Molecular Biology

Molecular Biology

Molecular biology is a branch of general biology that deals with DNA manipulation for the point of mutation. Cell biology is the one of the most important branches of general biology. Cell biology concentrates on studying the functions and structure of cells, which is the building blocks that make up all organisms. Combined, these two basics of biology concentrate on the molecular biology of the cell.

• The field of molecular biology was made in the 1930s but no real experimentation of molecular biology was made until the 1950s. When it began, research in molecular biology was done by using x-rays to view molecules within the cell. Studying the proteins within these cells helped scientists to determine how an organism works.

• Cell biology works closely with molecular biology. It deals with all information about the cell including structure, anatomy, death and respiration. The field of cell biology dates back to the 1650s, when Robert Hooke, a English physicist, first invented the term “cell” to describe the cell of a cork tree. Within molecular biology, cells are studied by their various molecules. Proteins are one of the most important molecules in a cell. Each protein functions a certain way and when combined, molecular biology and cell biology work together to determine what those functions are.

• Neither molecular biology or cell biology would be possible without the creation of the microscope. Today’s high-tech microscopes can see the tiniest details of cells.

• The cell theory determined that a cell is the building blocks of all living things. Within cell biology, the cell theory has changed over the years. Today, the cell theory states that all living things are made of cells, old cells diving in two creates new cells, and no two cells are identical. The molecular biology of the cell has created this entire branch of general biology, without which cell could not be studied. Molecular biology could not exist without cell biology, as the two are so closely linked together.

As more progress is made over the years by scientists to further develop the technology of cell biology and microbiology, the cell theory has remained the same for almost 200 years. Watching the cell functions in molecular biology has made it possible for diseases such as cancer to be studied in depth. Due to cell biology, many diseases can be studied and understood.

Genes and protein synthesis

There are many discussions between biologists to find a comprehensive definition of a gene, which is not easy, if possible at all. For our purposes
 

A gene is a continuous stretch of a genomic DNA molecule, from which a complex molecular machinery can read information (encoded as a string of A, T, G, and C) and make a particular type of a protein or a few different proteins.

This “definition” is not precise, and to better understand it we need to describe the molecular machinery making proteins based on the information encoded in genes. This process is called protein synthesis and has three essential stages: (1) transcription, (2) splicing, and (3) translation.

1. In transcription phase one strand of DNA molecule is copied into a complementary pre mRNA (pre stands for preliminary and m for messenger) by the protein complex RNA polymerase II (see section 2.2 and 2.4). In the process the two-stranded DNA double helix is unwound and information is read only from one strand (sometimes called the W-strand). 

2. Splicing removes some stretches of the pre mRNA, called introns, the remaining sections called exons are then joined together. Note that the removal of introns is a consequence of the way how eukaryote genomes are organised.   The genomic DNA that corresponds to the coding part of genes is not continuous, but consists of exons and introns. Exons are the part of the gene that code for proteins and they are interspersed with non coding introns which must be removed by splicing. The number and  size of introns and exons differs considerably between genes and also between species. Only very few genes in yeast have introns, while  for human threre are about 4 introns per gene on average, and the average size of exons is 150 bp and just above 3400 bp for introns. Prokaryote genes do not have introns and the splicing step is not present. The result of splicing is mRNA. Many eukaryote genes are known to have different alternative splice variants, i.e. the same pre-mRNA producing different mRNAs, known as alternative splicing.

(picture taken from  On-Line Biology Book )

3. Translation is the process of making proteins by joining together amino acids in order encoded in the mRNA. The order of the amino acids is determined by 3 adjacent nucleotides (triplets) in the DNA. This is known as the triplet or genetic code . Each triplet is called a codon and codes for one amino acid. As there are 64 codons and only 20 amino acids the code is redundant, for example histidine is encoded by CAT and CAC.  In cytoplasm the mRNA forms a complex with ribosomes, which are large complexes of proteins and RNA molecules. The precise interactions and functions of all protein in ribosomes are not yet fully understood.

(picture taken from  On-Line Biology Book )
Different transfer or tRNA molecules each carries one specific amino acid to the ribosome and specifically recognises one codon on the mRNA. The amino acid carried by the tRNA is added to the nascent (growing) protein. The translation is a complex process and not all the details are understood. Luckily most of these details are not crucial for understanding of bioinformatics. What is crucial however is to realise that there is nothing magical about proteins synthesis.
 

The end of translation is the final part of gene expression and the final product is a protein, the sequence of which corresponds to the sequence encoded by the mRNA. Proteins can be post-translationally modified e.g., by adding of sugars or cleavage (chopping), and this affects their location and function.

Biologists used to believe in paradigm - 'one gene - one protein'. Now this is known not to be true - due to alternative splicing and post-translational modifications one gene can produce a variety of proteins. There are also genes that do not encode proteins but encode RNA (for instance tRNA and ribosomal RNA).

Biotechnology and veterinary medicine

Biotechnology and veterinary medicine

While there have been many practical applications for bio technology and humans, there has also been extensive research in bio technology and animals. Biotech research in the field of veterinary medicine has helped to expand the healthy lifespan of our pets and to cure diseases that would have otherwise ended the life our pets prematurely. There is a very real difference between using animals in medical studies for the advancement of human medicine, and using animals in biotech research for the advancement of veterinary medicine. For example, scientists have used biotech research in order to make some dog breeds smaller in size. This may be a benefit to humans, such as city dwellers that don’t have the room for larger dogs. Shrinking breed size may also benefit the animals because it will eliminate such ailments as hip dysplesia.

Using bio technology in order to extend the life spans of our pets, is perhaps an accepted area of study. However, bio technology can also be used to produce a certain type of breed. Researchers could use genetic engineering to produce only the most vicious pit bulls and those dogs could be used for dogs fights or as guard dogs in dangerous parts of the world. There are advancements in bio technology that are meant to help our pets, and then there are those that would only be for human benefit. Scientists, for instance have genetically altered cows, so that they will produce more milk. There are many that feel animals are on the earth for human benefit. However, there are also those that fight for animal rights. Your opinion regarding the ethics of biotech research in regards to animals, would very much depend on where you stand with regard to animal rights.

Biotech research with animals has the possibility of helping both animals and humans. Bio technology can improve the health and lifespan of pets but it can also be used to alter the productivity of animals in the agricultural industry. The applications of bio technology and veterinary medicine are meant to improve the health of our companion animals. Research in the field of bio technology has found some success in curing cancer in dogs and in eliminating common problems associated with certain types of breeds, such as eye problems in Beagles.

Biotech research can produce cures for veterinary medicine that we may not otherwise discover. In striving to improve the lives of our companion animals, we must also remember to treat those animals we use in research humanely. If we don’t follow those principles, we eliminate the point of research meant to improve the quality of life for animals. Too often, scientists in the field of veterinary medicine treat animals in a disgusting manner and behave in a very hypocritical way. While we must realize that biotech research in the field of veterinary medicine is basically performed to sell food, medication or surgical procedures, we must also remember to think of the animals that we are trying to help.

DNA

DNA is the main information carrier molecule in a cell. DNA may be single or double stranded. A single stranded DNA molecule, also called a polynucleotide, is a chain of small molecules, called nucleotides . There are four different nucleotides grouped into two types, purines: adenosine and guanine and pyrimidines: cytosine and thymine. They are usually referred to as bases (in fact bases are the only distinguishing element between different nucleotides, see figure below) and denoted by their initial letters, A,C ,G and T (not to be confused with amino acids!).

(picture taken from  On-Line Biology Book )

Different nucleotides can be linked together in any order to form a polynucleotide, for instance, like this

     A-G-T-C-C-A-A-G-C-T-T

Polynucleotides can be of any length and can have any sequence. The two ends of this molecule are chemically different, i.e., the sequence has a directionality, like this

     A->G->T->C->C->A->A->G->C->T->T->

The end of the polynucleotide are marked either 5' and 3' (this has chemical reasons in the numbering of the –OH groups of the sugar ring); by convention DNA is usually written with 5' left and 3' right, with the coding strand at top. Two such strands are termed complementary , if one can be obtained from the other by mutually exchanging A with T and C with G, and changing the direction of the molecule to the opposite. For instance,

     <-T<-C<-A<-G<-G<-T<-T<-C<-G<-A<-A

is complementary to the polynucleotide given above.
Specific pairs of nucleotides can form weak bonds between them. A binds to T, C binds to G (to be more precise, two hydrogen bonds can be formed between each A-T pair, and three hydrogen bonds between each C-G pair). Although such interactions are individually weak, when two longer complementary polynucleotide chains meet, they tend to stick together, like this

      5' C-G-A-T-T-G-C-A-A-C-G-A-T-G-C 3'

         | | | | | | | | | | | | | | | 

      3' G-C-T-A-A-C-G-T-T-G-C-T-A-C-G 5'

Vertical lines between two strands represent the forces between them (to be more accurate we could draw triple lines between each C and G and double lines between A and T) as shown below. The A-T and G-C pairs are called base-pairs (bp). The length of a DNA molecule is usually measured in base-pairs or nucleotides (nt), which in this context is the same thing. 

 

(picture taken from  On-Line Biology Book )

Two complementary polynucleotide chains form a stable structure, which resembles a helix and is known as a the DNA double helix. About 10 bp in this structure takes a full turn, which is about 3.4 nm long.

(picture taken from  On-Line Biology Book )

This structure was first figured out in 1953 in Cambridge by Watson and Crick (with the help of others), and the birthplace of this structure is often thought to be the Eagle pub on Bene't street. Later they got the Nobel Prize for this discovery, for more see the book by Watson – The Double Helix.

Watson and Crick at their DNA model molecule

It is remarkable that two complementary DNA polypeptides form a stable double helix almost regardless of the sequence of the nucleotides. This makes the DNA molecule a perfect medium for information storage. Note that as the strands are complementary, each one of them fully determining the other, therefore for the information purposes it is enough to give only one strand of the genome molecules. Thus, for many information related purposes, the molecule used on the example above, can be represented as CGATTCAACGATGC. The maximal amount of information that can be encoded in such a molecule is therefore 2 bits times the length of the sequence. Noting that the distance between nucleotide pairs in a DNA is about 0.34 nm, we can calculate that the linear information storage density in DNA is about 6x10 ⁸ bits/cm, which is approximately 75 GB or 12.5 CD-Roms per cm.

Complementarity of two strands in the DNA is exploited for copying (multiplying) DNA molecules in a process known as the DNA replication , in which one double stranded DNA is replicated into two identical ones. (The DNA double helix unwinds and forks during the process, and a new complimentary strand is synthesised by specific molecular machinery on each branch of the fork. After the process is finished there are two DNA molecules identical to the original one.)   In a cell this happens during the cell division (see Section 1) and a copy identical to the original goes to each of the new cells.

Note that mismatched components between polynucleotide strands are possible, if the total sum of weak forces between the complementary nucleotides are strong enough. So the molecules like

 

C-G-A-T-T-G-C-C-A-C-G-A-T-G-C

| | | ~ | | | ~ | | | ~ | | |

G-C-T-T-A-C-G-T-T-G-C-A-A-C-G

are chemically possible, though they may be rare in a living cell. More bonds, i.e., more complementary pairs, makes the molecule more stable. If there are not enough bonds, the two stranded molecular structure may become weak and the strands may come apart. The number of links needed to keep the double-helix together depends on the temperature (so-called melting temperature) and other environmental factors. DNA which is no longer in the helical form is said to be denatured.

Molecules of life : Small molecules

These can be the building blocks of the macromolecules or they can have independent roles, such as signal transmission or being a source of energy or material for a cell. Some important examples besides water are sugars, fatty acids, amino acids and nucleotides. For instance, biological membranes are constructed from fatty acids, into which macromolecules are embedded. There are 20 different amino acid molecules, which are the building blocks for proteins (to be more precise, there are 19 amino acids and one which has a slightly different structure and therefore is called imino acid).
 

 

These are three examples of amino acid moleclues, there are 17 more. They differ by R side chains which determine their properties and the order of these different amino acids within the protein determines the three dimensional structure of the protein. There is a convention that each amino-acid is denoted by a letter in Latin alphabet, for instance arginine  is denoted by R, histidine by H, lysine by L and there are 20 such letters .

Proteins

Proteins are the main building blocks and functional molecules of the cell, taking up almost 20% of a eukaryotic cell’s weight, the largest contribution after water (70%). Among others, there are

• Structural proteins, which can be thought of as the organism's basic building blocks. An example is collagen, which is the major structural protein of connective tissue and bone.
• Enzymes, which perform (catalyse) a multitude of biochemical reactions, such as altering, joining together or chopping up other molecules. Together these reactions and the pathways they make up is called metabolism. For example the first step in the glycolysis pathway, which is the conversion of glucose to glucose 6-phosphate, is catalysed by the enzyme hexokinase. Usually enzymes are very specific and catalyse only a single type of reaction, however the same enzyme can play role in more than one pathway.
• Transmembrane proteins are key in maintenance of the cellular environment, regulating cell volume, extraction and concentration of small molceules from the extracellular environment and generation of ionic gradients essential for muscle and nerve cell function. An example is the sodium/potassium pump.

Proteins have complex three dimensional (3D) structure (see figure below). Four levels of protein structure are distinguishable:

1. Proteins are chains of 20 different types of amino acids, which in principle can be joined together in any linear order, sometimes called poly-peptide chains. This sequence of amino-acids is known as the primary structure, and it can be represented as a string of 20 different symbols  (i.e., a word over the common alphabet of 20 letters). Information about various protein sequences and the functional roles of the respective proteins, can be found in UniProtKB/Swiss-Prot database. UniProtKB/Swiss-Prot is a joint project between the EBI and the Swiss Institute of Bioinformatics (SIB). The length of the protein molecule can vary from few to many thousands of amino-acids. For example insulin is a small protein and it consists of 51 amino acids, while titin has ~28,000 amino acids.
2. Although the primary structure of a protein is linear, the molecule is not straight, and the sequence of the amino acids affects the folding. There are two common substructures often seen within folded chains - alpha-helices and beta-strands. They are typically joined by less regular structures, called  loops. These three are called secondary structure elements.
3. As the result of the folding, parts of a protein molecule chain come into contact with each other and various attractive or repulsive forces (hydrogen bonds, disulfide bridges, attractions between positive and negative charges, and hydrophobic and hydrophilic forces) between such parts cause the molecule to adopt a fixed relatively stable 3D structure. This is called tertiary structure. In many cases the 3D structure is quite compact.
4. A protein may be formed from more than one chain of amino-acids, in which case it is said to have quaternary structure. For example haemoglobin, is made up of four chains each of which is capable of binding an iron molecule.

Proteins are much too small to be seen in an optical microscope - a characteristic protein size varies from about 3 to 10 nanometers (nm), i.e., 3 to 10 times 10^-9 m, and solving (i.e., discovering) their structure is a difficult and expensive exercise (approximately €50,000 - €200,000 per novel structure), which is done by a variety of methods including X-ray crystallography, nuclar-magnetic resonance spectroscopy, and advanced electron microscopy. PDbe is a database of known protein structures, which is housed and developed at the EBI. The images below shows the structure of triosephosphate isomerase visualised by RasMol software package, a 3D viewer for PDBe structures.

          

In this image the magenta coloured bits are alpha-helices, while yellow bits are beta-strands.

An alternative view in which the two monomer units are highlighted. The size of this protein in a crystallised state is about 13 x 7 x 5 nm. The images above are only models of these molecules, as the molecules are two small to have a ‘real’ image. For instance they cannot have any conventional colour, they are in constant motion, and when we start zooming in into a finer structure, quantum effects, such as Heisenberg uncertainty principle start playing role. 

There are roughly 15,000 protein structures deposited in public databases, though many of them are very similar to each other. Whether to consider two protein structures  similar or different depends on the similarity threshold (as with cell types). Structural biologists think that currently there are about 1,500 different representative protein structures known. 

All four structural levels are essentially determined by the primary structure (i.e., the amino-acid sequence) plus the physico-chemical environment where the molecule is placed. Predicting protein structure from the amino-acid sequence is one of the most important problems of computational biology (another name for bioinformatics, though some try to make a distinction between these two terms) and is far from being solved. Characteristic, frequently reoccurring structural elements are called protein domains. Sometimes it is possible to identify these domains in proteins of unknown structure, if their sequence is similar to that of a known structural domain. Structural domains are often associated with a particular protein function. Protein similarity is also deemed to be the result of evolutionary relationship.

What are the comparative sizes of proteins and cells? There is a proverb saying that size does not matter. Still comparative sizes may matter, particularly if we try to imagine the cellular processes described in the next sections. A typical linear dimension (diameter) of a globular protein is about 5 x 10 ^-9 m, while of a eukaryotic cell about 5 x 10 ^-5 m. This means the a cell is about a 10,000 times larger than a protein linearly. Alternatively, if we estimate the average weight of a human cell as about 10 ^-9 g, and remember that proteins constitute about one fifth of cell mass, then assuming the weight of an average protein to be about 10 ^-19 g (say hemoglobin is 64,500 atomic units, each of which is 1.66 x 10 ^-24 g), we see that there are 0.2 x 10 ^-9 / 10 ^-19 proteins per cell, which equals two billion (2 x 10 ⁹ ). These of course are very rough estimates which would vary from cell to cell. If we remember that there are about 6 x 10 ¹³ cells, we see that there are 30,000 times more cells per human, than proteins per cell. This may be an indication of the relative complexity of a human compared to a single cellular organism (a similar estimate regarding the relative complexity of an elephant or dinosaur and human may not be flattering for a human). 

Although forces such as hydrogen bonds are weak individually, when two or more biological macromolecules with complementary shapes come close to each other, the sum of all such weak forces may cause the molecules interact rather strongly, e.g., to make them stick together. In fact, such weak inter-molecular forces and interactions play a fundamental role in life and are at the basis of virtually all biological processes. For instance many proteins can stick together to form large protein complexes such as yeast RNA polymerase II, which reads and transcribes the genetic information (see Section 3.3), and which has 10 subunits and for which the structure has been solved recently. These weak interactions also underlie how microarrays work, which is discussed in the last section.

Agricultural biotechnology and your dinner table

Plant Biotechnology

Agricultural biotechnology has successfully altered the food that we eat and even the way that we eat it. Scientists have produced cows that make more milk and in turn, are able to make that milk last longer in our refrigerators. They have used plant biotechnology to grow bigger, longer lasting vegetables. They have even produced plants that can fight diseases or environmental conditions that would have wiped out entire crops in the past. Agricultural biotechnology has improved the quality and quantity of food that we eat. For instance, our tomato plants are stronger and we have more varieties available, thanks in part to plant biotechnology.

Agricultural biotechnology has made advancements in the Health and productivity of farm animals. Thanks to research in these fields, chickens may produce more eggs, cows may have more offspring and sheep’s wool may grow faster. If the wait for sheep’s wool to grow is decreased, so to is the necessity for more sheep. By reducing the amount of sheep necessary to produce the needed wool, we are also decreasing the resources needed to sustain those animals. Those saved resources may be used for other farm animals or even for people.

Agricultural biotechnology has been able to prevent some starvation in third world countries. Through the study of plant biotechnology, they have found ways to make crops stronger. For instance, imagine a village that experiences extreme drought with great frequency throughout the year. In the past, it would have been difficult for that village to sustain life with the limited crops available for growth in drought areas. However, the study of plant biotechnology, has produced a varied array of plants that not only survive drought, but thrive in it. In the past, that village may have suffered from a deficiency in vitamin C because they had no food that contained that vitamin. However, advancements in agricultural biotechnology have produced plants that are more hardy, and therefore that village now has a wider array of vitamin rich food available to them. The village is now able to sustain life with the crops that they can grow on their own. They may even have enough to sell to other villages, thereby aiding their economy as well.

The field of agricultural biotechnology has made many advancements in enriching and protecting our food sources. Plant biotechnology has made plants stronger and we are now offered a wider variety of choice in fruits and vegetables. In the past you may not have been able to grow a certain crop in a certain geographical area, however that is becoming less true with advancements made in plant biotechnology. As a direct result of improved plant health, there is a positive impact on human health. It must also be noted, that a wider variety of crop choice may also increase production for farmers, thereby increasing their profits and helping the economy. The positive ripple effects of Agricultural biotechnology can be felt worldwide.