.

      is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme.
  • The induction of heat shock proteins in the fruit fly Drosophila melanogaster.
  • The Lac operon is an interesting example of how gene expression can be regulated.
  • Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV).
  • Gal4 is a transcriptional activator that controls the expression of GAL1, GAL7, and GAL10 (all of which code for the metabolic of galactose in yeast). The GAL4/UAS system has been used in a variety of organisms across various phyla to study gene expression. [37]
  • Developmental biology Edit

    A large number of studied regulatory systems come from developmental biology. Examples include:

    • The colinearity of the Hox gene cluster with their nested antero-posterior patterning
    • Pattern generation of the hand (digits - interdigits): the gradient of sonic hedgehog (secreted inducing factor) from the zone of polarizing activity in the limb, which creates a gradient of active Gli3, which activates Gremlin, which inhibits BMPs also secreted in the limb, results in the formation of an alternating pattern of activity as a result of this reaction-diffusion system.
    • Somitogenesis is the creation of segments (somites) from a uniform tissue (Pre-somitic Mesoderm). They are formed sequentially from anterior to posterior. This is achieved in amniotes possibly by means of two opposing gradients, Retinoic acid in the anterior (wavefront) and Wnt and Fgf in the posterior, coupled to an oscillating pattern (segmentation clock) composed of FGF + Notch and Wnt in antiphase. [38]
    • Sex determination in the soma of a Drosophila requires the sensing of the ratio of autosomal genes to sex chromosome-encoded genes, which results in the production of sexless splicing factor in females, resulting in the female isoform of doublesex. [39]

    Up-regulation and down-regulation Edit

    Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell), which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. Conversely, down-regulation is a process resulting in decreased gene and corresponding protein expression.

      occurs, for example, when a cell is deficient in some kind of receptor. In this case, more receptor protein is synthesized and transported to the membrane of the cell and, thus, the sensitivity of the cell is brought back to normal, reestablishing homeostasis. occurs, for example, when a cell is overstimulated by a neurotransmitter, hormone, or drug for a prolonged period of time, and the expression of the receptor protein is decreased in order to protect the cell (see also tachyphylaxis).

    Inducible vs. repressible systems Edit

    Gene Regulation can be summarized by the response of the respective system:

    • Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
    • Repressible systems - A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to "repress expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.

    The GAL4/UAS system is an example of both an inducible and repressible system. Gal4 binds an upstream activation sequence (UAS) to activate the transcription of the GAL1/GAL7/GAL10 cassette. On the other hand, a MIG1 response to the presence of glucose can inhibit GAL4 and therefore stop the expression of the GAL1/GAL7/GAL10 cassette. [40]

    Theoretical circuits Edit

    • Repressor/Inducer: an activation of a sensor results in the change of expression of a gene
    • negative feedback: the gene product downregulates its own production directly or indirectly, which can result in
      • keeping transcript levels constant/proportional to a factor
      • inhibition of run-away reactions when coupled with a positive feedback loop
      • creating an oscillator by taking advantage in the time delay of transcription and translation, given that the mRNA and protein half-life is shorter
      • signal amplification
      • bistable switches when two genes inhibit each other and both have positive feedback
      • pattern generation

      In general, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determine which genes changed and by how much. These are, however, not informative of where the regulation has occurred and may mask conflicting regulatory processes (see post-transcriptional regulation), but it is still the most commonly analysed (quantitative PCR and DNA microarray).

      When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:


      In the early days (1970s), scientists were not worried about having to align too many sequences. They wanted to find the best alignment between two sequences. Many bioinformatics courses start with learning these, although it is not the main focus of our course. We included two videos in case you are interested.

      The Needlemen-Wunsch algorithm is the earliest algorithm to find the alignment between two sequences and score their similarity.

      When two sequences are long, and only a portion of them can align well with each other, the Smith-Waterman algorithm can find the best local sequence alignment. It is still considered the best alignment approach, although it is slow.


      11.2: DNA Sequencing - Biology

      Google Classroom codes for second Semester

      Tests and Quiz

      Chapter 14 Test -EVEN ONLY

      Chapter 14 Test - ODD ONLY

      Assignment and Video Links

      Make Up Tests

      by Permission only during CAT30

      Extra Credit Assignments and Publisher Practice Tests

      Self-Check Quiz zes to help you study for your tests.

      An excellent way to study for your tests is to complete

      the chapters which we are covering this semester.

      Practice Test s to help you prepare for your tests and FINAL EXAM.

      Self-Check Quiz zes to help you study for your tests.

      Practice Test s to help you prepare for your tests and FINAL EXAM.

      Dear Biology Students,

      Welcome to second semester. Everyone begins with a 100 % and my goal is to help you keep an A grade in this class. You will see a sign up in my class that states "Have Fun but also get your work done" , I reall mean that. If you have any questions or need help please ask. Remember to Check Google Classroom for all your assignments and due dates and keep in mind that unexcused late work will no longer be accepted this semester.


      Table of Contents

      Foreword
      Acknowledgments
      1. The Basics of Molecular Biology
      2. The Tools of the Molecular Biologist
      3. General Preparations, Procedures, and Considerations for Use of Manual
      Section 3-1 Using This Manual
      3-2 Safety Considerations
      3-3 Equipment Needed for Molecular Biology Studies
      4. Cloning Vectors and Bacterial Cells
      Section 4-1 pBR322
      4-2 M13
      4-3 pUC
      4-4 λgtlO
      4-5 λgtll
      4-6 EMBL3 and EMBL4
      4-7 Charon 28
      4-8 Bacterial Strains
      5. Preparation of DNA from Eukaryotic Cells
      Section 5-1 Rapid DNA Preparation
      5-2 Preparation of DNA from Eukaryotic Cells: General Method
      5-3 DNA Preparation from Cultured Cells and Tissue
      5-4 Restriction Endonucleases (REs) and Their Use
      5-5 Agarose Gel Electrophoresis
      5-6 Southern Blot
      6. Probing Nucleic Acids with Labeled Synthetic Probes
      Section 6-1 Making Synthetic DNA Probes: General Description
      6-2 End Labeling of Synthetic Probes
      6-3 Hybridization with Synthetic 32P End-Labeled Probe
      7. Probing Nucleic Acids with Plasmid-Derived Probes
      Section 7-1 Nick Translation
      7-2 DNA Hybridization (Southern Blot Hybridization)
      8. Plasmid DNA Preparation
      Section 8-1 Transformation of Bacteria
      8-2 Plasmid DNA Preparation: Triton-Lysozyme Method
      8-3 Large-Scale Alkaline Lysis Method: Plasmid Purification
      8-4 Plasmid "Mini-Prep" Method
      9. DNA Restriction Fragment Preparation
      Section 9-1 Minigels 1
      9-2 Analysis of DNA Fragments After Enzymatic Cleavage: Agarose Gel Electrophoresis
      9-3 Electroelution
      9-4 Polyacrylamide Gel Electrophoresis of DNA Restriction Fragments
      10. Purification of DNA
      Section 10-1 Spermine Purification of DNA
      10-2 Glass Powder Elution of DNA
      10-3 Purification of DNA: Other Methods
      11. Preparation and Analysis of RNA from Eukaryotic Cells
      Section 11-1 Guanidine Isothiocyanate Preparation of Total RNA
      11-2 RNA Preparation: Mini Method
      11-3 Selection of Poly(A+) RNA on Oligo(dT) Cellulose
      11-4 Formaldehyde Gel for Electrophoretic Separation of RNA and Northern Blot
      11-5 "Dot Blot" Hybridization of Labeled Probe to DNA or RNA Samples
      11-6 Probing RNA Gels: General Notes
      11-7 Preparation of RNA Probes from DNA Cloned into Plasmids
      12. Preparation of DNA from Bacteriophage Clones
      Section 12-1 Growth and Preparation of Bacteriophage
      12-2 Large-Scale Preparation and Purification of DNA from Bacteriophage
      13. Cloning DNA from the Eukaryotic Genome
      Section 13-1 Cloning DNA from the Eukaryotic Genome: Introduction
      13-2 Preparation of Genomic DNA: Partial Mbol Digestion Method
      13-3 Preparation of Bacteriophage Vector for Genomic Cloning
      13-4 Ligation of Genomic DNA into Bacteriophage Arms and Packaging to Form Library
      13-5 Titering and Plating of Packaged Library
      13-6 Screening a Plated Library with Radiolabeled Probes
      13-7 Library Amplification
      14. cDNA Cloning into λgtlO and λgt11
      Section 14-1 Preparation of λgt10 and λgt11 cDNA Cloning Vectors
      14-2 Generation of cDNA Insert from Eukaryotic mRNA
      14-3 Ligation and Packaging of cDNA Library into λgt10 or λgt11 Arms
      14-4 Plating and Screening of λgt10 and λgt11 Packaged Inserts
      14-5 Preparation of DNA from λgt10 and λgt11 cDNA Clones
      15. Subcloning into Plasmids
      Section 15-1 Subcloning into Plasmids: General Notes
      15-2 Preparing pBR322 Plasmids for Subcloning and Ligation of Insert
      15-3 pBR322 Colony Hybridization
      15-4 Subcloning into pUC Plasmids
      16. M13 Cloning and Sequencing
      Section 16-1 M13 Cloning and Sequencing: General Notes
      16-2 Preparation of Insert for Cloning from Specific Restriction Sites
      16-3 Preparation of Insert for Μ13 Cloning by Successive BAL 31 Exonuclease Deletion
      16-4 Μ13 Vector Preparation and Ligation of Insert into Vector
      16-5 Transformation of M13 into JM103 E. coli Host
      16-6 Screening Μ13 Clones with a Radiolabeled Probe to Select Inserts for Sequencing
      16-7 Preparation of Single-Stranded Μ13 DNA for Sequencing
      16-8 Single-Lane Screen Analysis of Μ13 Clones
      16-9 Preparation of Polyacrylamide Sequencing Gel
      16-10 Sequencing Μ13 Clones
      17. Further Characterization of Cloned DNA
      Section 17-1 Si Nuclease Protection Assay
      18. Transfection of Mammalian Cells in Culture
      Section 18-1 Calcium Phosphate Transfection of Nonadherent and Adherent Cells with Purified Plasmids
      18-2 DEAE Dextran-Mediated Transfection of Nonadherent and Adherent Mammalian Cells
      18-3 Electroporation
      18-4 Selection of Transfected Mammalian Cells: The G418 Method
      18-5 Chloramphenicol Acetyltransferase (CAT) Assay
      19. Protein Methods
      Section 19-1 In Vitro Translation and Immunoprecipitation
      19-2 Polyacrylamide Gels for Protein Separation
      19-3 Western Blot Analysis
      19-4 Silver Staining of Gels for Proteins or RNA
      20. General Methods
      Section 20-1 DNA/RNA Extraction and Precipitation
      20-2 Plastic Bag Sealing
      20-3 Optical Density Analytical Measurements
      20-4 Photographing Gels or Autoradiograms
      20-5 Autoradiography
      20-6 Making Plates for Bacterial Growth
      20-7 Titering and Plating of Phage
      21. Specialized Methods
      Section 21-1 Transgenic Mouse Preparation
      21-2 Monoclonal Antibody Production: Hybridoma Fusion
      21-3 In Situ Hybridization of Labeled Probes to Tissue Sections
      21-4 Cloning into Yeast
      Appendix I Stock Solutions
      Appendix II Enzymes
      Appendix III Suppliers of Reagents and Equipment
      Index


      Watch the video: A2 Biology - DNA sequencing OCR A Chapter (September 2022).


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