Information

3.1.2: Archaea - Biology

3.1.2: Archaea - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Learning Objectives

  • Describe why the Archaea have been difficult to place on the tree of life
  • Explain what it means to be an extremophile

When these microscopic organisms were first separated from bacteria in 1977. When their ribosomal RNA was sequenced, it became obvious that they bore no close relationship to the bacteria and seemed to be more closely related to the eukaryotes. For a time they were referred to as archaebacteria, but to emphasize their distinctness, we now call them Archaea.

Diversity of Archaea

Though archaeans are involved in many important ecological processes and present across Earth's ecosystems, they are most known for being extremophiles, existing in conditions that prevent most organisms from functioning:

  • thermophiles live at high temperatures
  • hyperthermophiles live at really high temperatures (present record is 121°C!)
  • psychrophiles (also called cryophiles) like it cold (one in the Antarctic grows best at 4°C)
  • halophiles live in very saline environments (like the Dead Sea)
  • acidophiles live at low pH (as low as pH 1 and who die at pH 7!)
  • alkaliphiles thrive at a high pH.

Methanogens

Methanogens are chemoautotrophs that use hydrogen as a source of electrons for reducing carbon dioxide to food. This process produces methane ("marsh gas", CH4) as a byproduct. These are found living in such anaerobic environments as:

  • the muck of swamps and marshes
  • the rumen of cattle (where they live on the hydrogen and CO2 produced by other microbes living along with them)
  • our colon (large intestine)
  • sewage sludge
  • the gut of termites

Two methanogens that have had their complete genomes sequenced areMethanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus.

Crenarchaeota

The first members of this group to be discovered like it really hot and so are called hyperthermophiles. One can grow at 121°C (the same temperature in the autoclaves used to sterilize culture media, surgical instruments, etc.). Many like it acidic as well as hot and live in acidic sulfur springs at a pH as low as 1 (the equivalent of dilute sulfuric acid). These use hydrogen as a source of electrons to reduce sulfur in order to get the energy they need to synthesize their food (from CO2).

Aeropyrum pernix is one member of the group that has had its genome completely sequenced. Other members of this group seem to make up a large fraction of the plankton in cool, marine waters and the microbes in both soil and the ocean that convert ammonia into nitrites (nitrification).

Evolutionary Position of the Archaea

The Archaea have a curious mix of traits characteristic of bacteria as well as traits found in eukaryotes (discussed in Chapter 2.1). Table (PageIndex{1}) summarizes some of them.

Table (PageIndex{1}): Traits that the Archaea have in common with Eukarya (left) and Bacteria (right).

Eukaryotic TraitsBacterial Traits
  • DNA replication machinery
  • Histones
  • Nucleosome-like structures
  • Transcription machinery
    • RNA polymerase
    • TFIIB
    • TATA-binding protein (TBP)
  • Translation machinery
    • initiation factors
    • ribosomal proteins
    • elongation factors
    • poisoned by diphtheria toxin
  • Single, circular chromosome
  • Operons
  • No introns
  • Bacterial-type membrane transport channels
  • Many metabolic processes
    • energy production
    • nitrogen-fixation
    • polysaccharide synthesis

Economic Importance of the Archaea

Because they have enzymes that can function at high temperatures, considerable effort is being made to exploit the Archaea for commercial processes such as providing enzymes to be added to detergents (maintain their activity at high temperatures and pH) and an enzyme to covert corn starch into dextrins. Archaea may also be enlisted to aid in cleaning up contaminated sites, e.g., petroleum spills.

There are no known pathogenic Archaea.

Summary

Archaea are a group of prokaryotes that were distinguished from Bacteria in the late 1970s. These organisms often exist in extreme environments and have diverse metabolic processes, including anaerobes that produce methane. Archaea are difficult to place in the phylogenetic tree of life. Though they share many prokaryotic traits with Bacteria, they seem to be genetically closer to Eukarya, including the processes used to replicate DNA and synthesize proteins.


3.1.2: Archaea - Biology

Campbell Biology Chapter 27

Mycoplasmas are bacteria that lack cell walls. On the basis of this structural feature, which statement concerning mycoplasmas should be true?
A) They are gram-negative.
B) They are subject to lysis in hypotonic conditions.
C) They lack a cell membrane as well.
D) They should contain less cellulose than do bacteria that possess cell walls.
E) They possess typical prokaryotic flagella.

B) They are subject to lysis in hypotonic conditions.

Though plants, fungi, and prokaryotes all have cell walls, we place them in different taxa. Which of these observations comes closest to explaining the basis for placing these organisms in different taxa, well before relevant data from molecular systematics became available?
A) Some closely resemble animals, which lack cell walls.
B) Their cell walls are composed of very different biochemicals.
C) Some have cell walls only for support.
D) Some have cell walls only for protection from herbivores.
E) Some have cell walls only to control osmotic balance.

B) Their cell walls are composed of very different biochemicals.

Which statement about bacterial cell walls is false?
A) Bacterial cell walls differ in molecular composition from plant cell walls.
B) Cell walls prevent cells from bursting in hypotonic environments.
C) Cell walls prevent cells from dying in hypertonic conditions.
D) Bacterial cell walls are similar in function to the cell walls of many protists, fungi, and plants.
E) Cell walls provide the cell with a degree of physical protection from the environment.

C) Cell walls prevent cells from dying in hypertonic conditions.

The predatory bacterium, Bdellovibrio bacteriophorus, drills into a prey bacterium and, once inside, digests it. In an attack upon a gram-negative bacterium that has a slimy cell covering, what is the correct sequence of structures penetrated by B. bacteriophorus on its way to the prey's cytoplasm?

1. membrane composed mostly of lipopolysaccharide
2. membrane composed mostly of phospholipids
3. peptidoglycan
4. capsule

A) 2, 4, 3, 1
B) 1, 3, 4, 2
C) 1, 4, 3, 2
D) 4, 1, 3, 2
E) 4, 3, 1, 2

Jams, jellies, preserves, honey, and other foodstuffs with high sugar content hardly ever become contaminated by bacteria, even when the food containers are left open at room temperature. This is because bacteria that encounter such an environment

A) undergo death by plasmolysis.
B) are unable to metabolize the glucose or fructose, and thus starve to death.
C) experience lysis.
D) are obligate anaerobes.
E) are unable to swim through these thick and viscous materials.

A) undergo death by plasmolysis.

In a bacterium that possesses antibiotic resistance and the potential to persist through very adverse conditions, such as freezing, drying, or high temperatures, DNA should be located within, or be part of, which structures?
1. nucleoid region
2. endospore
3. fimbriae
4. plasmids

C) 1 and 4 only
D) 2 and 4 only

Which two structures play direct roles in permitting bacteria to adhere to each other, or to other surfaces?
1. capsules
2. endospores
3. fimbriae
4. plasmids
5. flagella

A) 1 and 2
B) 1 and 3
C) 2 and 3

The typical prokaryotic flagellum features
A) an internal 9 + 2 pattern of microtubules.
B) an external covering provided by the plasma membrane.
C) a complex "motor" embedded in the cell wall and plasma membrane.
D) a basal body that is similar in structure to the cell's centrioles.
E) a membrane-enclosed organelle with motor proteins

C) a complex "motor" embedded in the cell wall and plasma membrane.

Prokaryotic ribosomes differ from those present in eukaryotic cytosol. Because of this, which of the following is correct?
A) Some antibiotics can block protein synthesis in bacteria without effects in the eukaryotic host.
B) Eukaryotes did not evolve from prokaryotes.
C) Translation can occur at the same time as transcription in eukaryotes but not in prokaryotes.
D) Some antibiotics can block the synthesis of peptidoglycan in the walls of bacteria.
E) Prokaryotes are able to use a much greater variety of molecules as food sources than can eukaryotes.

A) Some antibiotics can block protein synthesis in bacteria without effects in the eukaryotic host.

Which statement about the genomes of prokaryotes is correct?
A) Prokaryotic genomes are diploid throughout most of the cell cycle.
B) Prokaryotic chromosomes are sometimes called plasmids.
C) Prokaryotic cells have multiple chromosomes, "packed" with a relatively large amount of protein.
D) The prokaryotic chromosome is not contained within a nucleus but, rather, is found at the nucleolus.
E) Prokaryotic genomes are composed of circular DNA.

E) Prokaryotic genomes are composed of circular DNA.

If a bacterium regenerates from an endospore that did not possess any of the plasmids that were contained in its original parent cell, the regenerated bacterium will probably also
A) lack antibiotic-resistant genes.
B) lack a cell wall.
C) lack a chromosome.
D) lack water in its cytoplasm.
E) be unable to survive in its normal environment.

A) lack antibiotic-resistant genes.

Although not present in all bacteria, this cell covering often enables cells that possess it to resist the defenses of host organisms, especially their phagocytic cells.
A) endospore
B) sex pilus
C) cell wall
D) capsule

Prokaryotes' essential genetic information is located in the
A) nucleolus.
B) nucleoid.
C) nucleosome.
D) plasmids.
E) exospore.

Which of the following is an important source of endotoxin in gram-negative species?
A) endospore
B) sex pilus
C) flagellum
D) cell wall
E) capsule

Chloramphenicol is an antibiotic that targets prokaryotic (70S) ribosomes, but not eukaryotic (80S) ribosomes. Which of these questions stems from this observation, plus an understanding of eukaryotic origins?

A) Can chloramphenicol also be used to control human diseases that are caused by archaeans?
B) Can chloramphenicol pass through the capsules possessed by many cyanobacteria?
C) If chloramphenicol inhibits prokaryotic ribosomes, should it not also inhibit mitochondrial ribosomes?
D) Why aren't prokaryotic ribosomes identical to eukaryotic ribosomes?
E) How is translation affected in ribosomes that are targeted by chloramphenicol?

C) If chloramphenicol inhibits prokaryotic ribosomes, should it not also inhibit mitochondrial ribosomes?

In a hypothetical situation, the genes for sex pilus construction and for tetracycline resistance are located together on the same plasmid within a particular bacterium. If this bacterium readily performs conjugation involving a copy of this plasmid, then the result should be
A) a bacterium that has undergone transduction.
B) the rapid spread of tetracycline resistance to other bacteria in that habitat.
C) the subsequent loss of tetracycline resistance from this bacterium.
D) the production of endospores among the bacterium's progeny.
E) the temporary possession by this bacterium of a completely diploid genome.

B) the rapid spread of tetracycline resistance to other bacteria in that habitat.

Regarding prokaryotic genetics, which statement is correct?
A) Crossing over during prophase I introduces some genetic variation.
B) Prokaryotes feature the union of haploid gametes, as do eukaryotes.
C) Prokaryotes exchange some of their genes by conjugation, the union of haploid gametes, and transduction.
D) Mutation is a primary source of variation in prokaryote populations.
E) Prokaryotes skip sexual life cycles because their life cycle is too short.

D) Mutation is a primary source of variation in prokaryote populations.

Which of these statements about prokaryotes is correct?
A) Bacterial cells conjugate to mutually exchange genetic material.
B) Their genetic material is confined within vesicles known as plasmids.
C) They divide by binary fission, without mitosis or meiosis.
D) The persistence of bacteria throughout evolutionary time is due to their genetic homogeneity (in other words, sameness).
E) Genetic variation in bacteria is not known to occur, because of their asexual mode of reproduction.

C) They divide by binary fission, without mitosis or meiosis.

Which of the following is least associated with the others?
A) horizontal gene transfer
B) genetic recombination
C) conjugation
D) transformation
E) binary fission

In Fred Griffith's experiments, harmless R strain pneumococcus
became lethal S strain pneumococcus as the result of which of the
following?
1. horizontal gene transfer
2. transduction
3. conjugation
4. transformation
5. genetic recombination
A) 2 only
B) 4 only
C) 2 and 5
D) 1, 3 and 5

Hershey and Chase performed an elegant experiment that convinced most biologists that DNA, rather than protein, was the genetic material. This experiment subjected bacteria to the same gene transfer mechanism as occurs in
A) transduction.
B) transformation.
C) conjugation.
D) binary fission.
E) endosymbiosis.

Match the numbered terms to the description that follows.
Choose all appropriate terms.
1. autotroph
2. heterotroph
3. phototroph
4. chemotroph
a prokaryote that obtains both energy and carbon as it decomposes
dead organisms
A) 1 only
B) 4 only
C) 1 and 3
D) 2 and 4

Match the numbered terms to the description that follows.
Choose all appropriate terms.
1. autotroph
2. heterotroph
3. phototroph
4. chemotroph
an organism that obtains both carbon and energy by ingesting
prey
A) 1 only
B) 4 only
C) 1 and 3
D) 2 and 4
E) 1, 3 and 4

Match the numbered terms to the description that follows.
Choose all appropriate terms.
1. autotroph
2. heterotroph
3. phototroph
4. chemotroph
an organism that relies on photons to excite electrons within its
membranes
A) 1 only
B) 3 only
C) 1 and 3
D) 2 and 4
E) 1 3 and 4

Which of the following obtain energy by oxidizing inorganic
substancesⷄenergy that is used, in part, to fix CO₂?
A) photoautotrophs
B) photoheterotrophs
C) chemoautotrophs
D) chemoheterotrophs that perform decomposition
E) parasitic chemoheterotrophs

Mitochondria are thought to be the descendants of certain
alpha proteobacteria. They are, however, no longer able to lead
independent lives because most genes originally present on their
chromosome have moved to the nuclear genome. Which
phenomenon accounts for the movement of these genes?
A) plasmolysis
B) conjugation
C) translation
D) endocytosis
E) horizontal gene transfer

E) horizontal gene transfer

Carl Woese and collaborators identified two major branches of
prokaryotic evolution. What was the basis for dividing prokaryotes into two
domains?
A) microscopic examination of staining characteristics of the cell wall
B) metabolic characteristics such as the production of methane gas
C) metabolic characteristics such as chemoautotrophy and photosynthesis
D) genetic characteristics such as ribosomal RNA sequences
E) ecological characteristics such as the ability to survive in extreme
environments

D) genetic characteristics such as ribosomal RNA sequences

Which statement about the domain Archaea is true?
A) Genetic prospecting has recently revealed the existence of
many previously unknown archaean species.
B) No archaeans can reduce CO₂ to methane.
C) The genomes of archaeans are unique, containing no genes that
originated within bacteria.
D) No archaeans can inhabit solutions that are nearly 30% salt.
E) No archaeans are adapted to waters with temperatures above the
boiling point.

A) Genetic prospecting has recently revealed the existence of
many previously unknown archaean species.

If archaeans are more closely related to eukaryotes than to
bacteria, then which of the following is a reasonable prediction?
A) Archaean DNA should have no introns.
B) Archaean chromosomes should have no protein bonded to
them.
C) Archaean DNA should be single-stranded.
D) Archaean ribosomes should be larger than typical prokaryotic
ribosomes.
E) Archaeans should lack cell walls.

D) Archaean ribosomes should be larger than typical prokaryotic
ribosomes.

Which of the following traits do archaeans and bacteria share?
1. composition of the cell wall
2. presence of plasma membrane
3. lack of a nuclear envelope
4. identical rRNA sequences
A) 1 only
B) 3 only
C) 1 and 3
D) 2 and 3
E) 2 and 4

Assuming that each of these possesses a cell wall, which
prokaryotes should be expected to be most strongly resistant to
plasmolysis in hypertonic environments?
A) extreme halophiles
B) extreme thermophiles
C) methanogens
D) cyanobacteria
E) nitrogen-fixing bacteria that live in root nodules

The thermoacidophile, Sulfolobus acidocaldarius, lacks peptidoglycan,
but still possesses a cell wall. What is likely to be true of this species?
1. It is a bacterium.
2. It is an archaean.
3. The optimal pH of its enzymes will lie above pH 7.
4. The optimal pH of its enzymes will lie below pH 7.
5. It could inhabit certain hydrothermal springs.
6. It could inhabit alkaline hot springs.
A) 1, 3, and 6
B) 2, 4, and 6
C) 2, 4, and 5
D) 1, 3, and 5
E) 1, 4, and 5

33) A fish that has been salt-cured subsequently develops a reddish color. You suspect that the fish has been contaminated by the extreme halophile, Halobacterium. Which of these features of cells removed from the surface of the fish, if confirmed, would support your suspicion?
1. the presence of the same photosynthetic pigments found in cyanobacteria
2. cell walls that lack peptidoglycan
3. cells that are isotonic to conditions on the surface of the fish
4. cells containing bacteriorhodopsin
5. the presence of very large numbers of ion pumps in its plasma membrane
A) 2 and 5
B) 3 and 4
C) 1, 4, and 5
D) 3, 4, and 5

34) The termite gut protist, Mixotricha paradoxa, has at least two kinds of bacteria attached to its outer surface. One kind is a spirochete that propels its host through the termite gut. A second type of bacteria synthesizes ATP, some of which is used by the spirochetes. The locomotion provided by the spirochetes introduces the ATP-producing bacteria to new food sources. Which term(s) is (are) applicable to the relationship between the two kinds of bacteria?
1. mutualism
2. parasitism
3. symbiosis
4. metabolic cooperation
A) 1 only
B) 1 and 2

35) In general, what is the primary ecological role of prokaryotes?
A) parasitizing eukaryotes, thus causing diseases
B) breaking down organic matter
C) metabolizing materials in extreme environments
D) adding methane to the atmosphere
E)serving as primary producers in terrestrial environments

B) breaking down organic matter

36) If all prokaryotes on Earth suddenly vanished, which of the
following would be the most likely and most direct result?
A) The number of organisms on Earth would decrease by 10ⷄ20%.
B) Human populations would thrive in the absence of disease.
C) Bacteriophage numbers would dramatically increase.
D) The recycling of nutrients would be greatly reduced, at least
initially.
E) There would be no more pathogens on Earth.

D) The recycling of nutrients would be greatly reduced, at least
initially.

37) In a hypothetical situation, a bacterium lives on the surface of a leaf, where it obtains nutrition from the leaf's nonliving, waxy covering while inhibiting the growth of other microbes that are plant pathogens. If this bacterium gains access to the inside of a leaf, however, it causes a fatal disease in the plant. Once the plant dies, the bacterium and its offspring decompose the plant. What is the correct sequence of ecological roles played by the bacterium in the situation described here? Use only those that apply.
1. nutrient recycler
2. mutualist
3. commensal
4.parasite
5. primary producer
A) 1, 3, 4

38) Foods can be preserved in many ways by slowing or preventing bacterial
growth. Which of these methods should be least effective at inhibiting
bacterial growth?
A) Refrigeration: slows bacterial metabolism and growth.
B) Closing previously opened containers: prevents more bacteria from
entering, and excludes O₂.
C) Pickling: creates a pH at which most bacterial enzymes cannot function.
D) Canning in heavy sugar syrup: creates osmotic conditions that remove
water from most bacterial cells.
E) Irradiation: kills bacteria by mutating their DNA to such an extent that
their DNA-repair enzymes are overwhelmed.

B) Closing previously opened containers: prevents more bacteria from
entering, and excludes O₂.

39) Broad-spectrum antibiotics inhibit the growth of most
intestinal bacteria. Consequently, assuming that nothing is done
to counter the reduction of intestinal bacteria, a hospital patient
who is receiving broad-spectrum antibiotics is most likely to
become
A) unable to fix carbon dioxide.
B) antibiotic resistant.
C) unable to fix nitrogen.
D) unable to synthesize peptidoglycan.
E) deficient in certain vitamins and nutrients.

E) deficient in certain vitamins and nutrients.

52) Consider the thermoacidophile, Sulfolobus acidocaldarius. Which of the following graphs most accurately depicts the expected temperature and pH profiles of its enzymes? (Note: The horizontal axes of these graphs are double, with pH above and temperature below.)

A. SEE IMAGE
B. SEE IMAGE
C. SEE IMAGE
D. SEE IMAGE

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

53) This bacterium's ability to survive in a human who is taking penicillin pills may be due to the presence of
1. penicillin-resistance genes
2. a secretory system that removes penicillin from the cell
3. a gram-positive cell wall
4. a gram-negative cell wall
5. an endospore

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

54) Adherence to the intestinal lining by this bacterium is due to its possession of
A) fimbriae.
B) pili.
C) a capsule.
D) a flagellum.
E) a cell wall with an outer lipopolysaccharide membrane.

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

55) What should be true of the cell wall of this bacterium?
A) Its innermost layer is composed of a phospholipid bilayer.
B) After it has been subjected to Gram staining, the cell should remain purple.
C) It has an outer membrane of lipopolysaccharide.
D) It is mostly composed of a complex, cross-linked polysaccharide.
E) Two of the responses above are correct.

C) It has an outer membrane of lipopolysaccharide.

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

56) Some of the proteins that allow this bacterium to swim are related (in an evolutionary sense) to proteins that
A) attach to the single chromosome.
B) act as restriction enzymes.
C) synthesize peptidoglycan for the cell wall.
D) move penicillin out of the cell.
E) comprise its ribosomes.

D) move penicillin out of the cell.

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.
57) In which feature(s) should one be able to locate a complete chromosome
of this bacterium?
1. nucleolus
2. prophage
3. endospore
4. nucleoid

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

58) The cell also lacks F factors and F plasmids. Upon its death, this bacterium should be able to participate in
A) conjugation.
B) transduction.
C) transformation.
D) Three of the responses above are correct.
E) Two of the responses above are correct.

A hypothetical bacterium swims among human intestinal contents until it finds a suitable location on the intestinal lining. It adheres to the intestinal lining using a feature that also protects it from phagocytes, bacteriophages, and dehydration. Fecal matter from a human in whose intestine this bacterium lives can spread the bacterium, even after being mixed with water and boiled. The bacterium is not susceptible to the penicillin family of antibiotics. It contains no plasmids and relatively little peptidoglycan.

59) This bacterium derives nutrition by digesting human intestinal contents (in other words, food). Thus, this bacterium should be an
A) aerobic chemoheterotroph.
B) aerobic chemoautotroph.
C) anaerobic chemoheterotroph.
D) anaerobic chemoautotroph.

C) anaerobic chemoheterotroph.

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

63) Which two species should have much more phospholipid, in the form of bilayers, in their cytoplasms than most other bacteria?
A) species A and B
B) species A and C
C) species B and E
D) species C and D
E) species C and E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

64) Which species should be able to respond most readily to taxes (plural of taxis)?
A) species A
B) species B
C) species C
D) species D
E) species E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

65) How many of these species probably have a cell wall that partly consists of an outer membrane of lipopolysaccharide?
A) only one species
B) two species
C) three species
D) four species
E) all five species

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

66) Gram-variable prokaryotes are, sometimes, those without any peptidoglycan. Which two species are most likely to be archaeans?
A) species A and B
B) species A and C
C) species B and E
D) species C and D
E) species C and E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

67) Species D is pathogenic if it gains access to the human intestine. Which other species, if it coinhabited a human intestine along with species D, is most likely to result in a recombinant species that is both pathogenic and resistant to some antibiotics?
A) species A
B) species B
C) species C
D) species D
E) species E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

68) Which species might be able to include Hfr cells?
A) species A
B) species B
C) species C
D) species D
E) species E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

69) Which species is most self-sustaining in terms of obtaining nutrition in environments containing little fixed nitrogen or carbon?
A) species A
B) species B
C) species C
D) species D
E) species E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

70) Which two species might be expected to cooperate metabolically, perhaps forming a biofilm wherein one species surrounds cells of the other species?
A) species A and B
B) species A and C
C) species B and E
D) species C and D
E) species C and E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

71) Which species is most likely to be found both in sewage treatment plants and in the guts of cattle?
A) species A
B) species B
C) species C
D) species D
E) species E

The following table depicts characteristics of five prokaryotic species (A—E). Use the information in the table to answer the following question.

72) Which species is probably an important contributor to the base of aquatic food chains as a primary producer?
A) species A
B) species B
C) species C
D) species D
E) species E

73) Genetic variation in bacterial populations cannot result from
A) transduction.
B) transformation
C) conjugation
D) mutation.
E) meiosis.

74) Photoautotrophs use
A) light as an energy source and CO₂ as a carbon source.
B) light as an energy source and methane as a carbon source.
C) N₂ as an energy source and CO₂ as a carbon source.
D) CO₂ as both an energy source and a carbon source.
E) H₂S as an energy source and CO₂ as a carbon source.

A) light as an energy source and CO₂ as a carbon source.

75) Which of the following statements is not true?
A) Archaea and bacteria have different membrane lipids.
B) Both archaea and bacteria generally lack membrane-enclosed organelles.
C) The cell walls of archaea lack peptidoglycan.
D) Only bacteria have histones associated with DNA.
E) Only some archaea use CO₂ to oxidize H₂, releasing methane.

D) Only bacteria have histones associated with DNA.

76) Which of the following involves metabolic cooperation among prokaryotic cells?
A) binary fission
B) endospore formation
C) endotoxin release
D) biofilms
E) photoautotrophy

77) Bacteria perform each of the following ecological roles. Which role typically does not involve a symbiosis?
A) skin commensalist
B) decomposer
C) aggregates with methane-consuming archaea
D) gut mutualist
E) pathogen

78) Plantlike photosynthesis that releases O₂ occurs in
A) cyanobacteria.
B) chlamydias.
C) archaea.
D) actinomycetes.
E) chemoautotrophic bacteria.


Introduction

Extremely halophilic archaea are a diverse group of euryarchaeota that inhabit hypersaline environments (3–5 M) such as salt lakes, salt ponds, and marine salterns. They are often referred to as “halobacteria,” named after the model organism Halobacterium salinarum, whose proton pump bacteriorhodopsin is one of the best-studied membrane proteins. Although haloarchaea share certain features in order to adapt to their extreme environment, i.e. acidic protein machineries, respiratory chains and rhodopsins, their metabolism is considerably different from each other. Although there are carbohydrate-utilizing species such as Haloferax mediterranei, Haloarcula marismortui, and Halococcus saccharolyticus, which catabolize hexoses (glucose, fructose), pentoses (arabinose, xylulose), sucrose, and lactose (Rawal et al. 1988 Altekar and Rangaswamy 1992 Johnsen et al. 2001 Johnsen and Schonheit 2004), other haloarchaea like H. salinarum are not capable of sugar degradation (Rawal et al. 1988). Instead, non-carbohydrate-utilizing species thrive on amino acids and typical compounds of hypersaline habitats. Haloferax volcanii, for example, is able to grow on glycerol and organic acids (Kauri et al. 1990) excreted by primary halophilic producers Dunaliella salina (Elevi Bardavid et al. 2006) and Microcoleus chthonoplastes (Zviagintseva et al. 1995), respectively. Haloarchaea differ not only in their catabolic pathways but also in their nutritional requirements. While simple growth media were described for Haloferax volcanii (Kauri et al. 1990) and Natronomonas pharaonis (Falb et al. 2005), H. salinarum exhibits complex nutritional demands. Growth of Halobacterium cells is often limited in synthetic media (Oesterhelt and Krippahl 1973 Grey and Fitt 1976), in spite of rich amino acid (at least 10 amino acids) and cofactor supplements (folate, biotin, thiamine).

The metabolic diversity of halophilic archaea has not yet been investigated at the genomic level by metabolic reconstruction and comparative analysis. The absence of enzyme genes for numerous metabolic reactions in archaeal genomes has limited reconstruction of metabolic pathways so far. However, many of these pathway gaps have been elucidated recently with the discovery of novel non-orthologous enzymes in archaea, e.g. for the de novo synthesis of mevalonate (Barkley et al. 2004 Grochowski et al. 2006b), purines (Graupner et al. 2002 Ownby et al. 2005), and cobamide (Woodson et al. 2003 Woodson and Escalante-Semerena 2004 Zayas et al. 2006). Archaea also employ novel enzymes and precursors for pentose formation (Grochowski et al. 2005) and aromatic amino acid synthesis (White 2004 Porat et al. 2006) circumventing absent enzymes of the classical pentose-phosphate pathway.

For this detailed review of haloarchaeal metabolism, metabolic pathways of halophilic archaea were systematically reconstructed. Currently, genome sequences of four diverse haloarchaeal species are available for comparative analysis (Table 1), namely that of the model organism H. salinarum (Ng et al. 2000 Pfeiffer et al. 2008 http://www.halolex.mpg.de), the metabolic-versatile H. marismortui (Baliga et al. 2004), the haloalkaliphile N. pharaonis (Falb et al. 2005), and the square-shaped Haloquadratum walsbyi (Bolhuis et al. 2006). The presented metabolic data will be a valuable resource for future system biology approaches, as each metabolic reaction has been carefully assessed and linked to experimental data from the literature.


Acknowledgements

We would like to thank the two anonymous reviewers whose suggestions and critical remarks improved the manuscript. This work was supported by the Wellcome Trust, BBSRC and EPSRC . S.K. is supported by the BBSRC and the EPSRC . Predicted protein datasets were obtained from the sources specified in the electronic supplementary material. We thank each of the organizations and the respective genome-sequencing projects for making the sequence, gene model and annotation data publicly available.


From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

2 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan

Abstract

DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures.

1. Introduction

DNA ligases are critical DNA replication and repair enzymes they have been widely used in molecular biology and biotechnology applications, such as cloning and next-generation DNA sequencing [1, 2]. DNA ligases catalyze the joining of adjacent 3′-hydroxyl and 5′-phosphorylated DNA termini in duplex DNA. All DNA ligases accept nicked dsDNA and homologous, cohesive ends as substrates, although the minimum length of the overlap required for efficient ligation varies widely. Some ligases, most notably T4 DNA ligase, also accept fully base-paired (blunt end) substrates for in vitro ligation reactions.

DNA ligases share a common mechanism and a high degree of structural similarity with other members of the nucleotidyltransferase superfamily, including RNA ligases and RNA-capping enzymes [3]. Like other nucleotidyltransferases, DNA ligases utilize an ATP molecule to activate the enzyme, and thus DNA ligases are prepared to react with DNA substrates [4].

The details of each step in the DNA ligation reaction sequence have been clarified by many efforts performed by numerous researchers. Above all, Shuman’s group has made extensive contributions toward the elucidation of the DNA ligation mechanisms [5]. We describe the exemplified DNA ligation schemes in particular, in the second section of this review.

Since the discovery of DNA ligase, this enzyme has been widely utilized in several types of molecular biology and biotechnology applications. DNA ligase is frequently utilized in combination with restriction enzymes in recombinant DNA experiments. The enzyme has been applied in DNA sequencing methods in diverse ways, by taking advantage of the fact that DNA ligase does not require dNTPs as substrates for its function, since large amounts of dNTPs in solution, a general situation in DNA polymerase reactions, sometimes inhibit effective measurements and cause misinterpretations. DNA ligase is also utilized in analytical methods for protein-protein interactions, as developed by Landegren et al. [6]. These methods are widely applied for quick and effective in situ investigations of protein-protein interactions [7]. We will describe the details of the utilization of DNA ligase in several aspects of genetic engineering technology and molecular and cell biology in the third section.

Since the first report of the X-ray crystal structure of the ATP-dependent DNA ligase from bacteriophage T7 [8], the crystallographic analyses of the archaeal and eukaryotic ATP-dependent DNA ligases were subsequently reported during the first decade of the new century [9–11]. The ATP-dependent DNA ligases from Archaea and Eukarya comprise three domains, but, surprisingly, the relative arrangements of the three domains were completely different from each other in these reports [9–11], reflecting the dynamic domain motion in the 3-step ligation sequence. Based on these structural transitions in the ligation sequence, several attempts toward improving the enzymatic reaction have been made by mutating the enzymes. In the fourth and the fifth sections, we summarize the structural information about DNA ligase and the mutation-based improvements of its enzymatic efficacy.

2. Basis of the DNA Ligase Reaction

The Gellert, Lehman, Richardson, and Hurwitz laboratories discovered DNA ligases in 1967 and 1968 [12–15]. By joining the 3′-OH and 5′-phosphate termini (nicks) to form a phosphodiester bond, DNA ligases play an important role in maintaining genome integrity. They are essential for DNA replication and repair in all organisms [16, 17]. Furthermore, DNA ligases have long been a critical reagent in the development of molecular cloning and many subsequent aspects of DNA biotechnology.

All DNA ligation reactions entail sequential nucleotidyltransfer steps [18]. The reaction mechanism can be divided into three distinct catalytic events: the first (step 1), the activation of the enzyme through the covalent addition of AMP to the conserved catalytic lysine of the ligase, accompanied by the release of PPi or nicotinamide mononucleotide from the cofactor (ATP or NAD + ) the second (step 2), the binding of the ligase-adenylylate to the substrate DNA and the transfer of AMP from the ligase to the 5′-phosphoryl group of the nick on the DNA and the third (step 3), the formation of the phosphodiester-bond with the concomitant release of free AMP from the DNA-adenylylate intermediate (Figure 1). All three chemical steps depend on a divalent cation. The DNA ligases are grouped into two families, according to the cofactor dependence for the reaction. ATP-dependent DNA ligases are found in viruses, bacteria, Eukarya, and Archaea [18, 19], whereas NAD + -dependent DNA ligases are primarily present in bacteria and entomopox viruses [19, 20].

Sequence analyses of DNA ligases revealed that they share six motifs (I, III, IIIa, IV, V, and VI), which are also conserved in the nucleotidyltransferase superfamily members, including RNA ligase [21], tRNA ligase [22], and mRNA-capping enzymes [23], except for motif VI. In the ATP-dependent DNA ligases, these six motifs align well among the enzymes from viruses, bacteriophages, Archaea, and Eukarya, without long gaps or insertions (Figure 2). The sequences of the ATP-dependent DNA ligases showed that the amino acids in motifs I, III, IIIa, IV, V, and VI contact the substrates, AMP or DNA. The important roles of the individual amino acids in these motifs were verified by alanine scanning mutational studies and confirmed as essential for one or more steps of the ligation pathway [24]. For example, motif I (KxDGxR) contains the lysine that becomes covalently linked to AMP in the first step of the ligase reaction [25, 26]. Furthermore, the arginine and lysine side chains in motif VI (RxDK) orient the PPi leaving group apically relative to the attacking motif I lysine, during step 1 enzyme nucleotidylation reaction [27, 28].

3. Utilization of DNA Ligases in Genetic Research Technologies

The in vitro manipulation of DNA has been broadly applied to studies in molecular genetics, microbiology, immunology, and oncology and also to practical uses in clinical assays [29]. This technology was developed with DNA-related enzymes, which were discovered by research on the molecular mechanisms of the replication and repair of DNA. DNA replication is initiated by DNA primase, which synthesizes small RNA primers that are subsequently elongated by DNA polymerases [30]. Based on its primer extension activity, a DNA polymerase can be utilized in the polymerase chain reaction (PCR), which amplifies a single copy of DNA by several orders of magnitude in a brief in vitro reaction [31]. In addition, DNA ligase plays a role in joining Okazaki fragments during lagging strand maturation [32]. Based on its activity to join two DNA strands in vitro, DNA ligase has been contributing to recombinant DNA technology, for example, DNA cloning [33]. In addition, DNA ligase is broadly utilized for ligase-mediated mutation detection methods and DNA sequencing.

3.1. Oligonucleotide Ligation Assay (OLA)

DNA ligase can only join two DNA strands when they are perfectly hybridized to a complementary DNA sequence. Even a single base pair mismatch between two strands significantly decreases the efficiency of the strand joining reaction [34]. In 1988, the Oligonucleotide Ligation Assay (OLA) was developed, as a useful method to detect the genotype of the target DNA by utilizing this characteristic [6, 35]. This was the first example of the development of a method for gene analysis by using DNA ligase.

OLA consists of two steps. First, the two probes to be hybridized with the target DNA at the sequence of interest must lie directly adjacent to one another. In this case, one probe must contain a reporter group, which is either 32 P- or fluorescently labeled, and the other must include a recognition group for immobilization, such as a biotin group, which could be captured by streptavidin immobilized on a solid support. Second, the neighboring probes that are perfectly hybridized to the target DNA at the sequence of interest are joined by DNA ligase. Then, the ligation signal is subsequently captured by streptavidin and detected by autoradiography or fluorography (Figure 3). This assay takes advantage of the enzymatic accuracy in two events, the hybridization of the probes and the joining by DNA ligase, resulting in the reliable analysis of the clinical genotype.

A breakthrough in OLA for practical use occurred in 1990, when PCR was coupled to the assay prior to the ligation reaction (PCR-OLA) [36]. PCR-OLA amplification only for the target DNA enhanced the sensitivity of the assay, enabling the nonradioactive detection of the OLA results. Recently, a sensitive, specific, and high-throughput OLA was developed for the detection of genotypic human immunodeficiency virus type 1 (HIV-1) resistance to a Food and Drug Administration-approved protease [37]. This report revealed that OLA can be used for the detection of the HIV-1 genotype (wild type or mutant) as a genetic diagnosis method.

3.2. DNA Ligase-Mediated Cycling of the Ligation Reaction

Further spreading of DNA ligase-mediated technologies was achieved by cycling the ligation reaction, like PCR, using a thermostable DNA ligase, which became available commercially in 1990. The thermostable DNA ligase enables the cycling OLA reaction. OLA reaction method with thermal cycling procedure developed by using thermostable DNA ligase is often specifically termed Ligation Detection Reaction (LDR) (Figure 4). Through repeated denaturation, annealing, and ligation, the target signal is amplified linearly [38–40]. Ligation Chain Reaction (LCR) [38, 39, 41] and Ligation Amplification Reaction (LAR) [42] are also performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of probes, and ligation processes. After annealing the first set of two adjacent oligonucleotide probes to the target DNA sequence in a unique manner, a second set of complementary oligonucleotide probes that hybridize to the sequence opposite to the target DNA sequence is applied (Figure 5). Thereafter, a thermostable DNA ligase will covalently link each pair of adjacent probes that are completely hybridized at the junction of the adjacent probes. Through the repeated sequential processes, including denaturation, annealing, and ligation, the target signal is amplified exponentially. These methods require a thermostable DNA ligase to allow the ligation to occur under temperature conditions that prevent mismatches from hybridizing to form acceptable substrates. The thermostable DNA ligase made this assay useful for DNA-based diagnostic tests for inherited diseases in clinical laboratories. The ligation-mediated detection of point mutations by thermostable DNA ligase is now used to detect hepatitis B virus (HBV) mutants [43–45], colon tumor microsatellite sequence alterations [46], the mutation responsible for bovine leukocyte adhesion deficiency [47], AZT-resistance HIV mutants [48], mutations in the ras family of oncogenes [49], extended spectrum β-lactamase resistance in bacteria [50], and ganciclovir-resistant cytomegalovirus mutants [51].

3.3. Padlock Probe and Rolling-Circle Amplification (RCA)

Padlock probes and RCA are alternative methods for detecting known sequence variants in DNA and particularly for detecting single nucleotide polymorphisms, by using a thermostable DNA ligase. The padlock probe has target recognition sequences situated at both the 5′- and 3′-ends, connected by an intervening sequence that can include the sequence for detection. When the probe is hybridized to a target sequence, the two ends are brought adjacent to each other, and then a thermostable DNA ligase covalently joins the two ends and circularizes the probe. This circularized probe is wound around the target strand in a manner similar to a padlock, driven by the helical nature of the double-stranded DNA [52] (Figure 6). This probe is used for the localization of signals in in situ analyses. For example, a padlock probe was able to detect repeated alphoid sequences in metaphase chromosomes in a living cell, demonstrating its utility for in situ studies [53–55]. RCA is also known as a simple and efficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases, like Phi29, Bst, and Vent exo-DNA polymerases for DNA and T7 RNA polymerase for RNA, to generate long single stranded DNAs and RNAs containing tens to hundreds of tandem repeats that are complementary to the circular template [56–58]. The padlock principle has been combined with RCA for mutation detection, in which a primer annealing to the linker region initiates rolling circle replication (Figure 6), because the padlock approach alone is not sufficient to detect single nucleotide differences in a single copy gene in situ [59–61]. Point mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), p53, BRCA1, and Gorlin syndrome genes were visualized in interphase nuclei and DNA fibers by RCA [62]. These results demonstrated that ligase-mediated mutation detection methods coupled with RCA are able to reveal single nucleotide differences in a single cell. The ability to detect mutations in a cellular milieu has important implications for cancer research and diagnosis.

In another aspect of the RCA with ligation methods, Proximity Ligation Assay (PLA) is known for one of the potent techniques for detecting individual proteins or protein complexes in situ, by using antibodies with attached DNA strands that participate in ligation and subsequent RCA reactions [63]. PLA can be used for detecting reactions in which identification of target molecules depends on two recognition events. Formation of a proper target complex results in the formation of a circular DNA strand by exogenously added DNA ligase. This circular DNA is used to template a locally restricted RCA reaction, which generates an elongating ssDNA rolling-up in a ball that can be detected when hybridized with fluorescence-labeled probes [7]. The binding to a target interacting complex by two different antibodies with attached oligonucleotides individually (referred to as proximity probes) is followed by the addition of two more oligonucleotides that are then ligated into a circular DNA strand by the function of DNA ligase. The circular DNA strand is templated by the oligonucleotides attached to antibodies (Figure 7). Next, one of the antibody-bound oligonucleotides is utilized as a primer of the succeeding RCA reaction, resulting in the generation of an ssDNA rolling circle product. The rolling circle is covalently attached to one of the proximity probes. A 60 min RCA by Phi29 DNA polymerase results in a 1000-fold amplification of the 100 nt DNA circle, producing an around 100 kb rolling circle product [63]. The rolling circle product is then visualized by hybridization of fluorescence-labeled complementary oligonucleotide detection probes in situ.

3.4. Next-Generation DNA Sequencing by Using DNA Ligase

Recently, Next Generation Sequencing (NGS) technologies, such as the 454 FLX pyrosequencer (Roche) [64], Illumina genome analyzer (Illumina) [65], and Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (Life Technologies) [66], have revolutionized genomic and genetic research. Although these platforms are quite diverse, in terms of their sequencing biochemistry and sequence detection, their workflows are conceptually similar to each other. The array is prepared by random fragmentation of DNA, followed by in vitro ligation of common adaptor sequences by DNA ligase. DNA fragments with adapters are amplified by DNA polymerase and are detected by each platform. The detection platforms rely on sequencing by DNA synthetic methods, that is, the serial extension of primed templates. The enzyme driving the DNA synthesis can be either a DNA polymerase or a DNA ligase. The 454 FLX Pyrosequencer and Illumina analyzer use DNA polymerase methods with pyrosequencing [67] and reversible dye terminator technology, respectively [68]. In contrast, SOLiD uses DNA ligase and a unique approach to sequence the amplified fragments [69]. A universal primer complementary to the adaptor sequence is hybridized to the array of amplicons. Each cycle of sequencing involves the ligation of a degenerate, fluorescently labeled 8-mer probe set (Figure 8). The octamer mixture is structured, and the type of nucleotide (A, T, G, or C) at the specific position within the 8-mer probe set correlates with the type of fluorescent label. After the ligation of the 8-mer probes, images are acquired in four channels, resulting in the effective collection of sequencing data for the 3′ end positions of the probes across all template-bearing beads. Then, the octamer is chemically cleaved between positions 3 and 4, to remove the fluorescent label. Progressive rounds of octamer ligation enable the sequencing of every 5th base (e.g., bases 1, 6, 11, and 16 of the template strand). After several cycles of probe ligation reactions, the extended primer is denatured to reset the system. Subsequent iterations of this process can be applied to a different set of positions (e.g., bases 0, 5, 10, and 15 of the template strand), by using different mixtures of octamers in which the nucleotides at the different position are correlated with the label colors. An additional feature of this platform involves the use of two-base encoding, an error-correction scheme in which two adjacent bases, rather than a single base, are correlated with the label. Each base position is then queried twice (in a set of 2 bp interrogated in a given cycle) and thus miscalls can be more readily identified [70].

4. Structural Transition of the DNA Ligase in the Reaction Sequence

In Section 2, we showed that the sequence alignments clearly revealed several homologous regions among ATP-dependent DNA ligases and RNA capping enzymes, indicating the presence of five conserved motifs (I, III, IIIa, IV, and V) in these proteins. This fact suggests that the nucleotidyltransferase superfamily members, including ATP-dependent DNA ligases, may share a similar protein fold and a common reaction mechanism. Next, we will present a series of crystal structures of ATP-dependent DNA ligases and describe the structural and functional insights into the mechanisms of the enzymes.

4.1. Structural Studies of ATP-Dependent DNA Ligase

ATP-dependent DNA ligases show considerable variation in their molecular sizes, which range from 41 kDa (bacteriophage T7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]. Despite this wide size variation, it is clear from the sequence alignments that the smaller enzymes constitute a common core structure that is conserved across all ATP-dependent ligases [73]. The crystal structures of the ATP-dependent DNA ligases, such as bacteriophage T7 DNA ligase (T7Lig) complexed with ATP [8, 74] and Chlorella virus DNA ligase (ChvLig) with covalently bound AMP [75], have been solved (Figure 9(a), top). These DNA ligases adopt a common architecture of two distinct domains: the adenylylation domain (AdD) and the oligonucleotide/oligosaccharide-binding-fold domain (OBD), which are jointly called the catalytic core domains. The catalytic core domains are the minimal entity for the nick-joining activity, as seen in the ChvLig and T7Lig enzymes [8, 75]. The AdD contains the catalytic lysine residue that forms a covalent bond with AMP, which is derived from the substrate ATP. The Lys238 and Lys240 residues of T7Lig (Lys188 and Lys186 of ChvLig) within the AdD are essential for the adenylylation and nick sealing activities [76, 77]. The Lys240 residue forms a photo-crosslinking adduct with the 5′-terminal nucleotide of the nick, implying its direct involvement in binding the phosphate of the nick [78]. The OBD is connected to the AdD via the conserved motif V [5, 79] and is similar to other DNA and RNA binding proteins [80, 81]. The OBD is observed in the related nucleotidyltransferase, the mRNA capping enzyme from Chlorella virus, which undergoes opened-closed conformational changes during catalysis. The OBD domain of the enzyme was found to move towards AdD and close the nucleotide-binding pocket [80, 81].

In contrast, three crystal structures of large ATP-dependent DNA ligases, which mainly ligate the nicks between Okazaki fragment in DNA replication in Eukarya and Archaea, with an additional N-terminal DNA-binding domain (DBD), have been reported (Figure 9(a), bottom) [9–11, 82, 83]. This extra domain is not essential for hLigI activity in vitro [84, 85] but is considered to be crucial for detecting a singly nicked dsDNA [9]. The structures of the two archaeal DNA ligases from Pyrococcus furiosus (PfuLig) and Sulfolobus solfataricus (SsoLig) were determined in the closed [11] and extended forms [10], respectively. The structure of hLigI in complex with DNA [9] revealed that the enzyme entirely encircles the nicked-DNA. All of these DNA ligases are commonly composed of three domains (DBD, AdD, and OBD) in the sequences from the N to C termini. Although the protein folding of each domain is strikingly similar among the three DNA ligases, the relative domain orientations within each enzyme are quite different.

4.2. Structural Transition of DNA Ligase Molecules in Each Reaction Step

Based on the crystal structures described above, a ligation mechanism was proposed, as schematically represented in Figure 9(b). In solution, a DNA ligase partly adopts the extended form, as expected from the SsoLig crystal structure and the small angle X-ray scattering (SAXS) analysis [10]. In the absence of DNA and AMP, the OBD of SsoLig is turned away from the AdD in an open conformation, resembling that seen in the crystal structures of the compact and two-domain (AdD-OBD) DNA ligases from Chlorella virus and T7 (Figure 9(a)). The closed conformation of the catalytic core domains, which represents the active conformation for step 1, is observed in the crystal structures of PfuLig, as proposed previously [10, 11]. In step 2, tight binding between the enzyme and the substrate DNA occurs, as revealed by the hLigI–DNA complex crystal structure [9]. These structures depict three different phosphoryl transfer reactions, and the flexible multidomain structure of DNA ligases facilitates the adoption of different enzyme conformations during the course of the reaction (Figure 9(b)) [10]. A superimposition of the AdDs in PfuLig, SsoLig, and hLigI revealed that the arrangements of the OBD relative to the AdD in each ligase are apparently different from each other. Notably, the OBD from PfuLig is closely bound to the AdD and is replaced by the bound-DNA substrate in hLigI (Figure 10(a)). Ribbon diagrams of the AdDs from hLigI and PfuLig, together with the adjacent surface representations of the substrate DNA (hLigI) and motif VI (PfuLig), are shown in Figure 10(b). The electrostatic distribution on the motif VI surface facing AdD is negative, suggesting that motif VI is a molecular mimic of the incoming DNA substrate [11]. This finding indicates that the ligation could be completed simply by the smooth replacement between the substrate DNA and motif VI. Indeed, in the closed structure of PfuLig, motif VI approaches the active site in the absence of DNA, whereas it occupies a position in the region upstream from the nicked DNA in the structure of hLigI.

4.3. Interface of the Mandatory Domains (AdD and OBD) for the Enzymatic Reaction

In the ATP-dependent DNA ligases, the enzyme captures ATP to adenylylate a Lys residue in the first step, in which motif VI is also indispensable [27]. Motif VI in PfuLig provides several basic residues located around the AMP binding pocket. This electron density contributes to forming the compartment that traps the AMP molecule (Figure 11). In particular, R531 and K534 in motif VI, specific to the DNA ligases in Archaea and Eukarya, contribute to the basic surface within the pocket (Figure 11) [11].

The electrostatic potential distributions of the AdD-OBD interacting surfaces (Figure 12) revealed that the predominant charge distribution on each surface was oppositely charged, and this may be involved in stabilizing the closed conformation of the two catalytic core domains [11]. There is a small exit of the pocket through the closed conformation of the two domains (Figure 11), and the diameter of the exit is about 5.5 Å and could accommodate the PPi molecule but not the AMP moiety. The exit may serve for the spontaneous release of the PPi product. This notion is consistent with the fact that the DNA ligase from Pyrococcus horikoshii, which is more than 90% identical to PfuLig, cannot use NAD + as a nucleotide cofactor [86], because the pocket is too small to accommodate the nicotinamide ring to be released from the active site. Presumably, this compact “reaction room” of PfuLig would have been specifically designed to complete the conversion process from ATP to AMP within the closed pocket [11].

4.4. Role of the C-Terminal Helix Commonly Observed in the Archaeal and Eukaryotic DNA Ligases

The overall architectures of the three domains in hLigI and PfuLig are similar to each other (Figure 9(a)), although the sequence identities between the corresponding fragments of hLigI and PfuLig are moderate (32%). The crystal structures and sequence analyses revealed that the eukaryotic and archaeal DNA ligases possess a C-terminal helix shortly after motif VI (Figures 9(a) and 13(a)). The sequence extension harboring the C-terminal helix is conserved among the eukaryotic and archaeal DNA ligases, whereas the ligases from viruses (Chlorella virus (ChV)) and bacteriophages (bacteriophage T7 (T7)) lack this long extension (Figure 13(a)). The C-terminal helix is located at the boundary of the AdD and the OBD in the closed structure of PfuLig [11]. In the case of hLigI, this helix is distant from the domain interface, because of the bound DNA substrate (Figure 9(a)). In fact, a structural comparison between the DNA-hLigI complex and PfuLig suggested that the C-terminal helix might play a crucial role in switching from the tightly closed form to the DNA-substrate-bound form. The C-terminal helix of PfuLig connects the AdD and the OBD through five polar or ionic interactions (Figure 13(b)). This feature suggests that the helix might play a critical role in stabilizing the closed conformation of the catalytic core domains during step 1.

5. Engineered DNA Ligases with Improved Abilities

As described in Section 3, DNA ligases are critical for many applications in molecular biology. Improvements in the enzymatic ability, such as the nick sealing efficiency or fidelity, will provide better performance in the current methods for ligase-mediated mutation detection and NGS, as described above. Numerous improvements of DNA polymerases have been reported [87–89], whereas fewer are known for DNA ligases. Here we describe some reports on improvements of the enzymatic performances of DNA ligases.

5.1. Improvement of the Nick-Sealing Activity by Fusion with the Archaeal DNA Binding Domain

The ATP-dependent DNA ligase from bacteriophage T4 (T4Lig) has evolved to be a nick-sealing enzyme. It can also join double-stranded DNA (dsDNA) fragments with cohesive ends [90]. Furthermore, it is the only commercially available DNA ligase that can join blunt-ended DNA duplexes in vitro, in the absence of macromolecular enhancers such as polyethylene glycol [91, 92]. The ligation reaction of cohesive or blunt-ended dsDNA fragments is prerequisite for NGS, which requires the in vitro ligation of common adaptor sequences. However, the turnover numbers for the fragment joining reactions of T4Lig are lower than those for nick sealing, and T4Lig is approximately five orders of magnitude less efficient in joining blunt-ended duplexes than nick-sealing [92, 93]. T4Lig is also inefficient for ligating fragments with single base overhangs [94]. The poor fragment joining activity of T4Lig often leads to NGS failures. In order to improve the efficiency of fragment joining by T4Lig, Wilson et al. constructed a variety of chimeras of T4Lig fused with the DNA-binding domains from other enzymes [92]. This mutational strategy was inspired by a previous report, in which the genetic fusion of a sequence of a nonspecific archaeal DNA binding protein (Sso7d from Sulfolobus solfataricus) to Pfu DNA polymerases resulted in considerably increased processivity and improved performance in polymerase chain reaction (PCR) amplifications [88]. The fusion of T4Lig with Sso7d showed a 60% increase in performance over T4Lig in the cloning of a blunt-ended fragment [92].

5.2. Improvement of the Fidelity of Thermostable DNA Ligase by Site-Directed Mutagenesis

The specificity of DNA ligase is exploited in LCR and LDR analyses to distinguish single base mutations associated with genetic diseases. The ligation fidelity of T4Lig is improved by the presence of spermidine and by high salt and low ligase concentrations [6, 34]. However, the increased fidelity of T4Lig by adjusting the reaction conditions is not sufficient for the ideal detection of SNPs [6, 34, 95, 96]. The thermostable DNA ligases from Thermus aquaticus (Taq) and Thermus thermophilus (Tth) exhibited far greater fidelity than that reported for T4Lig [39, 97]. The ligation reactions at the higher temperature prevented mismatched hybridizations in the substrates. Luo et al. reported the further improvement of the fidelity of TthLig by site-directed mutagenesis. The fidelity of DNA polymerases was decreased by site-directed mutagenesis at the motif associated with primer-template binding or the exoIII motif [98–100]. However, the exoIII motif mutants, also known as “antimutator” strains, showed increased fidelity in which the balanced activities of the polymerizing and

exonuclease reactions might improve the overall fidelity [99–102]. Two mutant ligases, K294R and K294P, were identified with fidelities increased by

4-fold and 11-fold, respectively, besides retaining their nick sealing activities [97].

5.3. Structure-Based Mutational Study of an Archaeal DNA Ligase towards Improvement of the Ligation Activity

As mentioned in the previous section, thermostable DNA ligases are utilized in LCR and LDR, which require a heat denaturation step. However, thermostable DNA ligases possess weaker ligation activity at lower temperatures (20–40°C), resulting in the decreased efficiency of the LCR and LDR procedures using short probes with low-melting temperatures. Here we present a structure-guided mutational analysis of the hyperthermostable DNA ligase from P. furiosus, in order to improve the ligation efficiency with the thermostability [103]. In Section 4.4, we showed that the C-terminal helix in the closed structure observed in PfuLig connects the AdD and the OBD via five polar or ionic interactions (Figure 13(b)). In contrast, the crystal structures of hLigI and SsoLig revealed that the relative arrangements of the OBD and the AdD are quite different from that of PfuLig (Figure 9(a)). Taken together, we hypothesized that the binding efficiency of the DNA ligase to the DNA substrate would increase by reducing the ionic interactions between the AdD and the OBD, resulting in improved ligation efficiency. The ionic residues involved in the interactions between the AdD and the OBD were selected and mutated to create a series of mutants in which certain ionic residues are replaced or deleted. First, the series of the alanine mutants (1ala, 2ala, and 3ala), in which the ionic residues at the C-terminal helix are replaced with alanine residues, were prepared, and the series of deletion mutants (d4, d8, and d15) were also created. Then the initial reaction rates of these mutants were measured at the maximum temperature (60°C). The initial reaction rates were increased according to the number of replaced or deleted ionic residues (Figure 14(a)) [104].

Next, we found that the initial reaction rate was largely improved when the fourth ionic residue (Asp540) from the C-terminus was mutated, in addition to the 3ala mutations (D540A/3ala). Therefore the single alanine mutant of Asp540 (D540A) was created and assessed the effect of the mutation on the ligation efficiency. The reaction rate of D540A was almost the same as that of D540A/3ala [103], suggesting that the effect of the mutation of Asp540 dominates the improvement of the alanine mutations of the ionic residues on the C-terminal helix. Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair with Arg414, in the vicinity of the AMP binding site in the AdD (Figure 12).

A series of Asp540 mutants, in which Asp540 was replaced with serine (D540S), lysine (D540K), and arginine (D540R), were also constructed, and their ligation efficiencies were evaluated. As a result, the initial reaction rates of D540K and D540R were similarly the fastest among all of the mutants, followed by D540S, then D540A, and finally the wild type (Figure 14(b)). The evaluation of the ligation efficiencies of these Asp540 mutants over the broad temperature range from 20 to 80°C revealed that D540K and D540R were also the most productive. Thus, we successfully generated the PfuLig mutant with highly improved ligation efficiency over a broad temperature range, while maintaining its useful thermostability, by replacing Asp540 with a basic residue such as lysine or arginine (Figures 14(c) and 14(d)) [103].

Further investigation into the effects of the replacement of Asp540 with a basic residue on the ligation process revealed that the replacement contributed to both the adenylylation and DNA binding steps. These results suggested that the increase in the number of basic residues in the proximity of the active site was advantageous for the adenylylation step, in which the negatively charged pyrophosphate is pulled away from the ATP molecule in the active site pocket and that the alteration to the basic residue was also efficient for the AdD/OBD domain opening, caused by the repulsion between either Arg540 or Lys 540 and Arg414, which formed the ion pair with Asp540 (Figure 12) [103].

6. Conclusions

Archaea live in extreme conditions, even in the temperature range of 80°C to 100°C, and have yielded many useful enzymes for gene technology, such as hyperthermostable DNA polymerases and DNA ligases. In this review, we have described the utilization of thermostable DNA ligases in common molecular biology protocols and the structural mechanism of DNA ligase and shown some examples of protein-engineered DNA ligases. Improvements of these enzymes are potentially effective for advancing the existing methods mentioned above and beneficial for constructing novel biotechnological methods. Several protein engineering strategies, such as fusion with other proteins or site-direct mutagenesis based on structural information, may facilitate the construction of application-specific DNA ligases in the future. The enzymes can be improved artificially, based on their three-dimensional structures, even if they have naturally evolved for a long period of time.

As many Archaea thrive in extreme environments, it will be very interesting to learn how the fidelity mechanisms of these extremophilic organisms have adapted to overcome these harsh conditions. Therefore, archaeal enzymes will continue to play an important role in future research and will be employed in numerous aspects of gene technology.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors thank Professor K. Morikawa (Kyoto University) and Dr. S. Ishino (Kyushu University) for their support. Yoshizumi Ishino was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant nos. 23310152 and 26242075). The authors are also grateful to John Wiley & Sons Ltd., for the permission for the reuse of the figure (Figure

References

  1. B. Weiss and C. C. Richardson, “Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage,” Proceedings of the National Academy of Sciences of the United States of America, vol. 57, no. 4, pp. 1021–1028, 1967. View at: Publisher Site | Google Scholar
  2. H.-M. Eun, “DNA ligases,” in Enzymology Primer for Recombinant DNA Technology, pp. 109–133, Academic Press, San Diego, Calif, USA, 1996. View at: Google Scholar
  3. J. M. Pascal, “DNA and RNA ligases: structural variations and shared mechanisms,” Current Opinion in Structural Biology, vol. 18, no. 1, pp. 96–105, 2008. View at: Publisher Site | Google Scholar
  4. S. Shuman and B. Schwer, “RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases,” Molecular Microbiology, vol. 17, no. 3, pp. 405–410, 1995. View at: Publisher Site | Google Scholar
  5. S. Shuman, “Closing the gap on DNA ligase,” Structure, vol. 4, no. 6, pp. 653–656, 1996. View at: Google Scholar
  6. U. Landegren, R. Kaiser, J. Sanders, and L. Hood, “A ligase-mediated gene detection technique,” Science, vol. 241, no. 4869, pp. 1077–1080, 1988. View at: Publisher Site | Google Scholar
  7. O. Srberg, M. Gullberg, M. Jarvius et al., “Direct observation of individual endogenous protein complexes in situ by proximity ligation,” Nature Methods, vol. 3, no. 12, pp. 995–1000, 2006. View at: Publisher Site | Google Scholar
  8. H. S. Subramanya, A. J. Doherty, S. R. Ashford, and D. B. Wigley, “Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7,” Cell, vol. 85, no. 4, pp. 607–615, 1996. View at: Publisher Site | Google Scholar
  9. J. M. Pascal, P. J. O'Brien, A. E. Tomkinson, and T. Ellenberger, “Human DNA ligase I completely encircles and partially unwinds nicked DNA,” Nature, vol. 432, no. 7016, pp. 473–478, 2004. View at: Publisher Site | Google Scholar
  10. J. M. Pascal, O. V. Tsodikov, G. L. Hura et al., “A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA,” Molecular Cell, vol. 24, no. 2, pp. 279–291, 2006. View at: Publisher Site | Google Scholar
  11. H. Nishida, S. Kiyonari, Y. Ishino, and K. Morikawa, “The closed structure of an archaeal DNA ligase from Pyrococcus furiosus,” Journal of Molecular Biology, vol. 360, no. 5, pp. 956–967, 2006. View at: Publisher Site | Google Scholar
  12. S. B. Zimmerman, J. W. Little, C. K. Oshinsky, and M. Gellert, “Enzymatic joining of DNA strands: a novel reaction of diphosphopyridine nucleotide,” Proceedings of the National Academy of Sciences of the United States of America, vol. 57, no. 6, pp. 1841–1848, 1967. View at: Google Scholar
  13. I. R. Lehman, “DNA ligase: structure, mechanism, and function,” Science, vol. 186, no. 4166, pp. 790–797, 1974. View at: Publisher Site | Google Scholar
  14. M. J. Engler and C. C. Richardson, “DNA ligases,” in The Enzymes, P. D. Boyer, Ed., vol. 15, pp. 3–29, Academic Press, New York, NY, USA, 1982. View at: Google Scholar
  15. P. Sadowski, B. Ginsberg, A. Yudelevich, L. Feiner, and J. Hurwitz, “Enzymatic mechanisms of the repair and breakage of DNA,” Cold Spring Harbor Symposia on Quantitative Biology, vol. 33, pp. 165–177, 1968. View at: Google Scholar
  16. T. Lindahl and R. D. Wood, “Quality control by DNA repair,” Science, vol. 286, no. 5446, pp. 1897–1905, 1999. View at: Publisher Site | Google Scholar
  17. S. Waga and B. Stillman, “The DNA replication fork in eukaryotic cells,” Annual Review of Biochemistry, vol. 67, pp. 721–751, 1998. View at: Publisher Site | Google Scholar
  18. T. Ellenberger and A. E. Tomkinson, “Eukaryotic DNA ligases: structural and functional insights,” Annual Review of Biochemistry, vol. 77, pp. 313–338, 2008. View at: Publisher Site | Google Scholar
  19. A. Wilkinson, J. Day, and R. Bowater, “Bacterial DNA ligases,” Molecular Microbiology, vol. 40, no. 6, pp. 1241–1248, 2001. View at: Publisher Site | Google Scholar
  20. V. Sriskanda, R. W. Moyer, and S. Shuman, “NAD + -dependent DNA ligase encoded by a eukaryotic virus,” The Journal of Biological Chemistry, vol. 276, no. 39, pp. 36100–36109, 2001. View at: Publisher Site | Google Scholar
  21. Z. A. Wood, R. S. Sabatini, and S. L. Hajduk, “RNA ligase: picking up the pieces,” Molecular Cell, vol. 13, no. 4, pp. 455–456, 2004. View at: Publisher Site | Google Scholar
  22. L. K. Wang and S. Shuman, “Structure-function analysis of yeast tRNA ligase,” RNA, vol. 11, no. 6, pp. 966–975, 2005. View at: Publisher Site | Google Scholar
  23. R. Sawaya and S. Shuman, “Mutational analysis of the guanylyltransferase component of mammalian mRNA capping enzyme,” Biochemistry, vol. 42, no. 27, pp. 8240–8249, 2003. View at: Publisher Site | Google Scholar
  24. S. Shuman, “DNA ligases: progress and prospects,” The Journal of Biological Chemistry, vol. 284, no. 26, pp. 17365–17369, 2009. View at: Publisher Site | Google Scholar
  25. V. Sriskanda and S. Shuman, “Chlorella virus DNA ligase: nick recognition and mutational analysis,” Nucleic Acids Research, vol. 26, no. 2, pp. 525–531, 1998. View at: Publisher Site | Google Scholar
  26. V. Sriskanda and S. Shuman, “Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase,” Nucleic Acids Research, vol. 30, no. 4, pp. 903–911, 2002. View at: Google Scholar
  27. V. Sriskanda and S. Shuman, “Mutational analysis of Chlorella virus DNA ligase: Catalytic roles of domain I and motif VI,” Nucleic Acids Research, vol. 26, no. 20, pp. 4618–4625, 1998. View at: Publisher Site | Google Scholar
  28. P. Samai and S. Shuman, “Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation,” The Journal of Biological Chemistry, vol. 287, no. 34, pp. 28609–28618, 2012. View at: Publisher Site | Google Scholar
  29. C. A. Foy and H. C. Parkes, “Emerging homogeneous DNA-based technologies in the clinical laboratory,” Clinical Chemistry, vol. 47, no. 6, pp. 990–1000, 2001. View at: Google Scholar
  30. R. C. Conaway and I. R. Lehman, “A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos,” Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 8, pp. 2523–2527, 1982. View at: Publisher Site | Google Scholar
  31. H. A. Erlich, “Polymerase chain reaction,” Journal of Clinical Immunology, vol. 9, no. 6, pp. 437–447, 1989. View at: Publisher Site | Google Scholar
  32. D. S. Levin, W. Bai, N. Yao, M. O'Donnell, and A. E. Tomkinson, “An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 24, pp. 12863–12868, 1997. View at: Publisher Site | Google Scholar
  33. N. G. Copeland, N. A. Jenkins, and D. L. Court, “Recombineering: a powerful new tool for mouse functional genomics,” Nature Reviews Genetics, vol. 2, no. 10, pp. 769–779, 2001. View at: Publisher Site | Google Scholar
  34. D. Y. Wu and R. B. Wallace, “Specificity of the nick-closing activity of bacteriophage T4 DNA ligase,” Gene, vol. 76, no. 2, pp. 245–254, 1989. View at: Publisher Site | Google Scholar
  35. A. M. Alves and F. J. Carr, “Dot blot detection of point mutations with adjacently hybridising synthetic oligonucleotide probes,” Nucleic Acids Research, vol. 16, no. 17, article 8723, 1988. View at: Publisher Site | Google Scholar
  36. D. A. Nickerson, R. Kaiser, S. Lappin, J. Stewart, L. Hood, and U. Landegren, “Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 22, pp. 8923–8927, 1990. View at: Publisher Site | Google Scholar
  37. I. A. Beck, M. Mahalanabis, G. Pepper et al., “Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors,” Journal of Clinical Microbiology, vol. 40, no. 4, pp. 1413–1419, 2002. View at: Publisher Site | Google Scholar
  38. F. Barany, “The ligase chain reaction in a PCR world,” Genome Research, vol. 1, no. 1, pp. 5–16, 1991. View at: Google Scholar
  39. F. Barany, “Genetic disease detection and DNA amplification using cloned thermostable ligase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 1, pp. 189–193, 1991. View at: Publisher Site | Google Scholar
  40. M. Khanna, W. Cao, M. Zirvi, P. Paty, and F. Barany, “Ligase detection reaction for identification of low abundance mutations,” Clinical Biochemistry, vol. 32, no. 4, pp. 287–290, 1999. View at: Publisher Site | Google Scholar
  41. H. H. Lee, “Ligase chain reaction,” Biologicals, vol. 24, no. 3, pp. 197–199, 1996. View at: Publisher Site | Google Scholar
  42. D. Y. Wu and R. B. Wallace, “The ligation amplification reaction (LAR)-amplification of specific DNA sequences using sequential rounds of template-dependent ligation,” Genomics, vol. 4, no. 4, pp. 560–569, 1989. View at: Publisher Site | Google Scholar
  43. S. Minamitani, S. Nishiguchi, T. Kuroki, S. Otani, and T. Monna, “Detection by ligase chain reaction of precore mutant of hepatitis B virus,” Hepatology, vol. 25, no. 1, pp. 216–222, 1997. View at: Publisher Site | Google Scholar
  44. V. D. Karthigesu, M. Mendy, M. Fortuin, H. C. Whittle, C. R. Howard, and L. M. C. Allison, “The ligase chain reaction distinguishes hepatitis B virus S-gene variants,” FEMS Microbiology Letters, vol. 131, no. 2, pp. 127–132, 1995. View at: Publisher Site | Google Scholar
  45. C. Osiowy, “Sensitive detection of HBsAG mutants by a gap ligase chain reaction assay,” Journal of Clinical Microbiology, vol. 40, no. 7, pp. 2566–2571, 2002. View at: Publisher Site | Google Scholar
  46. M. Zirvi, T. Nakayama, G. Newman, T. McCaffrey, P. Paty, and F. Barany, “Ligase-based detection of mononucleotide repeat sequences,” Nucleic Acids Research, vol. 27, no. 24, pp. e40i–e40viii, 1999. View at: Publisher Site | Google Scholar
  47. C. A. Batt, P. Wagner, M. Wiedmann, and R. Gilbert, “Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction,” Animal Genetics, vol. 25, no. 2, pp. 95–98, 1994. View at: Publisher Site | Google Scholar
  48. K. Abravaya, J. J. Carrino, S. Muldoon, and H. H. Lee, “Detection of point mutations with a modified ligase chain reaction (Gap-LCR),” Nucleic Acids Research, vol. 23, no. 4, pp. 675–682, 1995. View at: Publisher Site | Google Scholar
  49. V. L. Wilson, Q. Wei, K. R. Wade et al., “Needle-in-a-haystack detection and identification of base substitution mutations in human tissues,” Mutation Research, vol. 406, no. 2𠄴, pp. 79–100, 1999. View at: Publisher Site | Google Scholar
  50. C. Niederhauser, L. Kaempf, and I. Heinzer, “Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 19, no. 6, pp. 477–480, 2000. View at: Google Scholar
  51. C. Bourgeois, N. Sixt, J. B. Bour, and P. Pothier, “Value of a ligase chain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirus,” Journal of Virological Methods, vol. 67, no. 2, pp. 167–175, 1997. View at: Publisher Site | Google Scholar
  52. M. Szemes, P. Bonants, M. de Weerdt, J. Baner, U. Landegren, and C. D. Schoen, “Diagnostic application of padlock probes-multiplex detection of plant pathogens using universal microarrays,” Nucleic Acids Research, vol. 33, no. 8, article e70, 2005. View at: Google Scholar
  53. M. Nilsson, H. Malmgren, M. Samiotaki, M. Kwiatkowski, B. P. Chowdhary, and U. Landegren, “Padlock probes: circularizing oligonucleotides for localized DNA detection,” Science, vol. 265, no. 5181, pp. 2085–2088, 1994. View at: Publisher Site | Google Scholar
  54. M. Nilsson, K. Krejci, J. Koch, M. Kwiatkowski, P. Gustavsson, and U. Landegren, “Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21,” Nature Genetics, vol. 16, no. 3, pp. 252–255, 1997. View at: Publisher Site | Google Scholar
  55. D.-O. Antson, M. Mendel-Hartvig, U. Landegren, and M. Nilsson, “PCR-generated padlock probes distinguish homologous chromosomes through quantitative fluorescence analysis,” European Journal of Human Genetics, vol. 11, no. 5, pp. 357–363, 2003. View at: Publisher Site | Google Scholar
  56. A. Fire and S.-Q. Xu, “Rolling replication of short DNA circles,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 10, pp. 4641–4645, 1995. View at: Publisher Site | Google Scholar
  57. D. Liu, S. L. Daubendiek, M. A. Zillman, K. Ryan, and E. T. Kool, “Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases,” Journal of the American Chemical Society, vol. 118, no. 7, pp. 1587–1594, 1996. View at: Publisher Site | Google Scholar
  58. S. L. Daubendiek and E. T. Kool, “Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles,” Nature Biotechnology, vol. 15, no. 3, pp. 273–277, 1997. View at: Publisher Site | Google Scholar
  59. P. M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, and D. C. Ward, “Mutation detection and single-molecule counting using isothermal rolling-circle amplification,” Nature Genetics, vol. 19, no. 3, pp. 225–232, 1998. View at: Publisher Site | Google Scholar
  60. J. Banér, M. Nilsson, M. Mendel-Hartvig, and U. Landegren, “Signal amplification of padlock probes by rolling circle replication,” Nucleic Acids Research, vol. 26, no. 22, pp. 5073–5078, 1998. View at: Publisher Site | Google Scholar
  61. C. Larsson, J. Koch, A. Nygren et al., “In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes,” Nature Methods, vol. 1, no. 3, pp. 227–232, 2004. View at: Google Scholar
  62. X.-B. Zhong, P. M. Lizardi, X.-H. Huang, P. L. Bray-Ward, and D. C. Ward, “Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 7, pp. 3940–3945, 2001. View at: Publisher Site | Google Scholar
  63. O. Srberg, K.-J. Leuchowius, M. Gullberg et al., “Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay,” Methods, vol. 45, no. 3, pp. 227–232, 2008. View at: Publisher Site | Google Scholar
  64. M. Margulies, M. Egholm, W. E. Altman et al., “Genome sequencing in microfabricated high-density picolitre reactors,” Nature, vol. 437, no. 7057, pp. 376–380, 2005. View at: Publisher Site | Google Scholar
  65. D. R. Bentley, “Whole-genome re-sequencing,” Current Opinion in Genetics & Development, vol. 16, no. 6, pp. 545–552, 2006. View at: Publisher Site | Google Scholar
  66. E. R. Mardis, “Next-generation DNA sequencing methods,” Annual Review of Genomics and Human Genetics, vol. 9, pp. 387–402, 2008. View at: Publisher Site | Google Scholar
  67. M. Ronaghi, M. Uhlén, and P. Nyrén, “A sequencing method based on real-time pyrophosphate,” Science, vol. 281, no. 5375, pp. 363–365, 1998. View at: Publisher Site | Google Scholar
  68. O. Morozova and M. A. Marra, “Applications of next-generation sequencing technologies in functional genomics,” Genomics, vol. 92, no. 5, pp. 255–264, 2008. View at: Publisher Site | Google Scholar
  69. J. Shendure, G. J. Porreca, N. B. Reppas et al., “Accurate multiplex colony sequencing of an evolved bacterial genome,” Science, vol. 309, no. 5741, pp. 1728–1732, 2005. View at: Publisher Site | Google Scholar
  70. J. Shendure and H. Ji, “Next-generation DNA sequencing,” Nature Biotechnology, vol. 26, no. 10, pp. 1135–1145, 2008. View at: Publisher Site | Google Scholar
  71. J. J. Dunn and F. W. Studier, “Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4,” Journal of Molecular Biology, vol. 148, no. 4, pp. 303–330, 1981. View at: Publisher Site | Google Scholar
  72. D. E. Barnes, L. H. Johnston, K. I. Kodama, A. E. Tomkinson, D. D. Lasko, and T. Lindahl, “Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 17, pp. 6679–6683, 1990. View at: Google Scholar
  73. A. Kletzin, “Molecular characterisation of a DNA ligase gene of the extremely thermophilic archaeon Desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases,” Nucleic Acids Research, vol. 20, no. 20, pp. 5389–5396, 1992. View at: Publisher Site | Google Scholar
  74. A. J. Doherty, S. R. Ashford, H. S. Subramanya, and D. B. Wigley, “Bacteriophage T7 DNA ligase: overexpression, purification, crystallization, and characterization,” The Journal of Biological Chemistry, vol. 271, no. 19, pp. 11083–11089, 1996. View at: Publisher Site | Google Scholar
  75. M. Odell, V. Sriskanda, S. Shuman, and D. B. Nikolov, “Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining,” Molecular Cell, vol. 6, no. 5, pp. 1183–1193, 2000. View at: Publisher Site | Google Scholar
  76. A. J. Doherty and T. R. Dafforn, “Nick recognition by DNA ligases,” Journal of Molecular Biology, vol. 296, no. 1, pp. 43–56, 2000. View at: Publisher Site | Google Scholar
  77. V. Sriskanda and S. Shuman, “Role of nucleotidyl transferase motif V in strand joining by Chlorella virus DNA ligase,” The Journal of Biological Chemistry, vol. 277, no. 12, pp. 9661–9667, 2002. View at: Publisher Site | Google Scholar
  78. D. Suck, “Common fold, common function, common origin?” Nature Structural & Molecular Biology, vol. 4, no. 3, pp. 161–165, 1997. View at: Google Scholar
  79. A. G. Murzin, “OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences,” The EMBO Journal, vol. 12, no. 3, pp. 861–867, 1993. View at: Google Scholar
  80. K. Håkansson, A. J. Doherty, S. Shuman, and D. B. Wigley, “X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes,” Cell, vol. 89, no. 4, pp. 545–553, 1997. View at: Google Scholar
  81. A. V. Cherepanov and S. de Vries, “Dynamic mechanism of nick recognition by DNA ligase,” European Journal of Biochemistry, vol. 269, no. 24, pp. 5993–5999, 2002. View at: Publisher Site | Google Scholar
  82. D. J. Kim, O. Kim, H.-W. Kim, H. S. Kim, S. J. Lee, and S. W. Suh, “ATP-dependent DNA ligase from Archaeoglobus fulgidus displays a tightly closed conformation,” Acta Crystallographica Section F: Structural Biology and Crystallization Communications, vol. 65, no. 6, pp. 544–550, 2009. View at: Publisher Site | Google Scholar
  83. T. Petrova, E. Y. Bezsudnova, K. M. Boyko et al., “ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain,” Acta Crystallographica Section F: Structural Biology and Crystallization Communications, vol. 68, no. 12, pp. 1440–1447, 2012. View at: Publisher Site | Google Scholar
  84. M. C. Cardoso, C. Joseph, H. P. Rahn, R. Reusch, B. Nadal-Ginard, and H. Leonhardt, “Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo,” The Journal of Cell Biology, vol. 139, no. 3, pp. 579–587, 1997. View at: Publisher Site | Google Scholar
  85. C. Prigent, D. D. Lasko, K. Kodama, J. R. Woodgett, and T. Lindahl, “Activation of mammalian DNA ligase I through phosphorylation by casein kinase II,” The EMBO Journal, vol. 11, no. 8, pp. 2925–2933, 1992. View at: Google Scholar
  86. N. Keppetipola and S. Shuman, “Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii,” Journal of Bacteriology, vol. 187, no. 20, pp. 6902–6908, 2005. View at: Publisher Site | Google Scholar
  87. H. Echols and M. F. Goodman, “Fidelity mechanisms in DNA replication,” Annual Review of Biochemistry, vol. 60, pp. 477–511, 1991. View at: Google Scholar
  88. Y. Wang, D. E. Prosen, L. Mei, J. C. Sullivan, M. Finney, and P. B. Vander Horn, “A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro,” Nucleic Acids Research, vol. 32, no. 3, pp. 1197–1207, 2004. View at: Publisher Site | Google Scholar
  89. T. Kuroita, H. Matsumura, N. Yokota et al., “Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases,” Journal of Molecular Biology, vol. 351, no. 2, pp. 291–298, 2005. View at: Publisher Site | Google Scholar
  90. B. H. Pheiffer and S. B. Zimmerman, “Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonudcleotides by T4 DNA ugase in polymer solutions,” Nucleic Acids Research, vol. 11, no. 22, pp. 7853–7871, 1983. View at: Publisher Site | Google Scholar
  91. V. Sgaramella, J. H. van de Sande, and H. G. Khorana, “Studies on polynucleotides, C. A novel joining reaction catalyzed by the T4-polynucleotide ligase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 67, no. 3, pp. 1468–1475, 1970. View at: Google Scholar
  92. R. H. Wilson, S. K. Morton, H. Deiderick et al., “Engineered DNA ligases with improved activities in vitro,” Protein Engineering, Design & Selection, vol. 26, no. 7, pp. 471–478, 2013. View at: Publisher Site | Google Scholar
  93. A. Sugino, H. M. Goodman, H. L. Heyneker, J. Shine, H. W. Boyer, and N. R. Cozzarelli, “Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base paired ends,” The Journal of Biological Chemistry, vol. 252, no. 11, pp. 3987–3994, 1977. View at: Google Scholar
  94. G. J. S. Lohman, S. Tabor, and N. M. Nichols, “DNA ligases,” in Current Protocols in Molecular Biology, vol. 94, chapter 3, unit 3.14, pp. 1–7, 2011. View at: Google Scholar
  95. K. Harada and L. E. Orgel, “Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection,” Nucleic Acids Research, vol. 21, no. 10, pp. 2287–2291, 1993. View at: Publisher Site | Google Scholar
  96. C. Goffin, V. Bailly, and W. G. Verly, “Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase,” Nucleic Acids Research, vol. 15, no. 21, pp. 8755–8771, 1987. View at: Publisher Site | Google Scholar
  97. J. Luo, D. E. Bergstrom, and F. Barany, “Improving the fidelity of Thermus thermophilus DNA ligase,” Nucleic Acids Research, vol. 24, no. 15, pp. 3071–3078, 1996. View at: Publisher Site | Google Scholar
  98. W. A. Beard, S. J. Stahl, H.-R. Kim et al., “Structure/function studies of human immunodeficiency virus type 1 reverse transcriptase. Alanine scanning mutagenesis of an α -helix in the thumb subdomain,” The Journal of Biological Chemistry, vol. 269, no. 45, pp. 28091–28097, 1994. View at: Google Scholar
  99. L. J. Reha-Krantz, R. L. Nonay, and S. Stocki, “Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid,” Journal of Virology, vol. 67, no. 1, pp. 60–66, 1993. View at: Google Scholar
  100. L. J. Reha-Krantz and R. L. Nonay, “Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity,” The Journal of Biological Chemistry, vol. 269, no. 8, pp. 5635–5643, 1994. View at: Google Scholar
  101. Q. Dong, W. C. Copeland, and T. S.-F. Wang, “Mutational studies of human DNA polymerase,” The Journal of Biological Chemistry, vol. 268, no. 32, pp. 24163–24174, 1993. View at: Google Scholar
  102. W. C. Copeland, N. K. Lam, and T. S.-F. Wang, “Fidelity studies of the human DNA polymerase α. The most conserved region among α-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis,” The Journal of Biological Chemistry, vol. 268, no. 15, pp. 11041–11049, 1993. View at: Google Scholar
  103. M. Tanabe, S. Ishino, M. Yohda, K. Morikawa, Y. Ishino, and H. Nishida, “Structure-based mutational study of an archaeal DNA ligase towards improvement of ligation activity,” ChemBioChem, vol. 13, no. 17, pp. 2575–2582, 2012. View at: Publisher Site | Google Scholar
  104. M. Tanabe, S. Ishino, Y. Ishino, and H. Nishida, “Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity,” FEBS Letters, vol. 588, no. 2, pp. 230–235, 2014. View at: Publisher Site | Google Scholar
  105. E. F. Pettersen, T. D. Goddard, C. C. Huang et al., “UCSF Chimera𠅊 visualization system for exploratory research and analysis,” Journal of Computational Chemistry, vol. 25, no. 13, pp. 1605–1612, 2004. View at: Publisher Site | Google Scholar

Copyright

Copyright © 2015 Maiko Tanabe et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


3.1.2: Archaea - Biology

13) Prokaryotes' essential genetic information is located in the

14) Which of the following is an important source of endotoxin in gram-negative species?

15) Chloramphenicol is an antibiotic that targets prokaryotic (70S) ribosomes, but not eukaryotic (80S) ribosomes. Which of these questions stems from this observation, plus an understanding of eukaryotic origins?

A) Can chloramphenicol also be used to control human diseases that are caused by archaeans?

B) Can chloramphenicol pass through the capsules possessed by many cyanobacteria?

C) If chloramphenicol inhibits prokaryotic ribosomes, should it not also inhibit mitochondrial ribosomes?

D) Why aren't prokaryotic ribosomes identical to eukaryotic ribosomes?

E) How is translation affected in ribosomes that are targeted by chloramphenicol?

16) In a hypothetical situation, the genes for sex pilus construction and for tetracycline resistance are located together on the same plasmid within a particular bacterium. If this bacterium readily performs conjugation involving a copy of this plasmid, then the result should be

A) a bacterium that has undergone transduction.

B) the rapid spread of tetracycline resistance to other bacteria in that habitat.

C) the subsequent loss of tetracycline resistance from this bacterium.

D) the production of endospores among the bacterium's progeny.

E) the temporary possession by this bacterium of a completely diploid genome.

17) Regarding prokaryotic genetics, which statement is correct?

A) Crossing over during prophase I introduces some genetic variation.

B) Prokaryotes feature the union of haploid gametes, as do eukaryotes.

C) Prokaryotes exchange some of their genes by conjugation, the union of haploid gametes, and transduction.

D) Mutation is a primary source of variation in prokaryote populations.

E) Prokaryotes skip sexual life cycles because their life cycle is too short.

18) Which of these statements about prokaryotes is correct?

A) Bacterial cells conjugate to mutually exchange genetic material.

B) Their genetic material is confined within vesicles known as plasmids.

C) They divide by binary fission, without mitosis or meiosis.

D) The persistence of bacteria throughout evolutionary time is due to their genetic homogeneity (in other words, sameness).

E) Genetic variation in bacteria is not known to occur, because of their asexual mode of reproduction.

19) Which of the following is least associated with the others?

A) horizontal gene transfer

20) In Fred Griffith's experiments, harmless R strain pneumococcus became lethal S strain pneumococcus as the result of which of the following?

  1. horizontal gene transfer
  2. transduction
  3. conjugation
  4. transformation
  5. genetic recombination
  6. A) 2 only
  7. B) 4 only
  8. C) 2 and 5
  9. D) 1, 3, and 5
  10. E) 1, 4, and 5

21) Hershey and Chase performed an elegant experiment that convinced most biologists that DNA, rather than protein, was the genetic material. This experiment subjected bacteria to the same gene transfer mechanism as occurs in


Inhoud

Binaire deling Bewerken

Alle prokaryoten (archaea en bacteriën) planten zich ongeslachtelijk voort door middel van binaire deling. De cel splitst zich in tweeën en vormt daarbij twee genetisch identieke dochtercellen. Onder de juiste omstandigheden kan een bacterie op basis van binaire deling een kolonie van miljoenen identieke cellen creëren. [2]

Ook eencellige eukaryoten (protisten zoals algen en schimmels) kunnen zich op een vergelijkbare manier voortplanten door mitose (gewone celdeling). Hierbij is het mogelijk dat het moederorganisme eerst een aantal kerndelingen ondergaat, waarna het cytoplasma meermalen deelt tot een klompje cellen. Dit proces wordt meervoudige binaire deling genoemd. [3] [4]

In Apicomplexa, een groep microscopische parasieten, kunnen door meervoudige binaire deling verschillende celtypen ontstaan, die elk een eigen rol vervullen in de levenscyclus, zoals merozoïeten (algemene dochtercellen), sporozoïeten (infecteuze dochtercellen) en gametocyten (geslachtscellen). [5] [6]

Knopvorming Bewerken

Sommige cellen delen zich door middel van knopvorming, een proces waarbij de dochtercel zich afsnoert van de moedercel. [a] Een bekend voorbeeld is bakkersgist. Kleine uitstulpingen groeien uit tot een nieuwe gistcel. Ook veel meercellige organismen vertonen knopvorming, bijvoorbeeld rifkoralen en hydroïdpoliepen. Het nieuwe individu ontwikkelt zich dan als uitgroeisel op het ouderorganisme, en kan ofwel loskomen (zoals bij kwalletjes), of verbonden blijven aan de ouder en zo een grote kolonie vormen (zoals bij koraalriffen). Knopvorming is eveneens beschreven bij sponzen, enkele platwormen en bij de larven van stekelhuidigen.

Knopvorming kan in zeldzame gevallen ook intracellulair plaatsvinden, namelijk bij cellen die geïnfecteerd zijn met een parasiet zoals Toxoplasma gondii. Tijdens dit proces worden twee (endodyogenie) of meer (endopolygenie) dochtercellen gevormd binnen de moedercel, die vervolgens het cytoplasma van de moedercel opeten voordat ze zich splitsen. [7]

Vegetatieve vermeerdering Bewerken

Vegetatieve vermeerdering is een andere vorm van ongeslachtelijke voortplanting die voortkomt in planten. Kenmerkend voor vegetatieve vermeerdering is dat de plant nieuwe individuen voortbrengt zonder voorafgaande productie van zaden of sporen, en dus zonder bevruchting of meiose. Veel planten kunnen zich van nature vegetatief voortplanten, bijvoorbeeld via wortelstokken, uitlopers of broedbolletjes. Bij grazige vegetaties met wortelstok-vormende grassen is het dan moeilijk te onderscheiden of er sprake is van individuen of van spruiten van een exemplaar.

De mens maakt al duizenden jaren gebruik van het principe van vegetatieve vermeerdering, bijvoorbeeld door te stekken, af te leggen of te enten. Door planten via vegetatieve weg te vermenigvuldigen, kan men klonen verkrijgen die dezelfde eigenschappen hebben als de moederplant. Om deze reden speelt vegetatieve vermeerdering een belangrijke rol bij de teelt van cultuurgewassen (aardappel, tropische voedingsge­wassen), tuinbouwgewassen en in het bijzonder sierplanten en vruchtbomen. [8]

Een bijzondere manier van vegetatieve vermeerdering is weefselkweek. Dit is een techniek die wordt aangewend om plantencellen onder steriele omstandigheden te laten groeien op een voedingsmedium. Weefselkweek is gebaseerd op het feit dat veel plantencellen het vermogen hebben om te regenereren tot een volledig individu. [9]

Sporenvorming Bewerken

Veel meercellige organismen – met name planten, algen en schimmels – vormen sporen in een bepaalde fase van hun levenscyclus. [b] Een spore is een eencellig lichaam dat meestal inwendig in het ouderorganisme wordt aangelegd. Sporen zijn de media van ongeslachtelijke voortplanting: ze hoeven niet met elkaar te versmelten, maar ontwikkelen zich rechtstreeks tot een nieuw individu.

In het plantenrijk is sporenvorming voornamelijk duidelijk te zien bij soorten die zaadloos zijn, waaronder mossen en varens. De (meio-)sporen worden bij planten door reductiedeling (meiose) geproduceerd in speciale organen, de sporangiën. Na verspreiding en ontkieming groeien de sporen uit tot haploïde meercellige individuen, de gametofyten. Deze individuen vormen dan door middel van gewone celdeling (mitose) de gameten. Na de bevruchting (versmelting van de gameten) tot een zygote kan zich een nieuwe sporofyt ontwikkelen. Sporenvorming en gametenvorming vinden bij mossen en veel varens plaats in afzonderlijke generaties van de levenscyclus.

Bij schimmels vervullen sporen een functie die analoog is aan die van zaden. Schimmels kunnen zich ongeslachtelijk voortplanten door via mitotische weg sporen te vormen. Dit gebeurt bij hogere schimmels veelal in gespecialiseerde vruchtlichamen. Onder de juiste omstandigheden van vocht, temperatuur en voedselbeschikbaarheid groeien de sporen uit tot nieuwe individuen.

Sommige bacteriën kunnen endosporen vormen wanneer ze in ongunstige omstandigheden terechtkomen. In de spore is een van de twee DNA-strengen opgeslagen, omgeven door een sporemembraan en een laag peptidoglycaan. De spore is zeer goed bestand tegen de inwerking van chemicaliën (waaronder diverse desinfectantia) en hitte (denatureren pas boven 100 °C). Endosporen kunnen na maanden of jaren van dormantie in gunstig milieu ontkiemen. [10]


Watch the video: Πανελλαδικές 2021 - Αρχαία Ελληνικά- Βιολογία-Μαθηματικά 160621 (December 2022).