Oligonucleotides

The oligonucleotides are short nucleic acid polymer, normally composed by fifty bases or less.
Frequently they are used as probes to detect complementary DNA or RNA, because they connect whit their complementary nucleic acid chain, immediately.
On PCR's the oligonucleotides are used as a primer. The connect whit target DNA in order to create a spot which will allow polymerase to attach. Therefore these molecules are responsible for many copy processes of nucleic acids.

2µ circle

The 2µ circle is a circular plasmid composed of about 6.000 bp of double-stranded DNA, comprising approximately 1.5-5% of total nuclear DNA. It’s found in the nucleus in most strains of Saccharomyces cerevisiae at a copy number of about 60. No sequences homologous of 2µ circle have been found in chromosomal DNA.

2μ circle can also serve as a model system for specialized recombination in a eukaryotic cell with the possibility that this system will provide insights into an unusual form of gene regulation.

The principal element in the strategy of 2 μm circle for persistence as a parasitic DNA species is its ability to raise its copy number.

F-plasmid

A plasmid that gives a bacterium the ability of producing sexual pilus for conjugation. It is a fertility factor present in some bacteria that permits the transfer of genes from a bacterium with the factor to a bacterium that doesn't have this F-factor. This transfer process is achieved by conjugation.

Figure 1: F-plasmid map. The F plasmid has two IS3 elements and one IS2 element. A recombination event between any of the chromosomal IS2 or IS3 elements and the corresponding element on the F-plasmid will integrate the entire F-plasmid into the chromosome. Tn1000 is another insertion sequence, although not generally envolved in F-plasmid integration. OriT is the origin of transcription

Conjugation with F-plasmid:

 This animation describes the conjugation between two bacterial cells.

Alexandra Meira

Adriana Lima

Duarte Oliveira

T-4 DNA Ligase

This enzyme catalyzes the ligation of blunt and sticky ends of DNA, in only 5 minutes and PCR fragments with A overhangs, this reactions occur at the temperature of 25º. The T-4 DNA ligase catalyzes the formation of the phosphodiester bonds between the 3' hydroxyl of a chain and the 5' phophate of the other, in the presence of ATP. T-4 DNA ligase formulation was optimized so it can promote fast reaction, and a more convenient incubation. Single-strains nucleics acids aren´t substract to this enzyme.

Pedro Silva

Bacterial Artificial Chromosome (BAC)

BAC

 Bacterial Artificial Chromosomes (BAC) consist in genetic material created in laboratory that contain a gene of interest from a given species  cloned into a bacterial F-plasmid. At the end it will result in million copies of DNA.

The F-plasmids are very important since they promote the even distribution of plasmids after bacterial cell division, due to the activity of the genes oriS, parA and parB. 

Thus, BAC has common gene components as:

•          oriS and rep E-F, implicated on plasmid replication and posterior regulation of copy number;
•          parA and parB, for partitioning F plasmid DNA to daughter cells during division and ensure stable maintenance of the BAC;
•          lacZ, used for blue/white selection of recombinant bacterial colonies;
•          ampicillin resistance gene, used as a selectable marker for transformants;
•          gene inserted, part of the genome for cloning;
•         T7 & Sp6, which are phage promoters implicated on the transcription of the inserted genes.

The Bacterial Artificial Chromosomes are useful for gene insertion with length until 300 kb. For human genomic libraries, 32000 clones would be needed for a given gene to be represented (for P=95%) or 50000 (for P=99%).

Cláudio Oliveira

Hélder Badim

Liliane Barroso

Mariana Vieira

Fosmids

Fosmids are used when preparing genomic libraries for genome sequencing.  Fosmids are circular DNA of bacterial origin – technically plasmids – but where typical plasmids exist in high copy number (up to 100 copies per cell) and possess small (3 to 6 kb) inserts, fosmids are present as a single copy in a cell and may possess inserts upwards of 40 kb.  Fosmids are advantageous because they produce stable libraries for genome sequencing.  They have a tendency to provide fairly uniform coverage, so they are optimal for closing gaps in whole genome alignments.  In addition to genome sequencing, they have also been used for metagenomics and expression studies.

Furthermore, Fosmids contain the F plasmid (a fertility plasmid that directs conjugal transfer of DNA between bacteria) origin of replication and a cos site. They are similar to cosmids but have a lower copy number in E. coli, which means that they are less prone to instability problems.

Fosmid vectors are derived from random shearing – which yields more uniform coverage when comparing against other library cloning methods.

Histones

Histones are specific proteins that are found in eukariotic cells nuclei. In fact, their molecular weight is approximatedly the same as DNA's mass. DNA binds to these alkaline proteins, which play an extremely important role in gene regulation. In order to transcription occur properly, DNA requires specific proteins that release it form histones, otherwise transcription becomes impossible. As histones are so highly alkaline, negatively charged DNA easily binds with them, agreggating with 5 types of histones: H2A, H2B, H3, H4 and H1, creating a superstructure we define as nucleossomes.

Fig 1 - Nucleossome core particle bound to DNA (Science Photo)

Bibliography

 Cooper, G.M. , Hausman, R. E., 2009, The Cell, A Molecular Approach, 5th ed,Sinauer

Transformation; transduction; transfection

Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell. We can say that transformation occurs when the direct uptake of exogenous genetic material is incorporated and expressed in a cell resulting in a genetic alteration. Transformation may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells. 

The other two processes are conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).

Introduction of foreign DNA into eukaryotic cells is often called transfection. Transfection of animal cells typically involves opening "holes" in the cell membrane to allow the uptake of genetic material or even proteins such as antibodies.

Genomic Library

A genomic library is a collection of DNA from a single organism. The DNA from the source organism in study is divided into multiple fragments and packaged within cloning vectors such that each carries a portion of it. Different vectors can store differing amounts of DNA. Once the DNA has been inserted, the vector can be introduced into host organism (commonly a population of bacteria). Together, the collection of bacteria holds an organism's complete genome. Once a genomic library is produced, researchers can work with it in a number of different ways.

YAC - Yeast artificial chromosome

The YAC, defined by a high-capacity cloning (600-1400 kb) vector constructed from the components of a yeast chromosome, appeared when it was necessary to create vectors that would be able to clone DNA fragments longer than 50 kb.

This vector contains a centromere, telomeres and an autonomous replicating sequence (ARS). These structures are important requirements for replication and preservation of YAC in yeast cells. These vectors are propagated in Saccharomyces cerevisiae rather than in Escherichia coli and are based on chromosomes, rather than on plasmids or viruses.

Currently, this vector has the highest capacity of any type of cloning vector, and a lot of genome projects have used it.  Unfortunately, with some types of YAC there have been problems with insert stability, the cloned DNA becoming rearranged into new sequence combinations (Anderson, 1993). For this reason there is also great interest in other types of vectors, ones that cannot clone such large pieces of DNA but which suffer less from instability problems.

Bibliography:

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

Anderson, C., Genome shortcut leads to problems. Science. (1993); 259:1684–1687

Episomes

Epissomes are a genetical component that can either live freely and independently in the bacterial cytoplasm (having several copies in that environment) or be inserted in the main bacterial chromosome, thus, replicating with it. In other words, it is an equivalent of bacterial plasmids. In fact, plasmids are an example of this genetic element.

 In general, episomes are closed circular DNA molecules that are replicated in the nucleus of the host cell. Viruses are the most common examples of this (for example, viruses that perform the lysogenic cycle). 

Chromatin

It is a complex of genetic material (DNA and RNA) associated protein (histone) located in the cell nucleus and condenses during cell division in order to form chromosomes.

There are two states of chromatin organization:

- Euchromatin: slightly condensed chromatin. It is this state which is the cell nucleus and has enhanced activity because of its easy genetic manipulation.

- Heterochromatin: highly condensed chromatin. This is the state that is when the cell does not want particular gene is expressed, thereby reducing its genetic activity.

DNA Concatemer

A concatemer is an intermediate structure in the biosynthesis of certain DNA viruses, consisting of a number of genomes connecter together end to end and separated by cos site, and thought to be the intermediate precursor to the mature viral genome. Concatemers are frequently the result of rolling circle replication, and may be seen in the late stage of bacterial infection by phages. The term is now extended to include any linear or circular DNA structure composed of viral genomes joined end to end. The term is also used for circular DNAs which are physically inseparable, one being threaded through the other.

For example in the infection phenomenon of T-4 bacteriophages, when T-4 DNA enters a host cell, it is first replicated as a unit, and then several genomic units are recombined end to end to form a long DNA molecule, a concatemer. The concatemer is then cut by endonucleases in non-specific sites, without regard to the sequence in order to yield T4 genomes of the same size. This happens in order to yield DNA molecules just long enough to fill a phage head.

Opening post

This is a blog for my students to create a glossary of terms related to the scientific area of Genes and Genomes of the course on Applied Biology of the Department of Biology of the University of Minho in Braga, Portugal.

Although definitions are usually a simplification of concepts, they help us to integrate knowledge. Furthermore, when students seek for information to "create a definition" they are learning. And you, when you read these definitions you are also learning. So, everybody wins!

I wish a nice work for my students and pleasant and fruitful readings for everybody!

Rui Oliveira