Alternative DNA Structures

Gene expression and transcription can be influenced by changes of DNA topology. However, this type of control of gene expression is relatively universal and non specific.Thus, it is more suitable for permanent suppression of transcription, e.g., in genes that are expressed only in certain tissues or are active only during the embroyonic period and later become permanently inactive.

A. Three forms of DNA

Three forms of DNA
Three forms of DNA

The DNA double helix does not occur as a single structure, but rather represents a structural family of different types. The original classic form, determined by Watson and Crick in 1953, is B-DNA. The essential structural characteristic of B-DNA is the formation of two grooves, one large (major groove) and one small (minor groove). There are at least two further, alternative forms of the DNA double helix, Z-DNA and the rare form A-DNA. While B-DNA forms a right-handed helix, Z-DNA shows a left-handed conformation. This leads to a greater distance (0.77nm) between the base pairs than in B-DNA and a zig zag form(thus the designation Z-DNA). A-DNA is rare. It exists only in the dehydrated state and differs from the B form by a 20-degree rotation of the perpendicular axis of the helix. A-DNA has a deep major groove and a flat minor groove (Figures from Watson et al, 1987).

B. Major and minor grooves in B-DNA

The base pairing in DNA (adenine–thymine and guanine–cytosine) leads to the formation of a large and a small groove because the glycosidic bonds to deoxyribose (dRib) are not diametrically opposed. In B-DNA, the purine and pyrimidine rings lie 0.34 nm apart. DNA has ten base pairs per turn of the double helix. The distance from one complete turn to the next is 3.4 nm. In this way, localized curves arise in the double helix. The result is a somewhat larger and a somewhat smaller groove. <fn>Stryer, L.: Biochemistry, 4 th ed. W.H. Freeman & Co., New York, 1995.</fn>

C. Transition from B-DNA to Z-DNA

Transition from B-DNA to Z-DNA
Transition from B-DNA to Z-DNA

B-DNA is a perfect regular double helix except that the base pairs opposite each other do not lie exactly at the same level. They are twisted in a propeller-like manner. In this way, DNA can easily be bent without causing essential changes in the local structures. In Z-DNA the sugar–phosphate skeleton has a zigzag pattern; the single Z-DNA groove has a greater density of negatively charged molecules. Z-DNA may occur in limited segments in vivo. A segment of B-DNA consisting of GC pairs can be converted into Z-DNA when the bases are rotated 180 degrees. Normally, Z-DNA is thermodynamically relatively unstable. However, transition to Z-DNA is facilitated when cytosine is methylated in position 5 (C5). The modification of DNA by methylation of cytosine is frequent in certain regions of DNA of eukaryotes.Therearespecificproteinsthatbind to Z-DNA, but their significance for the regulation of transcription is not clear. <fn>Watson, J.D. et al.: Molecular Biology of the Gene. 3rd ed. Benjamin/Cummings Publishing Co., Menlo Park, California, 1987.</fn>




Chemical bounds

Some Types of Chemical Bonds close to 99% of the weight of a living cell is composed of just four elements: carbon (C), hydrogen (H), nitrogen (N), and oxygen (O). Almost 50% of the atoms are hydrogen atoms; about 25% are carbon, and 25% oxygen. Apart from water (about 70% of the weight of the cell) almost all components are carbon compounds. Carbon, a small atom with four electrons in its outer shell, can form four strong covalent bonds withotheratoms.But most importantly,carbon atoms can combine with each other to build chains and rings, and thus large complex molecules with specific biological properties.

A. Compounds of hydrogen (H), oxygen (O), and carbon (C)

Four simple combinations of these atoms occur frequently in biologically important molecules: hydroxyl (—OH; alcohols), methyl (—CH 3 ), carboxyl (—COOH), and carbonyl (C=O; aldehydes and ketones) groups. They impart to the molecules characteristic chemical properties, including possibilities to form compounds.

B. Acids and esters

Many biological substances contain a carbon– oxygen bond with weak acidic or basic (alkaline) properties. The degree of acidity is expressed by the pH value, which indicates the concentration of H + ions in a solution, ranging from 10 –1 mol/L (pH 1, strongly acidic) to 10 –14 mol/L (pH 14, strongly alkaline). Pure water contains 10 –7 moles H + per liter (pH 7.0). An ester is formed when an acid reacts with an alcohol. Esters are frequently found in lipids and phosphate compounds.

C. Carbon–nitrogen bonds (C—N)

C—N bonds occur in many biologically important molecules: in amino groups, amines, and amides, especially in proteins. Of paramount significance are the amino acids, which are the subunits of proteins. All proteins have a specific role in the functioning of an organism.

D. Phosphate compounds

Ionized phosphate compounds play an essential biological role. HPO 4 2– is a stable inorganic phosphate ion from ionized phosphoric acid. A phosphate ion and a free hydroxyl group can form a phosphate ester. Phosphate compounds playan important role in energy-rich molecules and numerous macromolecules because they can store energy.

E. Sulfur compounds

Sulfur often serves to bind biological molecules together, especially when two sulfhydryl groups(—SH)reacttoformadisulfidebridge(— S—S—). Sulfur is a component of two amino acids (cysteine and methionine) and of some polysaccharides and sugars. Disulfide bridges play an important role in many complex molecules, serving to stabilize and maintain particular three-dimensional structures.




Probiotics

The notion of probiotics has recently developed and most pharmacists have not been trained in these new food supplements.

From birth, our gastrointestinal tract is colonized by many microorganisms that constitute the digestive microbiota. This complex and diversified ecosystem, unique to each individual, contributes to the proper functioning of the intestine through the many activities it carries out. However, the balance of the microbiota is sensitive and its rupture occurs in the pathophysiology of various intestinal disorders, hence the idea of positively modulating a microbiota unbalanced by the administration of probiotics.

The term “probiotic” means “for life” and refers to living microorganisms that, when ingested in appropriate amounts, produce a benefit to the health of the host that goes beyond basic nutritional functions.

Probiotics are often lactic acid bacteria (lactobacilli and bifidobacteria) or yeasts introduced into the diet in the form of fermented milk products or food supplements.

These microorganisms strengthen the intestinal and vaginal flora. Their presence makes it possible to fight against the proliferation of pathogenic bacteria.

Several clinical studies have already demonstrated the efficacy of certain probiotics in the treatment of systemic and infectious diseases such as acute diarrhea and Crohn’s disease.

Other studies have suggested a potential application for the treatment of urogenital infections, colon cancer, atopic dermatitis and allergic diseases including food allergy such as lactose intolerance.

History

The definition of probiotics has evolved over time according to researchers, scientific knowledge and technological advances.

In the 20th century the Nobel Prize winner, Elie Metchnikoff, observed that a surprising number of people in Bulgaria lived for more than 100 years. This longevity could not be explained by the advances in modern medicine, because Bulgaria, one of the poorest countries in Europe at that time, did not benefit from such advances. Dr. Metchnikoff found that Bulgarians consume large quantities of yogurt, he associated the increase in longevity observed with the consumption of living microorganisms from fermented dairy products. Although Metchnikoff saw germs as rather harmful to human health, he considered it beneficial to replace bacteria in the gastrointestinal tract with yogurt, including the Bulgarian bacillus. He then explained the better beneficial effect of this bacteria by the absence of alcohol production (harmful to longevity), compared to bacteria present in other fermented milk such as kefir or kumys. In addition, he assumed that the lactic acid produced, as well as other unidentified factors, would act synergistically to inhibit the growth of putrefaction bacteria in the colon.

At the same time, in 1906, the French pediatrician Henry Tissier observed that the stools of children with diarrhea contained a small number of bifidobacteria compared to the stools of healthy children. He then suggested that these bacteria be administered to diarrheal patients to help them restore a healthy intestinal microbiota.

Metchnikoff and Tissier are therefore the first to put forward the idea of administering exogenous microorganisms to compensate for a possible dysfunction in our intestinal ecosystem. The concept of “probiotics” was born.

Nevertheless, it was not until 1954 that the term probiotics was introduced into the literature by Ferdinand Vergin in a paper entitled “Anti-und Probiotika”. This term derived from the Greek “pro bios”, which literally means “for life” as opposed to the harmful effects of antibiotics

In 1965, Lilly and Stilwell, in the journal Science, defined probiotics as substances produced by microorganisms capable of stimulating the growth of other microorganisms.

In 1989, Fuller highlighted the microbial nature of probiotics by redefining the term as a “living microbial nutritional supplement that has a positive effect on the host animal by improving its intestinal balance”.

In 1992, Havenaar and Huis in’t Velt further refined the term to “a viable culture composed of one or a mixture of bacteria that, when applied to animals or humans, has a beneficial effect on the host by improving the properties of native flora. ».

In 1998, Guarner and Schaafsmaa specified that probiotics are “living microorganisms, which, when consumed in adequate amounts, have a beneficial effect on the health of the host”.

In 2002, the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) formalized the definition of probiotics to avoid any drift.

Probiotics are therefore defined as “living organisms that, when ingested in sufficient quantities, have a beneficial effect on the health of the host”.

History, therefore, underlines that the current definition could still evolve, as there are still many fields of research to better understand and understand the action of probiotics.

Regulation

The conditions and marketing authorization of probiotics are defined according to their drug or food application. Most probiotics are functional foods or are used as food supplements. These “healthy foods” are at the border between the drug and the traditional food and are governed by food legislation.

Probiotic foods

The global market of probiotic foods has been growing rapidly since the early 2000s, particularly in Europe. This dynamic is supported in particular by the link between food and health benefits.

Probiotics used as food supplements, as well as functional foods, are considered as food and are governed by the relevant legislation. They are different from dietary foods that are intended for a particular food and require a specific formulation or manufacturing process to differentiate them from the common food, and from medicinal products.




Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is a heterogeneous group of genetically determined skin disorders due to unusual sensitivity to ultraviolet light. They are manifested by dryness and pigmentation of the exposed regions of skin (xeroderma pigmentosum=“dry, pigmented skin”). The exposed areas of skin also show a tendency to develop tumors. The causes are different genetic defects of DNA repair. Repair involves mechanisms similar to those involved in transcription and replication. The necessary enzymes are encoded by at least a dozen genes, which are highly conserved in bacteria, yeast, and mammals.

A. Clinical phenotype

The skin changes are limited to UV-exposed areas (2). Unexposed areas show no changes. Thus it is important to protect patients from UV light. An especially important feature is the tendency for multiple skin tumors to develop in the exposed areas (3). These may even occur in childhood or early adolescence. The types of tumors are the same as those occurring in healthy individuals after prolonged UV exposure.

Clinical phenotype
Clinical phenotype

B. Cellular phenotype

The UV sensitivity of cells can be demonstrated in vitro. When cultured fibroblasts from the skin of patients are exposed to UV light, the cells show a distinct dose-dependent decrease in survival rate compared with normal cells (1). Different degrees of UV sensitivity can be demonstrated. The short segment of new DNA normally formed during excision repair can be demonstrated by culturing cells in the presence of [ 3 H]thymidine and exposing them to UV light. The DNA synthesis induced for DNA repair can be made visible in autoradiographs. Since [ 3 H]thymidine is incorporated during DNA repair, these bases are visible as small dots caused by the isotope on the film (2). In contrast, xeroderma (XP) cells show markedly decreased or almost absent repair synthesis. (Photograph of Bootsma & Hoeijmakers, 1999).

Cellular phenotype
Cellular phenotype

C. Genetic complementation in cell hybrids

If skin cells (fibroblasts) from normal persons and from patients (XP) are fused (cell hybrids) in culture and exposed to UV light, the cellular XP phenotype will be corrected (1). Normal DNA repair occurs. Also, hybrid cells from two different forms of XP show normal DNA synthesis (2) because cells with different repair defects correct each other (genetic complementation). However, if the mutant cells have the same defect (3), they are not be able to correct each other (4) because they belong to the same complementation group. At present about ten complementation groups are known in xeroderma pigmentosum. They differ clinically in terms of severity and central nervous system involvement. Each complementation group is based on a mutation at a different gene locus. Several of these genes have been cloned and show homology with repair genes of other organisms, including yeast and bacteria.

Genetic complementation in cell hybrids
Genetic complementation in cell hybrids




Changes in DNA (Mutations)

When it was recognized that changes (mutations) in genes occur spontaneously (T. H. Morgan, 1910) and can be induced by X-rays (H. J. Muller, 1927), the mutation theory of heredity became a cornerstone of early genetics. Genes were defined as mutable units, but the question what genes and mutations are remained. Today we know that mutations are changes in the structure of DNA and their functional consequences. The study of mutations is important for several reasons. Mutations cause diseases, including all forms of cancer. They can be induced by chemicals and by irradiation. Thus, they represent a link between heredity and environment. And without mutations, well-organized forms of life would not have evolved.

The following two plates summarize the chemical nature of mutations.

A. Error in replication

The synthesis of a new strand of DNA occurs by semiconservative replication based on complementary base pairing. Errors in replication occur at a rate of about 1 in 10 5 . This rate is reduced to about 1 in 10 7 to 10 9 by proofreading mechanisms. When an error in replication occurs before the next cell division (here referred to as the first division after the mutation), e.g., a cytosine (C) might be incorporated instead of an adenine (A) at the fifth base pair as shown here, the resulting mismatch will be recognized and eliminated by mismatchre pair in most cases.

However, if the error is undetected and allowed tostand, the next(second) division will result in a mutant molecule containing a CG instead of an AT pair at this position. This mutation will be perpetuated in all daughter cells. Depending on its location within or outside of the coding region of a gene, functional consequences due to a change in a codon could result.

B. Mutagenic alteration of a nucleotide

A mutation may result when a structural change of a nucleotide affects its base-pairing capability. The altered nucleotide is usually present in one strand of the parent molecule. If this leads to incorporation of a wrong base, such as a C instead of a T in the fifth base pair as shown here, the next (second) round of replication will result in two mutant molecules.

C. Replication slippage

A different class of mutations does not involve an alteration of individual nucleotides, but results from incorrect alignment between allelic or nonallelic DNA sequences during replication. When the template strand contains short tandem repeats, e.g., CA repeats as in microsatellites (see DNA polymorphism and Part II, Genomics), the newly replicated strand and the template strand may shift their positions relative to each other. With replication or polymerase slippage, leading to incorrect pairing of repeats, some repeats are copied twice or not at all, depending on the direction of the shift. One can distinguish forward slippage (shown here) and backward slippage with respect to the newly replicated strand. If the newly synthesized DNA strand slips forward, a region of nonpairing remains in the parental strand. Forward slippage results in an insertion. Backward slippage of the new strand results in deletion. Microsatellite instability is a characteristic feature of hereditary nonpolyposis cancer of the colon (HNPCC). HNPCC genes are localized on human chromosomes at 2p15–22 and 3p21.3. About 15% of all colorectal, gastric, and endometrial carcinomas show microsatellite instability. Replication slippage must be distinguished from unequal crossing-over during meiosis. This is the result of recombination between adjacent, but not allelic, sequences on nonsister chromatids of homologous chromosomes (Figures redrawn from Brown, 1999).

Replication slippage
Replication slippage




The Strange world of Bionanomachines

Very remarkably, many of nanomachines will always perform their functions automatically, after they are isolated and purified, provided the environment is not too hard. They must not be sequestered inside the cells in the pure state. Each of them constitutes a self-sufficient molecular machine. Natural digestive enzymes such as pepsin and lysozyme are so hard that they can be added to the laundry detergent to help dissociate partial stains. Amylases are used on an industrial scale to convert pulverulent starch into corn syrup. The entire field of genetic engineering and biotechnology is made possible by a collection of DNA and manipulative nanomachines, now commercially available. Generally the natural bionanomachines are remarkably robust. This chapter explores the bionanomachines made by living cells. They are different from machines in our world.

Bionanomachines are also chosen by their very specific tasks in a given environment, and are also subject to unfamiliar forces exerted by this environment. It is imperative to keep these notions of difference in mind before embarking on the biotechnology study.

A strange world

Natural biomolecules have often unbelievable shapes, unlike designs arranged in bread grids and tractors. They perform their functions in a strange environment, where the thermal movements, the nervous movements constantly influenced by pushing and attracting forces on their constituents. They are linked by a complex set of links and external forces. The bionanomachines are almost immune to the laws of gravity and inertia which dominate our machines.

Gravity and inertia are negligible at the nano scale

Physical properties such as friction, tensile strength, adhesion, and resistance to disintegration are comparable in magnitude to the forces exerted by inertia and gravity. This balance changes, however, when we move to larger or smaller objects. As we move to larger objects, the graduation laws shift the balance. Mass increases with the cube of size of an object, and properties such as force and friction increase linearly with the square of the waist.

The graduation laws also go in the opposite direction, the grains of micrometer-sized sands, or the cells, act on each other differently from macroscopic objects. In fact, if we consider a bacterial cell that swims in a volume of water by its ciliature, it moves slowly, and stops at a given moment as does the submarine in the ocean. However, if one considers the inertial forces relative to the viscosity of the surrounding water, the cell would stop less than the diameter of an atom!

Gravity is neglected at the nano scale, so molecules dissociated in water or propagated in the air are in continuous motion, the forces of intermolecular attraction are more important than the force of gravity. Flies take advantage of these forces and can crawl up a wall, even the water droplets may remain adhered to the ceiling as a result of these forces.

Nanomachines can show the granularity of atoms

Objects on the nanoscale scale are constructed by discrete combinations of atoms, which act on each other by specific atom-atom interactions. They must consist of a fixed number of atoms (rotary engine at the nanometer scale). Similar engines already existing, such as adenosine triphosphate synthetase, the bacterial flagellar engine.

Due to atomic granularity, typical continuous representations are not used in technology because of physical properties; Such as viscosity and friction, are not defined for discrete atomic groups. Instead, various atomic substitutive properties are used, quantum mechanics in fact provides a deep understanding of atomic properties, but fortunately most of the properties of atoms can be understood qualitatively by using simple rules: 1. The links 2. Steric repulsion between unconnected atoms, electrostatic interactions, hydrogen bonds that allow the understanding of most aspects, structures, and molecular interactions.

Generally, biomolecules are considered as articulated chains of atoms that interact precisely with one another.

Heat flux is a significant force at the nano scale

Molecular nanotechnology seeks to create the environment of the “machine phase” with different organized nanomachines, in order to form objects on the micro or macroscopic scale. On the other hand, natural ionanomachinery takes a different approach because it requires the introduction of nanomachinery inside the cell. The various parts act upon each other by induced movements and diffusion.

Biomachines work in a chaotic environment, they are continually bombarded by molecules of water. They will disperse but always held in place. Biomachines can establish specific interactions with other biomachines, adapting together to the state of rest, and separating in the event of operation. If two closely matched molecules have a good complementarity of chemical groups, they will interact for long periods, but if the interactions are weak, they will establish temporary interactions before moving on to the next.

The careful design of the bionanomachines and the rigorous regulation of the forces of their interactions give them the character of being able to form stable molecular beams that can remain years or build biological Detectors capable of detecting molecular traces. Bionanomachines require water as an environment. The shape and function of bionanomolecules are dominated by two things: the chemistry of their component atoms and the irregular properties of the surrounding water molecules. The energy of these interactions is different from what is meant in the macroscopic world, the water molecules interact strongly with each other by hydrogen bonds. They do not separate and do not interact with other molecules unless they have something to offer. Bionanomolecules, which have an important value in terms of electron richness, nitrogen (N) and Oxygen (O), interact favorably with water molecules, and therefore have good solubility in water. The hydrophobicity of biomolecules strongly influences their function and shape. The geometry of the molecular chain alone, creates a large number of conformations, one would thus rarely find simple structures. Once placed in water, the biomolecules rapidly respond to the environment, folding into a conformation, so that the hydrophobic regions are stacked inwards, the surface being decorated by the H 2 O-hydrophilic molecules . For proteins: the chain is most often forced into a compact globule, for DNA the pairs of bases are sequestered without alteration inside, leaving the phosphates heavily loaded on the surface, for lipids, hydrophobic groups ( In the cell membranes) are directed inside the lamellae as well as the hydrophilic groups are directed outwards. If carefully constructed, one would obtain a unique structure with an appropriate conformation in order to perform its task properly.




DNA as Carrier of Genetic Information

Although DNA was discovered in 1869 by Friedrich Miescher as
a new, acidic, phosphorus containing substance made up of very large molecules that he named “nuclein”, its biological role was not recognized. In 1889 Richard Altmann introduced the term “nucleic acid”. By 1900 the purine and pyrimidine bases were known. Twenty years later, the two kinds of nucleic acids, RNA and DNA, were distinguished. An incidental but precise observation (1928) and relevant investigations (1944) indicated that DNA could be the carrier of genetic information.

A. The observation of Griffith

The observation of Griffith
The observation of Griffith

In 1928 the English microbiologist Fred Griffith made a remarkable observation. While investigating various strains of Pneumococcus, he determined that mice injected with strain S (smooth) died (1). On the other hand, animals injected with strain R (rough) lived (2). When he inactivated the lethal S strain by heat, there were no sequelae, and the animal survived (3). Surprisingly, a mixture of the nonlethal R strain and the heat-inactivated S strain had a lethal effect like the S strain (4). And he found normal living pneumococci of the S strain in the animal’s blood. Apparently, cells of the R strain were changed into cells of the S strain (transformed). For a time, this surprising result could not be explained and was met with skepticism. Its relevance for genetics was not apparent.

B. The transforming principle is DNA

The transforming principle is DNA
The transforming principle is DNA

Griffith’s findings formed the basis for investigations by Avery, MacLeod, and McCarty (1944). Avery and co-workers at the Rockefeller Institute in New York elucidated the chemical basis of the transforming principle. From cultures of an S strain (1) they produced an extract of lysed cells (cell-free extract) (2). After all its proteins, lipids, and polysaccharides had been removed, the extract still retained the ability to transform pneumococci of the R strain to pneumococci of the S strain (transforming principle) (3). With further studies, Avery and co-workers determined that this was attributed to the DNA alone. Thus, the DNA must contain the corresponding genetic information. This explained Griffith’s observation. Heat inactivation had left the DNA of the bacterial chromosomes intact. The section of the chromosome with the gene responsible for capsule formation (S gene) could be released from the destroyed S cells and be taken up by some R cells in subsequent cultures. After the S gene was incorporated into its DNA, an R cell was transformed into an S cell(4).

C. Genetic information is transmitted by DNA alone

The final evidence that DNA, and no other molecule, transmits genetic information was provided by Hershey and Chase in 1952.They labeled the capsular protein of bacteriophages (see p. 88) with radioactive sulfur ( 35 S) and the DNA with radioactive phosphorus ( 32 P). When bacteria were infected with the labeled bacteriophage, only 32 P (DNA) entered the cells, and not the 35 S (capsular protein). The subsequent formation of new, complete phage particles in the cell proved that DNA was the exclusive carrier of the genetic information needed to form new phage particles, including their capsular protein. Next, the structure and function of DNA needed to be clarified. The genes of all cells and some viruses consist of DNA, a long-chained threadlike molecule.




DNA Polymorphism

Genetic polymorphism is the existence of variants with respect to a gene locus (alleles), a chromosome structure(e.g., sizeofcentromeric heterochromatin), a gene product (variants in enzymatic activity or binding affinity), or a phenotype. The term DNA polymorphism refers to a wide range of variations in nucleotide base composition, length of nucleotide repeats, or single nucleotide variants. DNA polymorphisms are important as genetic markers to identify and distinguish alleles at agene locus and to determine their parental origin.

A. Single nucleotide polymorphism (SNP)

These allelic variants differ in a single nucleotide at a specific position. At least one in a thousand DNA bases differs among individuals (1). The detection of SNPs does not require gel electrophoresis. This facilitates large-scale detection. A SNP can be visualized in a Southern blot as a restriction fragment length polymorphism (RFLP) if the difference in the two alleles corresponds to a difference in the recognition site of a restriction enzyme (see Southern blot, p. 62). B. Simple sequence length polymorphism (SSLP) These allelic variants differ in the number of tandemly repeated short nucleotide sequences in noncoding DNA. Short tandem repeats (STRs) consist of units of 1,2,3,or 4 base pairs repeated from 3 to about 10 times. Typical short tandem repeats are CA repeats in the 5′ to 3′ strand, i.e., alternating CG and AT base pairs in the double strand. Each allele is defined by the number of CA repeats, e.g., 3 and 5, as shown (1). These are also called microsatellites. The size differences due to the number of repeats are determined by PCR. Variable number of tandem repeats (VNTR), also called minisatellites, consist of repeat units of 20–200 base pairs (2). C. Detection of SNP by oligonucleotide hybridization analysis Oligonucleotides, short stretches of about 20 nucleotides with a complementary sequence to the single-stranded DNA to be examined, will hybridize completely only if perfectly matched. If there is a difference of even one base, such as due to an SNP, the resulting mismatch can be detected because the DNA hybrid is unstable and gives no signal.

D. Detection of STRs by PCR

Short tandem repeats (STRs) can be detected by the polymerase chain reaction (PCR). The allelic regions of a stretch of DNA are amplified; the resulting DNA fragments of different sizes are subjected to electrophoresis; and their sizes are determined.

E. CEPH families

CEPH family
CEPH family

An important step in gene identification is the analysis of large families by linkage analysis of polymorphic marker loci on a specific chromosomal region near a locus of interest. Large families are of particular value. DNA from such families has been collected by the Centre pour l’Étude du Polymorphisme Humain (CEPH) in Paris, now called the Centre Jean Dausset, after the founder. Immortalized cell lines are stored from each family. A CEPH family consists of four grandparents, the two parents, and eight children. If four alleles are present at a given locus they are designated A, B, C, and D. Starting with the grandparents, the inheritance of each allele through the parents to the grandchildren can be traced (shown here as a schematic pattern in a Southern blot). Of the four grandparents shown, three are heterozygous (AB, CD, BC) and one is homozygous (CC). Since the parents are heterozygous for different alleles (AD the father and BC the mother), all eight children are heterozygous (BD, AB, AC, or CD).




The Cell and Its Components

Cells are the smallest organized structural units able to maintain an individual, albeit limited, life span while carrying out a wide variety of functions. Cells have evolved on earth during the past 3.5 billion years, presumably orginating from suitable early molecular aggregations. Each cell originates from another living cell as postulatedbyR.Virchowin1855(“omniscellula ecellula”).Thelivingworldconsistsoftwobasic types of cells: prokaryotic cells, which carry their functional information in a circular genome without a nucleus, and eukaryotic cells, which contain their genome in individual chromosomes in a nucleus and have a well-organized internal structure. Cells communicate with each other by means of a broad repertoire of molecular signals. Great progress has been made since 1839, when cells were first recognized as the “elementary particles of organisms” by M. Schleiden and T. Schwann. Today we understand most of the biological processes of cells at the molecular level.

Eukaryotic cells

A eukaryotic cell consists of cytoplasm and a nucleus. It is enclosed by a plasma membrane. The cytoplasm contains a complex system of inner membranes that form cellular structures (organelles). The main organelles are the mitochondria (in which important energy–delivering chemical reactions take place), the endoplasmic reticulum (consisting of a series of membranes in which glycoproteins and lipids are formed), the Golgi apparatus (for certain transport functions), and peroxisomes (for the formation or degradation of certain substances). Eukaryotic cells contain lysosomes, in which numerous proteins, nucleic acids, and lipids are broken down. Centrioles, small cylindrical particles made up of microtubules, play an essential role in cell division. Ribosomes are the sites of protein synthesis.

Nucleus

The eukaryotic cell nucleus contains the genetic information. It is enclosed by an inner and an outer membrane, which contain pores for the transport of substances between the nucleus and the cytoplasm. The nucleus contains a nucleolus and a fibrous matrix with different DNA–protein complexes.

Plasma membrane of the cell

Plasma membrane of the cell
Plasma membrane of the cell

The environment of cells, whether blood or other body fluids, is water-based, and the chemical processes inside a cell involve water soluble molecules.In order to maintain their integrity, cells must prevent water and other molecules from flowing in or out uncontrolled. This is accomplished by a water-resistant membrane composed of bipartite molecules of fatty acids, the plasma membrane. These molecules are phospholipids arranged in a double layer (bilayer) with a fatty interior. The plasma membrane itself contains numerous molecules that traverse the lipid bilayer once or many times to perform special functions. Different types of membrane proteins can be distinguished: (i) transmembrane proteins used as channels for transport of molecules into or out of the cell, (ii) proteins connected with each other to provide stability, (iii) receptor molecules involved in signal transduction, and (iv) molecules with enzyme function to catalyze internal chemical reactions in response to an external signal. (Figure redrawn from Alberts et al., 1998.)

Comparison of animal and plant cells

Eukartotic cell and neucleus
Eukartotic cell and neucleus

Plant and animal cells have many similar characteristics. One fundamental difference is that plant cells contain chloroplasts for photosynthesis. In addition, plant cells are surrounded by a rigid wall of cellulose and other polymeric molecules and contain vacuoles for water, ions, sugar, nitrogen–containing compounds,orwasteproducts.Vacuolesarepermeable to water but not to the other substances enclosed in the vacuoles. (Figures in A, B and D adapted from de Duve, 1984.) <fn>Alberts,B.etal.:EssentialCellBiology.An Introduction to the Molecular Biology of the Cell. Garland Publishing Co., New York, 1998.</fn> <fn>deDuve,C.:A Guided Tour of the Living Cell.Vol. I and II. Scientific American Books, Inc., New York, 1984.</fn> <fn>Lodish, H. et al.: Molecular Cell Biology (with an animatedCD-ROM).4thed.W.H.Freeman&Co., New York, 2000.</fn>

 

 




Impact of Particle Irradiation on the Immune System: From the Clinic to Mars

Despite the generalized use of photon-based radiation (i.e., gamma rays and X-rays) to treat different cancer types, particle radiotherapy (i.e., protons and carbon ions) is becoming a popular, and more effective tool to treat specific tumors due to the improved physical properties and biological effectiveness. Current scientific evidence indicates that conventional radiation therapy affects the tumor immunological profile in a particular manner, which in turn, might induce beneficial effects both at local and systemic (i.e., abscopal effects) levels. The interaction between radiotherapy and the immune system is being explored to combine immune and radiation (including particles) treatments, which in many cases have a greater clinical effect than any of the therapies alone. Contrary to localized, clinical irradiation, astronauts are exposed to whole body, chronic cosmic radiation, where protons and heavy ions are an important component. The effects of this extreme environment during long periods of time, e.g., a potential mission to Mars, will have an impact on the immune system that could jeopardize the health of the astronauts, hence the success of the mission. To this background, the purpose of this mini review is to briefly present the current knowledge in local and systemic immune alterations triggered by particle irradiation and to propose new lines of future research. Immune effects induced by particle radiation relevant to clinical applications will be covered, together with examples of combined radiotherapy and immunotherapy. Then, the focus will move to outer space, where the immune system alterations induced by cosmic radiation during spaceflight will be discussed.

Keywords :  protons, carbon ions, immunotherapy, space flight, cosmic radiation, immune response, cancer therapy.

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