Friday, November 30, 2012

PARANEMIC STRUCTURE


A paranemic structure is a type of DNA structure (double-stranded helix) that can be separated without uncoiling [1] [2].

One of the particularities of this structure is that it is seen as a possible solution to the topological problem generated from the structural double helix model of Watson and Crick. This problem arises during DNA replication, and relates to the need of unwinding the chains to make possible for this process. The paranemic structure may be a solution to this problem once the strands can be separated by movement, each one sideways, without unwinding, as previously referred [3].

Paranemic structure example, [1]



Bibliography:


[1] Yagil G., 1991, Paranemic Structures of DNA and their Role in DNA Unwinding, Critical Reviews in Biochemistry and Molecular Biology, [online] Available at: <http://informahealthcare.com/doi/pdf/10.3109/10409239109086791> [Accessed 28 November 2012]

[2] Cammack R. et al., 2006, Oxford Dictionary of Biochemistry and Molecular Biology, [online] Available at: <http://www.answers.com/topic/paranemic> [Accessed 28 November 2012]
[3] Brown, T. A., 2002, Genomes, 2nd ed., Oxford: Wiley-Liss.



Helicase


The helicase is an enzyme that promotes the separation of nucleic acid strands breaking the hydrogen bonds between base pairs so that replication may occur. This enzyme moves along the DNA double helix using energy from ATP hydrolysis. Helicases are also utilized to separate a self-annealed RNA molecule.
During this process helicases are helped by topoisomerases, enzymes that regulate the overwinding or underwinding of DNA. DNA becomes overwound ahead of a replication fork and if topoisomerase don’t act the replication process could get to a halt due to the tension.


Structure of E. coli's helicase RuvA

http://www.dnatube.com/video/56/Structure-of-DNA-helicase

ORC (Origin Replication Complex)






The ORC (Origin Replication Complex) is a proteic binding complex, made up of 6 subunits (ORC p1, ORC p2, ORC p3, ORC p4, ORC p5, ORC p6) that has a binding specificity to DNA, more specifically to the origins of replication, in the presence of ATP, in the genomes of the eukaryotes. Theoretically this binding complex’s function is the initiation and/or regulation of the DNA replication in eukaryotes.


For example, in the origin of replication ARS (Autonomously Replicating Sequence) of Saccharomyces cerevisiae, there are, as in the origin of replication of Escherichia coli, segments with different functional roles, and in both cases, these “subdomains” have similar sequences. 4 of these subdomains in Saccharomyces cerevisiae are known, and two of those – the subdomains A1 and B1 – constitute the origin recognition sequence, a segment of 40 bp that is the site where the ORC is going to bind to. The ORCs have been described as versions of the initiation proteins present in prokaryotes and viruses, such as the DnaA protein of Escherichia coli, which possess an initiation function in DNA replication by opening the double helix, but this interpretation is probably not entirely correct, because ORCs appear to remain attached to the yeast’s origins of replication throughout the cell cycle. 


This indicates that rather than being genuine initiation proteins, ORCs are more likely involved in the regulation of the genome’s replication, acting as mediators between the origins of replication and the regulating signals that coordinate the initiation of the replication of the DNA, with the cell cycle. More specifically, the ORC bound to the origins of replication act as the foundation for the assembly of the pre-replication complex (pre-RC), which is a proteic complex essential for the initiation of the replication, in other words, the pre-RCs act as licensing factor of the chromosomes. That’s why ORCs do not work directly in DNA replication initiation but indirectly instead.  In addition to this, the ORC is target of protein kinases which will phosphorylate some of it’s subunits in order to regulate DNA replication initiation and to block the reinitiation of the G2/M phases.



FEN1

Flap Endonucleases 1 (FEN1) is the recommended name for a class of nucleolytic enzymes that act towards 5’-3’ (in human beings), however it is also common to found other nomenclature such as “DNase IV”, “Flap structure-specific endonuclease 1” or “Maturation factor 1”. These enzymes are produced by E. Coli. FEN1 is a single polypeptide chain that contains about 380 amino acids and is implicated in biological processes, like DNA replication, recombination and repair. FEN1 splits 5’ flap single-stranded (ss) DNA at the single strand-double strand joint. It can as well displace and remove a defective nucleotide to a flap, in long-patch base excision repair. The endonuclease interacts with other nucleases and helicases that allow it to act efficiently on structured flaps. FEN1 is associated to FEN1 gene, wich one encodes a protein responsible for removing 5’ overhanging flaps in DNA repair and is required during the Okazaki fragment processing in DNA synthesis.

Stem cells


Stem cells are a peculiar group of cells whose cell fate is not yet determined. These are considered totipotent cells for their ability to originate any kind of cell by a process called cellular differentiation.

Early in development, cells of the morula are totipotent and, with the evolution of the embryo, they will differentiate into specialized cells that will result in the formation of all the different organs. However, there is a small amount of bone marrow stem cells in an adult individual destined to the production of other marrow cells and erythrocytes.


DNA Ligase

DNA ligase is a specific type of enzyme; that catalyzes the linkage between two free ends of double-stranded DNA chains by forming a phosphodiester bond between them, as in the repair of damaged DNA.
There are two classes of DNA ligases: 
·         The first uses NAD+ as a cofactor and only found in bacteria. 
·         The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages. 
Biologically, DNA ligases are essential for the joining of Okazaki fragments during replication, and for completing short-patch DNA synthesis occurring in DNA repair process. 
In addition, DNA ligase has extensive use in molecular biology laboratories for genetic recombination experiments. Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.



Fig. 1: DNA ligase mechanism


Thursday, November 29, 2012

Satellite DNA


Satellite DNA is a repetitive DNA sequence that originates a satellite band in a density gradient, consisting on non-coding DNA.

This is the major component of functional centromeres, being also the main structural component in heterochromatin.

Its GC content is different from the genome standard, which makes these fragments present different buoyant densities from those shown in single-copy DNA, therefore it migrates to different positions in the density gradient.


Satellite DNA is divided into 5 types:



α, which lenght is about 171 bp (base pairs) and is found in all chromossomes;


β, with 68 bp, is commonly found in centromeres of chromosomes 1, 9, 13, 14, 15, 21, 22 and Y;


Satellite 1, with 25-48 bp, is commonly present in centromeres and other regions in heterochromatin of most chromosomes;


Satellite 2 and 3 , both with 5 bp, can be found in most chromossomes.


Monday, November 26, 2012


Chromatin

The chromatin can be found in the cell’s nucleus. It is the combination between DNA and different proteins, the histones. Histones H2A, H2B, H3 and H4 attach each other to form an octamer or nucleosome, meanwhile the histone H1 attaches to the adjacent nucleosomes “condensing” the DNA and forming the chromosomes – this happens during prophase in which chromatin becomes coiled into chromosomes and each chromosome has two chromatids. Chromatin’s functions are to package DNA into a smaller volume so it can fit in the cell, it allows to have a higher control over these genes' expression.
 It’s called Euchromatin when DNA wraps around histone proteins to form the nucleossomes and it corresponds to a lightly packed form of chromatin . When multiple histones wrap into a 30 nm fibre, consisting in chromatin most compact form, it’s called heterochromatin, which can be constitutive or facultative. 
G4.2

Friday, November 23, 2012

Nucleosome


Nucleosome is considered the packaging unit of chromatin, its formed by the winding of DNA around four proteins designated Histones ( proteins essentially constituted by lysine and arginine that help in the winding of the DNA), this four proteins are H2A, H2B, H3 and H4.
Chromatin involves the histones in regular intervals, in other words, a nucleosome is formed every 200 base pairs, and around 56 nucleotides involve the histones, which means there are approximately 146 base pairs between each nucleosome.


CELL CYCLE


Cell cycle is a set of events that occur in order to a living cell divide in two, giving rise to a new cell with the same genetic material than the mother cell. This goes through two stages: interphase and mitosis.
G1 phase: Cell size grows due to an accumulation of nutrients. Transcription of DNA and translation of proteins are needed to make enzymes and other compounds that the next stage will need.
S phase: The DNA of the cell is replicated in this stage. The genetic material is doubled and each chromosome possesses two sister chromatids.
Mitosis (M phase): Division of the cell and nucleus by the order of prophase, metaphase, anaphase, telophase. It will end with de cytokinesis that is going to divide the cell in two.
The cell cycle


G2 phase: Cell grows because biosynthetic events happening in the cell like the formations of microtubules necessary to the mitosis for instance.
There must be some checkpoint preventing the occurrence of errors and stopping it’s proliferation. Cell cycle checkpoints are regulation mechanisms. Before entering in S phase there’s a checkpoint making sure that everything is set to DNA replication and before the M phase there’s one to prevent the damage in the DNA looking for errors. If this happens the cell may go under apoptosis preventing the passage of the error.




Bibliography:
Brown, T. A., 2002, Genomes, 2nd ed., Oxford: Wiley-Liss.

Ploidy/Aneuploidy/Polysomy


                 Ploidy is the number of chromosomes per set in a cell. In eukaryotes, we usually find two types of sets: Haploid organisms composed only for one chromosome per set and diploid organisms which have two chromosomes per set in each cell. Consequently, the germ cells of a diploid or a haploid organism will originate other organisms that will be diploid and haploid, respectively, at least in one stage of their life.

                Aneuploidy is a genetic disorder related with the number of chromosomes in a cell. This anomaly occurs, normally, in the development of the gametes, non-disjunction of homologous chromosomes during meiosis or problems in the random distribution of chromosomes or even in crossing-over. These mutated chromosomes have a number of chromosomes per set that weren’t “suppose” to have, more or less than expected.

               Polysomy is a type of aneuploidy in which a set of chromosomes has more chromosomes than it would be normal to have, for example, in a diploid organism, a set of chromosomes with more than a pair is a case of polysomy. It is usually classified by the number of chromosomes per set, for example, a polysomy with three chromosomes is called a trisomy, a polysomy with four chromosomes is called tetrasomy and so on.

Endogenous retroviruses (ERVs)

Endogenous retroviruses (ERVs) are LTR (Long Terminal Repeats) retroelements, present mostly in human and other mammal’s genomes, making up, for example, 4,7 % of the human genome. ERVs differ from the conventional LTR retroelements, because they resemble more decayed viral retroelements rather than true transposons.
ERVs are very similar to conventional retrotransposons, because in both cases there’s the involvement of a RNA intermediate. The difference is that in transcription mechanisms in retrotransposition the RNA molecule has an endogenous origin, being transcribed from its own genome, while in retroviral replication mechanisms, transcription occurs from an exogenous viral genome.
ERVs are permanent constituents of the host’s genome, and are inherited from generation to generation, like any other constituent of the host’s genome. Retroviral infections normally attack somatic cells, but in the ERV’s case, it is thought that this genomic integration is the result of retroviral infection of germ cells, which resulted in its integration and transmission to subsequent generations. That’s why the term “endogenous retroviruses” is used.
In the human genome, ERVs possess copies of the genes gag, pol and env, but however most ERVs suffered mutations or deletions which inactivated one or more of these genes, which resulted in the loss of its viral replication activity. The majority of ERVs are therefore inactive sequences that are not capable of additional proliferation as retrotransposons.


LINEs:



LINEs (long interspersed nuclear elements) are a non-LTR Retrotransposons, a type of genome-wide repeat, often with transposable activity.
In eukaryotes, these repetitive elements constitute a large portion of its genome. There are about 850,000 repetitions of these, constituting about 21% of human DNA.
Repetitive sequences of the genome are classified into sub-divisions based on their size and the SINEs (short interspersed elements), with a length between 100 and 300 pair of bases , and LINEs are the major classes of repeats.
LINEs have a length between 4-6 kb and many sequences, smaller, whit about 1kb, derive them.  They’re replicated and propagated genome’s components which propagate in genome by auto replication, moving to new locations of DNA by a process called transposition.
These sequences represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase II into mRNA and code for 2 proteins.
A typical LINE (fig 1) contains a 2 ORFs (open reading frames), a 3′ UTR (untranslated region), and 5′ UTR that contains an internal polymerase II promoter sequence, while the 3′ UTR contains a polyadenylation signal (AATAAA) and a poly-A tail.
LINE -1 is an example of a human LINE.
Their function in cell is not known, such as SINEs.


Fig 1-The components of a typical LINE. (5′ UTR, 2 ORFs and  3′ UTR with a polyadenylation signal (AATAAA) ).





Alu Elements


Alu elements are a short extension of DNA, classified as SINEs (Short INterspersed Elements), originally characterized by the action of the Alu restriction endonuclease. They are the most abundant mobile elements in the human genome, what make them an example of a "jumping gene", more exactly retrotransposons.
An Alu element has an independent transcription because it is transcribed into mRNA by RNA polymerase III and then converted into a double-stranded DNA molecule by a reverse transcriptase. The new double-stranded DNA molecule is then inserted into a new location in the genome.
The study of these elements is important to understand human population genetics and the evolution of primates and humans.

Thursday, November 22, 2012


SINES

·         SINE’s, Short INterspersed Elements, are, as the name suggests, short DNA sequences. They are genetic elements with the ability to replicate themselves in the genome of a host cell. These elements are reverse-transcripted molecules of RNA who were transcripted from tRNA, a reaction that is catalyzed by RNA polymerase III. But these elements cannot produce the polymerase due to their short size and therefore are dependent of other elements of transpositions present in the host cell.

·         SINE’s are retrotransposions without LTR group (Non-long terminal repeat). LTR’s are used by virus to insert their genomic sequences in to the host genome and since these elements do not poses these terminal, they must associate with LINE’s.

·         Retrotransposons without LTR have 3 subtypes: LINE’s, SINE’s and Composite SINE Transposons. While LINE’s have the ability to perform all the functions required to retrotranposition, SINE’s do not have such capability and, therefore, link themselves with LINE’s. As for Composite SINE Transposons, two SINE’s connect to each other to flank and mobilize an intervening single copy DNA sequence.

Karyogram


A karyogram, also known as an ideogram, is a diagram or photograph representation of a karyotype (total number and appearance of chromosomes in the nucleus of an eukaryotic cell) arranged in pairs of homologous chromosomes of the same size, ordered by size and position of centromere.
Some zones of the chromosome, like the centromere and telomeres, are visible in the karyogram. Chromosome’s coloration shows dark zones intercalated with light zones, pending on GC and AT percentage.
Therefore, karyograms are very useful in cytogenetics due to the practical application for genetic studies such as hereditary anomalies (p.e. trisomy 21).
Figure 1. Karyogram of a human male karyotype using Giemsa as colorant.

Figure
Retrovirus


Retrovirus consists of two molecules of single-stranded RNA connected together. The double strand allows a higher rate of recombination gene.
 Virus are morphologically very similar to each other and are composed of proteins, RNA and membrane lipids.
The main feature of this virus is the reverse transcriptase enzyme that produces DNA from an RNA molecule.
The virus recognizes the cell surface and fuses with the plasma membrane. After penetration of the viral nucleocapsid, occurs the reverse transcription of virus RNA into viral DNA that migrates to the nucleus of the cell and integrates in the host cell DNA. Then starts the transcription of this DNA into viral RNA, consequently proteins are also produced and hundreds of new virus. One of the most familiar types of retrovirus is HIV, which infects humans blood T lymphocytes.

Wednesday, November 21, 2012

Endogenous Retrovirus


Endogenous Retroviruses are retroviral genomes inserted in vertebrate chromossomes, that might induce the synthesis of exogenous viruses. However, the majority does not have the capacity to form new viruses. So, these inactive sequences represent genome wide-repeats that can not proceed with any further proliferation.


 Fig.1- Human endogenous retrovirus (TEM)

Operon


It’s a group of genes from the gDNA (genomic deoxyribonucleic acid) in prokaryotes and some eukaryote organisms. The transcription of the operon simply takes to a polycistronic mRNA (messenger ribonucleic acid) molecule wich is composed by an operator, a promoter and other structural genes. The promoter associated to the operator controls the structural genes expression. The translation of the operon rises to several independent proteins with related metabolic functions.

Tuesday, November 20, 2012

DNA Transposons



DNA Transposons

  A small mobile genetic element (DNA) that moves around the genome or to other genomes within the same cell, usually by copying itself to a second site but sometimes by splicing itself out of its original site and inserting in a new location within the same or another chromosome, plasmid, or cell and thereby transferring genetic properties.

  Transposons have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Consequently, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy.


Fig. 1: DNA transposon example

Friday, November 2, 2012

Cosmid


Cosmid


Figure 1 - A typical cosmid


                Cosmids are hybrid plasmid vectors that contain λ cos site. They were first described by Collins and Hohn in 1978. The λ cos site is the sequence needed so that the DNA molecule becomes recognized as part of the ‘λ genome’ by the proteins that package DNA into λ phage particles.

                Inside the cell, the cosmid DNA is incapable of directing the synthesis of new phage particles. As an alternative the cosmid DNA replicates as a plasmid.
The length of the cloned DNA can be as long as the available space within λ cos site. A cosmid can be 8 kb or less in size, so up to 44 kb of new DNA can be inserted before the packaging limit of the λ phage particle is reached. Recombinant DNA is obtained from colonies and can be used to build genomic libraries.