LIFE CYCLE OF CELLS. CELL DIVISION. MORPHOLOGY OF CHROMOSOMES. HUMAN KARYOTYPE.

CHARACTERISTIC OF NUCLEIC ACIDS.

 

Life cycle of cells. Cell division. There are 5 types of cell division: 1) amitosis; 2) mitosis; 3) meiosis; 4) polytenia; 5)  endomitosis.

 

Amitosis is an obsolete term describing eukaryotic cell division without nuclear envelope breakdown and formation of well-visible mitotic spindle and condensed chromosomes. The division of unicellular eukaryotes formerly regarded as amitosis is called today closed mitosis. An exception is the division of ciliate macronucleus, which is not mitotic, and the reference to this process as amitosis may be the only legitimate use of the term today. In animals and plants which normally have open mitosis, the microscopic picture described in the 19th century as amitosis most likely corresponded to apoptosis, a process of programmed cell death associated with fragmentation of the nucleus and cytoplasm. Actually, even in the late 19th century cytologists mentioned that in larger life forms, amitosis is a “forerunner of degeneration“

 

 

Cell (mitotic) cycle covers a time from one division of cells till other division or destruction (perish) of cell, is the repeating sequence of growth and division through which cells pass each generation.

The mitotic cycle occurs in two major stages: 1) mitosis, when the cell is actively dividing and 2) interphase – the period of tyme between cell divisions.

 

 

Mitosis (Gr. Mitos, “thread”) – is the form of cell division by which a somatic (nonsex) cell duplicates. One of the basic characteristic of mitotic cell division is that one maternal cell divides into two identical ”daughter” cells, each with its own set of the genetic material. After mitosis, the chromosome number in each daughter nucleus is the same as it was in the original dividing cell – it is the biological significance of mitosis.

Before the initiation of mitosis, the following events have occurred in the preceding interphase, which is divided into phases of gap (designed “G”) and synthesis “S”, that are of great importance for successful completion of the mitotic process.

 G₁ phase (lasts from a few hours to several days):

1) is the gap phase just after mitosis during which cell growth in size and the cellular organelles increase iumber;

2) RNA and protein synthesis occur.

 S phase (lasts 8-12 hours in most cells):

1) is the synthetic  phase during which DNA replication occur, resulting in duplication of the chromosomes: one replicated chromosome consisting of two chromatids;

2) is the period when centrioles are self-duplicated;

 G₂ phase (lasts 2 – 4 hours):

1) is the gap phase, which follows the S-phase and extends to mitosis;

2) is when the cell prepares to divide; the centrioles grow to maturity; energy required for the completion of mitosis is stored; RNA and proteins necessary for mitosis are synthesized.

 During interphase a nucleus is limited from the cytoplasm by nuclear envelope. There are one or two nucleoli in it. Chromosomes are not condensed, that’s why they are not visible.  The start of chromosome condensation at the end of G₂ also signals impending mitosis.

 Mitosis, or M phase (lasts 1-3 hours):

   1) involves division of the nucleus (karyokinesis) and division of the cytoplasm (cytokinesis), resulting in the production of two identical daughter cells;

    2) includes 4 major phases: : prophase, metaphase, anaphase and telophase;

 

 

 

Prophase (lasts 30-60 minutes) begins when

1) the chromosomes condense and become rodlike and distinct chromosomes suddenly appear under the light microscope.

2) nucleoli and nuclear envelope disappear;

3) a mitotic spindle forms from microtubules: a centrosome contains centrioles and is the principal microtubule – organizing center of the cell; centrioles migrates to opposite poles of the cell and give rise to the spindle fibers and astral rays of the mitotic spindle; kinetochores begin to develop at the centromere region.

 

Metaphase:

1) chromosomes are aligned in a plane on the metaphase plate at equator.  

2) each chromosome consists of two identical chromatids, held together at a single point, the centromere;

3) spindle microtubules attach to chromosomes by kinetochores, special sites located at the centromere of each chromosome.

 Anaphase begins as

1) kinetochores separate pulling sister chromatids apart (at this time they called chromosomes);

2) diploid set of daughter chromosomes move toward each opposite poles;

3) is associated with elongation of the spindle;

4) is also characterized ( in its later stages) by a cleavage furrow that begins to form the cell due to contraction of a band of actin filaments called the contractile ring (cytokinesis begins).

 Telophase (lasts 30-60 minutes) is opposite to prophase and characterized:

1) by a deeping of the cleavage furrow, which leaves the midbody (containing overlapping polar microtubules) between the newly forming two identical daughter cells;

2) reformation of the nuclear envelope around the condensed chromosomes in the daughter cells;

3) reappearance of nucleoli, which arise from specific nucleolar organizer regions (called secondary constriction sites) on the chromosomes;

4)    mitotic spindle dissolves;

5)    is completed as the daughter nuclei gradually enlarge and the dense chromosomes disperse to form the typical interphase nucleus with hetero- and euchromatin.

 

CELL DIVISION AMONG EUKARYOTES

 

The evolution of the eukaryotes introduced several additional factors into the process of cell division. Eukaryotic cells are much larger than bacteria, and they contain genomes with much larger quantities of DNA. This DNA, however, is located in a number of individual linear chromosomes, rather than in one single circular molecule. In these chromosomes, the DNA forms a complex with proteins and is wound into tightly condensed coils. The eukaryotic chromosome is a structure with complex organization, one that contrasts strongly with the single, circular DNA molecule that plays the same role in bacteria.

 

 

CHROMOSOMES: THE VEHICLES OF MENDELIAN INHERITANCE

Chromosomes are not the only kinds of organelles that segregate regularly when eukaryotic cells divide. Centrioles also divide and segregate in a regular fashion, as do the mitochondria and chloroplasts in the cytoplasm. Thus in the early twentieth century it was by no means obvious that chromosomes were the vehicles for the information of heredity. A central role for them was first suggested in 1900 by the German geneticist Karl Correns, in one of the papers announcing the rediscovery of Mendel’s work. Soon after, observations that similar chromosomes paired with one another in the process of meiosis led directly to the chromosomal theory of inheritance, first formulated by the American Walter Sutton in 1902. Sutton’s argument was as follows:

1.     Reproduction involves the initial union of only two cells, egg and sperm. If Mendel’s model is correct, then these two gametes must make equal hereditary contributions. Sperm, however, contain little cytoplasm. Therefore the hereditary material must reside within the nuclei of the gametes.

2. Chromosomes segregate during meiosis in a manner similar to that exhibited by the elements of Mendel’s model.       

3. Gametes have one copy of each pair of homologous chromosomes; diploid individuals, two copies. In Mendel’s model gametes have one copy of each element; diploid individuals, two copies.

 

4. During meiosis, each pair of homologous chromosomes orients on the metaphase plate independently of any other pair. This independent assortment of chromosomes is a process very reminiscent of the independent assortment of factors postulated by Mendel.

There was one problem with this theory, as many investigators soon pointed out. If Mendelian traits are determined by factors located on the chromosomes, and if the independent assortment of Mendelian traits reflects the independent assortment of these chromosomes in meiosis, why is it that the number of genes that assort independently of one another in a given kind of organism is often much greater than the number of chromosome pairs that the organism possesses? This seemed a fatal objection, and it led many early researchers to have serious reservations about Sutton’s theory.

 

The Structure of Eukaryotic Chromosomes

 Chromosomes were first observed by the German embryologist Walther Fleming in 1882, while he was examining the rapidly dividing cells of salamander larvae. When Fleming looked at these cells through what we would now regard as a rather primitive light microscope, he saw minute threads within the nuclei of the cells. These threads appeared to be dividing lengthwise, and Fleming called their division mitosis, basing his term on the Greek word mitos, meaning “thread.”

Since their initial discovery, chromosomes have proved to be present in the cells of all eukaryotes (Figure 24). Their number may vary enormously from one species to another. A few kinds of plants and animals – such as Haplopappus gracilis, a relative of the sunflower that grows in North American deserts, or the fungus Penicillium – have as few as 1 or 2 pairs of chromosomes, whereas some ferns have more than 500 pairs.

In the century since their discovery, we have learned a great deal about the structure and function of chromosomes. Eukaryotic chromosomes are composed of chromatin, a complex of DNA, and protein. Most eukaryotic chromosomes are about 60% protein and 40% DNA. A significant amount of RNA is also associated with chromosomes, because they are the sites of RNA synthesis. The DNA of a chromosome exists as one very long double-stranded fiber, a duplex, which extends unbroken through the entire length of the chromosome. A typical human chromosome contains about half a billion (5 X 10⁸) nucleotides in its DNA fiber. The amount of information one chromosome contains, therefore, would fill about 2000 printed books of 1000 pages each, assuming that the nucleotides were “words” and that each page had about 500 of them on it. If the strand of DNA from a single chromosome were laid out in a straight line, it would be about 5 centimeters (2 inches) long. This is much too long to fit into a cell. In the cell, however, the DNA is coiled, thus fitting into a much smaller space than would be possible if it were not.

How is the coiling of this long DNA fiber achieved? If we gently disrupt a eukaryotic nucleus and examine the DNA with an electron microscope, we find that it resembles a string of beads. Every 200 nucleotides, the DNA duplex is coiled about a complex of histones, which are small, very basic polypeptides, rich in the amino acids arginine and lysine. Eight of these histones form the core of an assembly called a nucleosome. Because so many of their amino acids are basic, histones are very positively charged. The DNA duplex, which is negatively charged, is strongly attracted to the histones and wraps tightly around the histone core of each nucleosome. The core thus acts as a “form” that promotes and guides the coiling of the DNA. Further coiling of the DNA occurs when the string of nucleosomes wraps up into higher-order coils called supercoils.

Highly condensed portions of the chromatin are called heterochromatin. Some remain condensed permanently, so that their DNA is never expressed. The remainder of the chromosome, called euchromatin, is not condensed except during cell division, when the movement of the chromosomes is facilitated by the compact packaging that occurs at that stage. At all other times, the euchromatin is present in an open configuration and its genes can be activated.

Chromosomes may differ widely from one another in appearance. They vary in such features as the location of a region called a centromere present on all chromosomes, the relative length of the two arms (regions on either side of the centromere), size, staining properties, and the position of constricted regions along the arms. The particular array of chromosomes that an individual possesses, called its karyotype, may differ greatly between different species, or sometimes even between particular individuals.

To examine human chromosomes, investigators collect a blood sample, add chemicals that induce the cells in the sample to divide, and then add other chemicals that stop cell division at metaphase. Metaphase is the stage of mitosis at which the chromosomes are most condensed and thus most easily distinguished from one another. After arresting the process of cell division in their sample at this stage, the biologists break the cells to spread out their contents, including the chromosomes, and then stain and examine the chromosomes. To facilitate the examination of the karyotype, the chromosomes are usually photographed, and then the outlines of each chromosome are cut out, like paper dolls, and arranged in order.

Karyotypes of individuals are often examined to detect genetic abnormalities, such as those arising from extra or lost chromosomes. The human congenital defect known as Down syndrome (or trisomy 21), for example, is associated with the presence of an extra copy of a particular segment of chromosome 21, usually the result of an extra chromosome 21, and can be recognized in photographs of the chromosomes.

 

How Many Chromosomes Are in a Cell?

          Each of the cells in your body, except your gametes (sex cells), is diploid and contains 46 chromosomes, consisting of two nearly identical copies of each of the basic set of 23 chromosomes. This basic set of 23 chromosomes, called the haploid complement, is present in all of your gametes, in eggs or sperm. The two nearly identical copies of each of the 23 different kinds of chromosomes are called homologous chromosomes or homologues (Greek, homologia, agreement). Before cell division, each of the two homologues replicates, producing in each case two identical copies called sister chromatids that remain joined together at the centromere. Thus at the beginning of cell division a body cell contains a total of 46 replicated chromosomes, each composed of two sister chromatids joined by one centromere. How many centromeres does the cell contain? 46. How many copies of the basic set of 23 chromosomes does it contain? Four, with a total of 92 chromosome copies (23 basic set x 2 homologues x 2 sister chromatids). The cell is said to contain 46 chromosomes and not 92 because by convention, the number of chromosomes is obtained by counting centromeres.

HUMAN CHROMOSOMES

 

The exact number of chromosomes that humans possess was not established accurately until 1956. At that time, appropriate techniques were developed that allowed investigators to determine accurately the number, shape, and form of human chromosomes, and those of other mammals. Earlier, the number of chromosomes characteristic of human beings had been known only approximately. We now know that each typical human cell has 46 chromosomes, which come together in meiosis to form 23 pairs.

By convention, the 23 different kinds of human chromosomes are arranged into seven groups, each characterized by a different size, shape, and appearance. These groups are designated A through G. Of the 23 pairs, 22 are perfectly matched in both males and females and are called autosomes. The remaining pair consists of two unlike members in males; in females, it consists of two similar members. The chromosomes that constitute this pair are called the sex chromosomes. Just as in Drosophila, females are designated XX and males XY, to indicate that the male pair of sex chromosomes contains one member (the Y chromosome) that bears few functional genes, and differs in this respect from all other chromosomes.

 Тhe human Y chromosome is found only in males, and exhibits surprisingly low levels of genetic diversity. This low diversity could result from neutral processes, for example, if there are fewer males successfully mating (and thus fewer Y chromosomes being inherited) relative to the number of females who successfully mate. Alternatively, natural selection may act on mutations on the Y chromosome to reduce genetic diversity. Because there is no recombination across most of the Y chromosome all sites on the Y are effectively linked together. Thus, selection acting on any one site will affect all sites on the Y indirectly. Here, studying the X, Y, autosomal and mitochondrial DNA, in combination with population genetic simulations, we show that low observed Y chromosome variability is consistent with models of purifying selection removing deleterious mutations and linked variation, although positive selection may also be acting. We further infer that the number of sites affected by selection likely includes some proportion of the highly repetitive ampliconic regions on the Y. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area.

 

Telomeres are the specialized ends of linear chromosomes that are involved in a variety of functions, including meiotic chromosome segregation, chromatin silencing, and protecting the ends of the chromosomes from degradation or end-to-end fusion (for review, see Blackburn 1994; Zakian 1995; Greider 1996). In most organisms, telomeres are composed of repetitive sequences in which the strand with its 3′ end at the terminus is G-rich and may extend beyond the DNA duplex to form a single-stranded G-rich overhang. In humans, telomeres contain up to several thousand repeats of the sequence TTAGGG. Because of the requirement for an RNA primer, DNA polymerases are unable to replicate the extreme 3′ end of a parental DNA strand in the absence of compensatory mechanisms, telomeres shorten with each cell division. The ribonucleoprotein telomerase provides such a compensatory mechanism. Telomerase contains reverse transcriptase motifs, and using its RNA component as a template, it can add repetitive sequences to the 3′ end of the chromosomes. Eliminating the RNA component of telomerase prevents this activity and results in telomere shortening in organisms ranging from yeast to humans (Singer and Gottschling 1994;Blasco et al. 1995; Feng et al. 1995). Telomerase activity can be detected in the vertebrate testis telomere length is maintained in the germ line, telomerase activity is repressed in most human tissues during development and progressive telomere shortening is then observed (Hastie et al. 1990; Lindsey et al. 1991). This shortening has been proposed to serve as a mitotic clock that counts cell divisions and ultimately results in cellular senescence (de Lange et al. 1990; Greider 1990; Harley et al. 1990; Harley 1991; Wright and Shay 1995). The ability to maintain telomere length may be important in cancer formation, as approximately 85% of all human primary tumors express telomerase activity (for review, see Shay and Bachetti 1997).

The detailed structure of telomeric ends has been determined in hypotrichous ciliates such as Oxytricha nova, where a double-stranded region of 28 bp of TTTTGGGG repeats is followed by 14 nucleotides of a G-rich single-stranded overhang (Klobutcher et al. 1981). In Saccharomyces cerevisiae, although a longer single-stranded region can be transiently observed in late S phase, during most of the cell cycle any G-rich overhangs that are present are shorter than a 30-nucleotide detection limit. The loss of ∼5 bp per division in yeast lacking telomerase RNA is consistent with a model in which both ends of the yeast telomere have an ∼10-nucleotide G-rich overhang. Recent models for the action of telomerase have emphasized the need for processing of the blunt end generated by leading strand synthesis so that it can be a substrate for telomerase, with subsequent processing events generating chromosomes with symmetrical telomeres containing short G-rich overhangs. These models have a working assumption that there is a primase activity that can position an RNA primer at the extreme 3′ end of the chromosome. Such a primase activity has been found inO. nova.

In contrast to yeast telomeres which lose only a few base pairs per division in the absence of telomerase, telomeres from normal diploid human cells have been found to shorten at rates varying between 40 and 200 bp per division (Harley et al. 1990; Counter et al. 1992; Shay et al. 1993; Vaziri et al. 1993). There are at least three hypotheses to explain the much greater losses in human cells. Exposure to oxygen levels >20% causes premature senescence in human fibroblasts, and it has been proposed that unrepaired oxidative damage causes the one-step loss of long stretches of telomeric repeats. This hypothesis predicts that the rate of loss of telomeric DNA under normoxic conditions would represent the average between slow rates of shortening on most chromosomes and rapid losses on some damaged chromosomes. A second hypothesis is that processing events involving the nucleolytic degradation of one or both strands would cause increased rates of shortening in human telomeres (Makarov et al. 1997). There is good evidence for a variety of processing mechanisms at telomeres. Different mutations in the yeast single-stranded telomeric binding protein cdc13p can cause the massive nucleolytic degradation of the C-rich strand (Garvik et al. 1995) or a failure of yeast telomerase to maintain telomere length. The appearance of transient ⩾30-nucleotide overhangs on both ends of yeast chromosomes does not require yeast telomerase (Wellinger et al. 1996), and a nuclease able to digest G4 tetrastrand structures has been identified (Liu and Gilbert 1994). These observations suggest that specific nucleolytic processing of telomeres occurs in yeast. Nucleolytic processing is also seen in ciliates. The G-rich strand added by telomerase to the newly fragmented macronuclear DNA in hypotrichous ciliates is initially longer than in mature telomeres (Roth and Prescott 1985; Vermeesch and Price 1994), and the preferential pause site used by telomerase in vitro is not found at the end of ciliate telomeres synthesized in vivo. A third hypothesis is that human cells lack the ability to position the final RNA priming event at the very end of the chromosome. RNA priming events are thought to occur about every 100–600 bp during lagging strand synthesis in mammals. This is roughly consistent with the rates of telomere shortening of 40–200 bp per cell division that has been observed in cultured human cells. The length of the single-stranded G-rich overhang might thus represent the distance between the last priming event during lagging strand synthesis and the end of the chromosome.

As a first step in distinguishing between these models, we have developed techniques for purifying human telomeres and examining their structure. Our results demonstrate that the telomeres generated by leading versus lagging strand DNA synthesis are different and suggest that each chromosome has one telomere with a long G-rich overhang and one that is either blunt or has a short G-rich overhang. We provide the first direct electron microscopic measurement of the single-stranded region in telomeres from normal diploid human cells and find a 200 ± 75-nucleotide overhang. The rate of telomere shortening of 50 bp per division in these cells is consistent with models in which shortening results from overhangs produced by lagging strand synthesis. Our results do not support models of telomere shortening in which the primary mechanism is either oxidative damage or nucleolytic processing.

 

WHERE DO CELLS STORE HEREDITARY INFORMATION?

         Perhaps the most basic question that one can ask about hereditary information is where it is stored in the cell. Of the many approaches that one might take to answer this question, let us start with a simple one: cut a cell into pieces and see which of the pieces are able to express hereditary information. For this experiment we will need a single-celled organism that is large enough to operate on conveniently and differentiated enough that the pieces can be distinguished.

An elegant experiment of this kind was performed by the Danish biologist Joachim Hammerling in the 1930s. As an experimental subject, Hammerling chose the large unicellular green alga Acetabularia. Individuals of this genus have distinct foot, stalk, and cap regions, all of which are differentiated parts of a single cell. The nucleus of this cell is located in the foot. As a preliminary experiment, Hammer-ling tried amputating the caps or feet of individual cells. He found that when the cap is amputated, a new cap regenerates from the remaining portions (foot and stalk) of the cell. When the foot is amputated and discarded, however, no new foot is regenerated from the cap or the stalk. Hammerling concluded that the hereditary information resided within the foot, or basal portion, of Acetabularia.

To test this hypothesis, Hammerling selected individuals from two species of the genus in which the caps looked very different from one another: Acetabularia mediterranea, which has a disk-shaped cap, and Acetabularia crenulata, which has a branched, flowerlike cap. Hammerling cut the stalk and cap away from an individual of A. mediterranea; to the remaining foot he grafted a stalk cut from a cell of A. crenulata. The cap that formed looked something like the flower-shaped one characteristic of A. crenulata, although it was not exactly the same.

Hammerling then cut off this regenerated cap and found that a disk-shaped one exactly like that of A. mediterranea formed in the second regeneration and in every regeneration thereafter. This experiment strengthened Hammerling’s earlier conclusion that the instructions which specify the kind of cap that is produced are stored in the foot of the cell – and probably therefore in the nucleus – and that these instructions must pass from the foot through the stalk to the cap. In his regeneration experiment the initial flower-shaped cap was formed as a result of the instructions that were already present in the transplanted stalk when it was excised from the original A. crenulata cell. In contrast, all subsequent caps used new information, derived from the foot of the A. mediterranea cell onto which the stalk had been grafted. In some unknown way the original instructions that had been present in the stalk were eventually “used up.”

Hammerling’s experiments identified the nucleus as the likely repository of the hereditary information but did not prove definitely that this was the case. To do that, isolated nuclei had to be transplanted. Such an experiment was carried out in 1952 by American embryologists Robert Briggs and Thomas King. Using a glass pipette drawn to a fine tip and working with a microscope, Briggs and King removed the nucleus from a frog egg; without the nucleus, the egg would not develop. They then replaced the absent nucleus with one that they had isolated from a cell of a more advanced frog embryo. The implant of this nucleus ultimately caused an adult frog to develop from the egg. Clearly the nucleus was directing the frog’s development.

 

Hereditary information is stored in the nucleus of eukaryotic cells.

Can each and every nucleus of an organism direct the development of an entire adult individual? The Briggs and King experiment did not answer this question definitively, since the nuclei that they took from more advanced frog embryos often caused the eggs into which the nuclei were transplanted to develop abnormally. But at Oxford and Yale, John Gurdon, working with another amphibian, was able to transplant nuclei isolated from developed tadpole tissue into eggs from which the nuclei had been removed and obtaiormal development.

Have the nuclei in the cells of adult animals lost their hereditary information? This question has proved very difficult to answer, since animal development is so complex. In plants, on the other hand, a simple experiment did yield a clear-cut answer. At Cornell University in 1958, plant physiologist F.C. Steward let fragments of fully developed carrot tissue (bits of conducting tissue called phloem) swirl around in a rotating flask containing liquid growth medium. Individual cells broke away and tumbled through the liquid. Steward observed that these cells often divided and differentiated into multicellular roots. If these roots were then immobilized by placing them in a gel, they would go on to develop into entire plants that could be transplanted to soil and develop normally into maturity. Steward’s experiment makes clear that in plants at least some of the cells present in adult individuals do contain a full complement of hereditary information.

 

With rare exceptions, the nuclei of all cells of multicellular eukaryotes contain a full complement of genetic information. In many of the tissues of adult animals, however, the expression of much of this information is blocked.

 

WHICH COMPONENT OF THE CHROMOSOMES CONTAINS THE HEREDITARY INFORMATION?

 

The identification of the nucleus as the source of hereditary information focused attention on the chromosomes, which were already suspected to be the vehicles of Mendelian inheritance. Specifically, biologists wondered how the actual hereditary information was arranged in the chromosomes. It was known that chromosomes contain both protein and DNA. On which of these was the hereditary information written?

Over a period of about 30 years, starting in the late 1920s, a series of investigators addressed this issue, resolving it clearly. We will describe three very different kinds of experiments, each of which yields a clear answer in a simple and elegant manner.

 

The Griffith-Avery Experiments: Transforming Principle is DNA

          As early as 1928, a British microbiologist, Frederick Griffith, made a series of unexpected observations while experimenting with pathogenic (disease-causing) bacteria.

For the past 2 years, first with MacLeod and now with Dr. McCarty, I have been trying to find out what is the chemical nature of the substance in the bacterial extract, which induces this specific change. The crude extract of Type III is full of capsular polysaccharide, C (somatic) carbohydrate, nucleoproteins, free nucleic acids of both the yeast and thymus type, lipids, and other cell constituents. Try to find in the complex mixtures the active principle! Try to isolate and chemically identify the particular substance that will by itself, when brought into contact with the R cell derived from Type II, cause it to elaborate Type III capsular polysaccharide and to acquire all the aristocratic distinctions of the same specific type of cells as that from which the extract was prepared! Some job, full of headaches and heartbreaks. But at last perhaps we have it.

If we prove to be right – and of course that is a big if – then it means that both the chemical nature of the inducing stimulus is known and the chemical structure of the substance produced is also known, the former being thymus nucleic acid, the latter Type III polysaccharide, and both are thereafter reduplicated in the daughter cells and after innumerable transfers without further addition of the inducing agent and the same active and specific transforming substance can be recovered far in excess of the amount originally used to induce the reaction. Sounds like a virus – may be a gene. But with mechanisms I am not now concerned. One step at a time and the first step is what is the chemical nature of the transforming principle? Someone else can work out the rest. Of course the problem bristles with implications. It touches the biochemistry of the thymus type of nucleic acids, which are known to constitute the major part of chromosomes but have been thought to be alike regardless of origin and species. It touches genetics, enzyme chemistry, cell metabolism and carbohydrate synthesis. But today it takes a lot of well documented evidence to convince anyone that the sodium salt of deoxyribose nucleic acid, protein free, could possibly be endowed with such biologically active and specific properties and that is the evidence we are now trying to get. It is lots of fun to blow bubbles but it is wiser to prick them yourself before someone else tries to.

When Griffith infected mice with a virulent strain of Pneumococcus bacteria, the mice died of blood poisoning, but when he infected similar mice with a strain of Pneumococcus that lacked a polysaccharide coat like that possessed by the virulent strain, the mice showed no ill effects. The coat was apparently necessary for successful infection.

As a control, Griffith injected normal but heat-killed bacteria into the mice to see if the polysaccharide coat itself had a toxic effect. The mice remained perfectly healthy. As a final control, he blended his two ineffective preparations – living bacteria whose coats had been removed and dead bacteria with intact coats – and injected the mixture into healthy mice. Unexpectedly, the injected mice developed disease symptoms, and many of them died. The blood of the dead mice was found to contain high levels of normal virulent Pneumococcus bacteria. Somehow the information specifying the polysaccharide coat had passed from the dead bacteria to the live but coatless ones in the control mixture, transforming them into normal virulent bacteria that infected and killed the mice.

The agent responsible for transforming Pneumococcus was not discovered until 1944. In an elegant series of experiments Oswald Avery and his coworkers characterized what they referred to as the “transforming principle”. Its properties resembled those of DNA rather than protein: the activity of the transforming principle was not affected by protein-destroying enzymes but was lost completely in the presence of the DNA-destroying enzyme DNase.

When the transforming principle was purified, it indeed consisted predominantly of DNA. Subsequently, it was shown that all but trace amounts of protein (0.02%) could be removed without reducing the transforming activity. The conclusion was inescapable: DNA is the hereditary material in bacteria. It has since proved possible to use purified DNA to change the genetic characteristics of eukaryotic cells in tissue culture and even possible to inject pure DNA into fertilized Drosophila eggs and thereby alter the genetic characteristics of the resulting adult.

 

The Hershey-Chase Experiment: Bacterial Viruses Direct Their Heredity with DNA

Avery’s results were not widely appreciated at first, many biologists preferring to believe that proteins were the depository of the hereditary information. Another very convincing experiment was soon performed, however, that was difficult to ignore. It was done in a very simple system – viruses – so that a very direct experimental question could be asked. Viruses consist of either RNA or DNA with a protein coat. These investigators focused on bacteriophages, viruses that attack bacteria, and carried out an experiment analogous to the transplant experiments described before. When a bacteriophage infects a bacterial cell, it first binds to the cell’s outer surface and then injects its hereditary information into the cell. There the hereditary information directs the production of thousands of new virus particles within the cell. The host bacterial cell eventually falls apart, or lyses, releasing the newly made viruses.

In 1952 Alfred Hershey and Martha Chase set out to identify the material injected into the bacterial cell at the start of an infection. They used a strain of bacteriophage known as T2, which contains DNA rather than RNA, and designed an experiment to distinguish between the alternative hypotheses that the genetic material was DNA or that it was protein. Hershey and Chase labeled the DNA of these T2 bacteriophages with a radioactive isotope of phosphorus, ³²P, and, at the same time, labeled their protein coats radioactively with an isotope of sulfur, ³⁵S. Since the radioactive ³²P and ³⁵S isotopes emit particles of very different energies when they decay, they are easily distinguished. The labeled viruses were permitted to infect bacteria. The bacterial cells were then agitated violently to shake the protein coats of the infecting viruses loose from the bacterial surfaces to which they were attached. Spinning the cells at high speed so that they were pulled from solution by the centrifugal forces (in this procedure, called centrifugation, the cells experience the same forces you do when pressed against the floor of a rapidly-rising elevator), Hershey and Chase found that the ³⁵S label (and thus the virus protein) was now predominantly in solution with the dissociated virus particles, whereas the ³²P label (and thus the DNA) had transferred to the interior of the cells. The viruses subsequently released from the infected bacteria contained the ³²P label. The hereditary information injected into the bacteria that specified the new generation of virus particles was DNA and not protein.

 

The Fraenkel-Conrat Experiment: Some Viruses Direct Their Heredity with RNA

One objection might still be raised about accepting the hypothesis that DNA was the genetic material. Some viruses contaio DNA and yet manage to reproduce themselves quite satisfactorily. What is the genetic material in this case?

In 1957 Heinz Fraenkel-Conrat and coworkers isolated tobacco mosaic virus (TMV) from tobacco leaves. From ribgrass (Plantago), a common weed, they isolated a second, rather similar kind of virus, Holmes ribgrass virus (HRV). In both TMV and HRV the viruses consist of a protein coat and a single strand of RNA. When they had isolated these viruses, the scientists chemically dissociated each of them, separating their protein from their RNA. By putting the protein component of one virus with the RNA of another, they were able to reconstitute hybrid virus particles.

The payoff of the experiment was in its next step. To choose between the alternative hypotheses “the genetic material of viruses is protein” and “the genetic material of viruses is RNA,” Fraenkel-Conrat now infected healthy tobacco plants with a hybrid virus composed of TMV protein capsules and HRV RNA, being careful not to include any nonhybrid virus particles. The tobacco leaves that were infected with the reconstituted hybrid virus particles developed the kinds of lesions that were characteristic of HRV and that normally formed on infected ribgrass. Clearly, the hereditary properties of the virus were determined by the nucleic acid in its core and not by the protein in its coat.

Later studies have shown that many virus particles contain RNA rather than the DNA found universally in cellular organisms. When these viruses infect a cell, they make DNA copies of themselves, which can then be inserted into the host DNA as if they were cellular genes.

 

Human chromosomes and chromatids during mitosis

 

Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to “reverse” the cycle. Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases, determine a cell’s progress through the cell cycle.

Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

 

Clinical considerations.

1) Transformed cells have lost their ability to respond to regulatory signals controlling the cell cycle. A disregulation of the cell cycle components may lead to tumor formation. Some genes like the cell cycle inhibitors, RB, p53 etc., when they mutate, may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G0 phase) in tumors is much higher than that iormal tissue. They may undergo cell division indefinitely, thus becoming cancerous. The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation.

2) Vinca alkaloids may arrest these cells in mitosis. Treatment with colchicine and treatment with Nocodazole halt the cell in M.

3) Oncogenes represent mutations of certain regulatory genes, called proto-oncogenes, which normally stimulate or inhibit cell proliferation and development. Bladder cancer and acute myelogenous leukemia are caused by oncogenes.

 

Meiosis is a special form of cell division on which the chromosome number is reduced from diploid (2n) to haploid (n). Occurs in developing germs (spermatozoa and oocytes) in preparation for sexual reproduction.

Meiosis is divided in the following stages:

 

 

Reductional division (meiosis I) – occurs following interphase during which the 46 chromosomes are duplicated.

Prophase I is divided into the following five stages:

Leptotene, during which the chromatin condenses into the visible chromosomes, each of which contain two chromatids joined at the centromere;

Zygotene, during which homologous maternal and paternal chromosomes pair and make physical contact (synapsis), forming a tetrad;

Pachytene, during which the chiasmata are formed and crossing over (random exchanging of genes between segments of homologous chromosomes) occurs – an event that is crucial for increasing generic diversity;

Diplotene, during which the chromosomes continue to condense and chiasmata can be observed, indicating where crossing over has taken place;

Diakinesis, during which the nucleolus disappears, chromosomes are condensed maximally, and the nuclear envelope disappears.

Metaphase I:

1) includes alignment of homologous chromosomes on the equatorial platter of the meiotic spindle in a random arrangement, thus facilitating genetic mixing;

2) includes attachment of spindle fibers from either pole to the kinetochore of any one of the chromosome pairs, thus assuring that genetic mixing takes place.

 

Anaphase I:

1) is similar to anaphase in mitosis except that each chromosome consists of two chromatids that remain held together;

2) involves migration of chromosomes to the poles.

Telophase I:

1) is similar to telophase in mitosis;

2) sncludes reinformation of the nucleus and cytokinesis, forming two daughter cells;

3) each daughter cell now contain 23 chromosomes (the haploid number – n), but has the diploid number – 2n of DNA;

4) each chromosome is composed of two sister chromatids, which are similar but not genetically identical.

 

Human chromosomes and chromatids during Meiosis I

 

 

Equatorial division (meiosis II) – begins soon after the completion of meiosis I, following a very brief interphase without DNA replication.

1) Involves separation of sister chromatids in the two daughter cells formed in meiosis I and their distribution as chromosomes into two daughter cells, each containing its own unique recombined generic material (the haploid number – of DNA);

2) Involves events similar to those in mitosis; thus the stages are named similarly (prophase II, metaphase II, anaphase II, and telophase II);

3) occurs more rapidly than mitosis.

 

Human chromosomes and chromatids during Meiosis II

 

The biological significance of meiosis:

 1.     Meiosis enables a species’ chromosome number to remain constant over generation.

2.     Meiosis produces novel combination of genes.

3.     Meiosis produces novel combination of non homologies chromosomes.

 

Gametogenesis refers to formation of mature ova and sperm, through a process involving meiosis. In human, the cells which will ultimately differentiate into eggs and sperm arise from primordial germ cells set aside from the somatic cells (oogonia and spermatogonia). The final products of gametogenesis are the large, sedentary egg cells, and the smaller, motile sperm cells. Each type of gamete is haploid. After fertilization and the formation of the polar bodies, the haploid sperm and egg nuclei (pronuclei) fuse, thus restoring the normal diploid complement of chromosomes.

 Spermatogenesis. In the mature male functional sperm cells are produced within the seminiferous tubules of the testes. Around the periphery of the seminiferous tubules are located specialized cells known as spermatogonia. Spermatogonia are diploid cells set aside early in embryonic development. They may divide by mitosis to generate more spermatogonia. Spermatogonia destined to undergo meiosis first differentiate into primary spermatocytes which undergo the two divisions of meiosis. After the first division the cells are referred to as secondary spermatocytes which in turn undergo the second division to become spermatids.

 

Schematic drawing of spermatogenesis

 1. Meiosis in males is a continual process from puberty until death.

2. When spermatogonia reproduce by mitosis and  becomes a spermatocyte, ready to undergo meiosis.

3. At the completion of meiosis, there are four haploid spermatids, which resemble other cells in having cytoplasm and nucleus.

4. By the process of spermiogenesis, the spermatids change their physical structure to become mature spermatozoa (sperm). This involves getting rid of cytoplasm and developing an acrosomal body and tail. The time required is about 50 days from primary spermatocyte to spermatozoon.

5. All four haploid products become functional sperm.

6. 200-500 million sperm/ejaculate several times per week; 2-5 trillion per lifetime.

 

 Oogenesis. Oogenesis is the process when haploid ova are formed and it occurs within the ovary. The cytoplasm of the primary oocyte increases greatly during the meiotic prophase and often contains large quantities of yolk accumulated from the blood. A diploid primary oocyte divides by meiosis producing, after the first division, a secondary oocyte and a polar body. In the second division the secondary oocyte divides giving rise to an ootid and another polar body. This second division does not occur unless fertilization of the secondary oocyte by a sperm occurs.

 

 

Schematic drawing of oogenesis

 1. Meiosis begins simultaneously in all primary oocytes in late embryonic/early fetal period, proceeding to late prophase I.

2. All oocytes then go into a resting phase until an ovarian follicle develops further.

3. At that point, meiosis resumes. The mature ovum that is released is a secondary oocyte.

4. In telophase I, one of the meiotic products becomes a polar body, with very little cytoplasm; the other product receives virtually all the cytoplasm.

5. If fertilization occurs, MII (meiosis II) is completed, with formation of a second polar body.

6. Only ovum of the four haploid cells is functional.

7. The polar bodies play no role in the formation of the embryo.

8. In female embryos, there are several million ovarian follicles; at birth, only 2 million; only 400 mature during lifetime.

 

Amitosis (direct cell division) is a type of cell division in which splitting of nucleus is followed by cytoplasm constriction. It is the mean of asexual reproduction in unicellular organisms like bacteria and protozoans and also a method of multiplication or growth in fetal membranes of some vertebrates.

 Endomitosis is such kind of cell division in which chromosome reproduction doesn‘t lead to to nucleus division. Whole diploid sets of chromosomes may be multiplied; an individual tissue, or cell that has three or more sets of chromosomes is said o be polyploid (it‘s leads to polyploidy). Endomitosis is present in liver cells.

 

 Polythenia is a chromonemas reproduction in the chromosomes. The chromosome number doesn‘t change (remains still). But the chromosomes increase in sizes considerably. It is observes in some special cells.

 Cell populations are certain proportions of cells in a particular stage of the cell cycle in a tissue. In a renewal cell population the cells are actively dividing (epidermis of skin and epithelium GI tract). In the human body, renewal cell populations’ replace many trillions of damaged cells each day.

In the expanding cell population, up to 3% of the cells are dividing. The remaining cells of the expanding population are not actively dividing, but they can enter mitosis when a tissue is injured and new cells are required to repair it. Fast-growing tissues of young organisms, as well as kidney, pancreas, and bone marrow tissues, consist of expanding cell populations.

 

Static cell populations are inactive and don‘t contain dividing cells. Nerve cells form this population. These cells grow by enlarging rather than dividing. A single nerve cell may grow to a meter in length, but it cannot divide.

 

Morphology of chromosomes. Human karyotype.

 

1.     Structural and functional states of the chromosomes.

2.     Euchromatin and heterochromatin.

3.     The levels of organization of eukariotic chromosomes.

4.     Сhromosomes types: metacentric, submetacentric, acrocentric, telocentric chromosomes.

5.     Haploid and diploid chromosome number (set up). Autosomes and sex chromosomes.

6.     Normal human karyotype characteristics.

7.     Human chromosomes ideogram.

Hereditary information lies in chromosomes. The eukaryotic chromosomes are located within the nucleus of the cell.

 

Chromosomes serve important functions:

 1.   They carry hereditary characters from parents to offspring.

2.   They help the cell to grow, to divide and to maintain itself by directing the synthesis of structural proteins.

3.   They control metabolism by directing the formation of necessary enzymatic proteins.

4.   They undergo mutation and thus contribute to the evolution of the animal.

5.   They guide cell differentiation during development.

6.   They form nucleoli in daughter cells at nucleolar organizing sites.

7.   They bring about continuity of life by replication.

8.   They play a role in sex determination.

 

 Each species has a characteristic number of chromosomes: 1) Haploid number (n) is the number of chromosomes in germ cells (23 in humans). It forms during meiosis. 2) Diploid number (2n) is the number of chromosomes in somatic cells (46 in humans). It forms during mitosis.

 Karyotype is a diploid number of chromosomes and it is a characteristics of the number and morphology of chromosomes, that is peculiarities of each species. Karyotype is represented in humans by the 22 pairs of autosomes and the 1 pair of sex chromosomes (either XX or XY) totaling 46 chromosomes. Pair of chromosomes, with the same gene loci in the same order, are known as the homologous chromosomes. The chromosomes of each pair have characteristic size and shape. Thus, the chromosomes have individuality and are recognizable.

        An ideogram is a karyotype, which displays chromosomes arranged in pairs in descending size order. Except Denver‘s classification, in which chromosomes are distinguish by their size and the position of centromere on 7 groups, there are such methods of chromosome identification as using of different methods of chromosome staining, among them fluorescent stains and laser using.   

 

 

Metaphase chromosome structure.

At first appearance, the chromosomes have already doubled, and each now consists of two identical rods, called sister chromatids. The chromatid is composed of a very fine filament, called as chromonema. The chromonema is a single double-stranded DNA molecule with a protein coat. The two chromatids remain attached to each other at a point of primary constriction, the centromere.  The centromere is a specific DNA sequence of about 220 nucleotides, to which is bound a disk of protein called a kinetochore. It is a place, where the spindle fibers attach during cell division. Regions on either sides of centromere are called arms. The long arm of a chromosome is designated “q” and the short arm  – “p”. Some chromosomes (13, 14, 15, 21, 22) have secondary constriction site, which is a specific nucleolar organizer region, where rRNA genes located. Secondary constriction site divides arm of chromosome on satellites (the rest of the chromosome). The ends of the chromosome arms called telomeres, or tips. Telomeres keep chromosomes individuality. Satellites promote chromosomes sticking together. Chromosomes vary in the location of a centromere, and that‘s why they may be such types as: 1. Metacentric – if the centromere divides it into two equal arms. 2. Submetacentric – if the centromere is slightly displace from the center of chromosome. 3. Acrocentric – if the centromere establishes one long arm and one short arm. 4. Telocentric – if the centromere is in the end of the chromosome and only one arm presents.

 

 

Eukariotic chromosomes have several levels of organization:

1. The DNA is associated with basic proteins called histones to form nucleosomes, each of which consists of 8 histones bead with DNA wrapped around it, plus an adjacent linker DNA with a histone attached.

2. The nucleosomes are organized into large coiled loops held together by nonhistone scaffolding proteins.

4.     DNA molecules are much longer than the nuclei or the cells that contain them. The organization of DNA into chromosomes allows the DNA to be accurately replicated and segregated into daughter cells without tangling.

 

Chromosome functional states:

 1.     Extended chromatin – chromosomes are not visible under the light microscope during interphase, when chromatin is scattered throughout the nucleoplasm.

2.     Condensed chromatin – chromosomes are visible under the light microscope during mitosis and meiosis.

 

Heterochromatin is a highly condensed portion of chromatin, which remains permanently condensed during interphase. Its major characteristic is that transcription is limited. As such, it is a means to control gene expression, through regulation of the transcription initiation. Heterochromatin is not transcribed into RNA and appears in the light microscope as basophilic clumps of nucleoprotein. Heterochromatin is usually localized to the periphery of the nucleus.

Heterochromatin mainly consists of genetically inactive satellite sequences. Heterochromatin also replicates later in S phase of the cell cycle than euchromatin, and is found only in eukaryotes. Both centromeres and telomeres are heterochromatic.

Heterochromatin may be constitutive and facultative. Constitutiv – maintains structural integrity. Human chromosomes 1, 9, 16, and the Y chromosome contain large regions of constitutive heterochromatin. Facultative – corresponding to one of two X- chromosomes, is present in somatic cells of female mammals. X-chromosome is visible as dark-staining evagination protruding from the nucleus. This structure as called the Barr body, or sex chromatin of the second inactivated X chromosome in a female. It is situated in the nucleoplasm, near the nuclear envelope from internal side.

 Euchromatin is a lightly packed (isn’t condensed) form of chromatin during interphase that is rich in gene concentration, and is often under active transcription. It is found in both eukaryotes and prokaryotes. Euchromatin comprises the most active portion of the genome within the cell nucleus. Euchromatin participates in the active transcription of DNA to mRNA products. The unfolded structure allows gene regulatory proteins and RNA polymerase complexes to bind to the DNA sequence, which can then initiate the transcription process. Euchromatin that is “always turned on” is housekeeping genes, which codes for the proteins needed for basic functions of cell survival.

Euchromatin generally appears as light-colored bands and observed under an optical microscope; in contrast to heterochromatin, which stains darkly. This lighter staining is due to the less compact structure of euchromatin.

  

Antibodies to certain types of chromatin organization, particularly nucleosomes, have been associated with a number of autoimmune diseases, such as systemic lupus erythematosus. These are known as anti-nuclear antibodies (ANA) and have also been observed in concert with multiple sclerosis as part of general immune system dysfunction.

 

 Polytene chromosomes – giant bundles of unsepapated chromonemata occuring especially in the salivary glands of some insects. They are many (100-200) times longer than the chromosomes found in other somatic cells or germinal cells. Being unusually large, they are visiable under a light microscope.Polytene chromosomes of fruit fly have numerous (512-1024) chromonemata. They result from repeated replication of DNA without separation into daughter chromosomes.

Polytene chromosomes are specific interphase chromosomes consisting of thousands of deoxyribonucleic acid (DNA) strands. For this reason they are very large and display a characteristic band–interband morphology. Polyteny arises in tissues, organs and at developmental stages when there is need for the rapid development of an organ at an unaltered high level of function. Organs containing cells with polytene chromosomes are, as a rule, involved in intense secretory functions accomplished during a short time against a background of rapid growth. Chromosome rearrangements and in situ hybridization on polytene chromosomes allow genes to be mapped to a resolution of a few tens of kilobases. Polytene chromosomes allow a specific narrow region to be dissected out with a micromanipulator and a library of DNA clones to be derived from the region.

 

Function

In addition to increasing the volume of the cells’ nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as multiple copies of genes permits a high level of gene expression. In Drosophila melanogaster, for example, the chromosomes of the larval salivary glands undergo many rounds of endoreduplication, to produce large amounts of glue before pupation. Another example, within the organism itself is the tandem duplication of various polytene bands located near the centromere of the X chromosome which results in the Bar phenotype of kidney-shaped eyes.

Structure

Polytene chromosomes have characteristic light and dark banding patterns that can be used to identify chromosomal rearrangements and deletions. Dark banding frequently corresponds to inactive chromatin, whereas light banding is usually found at areas with higher transcriptional activity. The banding patterns of the polytene chromosomes of Drosophila melanogaster were sketched in 1935 by Calvin B. Bridges, in such detail that his maps are still widely used today. The banding patterns of the chromosomes are especially helpful in research, as they provide an excellent visualization of transcriptionally active chromatin and general chromatin structure. For example, the polytene chromosomes in Drosophila have been used to support the theory of genomic equivalence, which states that all of the cells in the body maintain the same genome.^[2] Chromosome puffs are diffused uncoiled regions of the polytene chromosome that are sites of RNA transcription. A Balbiani ring is a large chromosome puff. Polytene chromosomes are about 200 µm in length. The chromonema of these chromosomes divide but do not separate. Therefore, they remain together to become large in size. Another form of chromosomal enlargement that provides for increased transcription is the lampbrush chromosome.

 

Characteristic of nucleic acids.

 1. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA): ribosomal RNA (rRNA), messenger RNA (mRNA) and transport RNA (tRNA); structure and role in protein synthesis. Introns and exons.

2. Watson and Crick model of DNA.

3. Chargaff’s rules.

4. Replication of DNA.

5. Reparation of DNA.

6. Point Mutations.

 

 

The DNA chains in the organisms exist not as a single chain folded into complex shapes, as in proteins, but rather as double chains. Two of the polymeres winds around each other like the outside and inside rails of a circular staircase. Such a winding shape is called a helix, and one composed of two molecules winding in concert as DNA does is called a double helix. In a double helix the nucleоtide bases sticking out from each of two chains point toward each other, and hydrogen bonds between opposite bases hold the two chains together. To fit into the chain, a large base (A, G) must always be opposite a small one (T, C). Also, the two opposite bases must be able to form loose hydrogen bonds. Because of these size and bonding restrictions, A and T can pair only with one another within a DNA double helix, and only G and C can pair with one another. A sequence of bases on one strand, that also serves to fully specify the other strand. Wherever base A occurs on one strand, the other strand must have a T, wherever G occurs, the other strand must have C, and so on… One strand is said to be complementary to the other. It is this feature of the DNA molecule that makes possible the precise duplication of the hereditary information. 

DNA adenine thymine

 

 

Watson and Crick model shows that DNA is a double helix with sugar-phosphate backbones on the outside and paired bases on the inside. This arrangement fits the mathematical measurements provided by the X-ray diffraction data for the spacing between the base pairs (0.34 nm) and for a complete turn of the double helix (3.4 nm).      

In 1962 James Watson (b. 1928), Francis Crick (1916–2004), and Maurice Wilkins (1916–2004) jointly received the Nobel Prize in physiology or medicine for their 1953 determination of the structure of deoxyribonucleic acid (DNA). Because the Nobel Prize can be awarded only to the living, Wilkins’s colleague Rosalind Franklin (1920–1958), who died of cancer at the age of 37, could not be honored.

The molecule that is the basis for heredity, DNA, contains the patterns for constructing proteins in the body, including the various enzymes. A new understanding of heredity and hereditary disease was possible once it was determined that DNA consists of two chains twisted around each other, or double helixes, of alternating phosphate and sugar groups, and that the two chains are held together by hydrogen bonds between pairs of organic bases—adenine (A) with thymine (T), and guanine (G) with cytosine (C). Modern biotechnology also has its basis in the structural knowledge of DNA—in this case the scientist’s ability to modify the DNA of host cells that will then produce a desired product, for example, insulin.

 

Rosalind Franklin in Paris. Courtesy Vittorio Luzzati.

The background for the work of the four scientists was formed by several scientific breakthroughs: the progress made by X-ray crystallographers in studying organic macromolecules; the growing evidence supplied by geneticists that it was DNA, not protein, in chromosomes that was responsible for heredity; Erwin Chargaff’s experimental finding that there are equal numbers of A and T bases and of G and C bases in DNA; andLinus Pauling’s discovery that the molecules of some proteins have helical shapes—arrived at through the use of atomic models and a keen knowledge of the possible disposition of various atoms.

Of the four DNA researchers, only Rosalind Franklin had any degrees in chemistry. She was born into a prominent London banking family, where all the children—girls and boys—were encouraged to develop their individual aptitudes. She attended St. Paul’s Girls School, one of the few schools in London where girls were taught science. Then she proceeded to Newnham College, one of the women’s colleges at Cambridge University. She completed her degree in 1941 in the middle of World War II and undertook graduate work at Cambridge with Ronald Norrish, a future Nobel Prize winner. She resigned her research scholarship in just one year to contribute to the war effort at the British Coal Utilization Research Association. There she performed fundamental investigations on the properties of coal and graphite. She returned briefly to Cambridge, where she presented a dissertation based on this work and was granted a Ph.D. in physical chemistry. After the war, through a French friend, she gained an appointment at the Laboratoire Centrale des Services Chimiques de l’Etat in Paris, where she was introduced to the technique of X-ray crystallography (see video below) and rapidly became a respected authority in this field. In 1951 she returned to England to King’s College London, where her charge was to upgrade the X-ray crystallographic laboratory there for work with DNA.

 

James Watson and Francis Crick with their DNA model at the Cavendish Laboratories in 1953. Photograph copyright A. Barrington Brown. To request permission to use this photo, please visit the Science Photo Library Web (site at www.photoresearchers.com.)

 

Already at work at King’s College was Maurice Wilkins, a New Zealand–born but Cambridge-educated physicist. As a new Ph.D. he worked during World War II on the improvement of cathode-ray tube screens for use in radar and then was shipped out to the United States to work on the Manhattan Project. Like many other nuclear physicists, he became disillusioned with his subject when it was applied to the creation of the atomic bomb; he turned instead to biophysics, working with his Cambridge mentor, John T. Randall—who had undergone a similar conversion—first at the University of St. Andrews in Scotland and then at King’s College London. It was Wilkins’s idea to study DNA by X-ray crystallographic techniques, which he had already begun to implement when Franklin was appointed by Randall. The relationship between Wilkins and Franklin was unfortunately a poor one and probably slowed their progress.

Meanwhile, in 1951, 23-year-old James Watson, a Chicago-born American, arrived at the Cavendish Laboratory in Cambridge. Watson had two degrees in zoology: a bachelor’s degree from the University of Chicago and a doctorate from Indiana University, where he became interested in genetics. He had worked under Salvador E. Luria at Indiana on bacteriophages, the viruses that invade bacteria in order to reproduce—a topic for which Luria received a Nobel Prize in physiology or medicine in 1969. Watson went to Denmark for postdoctoral work, to continue studying viruses and to remedy his relative ignorance of chemistry. At a conference in the spring of 1951 at the Zoological Station at Naples, Watson heard Wilkins talk on the molecular structure of DNA and saw his recent X-ray crystallographic photographs of DNA. He was hooked.

Watson soon moved to the Cavendish Laboratory, where several important X-ray crystallographic projects were in progress. Under the leadership of William Lawrence Bragg, Max Perutz was investigating hemoglobin and John Kendrew was studying myoglobin, a protein in muscle tissue that stores oxygen. (Perutz and Kendrew received the Nobel Prize in chemistry for their work in the same year that the prize was awarded to the DNA researchers—1962.) Working under Perutz was Francis Crick, who had earned a bachelor’s degree in physics from University College London and had helped develop radar and magnetic mines during World War II. Crick, another physicist in biology, was supposed to be writing a dissertation on the X-ray crystallography of hemoglobin when Watson arrived, eager to recruit a colleague for work on DNA. Inspired by Pauling’s success in working with molecular models, Watson and Crick rapidly put together several models of DNA and attempted to incorporate all the evidence they could gather. Franklin’s excellent X-ray photographs, to which they had gained access without her permission, were critical to the correct solution. The four scientists announced the structure of DNA in articles that appeared together in the same issue of Nature.

Then they moved off in different directions. Franklin went to Birkbeck College, London, to work in J. D. Bernal’s laboratory, a much more congenial setting for her than King’s College. Before her untimely death from cancer, she made important contributions to the X-ray crystallographic analysis of the structure of the tobacco mosaic virus, a landmark in the field. By the end of her life, she had become friends with Francis Crick and his wife and had moved her laboratory to Cambridge, where she undertook dangerous work on the poliovirus. Wilkins applied X-ray techniques to the structural determination of nerve cell membranes and of ribonucleic acid (RNA)—a molecule that is associated with chemical synthesis in the living cell—while rising in rank and responsibility at King’s College. Watson’s subsequent career eventually took him to the Cold Spring Harbor Laboratory (CSHL) of Quantitative Biology on Long Island, New York, where as director from 1968 onward he led it to new heights as a center of research in molecular biology. From 1988 to 1992 he headed the National Center for Human Genome Research at the National Institutes of Health. Afterwards he returned to CSHL, from which he retired in 2007. During Crick’s long tenure at Cambridge, he made fundamental contributions to unlocking the genetic code. He and Sydney Brenner demonstrated that each group of three adjacent bases on a single DNA strand codes for one specific amino acid. He also correctly hypothesized the existence of “transfer” RNA, which mediates between “messenger” RNA and amino acids. After 20 years at Cambridge, with several visiting professorships in the United States, Crick joined the Salk Institute for Biological Studies in La Jolla, California.

 

Chargaff’s rules said that A = T and G = C. The model shows that A is hydrogen bonded to T and G is hydrogen bonded to C. This so-called complementary base pairing means that a purine is always bonded to a pyrimidine. Only in this way will the molecule have the width (2 nm) dictated by its X-ray diffraction pattern, since 2 pyrimidines together are too narrow and 2 purines together are too wide.

Exons are regions of the DNA molecule that code for specific RNAs.

Introns are regions of the DNA molecule, between exons, that do not code for RNAs.

A codon is a sequence of three bases in the DNA molecule that codes for a single amino acid.

A gene is a segment of the DNA molecule that is responsible for the formation of a single RNA molecule.

 

Organization of DNA in Cells. Classical geneticists who made chromosome maps would have predicted that the genome (all the genes of an organism) consists of DNA that directs the synthesis of cytoplasmic proteins. It appears, however, that at most 70% of eukaryotic DNA seems to function in this way. The rest either has no function or has a function we haven’t been able to determine yet.

 

The main DNA functions:

1.     DNA stores hereditary information in the order of its bases. The order of the bases specifies the order of amino acids in polypeptides.

 

2.     DNA-replication – maintaining genetic information. DNA-replication occurs in three stages: 1) enzymes unwind the two chains of the double helix from each other; 2) bases float in and pair with their complements to form a new chain; 3) an enzyme called DNA-polymerase joints the bases in the new chain together – sugar to phosphate to sugar.

  

3.     The transmission of hereditary information during the transcription. It is the RNA-polymerase – catalyzed assembly of an mRNA molecule on a DNA template (or complementary to a strand of DNA).

       

Repetitive DNA Sequences. Genes that direct the synthesis of cytoplasmic proteins are sepa­rated from one another by repetitive DNA. Repetitive DNA contains the same sequence of base pairs repeated many times. In highly repetitive DNA, the same sequence of 5-15 base pairs is repeated 100,000-1 million times. This number of base pairs is much too short to be multiple copies of the same gene. Since highly repetitive DNA is found in the region of the centromere, it speculated it has a structural role here and in all places where it ь found. One interesting aspect of highly repetitive DNA is that the number of repeats between genes is inherited in a Mendelian fashion and is unique to the person. One’s so-called DNA finger­print is based on this finding. In moderately repetitive DNA, the same sequence of 1,000-1,500 base pairs is repeated only 10 3,000 times. Some of these sequences seem to be multiple copies of genes for ribosomal RNA, ribosomal proteins, and histones. It can be speculated that the many copies of these genes allow a faster production rate of these substances than would otherwise be possible.

 

RNA – a polymer of ribose-containing nucleotides, with forms single-strand. RNA contains Adenine, Guanine, Cytosine, Uracil.

 

There are three types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). RNA is synthesized by transcription of DNA. Transcription is catalyzed by three RNA polymerases: I for rRNA, II for mRNA, and III for tRNA.

 

Messenger RNA carries the genetic code to the cytoplasm to direct protein synthesis.

1. This single-stranded molecule consists of hundreds to thousands of nucleotides.

2.  mRNA contains codons that are complementary to the DNA codons from which it was transcribed, including one codon (AUG) for initiating protein synthesis and one of three codons (UAA, UAG, or UGA) for terminating protein synthesis.

 

Transfer RNA is folded into a cloverleaf shape and contains about 80 nucleo­tides, terminating in adenylic acid (where amino acids attach).

1.  Each tRNA combines with a specific amino acid that has been activated by an enzyme.

2.  One end of the tRNA molecule possesses an anticodon, a triplet of nucleo­tides that recognizes the complementary codon in mRNA. If recognition occurs, the anticodon insures that the tRNA transfers its activated amino acid molecule in the proper sequence to the growing polypeptide chain.

 

 

Ribosomal RNA associates with many different proteins (including enzymes) to form ribosomes.

1.  rRNA associates with mRNA and tRNA during protein synthesis.

2.  rRNA synthesis takes place in the nucleolus and is catalyzed by RNA polymerase.

 

The major types of RNA and their function:

 

 

DNA Replication. The Watson and Crick model suggests that DNA can be replicated by means of complementary base pairing. During replication, each old DNA strand of the parent molecule serves as a template for a new strand in a daughter molecule. A template is most often a mold used to produce a shape complementary to itself.

 

 

Replication requires the following steps:

 1. Unwinding. The old strands that make up the parent DNA molecule are unwound and “unzipped” (i.e., the weak hydrogen bonds between the paired bases are broken). There is a special enzyme called helicase that unwinds the molecule.

2. Complementary base pairing. New complementary nucleotides, always present in the nucleus, are positioned by the process of complementary base pairing.

3. Joining. The complementary nucleotides become joined together to form new strands. Each daughter DNA mol­ecule contains an old strand and a new strand. Steps 2 and 3 are carried out by the enzyme DNA polymerase.

DNA replication is termed semiconservative replication because one of the old strands is conserved, or present, in each daughter double helix. Semiconservative replication was experimentally con­firmed by Matthew Meselson and Franklin Stahl in 1958.

Accuracy of Replication. The mismatched nucleotide causes a pause in replication, and during this time, the mismatched nucleotide is excised from the daughter strand. The errors that slip through nucleotide selection and proofreading cause a gene mutation to occur. Actually it is of benefit for mutations to occur occasionally because variation is the raw material for the evolutionary process.

 

Point Mutations. A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. One can categorize point mutations as follows: 1) transitions: replacement of a purine base with another purine or replacement of a pyrimidine with another pyrimidine; 2) transversions: replacement of a purine with a pyrimidine or vice versa.

Transition mutations are about an order of magnitude more common than transversions. Point mutations can also be categorized functionally:

nonsense mutations: code for a stop, which can truncate the protein

missense mutations: code for a different amino acid

silent mutations: code for the same or a different amino acid but without any functional change in the protein

For example, sickle-cell disease is caused by a single point mutation (a missense mutation) in the beta-hemoglobin gene that converts a GAG codon into GTG, which encodes the amino acid valine rather than glutamic acid.

Point mutations may arise from spontaneous mutations that occur during DNA replication. The rate of mutation may be increased by mutagens. Mutagens can be physical, such as radiation from UV rays, X-rays or extreme heat, or chemical (molecules that misplace base pairs or disrupt the helical shape of DNA). Mutagens associated with cancers are often studied to learn about cancer and its prevention.