Biotechnology-3- Dr . Mayssaa - PowerPoint Presentation

Slide1

Biotechnology-3-

Dr

.

Mayssaa

Essam

Replication of DNA in eukaryotic cells

The essential steps of replication are the same as in prokaryotes. Starting replication is more complex in eukaryotes. At the origin of replication, a pre-replication complex is made with other initiator proteins. Other proteins are then recruited to start the replication process. The overall process is the same, although differently named enzymes fulfill the same function. The first important difference is in the polymerases. So far, 15 kinds of polymerases were isolated from the eukaryotic cells. Amongst the most important are:*

DNA polymerase

δ

catalyzes

the synthesis of leading strand and finishes the synthesis

of lagging

strand;

*

DNA polymerase

α

(

DNA primase) catalyzes the synthesis of Okazaki fragments;

*

DNA polymerase

β

catalyzes the synthesis of short fragments during DNA reparation;

*

DNA

polymerase

ϒ

catalyzes the formation of mitochondrial DNA in the mitochondria

The

important differences between prokaryotic and eukaryotic replication:

*

Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. This means that there must be multiple origins of replication on the eukaryotic chromosome in order for the entire DNA to be replicated in a timely manner; humans can have up to 100,000 origins of replication.

DNA Packaging

*Eukaryotic

DNA is wound around proteins known as histones to form structures called

nucleosomes

. The DNA must be made accessible in order for DNA replication to proceed. The

chromatin

(the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the histones or otherwise be accessible to the enzymes of the DNA replication machinery. Prokaryotes do not package their DNA by wrapping it around histones.

*

Gene A gene is a basic functional and structural unit of genetic information. In meaning of molecular genetics gene is a segment of DNA codes for a protein and contain regulatory and coding parts. According to the genetic information that they carry, we can divide genes into three groups: structural genes, regulatory genes, and genes for the RNA molecules, except mRNA.

A Structural Gene is a part of the DNA chain, which codes for the primary structure of the proteins. The size of the genes is expressed in the number of base pairs (

bp

) in the DNA, it contains

.

The structural gene consists of two parts: regulatory and coding part. The regulatory part is called the promoter. It contains important sequences (parts of base sequences, the so called boxes), for example TATA box, CAAT box .

In the coding areas the eukaryotic genes often contain noncoding parts (introns) in between the coding parts (exons). The coding part of the gene starts with the untranslated region (UTR) on the 5´ end, which serves as a connection of the mRNA to a small ribosomal subunit. Immediately after it follows the first exon, which starts with the so called start triplet (ATG). The first exon is followed by the first intron. Then proceeds another exon etc. This area therefore contains alternating exons and introns. The last exon ends with the so called stop triplet (TAA, TAG or TGA). Then untranslated region (UTR) on the 3´ end

.

The regulatory gene

is a part of the DNA which codes for the primary structure of the regulatory protein, which function is usually the induction or repression of the other genes expression.

The RNA coding genes

are responsible for the primary structure of the ribosomal and transfer RNA and other types of smaller molecules of RNA.

Figure: Structure of the Eukaryotic gene

Gen Expression

Gene expression is the process by which the genetic code - the nucleotide sequence - of a gene is used to direct protein synthesis and produce the structures of the cell. Genes that code for amino acid sequences are known as 'structural genes'.The process of gene expression involves two main stages:Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase, and the processing of the resulting mRNA molecule.Translation: the use of mRNA to direct protein synthesis, and the subsequent post-translational processing of the protein molecule.

Genetic code

The set of correspondences between nucleotide pair triplets in DNA and amino acids in protein.A group of three following bases (nucleotides) is called

triplet (codon)

. A triplet is always read from the 5´ end of the mRNA, so for example the triplet for

tryptophane

is: 5´– U G

G

– 3

´.

There are 64 types of codons, from these only 61 code amino acids. The first, Start triplet, in mRNA is always AUG, which codes for methionine. Three of these triplets don’t code for any amino acid and have an important role during the termination of synthesis of the polypeptide chain. Those are the so called

termination codons

or stop codons. They are: UAA, UAG and UGA

.

Recombinant DNA, rDNA

, is a piece of DNA artificially created in vitro which contains DNA (natural or synthetic) obtained from two or more sources.



Recombinant DNA technology has had a fourfold positive impact upon the production of pharmaceutically important proteins:

It

overcomes the problem of source availability

.

It overcomes problems of product safety

.

It provides an alternative to direct extraction from inappropriate/dangerous source material.

It facilitates the generation of engineered therapeutic proteins displaying some clinical

advantage.

Gene therapy

Gene therapy

Is

a novel treatment method which utilizes genes or short oligonucleotide sequences as therapeutic molecules, instead of conventional drug compounds. This technique is widely used to treat those defective genes which contribute to disease development. Gene therapy involves the introduction of one or more foreign genes into an organism to treat hereditary or acquired genetic defects. In gene therapy, DNA encoding a therapeutic protein is packaged within a "vector", which transports the DNA inside cells within the body. The disease is treated with minimal toxicity, by the expression of the inserted DNA by the cell machinery

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