Genetic material

3rd Lecture Medical students Medical Biology

Genetic Material

In this section, we will focus on some techniques used in molecular genetics.

Nucleic acid hybridization

Extremely sensitive detection method, capable of picking out specific DNA sequences from complex mixtures. Usually a single pure sequence is labeled with P³² and used as a probe. The probe is denatured before use so that the strands are free to base-pair with their complements. When DNA is hybridized with a RNA probe, the DNA-RNA hybrids are immobilized onto a plate using anti-DNA-RNA antibodies. Antihybrid antibodies conjugated to an enzyme are used for detection in combination with appropriate chemiluminescent substrates.

Gel electrophoresis

The technique of gel electrophoresis is vital to the genetic engineer, as it represents the main way by which nucleic acid fragments may be visualized directly. The technique is carried out using a gel matrix, which separates the nucleic acid molecules according to size. A typical nucleic acid electrophoresis. Two gel types are commonly used: agarose and polyacrylamide. The data can be used to estimate the sizes of unknown fragments by construction of a calibration curve using standards of known size, as migration is inversely proportional to the log10 of the number of base pairs. This is particularly useful in the technique of restriction mapping. In addition to its use in the analysis of nucleic acids, PAGE is used extensively for the analysis of proteins. The methodology is different from that used for nucleic acids, but the basic principles are similar. Proteins can be separated according to their size in a similar way to DNA molecules.

DNA sequencing

The ability to determine the sequence of bases in DNA is a central part of modern molecular biology and provides what might be considered the ultimate structural information. Rapid methods for sequence analysis were developed in the late 1970s, and the technique is now used in laboratories worldwide. In recent years the basic techniques have been revolutionized by automation. the determination of a DNA sequence requires that the bases are identified in a sequential technique that enables the processive identification of each base in turn. There are two main methods for sequencing DNA. In one method, developed by Allan Maxam and Walter Gilbert, chemicals are used to cleave the DNA at certain positions, generating a set of fragments that differ by one nucleotide. The same result is achieved in a different way
in the second method, developed by Fred Sanger and Alan Coulson, which involves enzymatic synthesis of DNA strands that terminate in a modified nucleotide. Analysis of fragments is similar for both methods and involves gel electrophoresis and autoradiography (assuming that a radioactive label has been used). The difficulty of preparing a fragment for sequencing is largely dependent on the scale of the sequencing project. If the aim is to sequence part of a gene that has already been isolated and identified, the process is relatively straightforward and usually requires that the fragment is of a suitable length and in a suitable form for the sequencing procedure in use. If, however, the aim is to sequence a much larger piece of DNA (such as an entire chromosome), the problem is much greater.

The polymerase chain reaction

The polymerase chain reaction (PCR), which was discovered by Kary Mullis and for which he was awarded the Nobel prize in Chemistry in 1993. The PCR technique produces a similar result to DNA cloning the selective amplification of a DNA sequence and has become such an important part of the genetic engineer’s toolkit that in many situations it has essentially replaced traditional cloning methodology. In this technique, the DNA fragments separate according to their size.


REGULAT ION OF GENE TRANSCRIPTION
Promoters
The promoter region of a gene is usually several hundred nucleotides long and immediately upstream from the transcription initiation site. The promoter constitutes the binding site for the enzyme machinery that is responsible for the transcription of DNA to RNA, the RNA polymerase. In eukaryotic cells several RNA polymerases are present, the most prominent one, that is responsible for the transcription of protein coding genes, being RNA polymerase II. There are different types of promoters for different RNA polymerases. Promoters for RNA polymerase II, the polymerase that transcribes protein-coding genes into mRNA, often contain the consensus sequence 5´-TATA-3´, 30 to 50 bp upstream of the site at which transcription begins. Many eukaryotic promoters also have a so-called CAAT box with a GGNCAATCT consensus sequence centered about 75 bp upstream of the initiation start site (with N representing any of the four bases). RNA polymerases I and III are mainly responsible for the transcription of RNA molecules that possess an intrinsic function as catalytic or structural molecules, such as tRNA and rRNA, and that are not translated into proteins. In general, the promoter region has a high importance for the regulation of expression of any gene. This concept will come up again later on in this module when the production of transgenic animals is introduced. Careful choice of promoters to drive gene expression in transgenic organisms is very important to ensure the transgenic organism possesses the desired characteristics.

Enhancers

Enhancers were first described as sequences that increase transcription initiation but, unlike promoters, were not dependent on their orientation or the distance from the transcription start site. It is now apparent that enhancers are generally short sequences (less than 20 to 30 base pairs) that bind specific transcription factors, which then facilitate the assembly of an activated transcriptional complex (i.e. the RNA polymerase) at the promoter. Most enhancers function both on the coding and non-coding strand of the DNA (i.e. in either orientation), can act up to several thousand base pairs distant from their target promoter, and are a rather unspecific form of regulatory element. This implies that an enhancer element may influence several, possibly very distant, promoters. Most enhancers are only active in specific cell types and therefore play a central role in regulating tissue specificity of gene expression. Some regulatory elements bind transcription factors that act to reduce the efficiency of transcriptional initiation, and many genes contain a combination of both positive and negative upstream regulatory elements, which then act in concert on a single promoter. This allows gene expression to be controlled very precisely in a temporal and spatial manner with regard to cell type, developmental stage and environmental conditions. Mutations of promoters or enhancers can significantly alter the expression pattern, but not the structure of a particular gene product.
Operators
Operators are nucleotide sequences that are positioned between the promoter and the structural gene. They constitute the region of DNA to which repressor proteins bind and thereby prevent transcription. Repressor proteins have a very high affinity for operator sequences. Repression of transcription is accomplished by the repressor protein attaching to the operator sequence downstream of the promoter sequence (the point of attachment of the RNA polymerase). The enzyme must pass the operator sequence to reach the structural genes start site. The repressor protein bound to the operator physically prevents this passage and, as a result, transcription by the polymerase cannot occur. Repressor proteins themselves can be affected by a variety of other proteins or small molecules, e.g. metabolites, that affect their affinity for the operator sequence. This allows a further level of gene expression regulation to be accomplished.

Genes Made of RNA

Hershey & Chase investigated bacteriophage, virus particle by itself, a package of genes
This has no metabolic activity of its own.
When virus infects a host cell, the cell begins to make viral proteins.
Viral genes are replicated and newly made genes with viral protein assemble into virus particles.
Some viruses contain DNA genes, but some viruses have RNA genes, either double- or single-stranded.

Biotechnology

is defined as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for
specific use”. Several branches of industry rely on biotechnological tools for the production of food, beverages, pharmaceuticals and biomedicals. The CBD definition is applicable to both “traditional” or “old” and “new” or “modern” biotechnology. Since the middle of the twentieth century biotechnology has rapidly progressed and expanded. In the mid-1940s, scale-up and commercial production of antibiotics such as penicillin occurred. The techniques used for this development were:
» isolation of an organism producing the chemical of interest using screening/ selection procedures, and improvement of production yields via mutagenesis of the organism or optimization of media and fermentation conditions. This type of biotechnology is limited to chemicals occurring in nature. It is also limited by its trial-and-error approach, and requires a lengthy procedure over years or even decades to improve yields. The techniques used for this purpose include:
» isolation of the gene coding for a protein of interest.
» cloning (i.e. transfer) of this gene into an appropriate production host.
» improving gene and protein expression by using stronger promoters, improving
fermentation conditions etc. Together, these techniques are known as recombinant DNA technology.



لكل انسان الحق في التعبير عن رأيه لكن بأسلوب !

Mohammed J AlRawi