# 4.2: Density-enhanced growth - Biology

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Darwin made unparalleled use of a model that failed, but how can the model be improved so that it does not fail?

Think of only three Black-Eyed Susan plants (Rudbeckia hirta) becoming established in Yellowstone National Park, one near the north-east entrance, one in the center, and a third near the south entrance—the plants thus separated by over 30 miles. How often would the same pollinator be able to visit two of the plants so the plants could reproduce? Rarely or never, because these pollinators travel limited distances. The plant’s growth rate will thus be 0. (In fact, it will be negative, since the three plants will eventually die.)

Suppose instead that 1000 of these plants were scattered about the park, making them about 2 miles apart. Occasionally a pollinator might happen by, though the chance of it visiting one of the other Black-Eyed Susans would be very low. Still, with 1000 plants in the area, the growth rate could be slightly positive.

Now consider 1,000,000 of those plants, making them about 100 meters apart. Pollination would now become relatively frequent. The growth rate of the population thus depends on the number of plants in the vicinity, meaning that this number must be part of the equation used to calculate the population growth rate.

We can use the equation introduced earlier to calculate this rate. First, put a parameter in place of the 1, like this.

(frac{1}{N}, frac{∆N}{∆t}, =,r), where r formerly was 1

Then attach a term that recognizes the density of other members of the population, N.

(frac{1}{N}, frac{∆N}{∆t}, =,r,+,sN),

Here r is related to the number of offspring each plant will produce if it is alone in the world or in the area, and s is the number of additional offspring it will produce for each additional plant that appears in its vicinity.

Suppose r = 0 and s = 1/20, just for illustration, and start with three plants, so N (0) = 3. That is

(frac{1}{N}, frac{∆N}{∆t}, =,0,+,0.05,N),

For watching the dynamics of this, multiply it out again

(frac{∆N}{∆t}, =,(0,+,0.05,N),N),

and convert the model to computer code, like this.
r=0; s=0.05; dt=1; t=0; N=3; print(N);

while(t<=14)

{ dN=(r+s*N)*N*dt; N=N+dN; t=t+dt; print(N); }

If you run this model in R (or other languages in which this code works, like C or AWK), you will see the numbers below.

3

3.45

4.045125

4.863277

6.045850

7.873465

407.5176

8,711.049

3,802,830

723,079,533,905

26,142,200,000,000,000,000,000

Graph these, and you will see the numbers expand past all bounds, vertically off the page.

The blue line shows the unlimited bacterial growth (exponential growth) that helped lead Darwin to his idea of natural selection. The red line illustrates the new “density-enhanced growth” just being considered, where growth rate increases with density.

Because it approaches a line that is orthogonal to the line approached by the logistic model, described later, we call this an “orthologistic model.” It runs away to infinity so quickly that it essentially gets there in a finite amount of time. In physics and mathematics this situation is called a “singularity”—a place where the rules break down. To understand this, it is important to remember that all models are simplifications and therefore approximations, and apply in their specific range. The orthologistic model applies well at low densities, where greater densities mean greater growth. But a different model will take over when the densities get too high. In fact, if a population is following an orthologistic model, the model predicts that there will be some great change that will occur in the near future—before the time of the singularity.

In physics, models with singularities command special attention, for they can reveal previously unknown phenomena. Black holes are one example, while a more mundane one from physics is familiar to all. Consider a spinning coin with one point touching the table, spinning ever more rapidly as friction and gravity compel the angle between the coin and the table to shrink with time. It turns out that the physical equations that quite accurately model this spinning coin include a singularity—a place where the spinning of the coin becomes infinitely fast at a definite calculable time. Of course, the spinning cannot actually become infinitely fast. As the coin gets too close to the singularity—as its angle dips too near the table—it merely switches to a different model. That different model is a stationary coin. The exact nature of the transition between the spinning and stationary states is complex and debated, but the inevitability of the transition is not.

It is no different in ecology. Reasonable models leading to singularities are not to be discounted, but rather considered admissible where they apply. They arise inescapably in human population growth, considered in the next chapter.

## Population Growth

The two simplest models of population growth use deterministic equations (equations that do not account for random events) to describe the rate of change in the size of a population over time. The first of these models, exponential growth, describes populations that increase in numbers without any limits to their growth. The second model, logistic growth, introduces limits to reproductive growth that become more intense as the population size increases. Neither model adequately describes natural populations, but they provide points of comparison.

## 4.2: Density-enhanced growth - Biology

Other Population Growth Factors
Populations can also change size if organisms move in (immigration) or leave (emigration)

Putting It All Together
We can write a simple equation to show population growth as:

Change in Population Size = (Births + Immigration) - (Deaths + Emigration)

Expressing Population Changes as a Percentage
Suppose we had a population of 100,000 individuals. Suppose in one year there were 1000 births, and 500 deaths.
What percentage of the population were births?
1000/100,000 = 0.01, or in percentage terms, this is 1% of the population.

What percentage of the population were deaths?
500/100,000 = 0.005, or in percentage terms, this is 0.5% of the population.

Assume immigration equals emigration. If so, then they cancel out of our population equation. We'll come back to
this assumption later.

Now, subtract deaths from births but express as a percentage:
1000-500/100,000 = 500/100,000 = 0.005, or 0.5% net growth

Thus, this population would be growing by 0.5% this first year. That means that after one year, there will be 500 more
individuals than the previous year. So, after one year, the population would be 100,500 individuals.

The Net Reproductive Rate
The net reproductive rate (r) is the percentage growth after accounting for births and deaths. In the example above, the population reproductive rate is 0.5%/yr.

Net reproductive rate (r) is calculated as: r = (births-deaths)/population size or to get in percentage terms, just multiply by 100.

Suppose we came back many years later, the net reproductive rate was still the same, but now the population had grown to 1,000,000. How many new individuals would be added each year now? Simply multiply the population by the reproductive rate:
1,000,000 x 0.05 (which is 0.5%) = 50,000

This means that now 50,000 new individuals are added in one year!! The net reproductive rate is the same as before, but because
the population is so much bigger, many more individuals are added.

See figure to right - the curve sweeping upwards is the exponential growth curve.

Some Population Statistics for Humans
At the end of the 1700s, Robert Malthus, a priest, wrote one of the most influential essays in the world - He was pondering why there was so much suffering among humans, and came to the conclusion that human population growth tended to always outstrip food supply. The Core Principles of Malthus are:

1.Food is necessary for human existence.
2.Human population tends to grow faster than the power in the earth to produce subsistence, and that
3.The effects of these two unequal powers must be kept equal.
4.Since humans tend not to limit their population size voluntarily, population reduction tends to be accomplished through the
"positive" checks of famine, disease, poverty and war.

Darwin used this information to help develop his theory of natural selection by assuming that this situation occurs for all living organisms, not just humans!!

So, does it work? Consider that in a typical day, 35,000 humans starve to death around the world . Most in developing countries.

What is the Current Population of the Earth?
The current population of the earth is about 6.2 Billion people ! This is more humans alive than at any time in human history.
To see how fast the world population is growing, click on this to see a clock of human population growth.
For interesting facts on worldwide population growth, and the factors affecting it, click here. Recommended web site!!
For even more interesting facts, click here. The Population Reference Bureau has some of the best information on human population growth of anywhere!

What is Current Net Reproductive Rate of Humans Worldwide?
The current percentage increase in the human population (as of 2000) is about 1.3%, or 0.013 per year.
If we multiply this, as shown above, by the current population, we get the increase in humans per year:

0.013 x 6.2 B = 80,600,000 new people per year, or 80.6 million new humans each year!!

That is the equivalent of 2.5 California's per year, or 1 new Germany per year. It is 1.6 million people per week (one New Mexico per week), or 221,000 people per day (one Charlotte, NC added per day!).

An astounding growth rate, even though the net reproductive rate is actually quite small. But growth is not evenly distributed around the world. Certain countries are growing faster than others, while some are actually losing growth (deaths and emigration exceed births plus immigration - Albania is an example).

Why the Increase in Human Population Growth Rates This Century?
Remember, only two things affect population growth: births and deaths. So, have these changed?
Birth rates: have been constant for many years at about 22 babies/1000 people/year
Death rates: have declined dramatically due to more food, less disease, more social structure
Death Rates in 1900: 20/1000/year Net Reproductive Rate 1900: 22-20/1000 = 0.002 or 0.2%
Death Rates in 2000: 9/1000/year Net Reproductive Rate 2000: 22-9/1000 = 0.013 or 0.13%

Due to decline in death rates, r for humans has risen nearly 6 fold.

Some Representative Growth Rates for Countries Around the World
Consider this statistic: 90% of all world population growth occurs in developing countries!!
Go to this website to see net reproductive rates for all countries: http://www.prb.org/Content/NavigationMenu/Other_reports/2000-2002/sheet1.html

Net Reproductive Rates
World 1.3%
More Developed Countries 0.1%
Less Developed Countries 1.6%
Africa 2.9% !!
Liberia 3.1% .
United States 0.6% (much of it immigration, about 1/3!)
Mexico 1.9%
Europe -0.1% (population is declining!!)
England 0.1
France 0.4
Latvia -0.6%

You can determine the population doubling times for the world and countries by dividing 69.3 by the growth rate. For example, if the world growth rate is 1.3%, then the time it takes to double the population is:

Thus, if things don't change, the world population could rise to 12.4 Billion in the year 2055!! When I was born, the population was about 2 Billion in 1952. It is now 50 years later, and the population is 6.2 Billion. That is nearly a tripling!! Why? The world population growth rate was much higher in the past 50 years than it currently is. When I was born, the population growth rate was over 2% per year, and the doubling time was down to 42 years!!

Why Do Growth Rates Differ Between Countries?
Demographics!! If you have more young people, then you have more opportunity to make babies!! Developing countries have more young people than developed ones. Why? In developed countries, couples wait longer to have babies, and, they tend to have fewer per couple. In undeveloped countries, children are produced sooner, and couples have larger families than in developed countries.
In Mexico, 50% of the population is age 15 or younger!
In the United States, only about 25% of the population is this young.

What Can Be Done to Control Population Growth?
There are two simple ways to lower population growth: increase the number of deaths, or, decrease the number of births. I think for most of us, we would opt for the latter solution. How to do that?
1. Family Planning - have babies at a later age, use contraception (birth control), limit number of babies per family
2. Education - the best correlate of lowering the number of babies per family is the educational status of the females
The more education the females have, the more control they have over their reproductive lives
3. Better social security - in developing countries, large families are a form of social security. If poverty can be reduced,
then the need for large families is lowered (hard to do though!)

The Future - How Large Will the Population Become?
In your lifetime, the population could approach or exceed 14 Billion people. Can we feed that many? Not likely. Is there enough water for that many? Probably not. Enough habitat? Probably not.
So, what will the population stabilize at? Best guesses are between 7-10 Billion people. Will the world still be a great place with that many people? Unlikely.
There is a great need to reduce population growth, starting now!! You can help a great deal.

## 4.2: Density-enhanced growth - Biology

The Bacterial Growth Curve

In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 2 0 , 2 1 , 2 2 , 2 3 . 2 n (where n = the number of generations). This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature.

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve (Figure 3 below).

Figure 3. The typical bacterial growth curve. When bacteria are grown in a closed system (also called a batch culture), like a test tube, the population of cells almost always exhibits these growth dynamics: cells initially adjust to the new medium (lag phase) until they can start dividing regularly by the process of binary fission (exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Generation times are calculated during the exponential phase of growth. Time measurements are in hours for bacteria with short generation times.

Four characteristic phases of the growth cycle are recognized.

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum time necessary to recover from physical damage or shock in the transfer time required for synthesis of essential coenzymes or division factors and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time (below) derive.

3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients 2. accumulation of inhibitory metabolites or end products 3. exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.

4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

Growth Rate and Generation Time

As mentioned above, bacterial growth rates during the phase of exponential growth, under standard nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation time. Generation times for bacteria vary from about 12 minutes to 24 hours or more. The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform's generation time is estimated to be 12-24 hours. For most known bacteria that can be cultured, generation times range from about 15 minutes to 1 hour. Symbionts such as Rhizobium tend to have longer generation times. Many lithotrophs, such as the nitrifying bacteria, also have long generation times. Some bacteria that are pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long generation times, and this is thought to be an advantage in their virulence. Generation times for a few bacteria are are shown in Table 2.

Table 2. Generation times for some common bacteria under optimal conditions of growth.

 Bacterium Medium Generation Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Streptococcus lactis Milk 26 Streptococcus lactis Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30 Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract 344-461 Mycobacterium tuberculosis Synthetic 792-932 Treponema pallidum Rabbit testes 1980

Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)

t = time interval in hours or minutes

B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

n = number of generations (number of times the cell population doubles during the time interval)

b = B x 2 n (This equation is an expression of growth by binary fission)

Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth?

## Contents

DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together. [9] [12] These two long strands coil around each other, in the shape of a double helix. The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide. [13]

The backbone of the DNA strand is made from alternating phosphate and sugar groups. [14] The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end (three prime end), and 5′-end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond. Therefore, any DNA strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA. [12]

### Non-canonical bases

Modified bases occur in DNA. The first of these recognised was 5-methylcytosine, which was found in the genome of Mycobacterium tuberculosis in 1925. [20] The reason for the presence of these noncanonical bases in bacterial viruses (bacteriophages) is to avoid the restriction enzymes present in bacteria. This enzyme system acts at least in part as a molecular immune system protecting bacteria from infection by viruses. [21] Modifications of the bases cytosine and adenine, the more common and modified DNA bases, plays vital roles in the epigenetic control of gene expression in plants and animals. [22]

### Listing of non-canonical bases found in DNA

A number of non canonical bases are known to occur in DNA. [23] Most of these are modifications of the canonical bases plus uracil.

• 7-Deazaguanine
• 7-Methylguanine
• N4-Methylcytosine
• 5-Carboxylcytosine
• 5-Formylcytosine
• 5-Glycosylhydroxymethylcytosine
• 5-Hydroxycytosine
• 5-Methylcytosine
• α-Glutamythymidine
• α-Putrescinylthymine
• Base J
• Uracil
• 5-Dihydroxypentauracil
• 5-Hydroxymethyldeoxyuracil
• Deoxyarchaeosine

### Grooves

Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. One groove, the major groove, is 22 ångströms (2.2 nm) wide and the other, the minor groove, is 12 Å (1.2 nm) wide. [24] The width of the major groove means that the edges of the bases are more accessible in the major groove than in the minor groove. As a result, proteins such as transcription factors that can bind to specific sequences in double-stranded DNA usually make contact with the sides of the bases exposed in the major groove. [25] This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.

### Base pairing

#### SsDNA vs. dsDNA

In the laboratory, the strength of this interaction can be measured by finding the temperature necessary to break half of the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others. [30]

### Sense and antisense

A DNA sequence is called a "sense" sequence if it is the same as that of a messenger RNA copy that is translated into protein. [31] The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear. [32] One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing. [33]

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. [34] In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, [35] while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome. [36]

### Supercoiling

DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. [37] If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases. [38] These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication. [39]

### Alternative DNA structures

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms. [14] The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, and the presence of polyamines in solution. [40]

The first published reports of A-DNA X-ray diffraction patterns—and also B-DNA—used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA. [41] [42] An alternative analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. [43] In the same journal, James Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix. [9]

Although the B-DNA form is most common under the conditions found in cells, [44] it is not a well-defined conformation but a family of related DNA conformations [45] that occur at the high hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder. [46] [47]

Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, and in enzyme-DNA complexes. [48] [49] Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form. [50] These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription. [51]

### Alternative DNA chemistry

For many years, exobiologists have proposed the existence of a shadow biosphere, a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA. A report in 2010 of the possibility in the bacterium GFAJ-1, was announced, [52] [53] though the research was disputed, [53] [54] and evidence suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other biomolecules. [55]

At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes. [56] These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. [57] In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence. [58]

These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases, known as a guanine tetrad, form a flat plate. These flat four-base units then stack on top of each other to form a stable G-quadruplex structure. [60] These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit. [61] Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.

In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins. [62] At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop. [60]

### Branched DNA

In DNA, fraying occurs when non-complementary regions exist at the end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible. [63] Branched DNA can be used in nanotechnology to construct geometric shapes, see the section on uses in technology below.

### Artificial bases

Several artificial nucleobases have been synthesized, and successfully incorporated in the eight-base DNA analogue named Hachimoji DNA. Dubbed S, B, P, and Z, these artificial bases are capable of bonding with each other in a predictable way (S–B and P–Z), maintain the double helix structure of DNA, and be transcribed to RNA. Their existence could be seen as an indication that there is nothing special about the four natural nucleobases that evolved on Earth. [64] [65] On the other hand, DNA is tightly related to RNA which does not only act as a transcript of DNA but also performs as moleular machines many tasks in cells. For this purpose it has to fold into a structure. It has been shown that to allow to create all possible structures at least four bases are required for the corresponding RNA, [66] while a higher number is also possible but this would be against the natural Principle of least effort.

### Base modifications and DNA packaging

The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin. Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling). There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression. [67]

For one example, cytosine methylation produces 5-methylcytosine, which is important for X-inactivation of chromosomes. [68] The average level of methylation varies between organisms—the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. [69] Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations. [70] Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain, [71] and the glycosylation of uracil to produce the "J-base" in kinetoplastids. [72] [73]

### Damage

DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases. [75] On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. [76] A typical human cell contains about 150,000 bases that have suffered oxidative damage. [77] Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions, deletions from the DNA sequence, and chromosomal translocations. [78] These mutations can cause cancer. Because of inherent limits in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. [79] [80] DNA damages that are naturally occurring, due to normal cellular processes that produce reactive oxygen species, the hydrolytic activities of cellular water, etc., also occur frequently. Although most of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes. These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging. [81] [82] [83]

Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. [84] As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen. [85] Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication. [86] Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells. [87]

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. [88] The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles here the focus is on the interactions between DNA and other molecules that mediate the function of the genome.

### Genes and genomes

Genomic DNA is tightly and orderly packed in the process called DNA condensation, to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, with small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. [89] The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, and regulatory sequences such as promoters and enhancers, which control transcription of the open reading frame.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. [90] The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species, represent a long-standing puzzle known as the "C-value enigma". [91] However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression. [92]

Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. [57] [94] An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation. [95] These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence. [96]

### Transcription and translation

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (4 3 combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region these are the TAA, TGA, and TAG codons.

### Replication

Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. [97] In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.

### Extracellular nucleic acids

Naked extracellular DNA (eDNA), most of it released by cell death, is nearly ubiquitous in the environment. Its concentration in soil may be as high as 2 μg/L, and its concentration in natural aquatic environments may be as high at 88 μg/L. [98] Various possible functions have been proposed for eDNA: it may be involved in horizontal gene transfer [99] it may provide nutrients [100] and it may act as a buffer to recruit or titrate ions or antibiotics. [101] Extracellular DNA acts as a functional extracellular matrix component in the biofilms of several bacterial species. It may act as a recognition factor to regulate the attachment and dispersal of specific cell types in the biofilm [102] it may contribute to biofilm formation [103] and it may contribute to the biofilm's physical strength and resistance to biological stress. [104]

Cell-free fetal DNA is found in the blood of the mother, and can be sequenced to determine a great deal of information about the developing fetus. [105]

Under the name of environmental DNA eDNA has seen increased use in the natural sciences as a survey tool for ecology, monitoring the movements and presence of species in water, air, or on land, and assessing an area's biodiversity. [106] [107]

All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.

### DNA-binding proteins

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes, this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. [108] [109] The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence. [110] Chemical modifications of these basic amino acid residues include methylation, phosphorylation, and acetylation. [111] These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. [112] Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. [113] These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes. [114]

A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination, and DNA repair. [115] These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.

In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins this locates the polymerase at the promoter and allows it to begin transcription. [117] Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase. [118]

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. [119] Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible. [25]

### DNA-modifying enzymes

#### Nucleases and ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the horizontal line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. [121] In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Enzymes called DNA ligases can rejoin cut or broken DNA strands. [122] Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination. [122]

#### Topoisomerases and helicases

Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling the enzyme then seals the DNA break. [38] Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix. [123] Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription. [39]

Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly adenosine triphosphate (ATP), to break hydrogen bonds between bases and unwind the DNA double helix into single strands. [124] These enzymes are essential for most processes where enzymes need to access the DNA bases.

#### Polymerases

Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products is created based on existing polynucleotide chains—which are called templates. These enzymes function by repeatedly adding a nucleotide to the 3′ hydroxyl group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction. [125] In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.

In DNA replication, DNA-dependent DNA polymerases make copies of DNA polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many DNA polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed. [126] In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases. [127]

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres. [56] [128] For example, HIV reverse transcriptase is an enzyme for AIDS virus replication. [128] Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes telomeres at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage. [57]

Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits. [129]

A DNA helix usually does not interact with other segments of DNA, and in human cells, the different chromosomes even occupy separate areas in the nucleus called "chromosome territories". [131] This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is in chromosomal crossover which occurs during sexual reproduction, when genetic recombination occurs. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.

Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. [132] Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks. [133]

The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51. [134] The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA. [135] A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA. [136] Only strands of like polarity exchange DNA during recombination. There are two types of cleavage: east-west cleavage and north–south cleavage. The north–south cleavage nicks both strands of DNA, while the east–west cleavage has one strand of DNA intact. The formation of a Holliday junction during recombination makes it possible for genetic diversity, genes to exchange on chromosomes, and expression of wild-type viral genomes.

DNA contains the genetic information that allows all forms of life to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. [137] [138] RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes. [139] This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. [140] However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution. [141] Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, [142] but these claims are controversial. [143] [144]

Building blocks of DNA (adenine, guanine, and related organic molecules) may have been formed extraterrestrially in outer space. [145] [146] [147] Complex DNA and RNA organic compounds of life, including uracil, cytosine, and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar cosmic dust and gas clouds. [148]

In February 2021, scientists reported, for the first time, the sequencing of DNA from animal remains, a mammoth in this instance over a million years old, the oldest DNA sequenced to date. [149] [150]

### Genetic engineering

Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector. [151] The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, [152] or be grown in agriculture. [153] [154]

### DNA profiling

Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. [155] This process is formally termed DNA profiling, also called DNA fingerprinting. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. [156] However, identification can be complicated if the scene is contaminated with DNA from several people. [157] DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, [158] and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case. [159]

The development of forensic science and the ability to now obtain genetic matching on minute samples of blood, skin, saliva, or hair has led to re-examining many cases. Evidence can now be uncovered that was scientifically impossible at the time of the original examination. Combined with the removal of the double jeopardy law in some places, this can allow cases to be reopened where prior trials have failed to produce sufficient evidence to convince a jury. People charged with serious crimes may be required to provide a sample of DNA for matching purposes. The most obvious defense to DNA matches obtained forensically is to claim that cross-contamination of evidence has occurred. This has resulted in meticulous strict handling procedures with new cases of serious crime.

DNA profiling is also used successfully to positively identify victims of mass casualty incidents, [160] bodies or body parts in serious accidents, and individual victims in mass war graves, via matching to family members.

DNA profiling is also used in DNA paternity testing to determine if someone is the biological parent or grandparent of a child with the probability of parentage is typically 99.99% when the alleged parent is biologically related to the child. Normal DNA sequencing methods happen after birth, but there are new methods to test paternity while a mother is still pregnant. [161]

### DNA enzymes or catalytic DNA

Deoxyribozymes, also called DNAzymes or catalytic DNA, were first discovered in 1994. [162] They are mostly single stranded DNA sequences isolated from a large pool of random DNA sequences through a combinatorial approach called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). DNAzymes catalyze variety of chemical reactions including RNA-DNA cleavage, RNA-DNA ligation, amino acids phosphorylation-dephosphorylation, carbon-carbon bond formation, etc. DNAzymes can enhance catalytic rate of chemical reactions up to 100,000,000,000-fold over the uncatalyzed reaction. [163] The most extensively studied class of DNAzymes is RNA-cleaving types which have been used to detect different metal ions and designing therapeutic agents. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), [162] the CA1-3 DNAzymes (copper-specific), [164] the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific). [165] The NaA43 DNAzyme, which is reported to be more than 10,000-fold selective for sodium over other metal ions, was used to make a real-time sodium sensor in cells.

### Bioinformatics

Bioinformatics involves the development of techniques to store, data mine, search and manipulate biological data, including DNA nucleic acid sequence data. These have led to widely applied advances in computer science, especially string searching algorithms, machine learning, and database theory. [166] String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. [167] The DNA sequence may be aligned with other DNA sequences to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. [168] Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally. [169] Entire genomes may also be compared, which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events.

### DNA nanotechnology

DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties. [170] DNA is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based and using the DNA origami method) and three-dimensional structures in the shapes of polyhedra. [171] Nanomechanical devices and algorithmic self-assembly have also been demonstrated, [172] and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins. [173]

### History and anthropology

Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny. [174] This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology.

### Information storage

DNA as a storage device for information has enormous potential since it has much higher storage density compared to electronic devices. However, high costs, slow read and write times (memory latency), and insufficient reliability has prevented its practical use. [175] [176]

DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein". [177] [178] In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases. [179] [180]

In 1909, Phoebus Levene identified the base, sugar, and phosphate nucleotide unit of the RNA (then named "yeast nucleic acid"). [181] [182] [183] In 1929, Levene identified deoxyribose sugar in "thymus nucleic acid" (DNA). [184] Levene suggested that DNA consisted of a string of four nucleotide units linked together through the phosphate groups ("tetranucleotide hypothesis"). Levene thought the chain was short and the bases repeated in a fixed order. In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". [185] [186] In 1928, Frederick Griffith in his experiment discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form. [187] [188] This system provided the first clear suggestion that DNA carries genetic information.

In 1933, while studying virgin sea urchin eggs, Jean Brachet suggested that DNA is found in the cell nucleus and that RNA is present exclusively in the cytoplasm. At the time, "yeast nucleic acid" (RNA) was thought to occur only in plants, while "thymus nucleic acid" (DNA) only in animals. The latter was thought to be a tetramer, with the function of buffering cellular pH. [189] [190]

In 1937, William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure. [191]

In 1943, Oswald Avery, along with co-workers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle, supporting Griffith's suggestion (Avery–MacLeod–McCarty experiment). [192] Late in 1951, Francis Crick started working with James Watson at the Cavendish Laboratory within the University of Cambridge. DNA's role in heredity was confirmed in 1952 when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that DNA is the genetic material of the enterobacteria phage T2. [193]

In May 1952, Raymond Gosling, a graduate student working under the supervision of Rosalind Franklin, took an X-ray diffraction image, labeled as "Photo 51", [194] at high hydration levels of DNA. This photo was given to Watson and Crick by Maurice Wilkins and was critical to their obtaining the correct structure of DNA. Franklin told Crick and Watson that the backbones had to be on the outside. Before then, Linus Pauling, and Watson and Crick, had erroneous models with the chains inside and the bases pointing outwards. Her identification of the space group for DNA crystals revealed to Crick that the two DNA strands were antiparallel. [195]

In February 1953, Linus Pauling and Robert Corey proposed a model for nucleic acids containing three intertwined chains, with the phosphates near the axis, and the bases on the outside. [196] Watson and Crick completed their model, which is now accepted as the first correct model of the double-helix of DNA. On 28 February 1953 Crick interrupted patrons' lunchtime at The Eagle pub in Cambridge to announce that he and Watson had "discovered the secret of life". [197]

The 25 April 1953 issue of the journal Nature published a series of five articles giving the Watson and Crick double-helix structure DNA and evidence supporting it. [198] The structure was reported in a letter titled "MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid", in which they said, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." [9] This letter was followed by a letter from Franklin and Gosling, which was the first publication of their own X-ray diffraction data and of their original analysis method. [42] [199] Then followed a letter by Wilkins and two of his colleagues, which contained an analysis of in vivo B-DNA X-ray patterns, and which supported the presence in vivo of the Watson and Crick structure. [43]

In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. [200] Nobel Prizes are awarded only to living recipients. A debate continues about who should receive credit for the discovery. [201]

In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis". [202] Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment. [203] Further work by Crick and co-workers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley, and Marshall Warren Nirenberg to decipher the genetic code. [204] These findings represent the birth of molecular biology. [205]

## 4.2: Density-enhanced growth - Biology

Bone, or osseous tissue, is a connective tissue that constitutes the endoskeleton. It contains specialized cells and a matrix of mineral salts and collagen fibers.

The mineral salts primarily include hydroxyapatite, a mineral formed from calcium phosphate. Calcification is the process of deposition of mineral salts on the collagen fiber matrix that crystallizes and hardens the tissue. The process of calcification only occurs in the presence of collagen fibers.

The bones of the human skeleton are classified by their shape: long bones, short bones, flat bones, sutural bones, sesamoid bones, and irregular bones (Figure 1).

Figure 1. Shown are different types of bones: flat, irregular, long, short, and sesamoid.

Figure 2. The long bone is covered by articular cartilage at either end and contains bone marrow (shown in yellow in this illustration) in the marrow cavity.

Long bones are longer than they are wide and have a shaft and two ends. The diaphysis, or central shaft, contains bone marrow in a marrow cavity. The rounded ends, the epiphyses, are covered with articular cartilage and are filled with red bone marrow, which produces blood cells (Figure 2). Most of the limb bones are long bones—for example, the femur, tibia, ulna, and radius. Exceptions to this include the patella and the bones of the wrist and ankle.

Short bones, or cuboidal bones, are bones that are the same width and length, giving them a cube-like shape. For example, the bones of the wrist (carpals) and ankle (tarsals) are short bones (Figure 1).

Flat bones are thin and relatively broad bones that are found where extensive protection of organs is required or where broad surfaces of muscle attachment are required. Examples of flat bones are the sternum (breast bone), ribs, scapulae (shoulder blades), and the roof of the skull (Figure 1).

Irregular bones are bones with complex shapes. These bones may have short, flat, notched, or ridged surfaces. Examples of irregular bones are the vertebrae, hip bones, and several skull bones.

Sesamoid bones are small, flat bones and are shaped similarly to a sesame seed. The patellae are sesamoid bones. Sesamoid bones develop inside tendons and may be found near joints at the knees, hands, and feet (see Figure 3).

Figure 3. The patella of the knee is an example of a sesamoid bone.

Sutural bones are small, flat, irregularly shaped bones. They may be found between the flat bones of the skull. They vary in number, shape, size, and position.

## 4.5 Other Techniques

Staining can be used to intensify structural discontinuities or to make visible those not available by ordinary light. Galstaff (1952) describes a method of staining tuna vertebrae with alizarin but it takes up to 12 days' preparation. This demonstrates the disadvantage of methods based upon staining they are too time-consuming for processing large amounts of material, even though batch processing will allow a quicker throughput. However, if staining is the only method which will show discontinuities then it must be used, even if it does result in smaller numbers of age determinations.

Polarized light and phase differentiation are also techniques that can be used.

Manuel J Santos, Editor-in-Chief

Dr Santos is an Associate Professor in the Faculty of Biological Sciences and Medicine at the Pontificia Catholic University of Chile.

Dr Santos received his MD from the University of Chile and his PhD in Cell and Molecular Biology from the Pontificia Catholic University of Chile. He majored in Medical Genetics at The John Hopkins University (USA) and The René Descartes University of Paris (France), and held a post doctorate position in Cell Biology and Genetics at the Rockefeller University (USA).

His research has focused on the biogenesis of cellular organelles, particularly peroxisomes. A pioneer in this field, his research lead him to discover a new type of human genetic disease, the peroxisomal biogenesis disorders, which include Zellweger Syndrome. More recently his research has centered on studying the role of peroxisomes in Alzheimer’s disease, and he also works in the field of bioethics.

Over the span of his career, Dr Santos has published more than 70 peer reviewed papers and been the President of the Society of Biology of Chile, the Genetics Society of Chile and the Bioethical Society of Chile.

A dependent variable is the measurement that changes in response to what you changed in the experiment. This variable is dependent on other variables hence the name! For example, in the plant growth experiment, the dependent variable would be plant growth.

You could measure this by measuring how much the plant grows every two days. You could also measure it by measuring the rate of photosynthesis. Either of these measurements are dependent upon the kind of light you give the plant.

## Case Study: Hepatitis B vaccines: a product of rDNA techniques

Open universiteit , Thames Polytechnic , in Biotechnological Innovations in Health Care , 1991

### Quality control of cells

A stock seed vial derived from a single colony is grown in fermentation broth for a period of about two days. During this interval, the yeast continually manufactures and stores HBsAg. Because many cell divisions occur during the large-scale fermentation, several parameters are considered important in the evaluation and comparison of plasmids within the master seed vial with plasmids within cells at the end of fermentation.

overall structural stability

intactness of the S-gene sequence

### Structural stability

The overall structural stability is assessed by restriction endonuclease mapping. This assay will detect (more than 0.1 kilo base pairs) deletions or rearrangements. With a panel of restriction endonucleases , it is possible to confirm identity in the sizes of restriction fragments of purified plasmid DNA isolated from cells either in the master seed or at the end of full-scale fermentation.

### Intactness of the S gene in master seed

To confirm the intactness of the coding region for the S gene, DNA sequence analysis is performed both for the master seed and for cells from a final time sample of a full-scale fermentation.

### Mitotic stability

At mitosis, yeast plasmids can segregate asymmetrically to the daughter cells. This segregation process is influenced by gene products encoded by the 2μ plasmid. In the course of large-scale fermentation, some plasmid loss may occur. Because it is desirable that a large and consistent percentage of the cells should retain plasmids, the degree of loss is measured by replica plating assays. The fraction of cells which retain the plasmid can be determined by testing the cell growth on leucine-deficient medium cells which have lost the expression plasmid are unable to grow on this medium.

### Copy number

A final parameter is the calculation of the number of expression plasmids in cells of the master seed compared with the number in cells after large-scale fermentation. After extraction of cells and restriction endonuclease digestion of DNA followed by agarose gel electrophoresis, the number of plasmids can be quantified relative to an appropriate internal control by densitometric scanning following staining or Southern blot hybridisation.

During the early stages of fermentation it is desirable to maintain a high plasmid number. This is achieved by using a defined selective medium, eg a leucine-deficient medium. During the later stages, in the larger fermentation vessels, it is desirable to use a more enriched medium which will favour the build-up of a high cell density and large quantities of HBsAg. The maintenance of plasmid numbers in the final fermentation as high as in the master seed is not critical, but it is important from the perspective of regulatory agencies that the copy number is consistent among all fermentations.

### Process control

In order to develop an efficient fermentation process, attention must be paid to:

the method by which cells in the stock seed are propagated through initial stages of growth to the stage of growth in the large fermentation

the growth media, both selective and enriched

cell growth curves, for maintaining cells for significant periods of time in their log phase

defined volumes transferred during scale-up

physical parameters of fermentation, eg pH dissolved oxygen agitation rate.

All of the above must be controlled to ensure high cellular viability, good production of HBsAg, and reproducibility of the process.

In comparing the stock seed vial and the large scale fermentation used in the production of hepatitis B vaccine, select the most appropriate techniques from the list provided to achieve the following objectives: i)

a comparison of the proportion of cells carrying the desired plasmids

a comparison of the number of plasmids per cell

a comparison of the coding region of the gene of the desired product

a comparison of the overall structure of the plasmids

Which of the techniques described would be suitable to determine the quality of the protein product?

## Watch the video: Exponential vs Logistic Growth (July 2022).

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