Embryo

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https://en.wikipedia.org/wiki/Embryo

 

Embryo
A human embryo seven weeks after conception (nine weeks gestational age)

An embryo (/ˈɛmbrioʊ/ EM-bree-oh) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sperm cell. The resulting fusion of these two cells produces a single-celled zygote that undergoes many cell divisions that produce cells known as blastomeres. The blastomeres are arranged as a solid ball that when reaching a certain size, called a morula, takes in fluid to create a cavity called a blastocoel. The structure is then termed a blastula, or a blastocyst in mammals.

The mammalian blastocyst hatches before implantating into the endometrial lining of the womb. Once implanted the embryo will continue its development through the next stages of gastrulation, neurulation, and organogenesis. Gastrulation, possibly induced in part by paracrine signalling from the amnion, is the formation of the three germ layers that will form all of the different parts of the body. Neurulation forms the nervous system, and organogenesis is the development of all the various tissues and organs of the body.

A newly developing human is typically referred to as an embryo until the ninth week after conception, when it is then referred to as a fetus. In other multicellular organisms, the word embryo can be used more broadly to any early developmental or life cycle stage prior to birth or hatching.

Etymology

First attested in English in the mid-14th century, the word embryon derives from Medieval Latin embryo, itself from Greek ἔμβρυον (émbryon) 'young one', which is the neuter form of ἔμβρυος (émbryos) 'growing in'. Morphologically it is derived from ἐν (en) 'in' and βρύω (brýō) 'to swell, to be full'. The proper Latinized form of the Greek term would be embryum.

Development

Animal embryos

Main article: Animal embryonic development

Embryos (and one tadpole) of the wrinkled frog (Rana rugosa)

Mouse and snake embryos

In animals, fertilization begins the process of embryonic development with the creation of a zygote, a single cell resulting from the fusion of gametes (e.g. egg and sperm). The development of a zygote into a multicellular embryo proceeds through a series of recognizable stages, often divided into cleavage, blastula, gastrulation, and organogenesis.

Cleavage is the period of rapid mitotic cell divisions that occur after fertilization. During cleavage, the overall size of the embryo does not change, but the size of individual cells decrease rapidly, as they divide to increase the total number of cells. Cleavage results in a blastula.

Depending on the species, a blastula or blastocyst stage embryo can appear as a ball of cells on top of yolk, or as a hollow sphere of cells surrounding a middle cavity. The embryo's cells continue to divide and increase in number, while molecules within the cells such as RNAs and proteins actively promote key developmental processes such as gene expression, cell fate specification, and polarity. Before implanting into the uterine wall the embryo is sometimes known as the pre-implantation embryo or pre-implantation conceptus. Sometimes this is called the pre-embryo, a term employed to differentiate from an embryo proper in relation to embryonic stem cell discourses.

Gastrulation is the next phase of embryonic development, and involves the development of two or more layers of cells (germinal layers). Animals that form two layers (such as Cnidaria) are called diploblastic, and those that form three (most other animals, from flatworms to humans) are called triploblastic. During gastrulation of triploblastic animals, the three germinal layers that form are called the ectoderm, mesoderm, and endoderm. All tissues and organs of a mature animal can trace their origin back to one of these layers. For example, the ectoderm will give rise to the skin epidermis and the nervous system, the mesoderm will give rise to the vascular system, muscles, bone, and connective tissues, and the endoderm will give rise to organs of the digestive system and epithelium of the digestive system and respiratory system. Many visible changes in embryonic structure happen throughout gastrulation as the cells that make up the different germ layers migrate and cause the previously round embryo to fold or invaginate into a cup-like appearance.

Past gastrulation, an embryo continues to develop into a mature multicellular organism by forming structures necessary for life outside of the womb or egg. As the name suggests, organogenesis is the stage of embryonic development when organs form. During organogenesis, molecular and cellular interactions prompt certain populations of cells from the different germ layers to differentiate into organ-specific cell types. For example, in neurogenesis, a subpopulation of cells from the ectoderm segregate from other cells and further specialize to become the brain, spinal cord, or peripheral nerves.

The embryonic period varies from species to species. In human development, the term fetus is used instead of embryo after the ninth week after conception, whereas in zebrafish, embryonic development is considered finished when a bone called the cleithrum becomes visible. In animals that hatch from an egg, such as birds, a young animal is typically no longer referred to as an embryo once it has hatched. In viviparous animals (animals whose offspring spend at least some time developing within a parent's body), the offspring is typically referred to as an embryo while inside of the parent, and is no longer considered an embryo after birth or exit from the parent. However, the extent of development and growth accomplished while inside of an egg or parent varies significantly from species to species, so much so that the processes that take place after hatching or birth in one species may take place well before those events in another. Therefore, according to one textbook, it is common for scientists to interpret the scope of embryology broadly as the study of the development of animals.

Plant embryos

Main article: Plant embryonic development

Further information: Sporophyte

The inside of a Ginkgo seed, showing the embryo

Flowering plants (angiosperms) create embryos after the fertilization of a haploid ovule by pollen. The DNA from the ovule and pollen combine to form a diploid, single-cell zygote that will develop into an embryo. The zygote, which will divide multiple times as it progresses throughout embryonic development, is one part of a seed. Other seed components include the endosperm, which is tissue rich in nutrients that will help support the growing plant embryo, and the seed coat, which is a protective outer covering. The first cell division of a zygote is asymmetric, resulting in an embryo with one small cell (the apical cell) and one large cell (the basal cell). The small, apical cell will eventually give rise to most of the structures of the mature plant, such as the stem, leaves, and roots. The larger basal cell will give rise to the suspensor, which connects the embryo to the endosperm so that nutrients can pass between them. The plant embryo cells continue to divide and progress through developmental stages named for their general appearance: globular, heart, and torpedo. In the globular stage, three basic tissue types (dermal, ground, and vascular) can be recognized. The dermal tissue will give rise to the epidermis or outer covering of a plant, ground tissue will give rise to inner plant material that functions in photosynthesis, resource storage, and physical support, and vascular tissue will give rise to connective tissue like the xylem and phloem that transport fluid, nutrients, and minerals throughout the plant. In heart stage, one or two cotyledons (embryonic leaves) will form. Meristems (centers of stem cell activity) develop during the torpedo stage, and will eventually produce many of the mature tissues of the adult plant throughout its life. At the end of embryonic growth, the seed will usually go dormant until germination. Once the embryo begins to germinate (grow out from the seed) and forms its first true leaf, it is called a seedling or plantlet.

Plants that produce spores instead of seeds, like bryophytes and ferns, also produce embryos. In these plants, the embryo begins its existence attached to the inside of the archegonium on a parental gametophyte from which the egg cell was generated. The inner wall of the archegonium lies in close contact with the "foot" of the developing embryo; this "foot" consists of a bulbous mass of cells at the base of the embryo which may receive nutrition from its parent gametophyte. The structure and development of the rest of the embryo varies by group of plants.

Since all land plants create embryos, they are collectively referred to as embryophytes (or by their scientific name, Embryophyta). This, along with other characteristics, distinguishes land plants from other types of plants, such as algae, which do not produce embryos.

Research and technology

Biological processes

Embryos from numerous plant and animal species are studied in biological research laboratories across the world to learn about topics such as stem cells, evolution and development, cell division, and gene expression. Examples of scientific discoveries made while studying embryos that were awarded the Nobel Prize in Physiology or Medicine include the Spemann-Mangold organizer, a group of cells originally discovered in amphibian embryos that give rise to neural tissues, and genes that give rise to body segments discovered in Drosophila fly embryos by Christiane Nüsslein-Volhard and Eric Wieschaus.

Assisted reproductive technology

Creating and/or manipulating embryos via assisted reproductive technology (ART) is used for addressing fertility concerns in humans and other animals, and for selective breeding in agricultural species. Between the years 1987 and 2015, ART techniques including in vitro fertilization (IVF) were responsible for an estimated one million human births in the United States alone. Other clinical technologies include preimplantation genetic diagnosis (PGD), which can identify certain serious genetic abnormalities, such as aneuploidy, prior to selecting embryos for use in IVF. Some have proposed (or even attempted—see He Jiankui affair) genetic editing of human embryos via CRISPR-Cas9 as a potential avenue for preventing disease; however, this has been met with widespread condemnation from the scientific community.

ART techniques are also used to improve the profitability of agricultural animal species such as cows and pigs by enabling selective breeding for desired traits and/or to increase numbers of offspring. For example, when allowed to breed naturally, cows typically produce one calf per year, whereas IVF increases offspring yield to 9–12 calves per year. IVF and other ART techniques, including cloning via interspecies somatic cell nuclear transfer (iSCNT), are also used in attempts to increase the numbers of endangered or vulnerable species, such as Northern white rhinos, cheetahs, and sturgeons.

Cryoconservation of plant and animal biodiversity

Cryoconservation of genetic resources involves collecting and storing the reproductive materials, such as embryos, seeds, or gametes, from animal or plant species at low temperatures in order to preserve them for future use. Some large-scale animal species cryoconservation efforts include "frozen zoos" in various places around the world, including in the UK's Frozen Ark, the Breeding Centre for Endangered Arabian Wildlife (BCEAW) in the United Arab Emirates, and the San Diego Zoo Institute for Conservation in the United States. As of 2018, there were approximately 1,700 seed banks used to store and protect plant biodiversity, particularly in the event of mass extinction or other global emergencies. The Svalbard Global Seed Vault in Norway maintains the largest collection of plant reproductive tissue, with more than a million samples stored at −18 °C (0 °F).

Fossilized embryos

Main article: Fossil embryos

Fossilized animal embryos are known from the Precambrian, and are found in great numbers during the Cambrian period. Even fossilized dinosaur embryos have been discovered.

Mutagenesis

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Mutagenesis

Mutagenesis (/mjuːtəˈdʒɛnɪsɪs/) is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. A mutagen is a mutation-causing agent, be it chemical or physical, which results in an increased rate of mutations in an organism's genetic code. In nature, mutagenesis can lead to cancer and various heritable diseases, and it is also a driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.

History

DNA may be modified, either naturally or artificially, by a number of physical, chemical and biological agents, resulting in mutations. Hermann Muller found that "high temperatures" have the ability to mutate genes in the early 1920s, and in 1927, demonstrated a causal link to mutation upon experimenting with an x-ray machine, noting phylogenetic changes when irradiating fruit flies with relatively high dose of X-rays. Muller observed a number of chromosome rearrangements in his experiments, and suggested mutation as a cause of cancer. The association of exposure to radiation and cancer had been observed as early as 1902, six years after the discovery of X-ray by Wilhelm Röntgen, and the discovery of radioactivity by Henri Becquerel. Lewis Stadler, Muller's contemporary, also showed the effect of X-rays on mutations in barley in 1928, and of ultraviolet (UV) radiation on maize in 1936. In 1940s, Charlotte Auerbach and J. M. Robson found that mustard gas can also cause mutations in fruit flies.

While changes to the chromosome caused by X-ray and mustard gas were readily observable to early researchers, other changes to the DNA induced by other mutagens were not so easily observable; the mechanism by which they occur may be complex, and take longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775, and coal tar was demonstrated to cause cancer in 1915. The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbons (PAH). PAHs by themselves are not carcinogenic, and it was proposed in 1950 that the carcinogenic forms of PAHs are the oxides produced as metabolites from cellular processes. The metabolic process was identified in 1960s as catalysis by cytochrome P450, which produces reactive species that can interact with the DNA to form adducts, or product molecules resulting from the reaction of DNA and, in this case, cytochrome P450; the mechanism by which the PAH adducts give rise to mutation, however, is still under investigation.

Distinction between a mutation and DNA damage

DNA damage is an abnormal alteration in the structure of DNA that cannot, itself, be replicated when DNA replicates. In contrast, a mutation is a change in the nucleic acid sequence that can be replicated; hence, a mutation can be inherited from one generation to the next. Damage can occur from chemical addition (adduct), or structural disruption to a base of DNA (creating an abnormal nucleotide or nucleotide fragment), or a break in one or both DNA strands. Such DNA damage may result in mutation. When DNA containing damage is replicated, an incorrect base may be inserted in the new complementary strand as it is being synthesized (see DNA repair § Translesion synthesis). The incorrect insertion in the new strand will occur opposite the damaged site in the template strand, and this incorrect insertion can become a mutation (i.e. a changed base pair) in the next round of replication. Furthermore, double-strand breaks in DNA may be repaired by an inaccurate repair process, non-homologous end joining, which produces mutations. Mutations can ordinarily be avoided if accurate DNA repair systems recognize DNA damage and repair it prior to completion of the next round of replication. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. Of these, 83 are directly employed in the 5 types of DNA repair processes indicated in the chart shown in the article DNA repair.

Mammalian nuclear DNA may sustain more than 60,000 damage episodes per cell per day, as listed with references in DNA damage (naturally occurring). If left uncorrected, these adducts, after misreplication past the damaged sites, can give rise to mutations. In nature, the mutations that arise may be beneficial or deleterious—this is the driving force of evolution. An organism may acquire new traits through genetic mutation, but mutation may also result in impaired function of the genes and, in severe cases, causes the death of the organism. Mutation is also a major source for acquisition of resistance to antibiotics in bacteria, and to antifungal agents in yeasts and molds. In a laboratory setting, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel functions, as well as mutant strains with useful properties. Initially, the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, but later techniques were developed to introduce specific mutations.

In humans, an average of 60 new mutations are transmitted from parent to offspring. Human males, however, tend to pass on more mutations depending on their age, transmitting an average of two new mutations to their progeny with every additional year of their age.

Mechanisms

Mutagenesis may occur endogenously (e.g. spontaneous hydrolysis), through normal cellular processes that can generate reactive oxygen species and DNA adducts, or through error in DNA replication and repair. Mutagenesis may also occur as a result of the presence of environmental mutagens that induce changes to an organism's DNA, like radiation and/or radioactivity. The mechanism by which mutation occurs varies according to the mutagen, or the causative agent, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on an organism's DNA, producing lesions. Some mutagens, however, may affect the replication or chromosomal partition mechanism, and other cellular processes.

Mutagenesis may also be self-induced by unicellular organisms when environmental conditions are restrictive to the organism's growth, such as bacteria growing in the presence of antibiotics, yeast growing in the presence of an antifungal agent, or other unicellular organisms growing in an environment lacking in an essential nutrient.

Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450. Other enzymes that may also produce mutagenic metabolites include glutathione S-transferase and microsomal epoxide hydrolase. Mutagens that are not mutagenic by themselves but require biological activation are called promutagens.

While most mutagens produce effects that ultimately result in errors in replication, for example creating adducts that interfere with replication, some mutagens may directly affect the replication process or reduce its fidelity. Base analog such as 5-bromouracil may substitute for thymine in replication. Metals such as cadmium, chromium, and nickel can increase mutagenesis in a number of ways in addition to direct DNA damage, for example reducing the ability to repair errors, as well as producing epigenetic changes.

Mutations often arise as a result of problems caused by DNA lesions during replication, resulting in errors in replication. In bacteria, extensive damage to DNA due to mutagens results in single-stranded DNA gaps during replication. This induces the SOS response, an emergency repair process that is also error-prone, thereby generating mutations. In mammalian cells, stalling of replication at damaged sites induces a number of rescue mechanisms that help bypass DNA lesions, however, this may also result in errors. The Y family of DNA polymerases specializes in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymerase, transit the lesion and extend the DNA until the lesion has been passed so that normal replication can resume; these processes may be error-prone or error-free.

Endogenous DNA damage

Endogenous DNA damage is caused by internal cellular processes rather than external agents. Cellular processes can generate reactive oxygen species that can modify the DNA, and DNA can also undergo spontaneous hydrolysis, while errors in replication can result in mutations.

DNA damage and spontaneous mutation

The number of DNA damage episodes occurring in a mammalian cell per day is high (more than 60,000 per day). Frequent occurrence of DNA damage is likely a problem for all DNA- containing organisms, and the need to cope with DNA damage and minimize their deleterious effects is likely a fundamental problem for life.

Most spontaneous mutations likely arise from error-prone trans-lesion synthesis past a DNA damage site in the template strand during DNA replication. This process can overcome potentially lethal blockages, but at the cost of introducing inaccuracies in daughter DNA. The causal relationship of DNA damage to spontaneous mutation is illustrated by aerobically growing E. coli bacteria, in which 89% of spontaneously occurring base substitution mutations are caused by DNA damage induced by reactive oxygen species. In yeast, more than 60% of spontaneous single-base pair substitutions and deletions are likely caused by trans-lesion synthesis.

An additional significant source of mutations in eukaryotes is the inaccurate DNA repair process non-homologous end joining, that is often employed in repair of double strand breaks.

In general, it appears that the main underlying cause of spontaneous mutation is error-prone trans-lesion synthesis during DNA replication and that the error-prone non-homologous end-joining repair pathway may also be an important contributor in eukaryotes.

Reactive oxygen species and oxidative damages

Reactive oxygen species is the typical byproducts of the electron transport chain during cellular respiration. Low levels of reactive oxygen species can function in cellular signaling and immune defenses, but excessive levels of reactive oxygen species can damage bases and the sugar phosphate backbone of the DNA. 8-oxo-guanine, an oxidized formed of guanine mispair with Adenine during replication instead of cytosine, causing an G:C to T:A mutation if left unrepaired.

Spontaneous hydrolysis

Base deamination

Base deamination is a major source of spontaneous mutagenesis happening in the human cells, and it is the loss of amine groups from a DNA base such as cytosine (C), adenine (A), guanine(G), and 5-methylcytosine. It changes the base pairing behavior so that cytosine (C) becomes Uracil (U) , adenine(A) becomes hypoxanthine, guanine (G) becomes xanthine, and 5-methylcytosine becomes thymine (T).

Cytosine and 5-methylcytosine deamination is the most frequently deaminated DNA bases, with 5-methylcytosine being three to four times more deaminated than Cytosine. For cytosine deamination, in DNA, cytosine (C) is usually paired with guanine (G). But after the deamination has happened, it creates a uracil (U) and Guanine (G) mismatch, and if the mismatch is not properly repaired, uracil can pair with adenine, creating a C:G to T:A mutation. While the deamination of 5-methylcytosine generates thymine (T) instead of uracil (U), creating a G:T mismatch.

Depurination

DNA is not entirely stable in aqueous solution, and depurination of the DNA can occur. Under physiological conditions the glycosidic bond may be hydrolyzed spontaneously and 5000 purine sites in DNA are estimated to be depurinated each day in a cell. Numerous DNA repair pathways exist for DNA; however, if the apurinic site is not repaired, misincorporation of nucleotides may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site.

Tautomerism

Main article: Tautomer

Tautomerization is the process by which compounds spontaneously rearrange themselves to assume their structural isomer forms. For example, the keto (C=O) forms of guanine and thymine can rearrange into their rare enol (-OH) forms, while the amino (-NH₂ ) forms of adenine and cytosine can result in the rarer imino (=NH) forms. In DNA replication, tautomerization alters the base-pairing sites and can cause the improper pairing of nucleic acid bases.

Modification of bases

Bases may be modified endogenously by normal cellular molecules. For example, DNA may be methylated by S-adenosylmethionine, thus altering the expression of the marked gene without incurring a mutation to the DNA sequence itself. Histone modification is a related process in which the histone proteins around which DNA coils can be similarly modified via methylation, phosphorylation, or acetylation; these modifications may act to alter gene expression of the local DNA, and may also act to denote locations of damaged DNA in need of repair. DNA may also be glycosylated by reducing sugars.

Many compounds, such as PAHs, aromatic amines, aflatoxin and pyrrolizidine alkaloids, may form reactive oxygen species catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication, and the bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination of the DNA; it is, however, uncertain how significant such depurination as caused by the adducts is in generating mutation.

Alkylation and arylation of bases can cause errors in replication. Some alkylating agents such as N-nitrosamines may require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. N⁷ and O⁶ of guanine and the N³ and N⁷ of adenine are most susceptible to attack. N⁷-guanine adducts form the bulk of DNA adducts, but they appear to be non-mutagenic. Alkylation at O⁶ of guanine, however, is harmful because excision repair of O⁶-adduct of guanine may be poor in some tissues such as the brain. The O⁶ methylation of guanine can result in G to A transition, while O⁴-methylthymine can be mispaired with guanine. The type of the mutation generated, however, may be dependent on the size and type of the adduct as well as the DNA sequence.

Ionizing radiation and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.

See also: Epigenetics

Exogenous DNA damage

Arrows indicates chromosomal breakages due to DNA damage.

Exogenous DNA damage is structural alteration of DNA caused by external environmental agents, including UV radiation, ionizing radiation, and chemical toxins.

Backbone damage

Ionizing radiation may produce highly reactive free radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation and deletion of part of a chromosome. Alkylating agents like mustard gas, diet, tobacco smoke may also cause breakages in the DNA backbone. Endogenous processes may also such as oxidative stress may also generate highly reactive oxygen species that can damage the DNA.

Crosslinking

Main article: Crosslinking of DNA

Covalent bonds between the bases of nucleotides in DNA, be they in the same strand or opposing strands, is referred to as crosslinking of DNA; crosslinking of DNA may affect both the replication and the transcription of DNA, and it may be caused by exposure to a variety of agents. Some naturally occurring chemicals may also promote crosslinking, such as psoralens after activation by UV radiation, and nitrous acid. Interstrand cross-linking (between two strands) causes more damage, as it blocks replication and transcription and can cause chromosomal breakages and rearrangements. Some crosslinkers such as cyclophosphamide, mitomycin C and cisplatin are used as anticancer chemotherapeutic because of their high degree of toxicity to proliferating cells.

Dimerization

Main article: Dimer

Dimerization consists of the bonding of two monomers to form an oligomer, such as the formation of pyrimidine dimers as a result of exposure to UV radiation, which promotes the formation of a cyclobutyl ring between adjacent thymines in DNA . These bulky bases would cause a distortion in the DNA helixes and can interfere with DNA replication and transcription. In human skin cells, thousands of dimers may be formed in a day due to normal exposure to sunlight. DNA polymerase η may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as those with xeroderma pigmentosum, are sensitive to sunlight and may be prone to skin cancer.

Ethidium intercalated between two adenine-thymine base pairs

Clinically, whether a tumor has formed as a direct consequence of UV radiation is discernible via DNA sequencing analysis for the characteristic context-specific dimerization pattern that occurs due to excessive exposure to sunlight.

Intercalation between bases

Main article: Intercalation (biochemistry)

The planar structure of chemicals such as ethidium bromide and proflavine allows them to insert between bases in DNA. This insert causes the DNA's backbone to stretch and makes slippage in DNA during replication more likely to occur since the bonding between the strands is made less stable by the stretching. Forward slippage will result in deletion mutation, while reverse slippage will result in an insertion mutation. Also, the intercalation into DNA of anthracyclines such as daunorubicin and doxorubicin interferes with the functioning of the enzyme topoisomerase II, blocking replication as well as causing mitotic homologous recombination.^{[citation needed]}

Insertional mutagenesis

Main article: Insertional mutagenesis

Transposons and viruses or retrotransposons may insert DNA sequences into coding regions or functional elements of a gene and result in inactivation of the gene.^[40]

DNA repair pathway

DNA damage happens frequently, but DNA damage does not always become a mutation. Only after the repair mechanism has failed or inaccurate, allowing the damages to bypass then the mutation would happen, and potentially lead to diseases like cancer.^{[citation needed]}

Base excision repair

Base excision repair corrects the small, non-helix distorting lesions of DNA helix such as oxidative, deaminated, alkylation, as well as basic single abase damages.^[30] DNA glycosylase within the base excision repair mechanism recognizes those damages and cleaves the N-glycosidic bond, leave behind an abasic sites. DNA backbone would then be cut at that site by AP endonuclease, after that DNA polymerase fills the gap.

Nucleotide excision repair

Nucleotide excision repair removes bulky lesion such as CPDs^{[clarification needed]} and (6-4) pp from UV radiation, or damage from chemotherapeutic agents. There are two major branches of nucleotide excision repair: Globular genome nucleotide excision repair and transcription-coupled nucleotide excision repair. Globular Genome NER, the XPC, RAD23B and CETN2 protein complex scans for whole genome damage, once the damage is found, endonuclease such as XPF–ERCC1 and XPG will cut out the lesion from 5' to 3', and POL ε or XRCC1–LIG3 will carry out the gap filling synthesis and ligation.^[30]

Mismatch repair

Mismatch repair removes base mismatch that have arisen during replication and the insertion-deletion loop.^[41] Humans employ the MutSα heterodimer (MSH2/MSH6) to recognize the base mismatch.^[42] Once the mismatch is found, Exo1 carries out the 5' directed mismatch excision, which creates a gap later being filled by Polδ, RFC, and HMGB.^[43]

Adaptive mutagenesis mechanisms

Main article: Adaptive mutation

Adaptive mutagenesis has been defined as mutagenesis mechanisms that enable an organism to adapt to an environmental stress. Since the variety of environmental stresses is very broad, the mechanisms that enable it are also quite broad, as far as research on the field has shown. For instance, in bacteria, while modulation of the SOS response and endogenous prophage DNA synthesis has been shown to increase Acinetobacter baumannii resistance to ciprofloxacin. Resistance mechanisms are presumed to be linked to chromosomal mutation untransferable via horizontal gene transfer in some members of family Enterobacteriaceae, such as E. coli, Salmonella spp., Klebsiella spp., and Enterobacter spp. Chromosomal events, specially gene amplification, seem also to be relevant to this adaptive mutagenesis in bacteria.

Research in eukaryotic cells is much scarcer, but chromosomal events seem also to be rather relevant: while an ectopic intrachromosomal recombination has been reported to be involved in acquisition of resistance to 5-fluorocytosine in Saccharomyces cerevisiae, genome duplications have been found to confer resistance in S. cerevisiae to nutrient-poor environments.

Laboratory applications

Main article: Mutagenesis (molecular biology technique)

In the laboratory, mutagenesis is a technique by which DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the function of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with altered properties, or enhanced or novel functions that may prove to be of use commercially. Mutant strains of organisms that have practical applications, or allow the molecular basis of particular cell function to be investigated, may also be produced.

Early methods of mutagenesis produced entirely random mutations; however, modern methods of mutagenesis are capable of producing site-specific mutations. Modern laboratory techniques used to generate these mutations include:

• Directed mutagenesis
• Site-directed mutagenesis/ PCR mutagenesis
• Insertional mutagenesis
• Signature tagged mutagenesis
• Transposon mutagenesis
• Sequence saturation mutagenesis

Molecular genetics

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Molecular_genetics

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens.

The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. It integrates these disciplines to explore things like genetic inheritance, gene regulation and expression, and the molecular mechanism behind various life processes.

A key goal of molecular genetics is to identify and study genetic mutations. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Therefore molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments of various genetics diseases.

History

For a detailled timeline, see Timeline of the history of genetics.

The discovery of DNA as the blueprint for life and breakthroughs in molecular genetics research came from the combined works of many scientists. In 1869, chemist Johann Friedrich Miescher, who was researching the composition of white blood cells, discovered and isolated a new molecule that he named nuclein from the cell nucleus, which would ultimately be the first discovery of the molecule DNA that was later determined to be the molecular basis of life. He determined it was composed of hydrogen, oxygen, nitrogen and phosphorus. Biochemist Albrecht Kossel identified nuclein as a nucleic acid and provided its name deoxyribonucleic acid (DNA). He continued to build on that by isolating the basic building blocks of DNA and RNA; made up of the nucleotides: adenine, guanine, thymine, cytosine, and uracil. His work on nucleotides earned him a Nobel Prize in Physiology.

In the early 1800s, Gregor Mendel, who became known as one of the fathers of genetics, made great contributions to the field of genetics through his various experiments with pea plants where he was able to discover the principles of inheritance such as recessive and dominant traits, without knowing what genes where composed of. In the mid 19th century, anatomist Walther Flemming discovered what we now know as chromosomes and the separation process they undergo through mitosis. His work along with Theodor Boveri first came up with the chromosomal theory of inheritance, which helped explain some of the patterns Mendel had observed much earlier.

For molecular genetics to develop as a discipline, several scientific discoveries were necessary.  The discovery of DNA as a means to transfer the genetic code of life from one cell to another and between generations was essential for identifying the molecule responsible for heredity. Molecular genetics arose initially from studies involving genetic transformation in bacteria. In 1944 Avery, McLeod and McCarthy^[8] isolated DNA from a virulent strain of S. pneumoniae, and using just this DNA were able to convert a harmless strain to virulence. They called the uptake, incorporation and expression of DNA by bacteria "transformation". This finding suggested that DNA is the genetic material of bacteria.^[9] Bacterial transformation is often induced by conditions of stress, and the function of transformation appears to be repair of genomic damage.

In 1950, Erwin Chargaff derived rules that offered evidence of DNA being the genetic material of life. These were "1) that the base composition of DNA varies between species and 2) in natural DNA molecules, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine (C)." These rules, known as Chargaff's rules, helped to understand of molecular genetics. In 1953 Francis Crick and James Watson, building upon the X-ray crystallography work done by Rosalind Franklin and Maurice Wilkins, were able to derive the 3-D double helix structure of DNA.

The phage group was an informal network of biologists centered on Max Delbrück that contributed substantially to molecular genetics and the origins of molecular biology during the period from about 1945 to 1970. The phage group took its name from bacteriophages, the bacteria-infecting viruses that the group used as experimental model organisms. Studies by molecular geneticists affiliated with this group contributed to understanding how gene-encoded proteins function in DNA replication, DNA repair and DNA recombination, and on how viruses are assembled from protein and nucleic acid components (molecular morphogenesis). Furthermore, the role of chain terminating codons was elucidated. One noteworthy study was performed by Sydney Brenner and collaborators using "amber" mutants defective in the gene encoding the major head protein of bacteriophage T4. This study demonstrated the co-linearity of the gene with its encoded polypeptide, thus providing strong evidence for the "sequence hypothesis" that the amino acid sequence of a protein is specified by the nucleotide sequence of the gene determining the protein. 

The isolation of a restriction endonuclease in E. coli by Arber and Linn in 1969 opened the field of genetic engineering. Restriction enzymes were used to linearize DNA for separation by electrophoresis and Southern blotting allowed for the identification of specific DNA segments via hybridization probes. In 1971, Berg utilized restriction enzymes to create the first recombinant DNA molecule and first recombinant DNA plasmid.  In 1972, Cohen and Boyer created the first recombinant DNA organism by inserting recombinant DNA plasmids into E. coli, now known as bacterial transformation, and paved the way for molecular cloning.  The development of DNA sequencing techniques in the late 1970s, first by Maxam and Gilbert, and then by Frederick Sanger, was pivotal to molecular genetic research and enabled scientists to begin conducting genetic screens to relate genotypic sequences to phenotypes. Polymerase chain reaction (PCR) using Taq polymerase, invented by Mullis in 1985, enabled scientists to create millions of copies of a specific DNA sequence that could be used for transformation or manipulated using agarose gel separation. A decade later, the first whole genome was sequenced (Haemophilus influenzae), followed by the eventual sequencing of the human genome via the Human Genome Project in 2001. The culmination of all of those discoveries was a new field called genomics that links the molecular structure of a gene to the protein or RNA encoded by that segment of DNA and the functional expression of that protein within an organism. Today, through the application of molecular genetic techniques, genomics is being studied in many model organisms and data is being collected in computer databases like NCBI and Ensembl. The computer analysis and comparison of genes within and between different species is called bioinformatics, and links genetic mutations on an evolutionary scale.

Central dogma

This image shows an example of the central dogma using a DNA strand being transcribed then translated and showing important enzymes used in the processes.

The central dogma plays a key role in the study of molecular genetics. The central dogma states that DNA replicates itself, DNA is transcribed into RNA, and RNA is translated into proteins. Along with the central dogma, the genetic code is used in understanding how RNA is translated into proteins. Replication of DNA and transcription from DNA to mRNA occurs in the nucleus while translation from RNA to proteins occurs in the ribosome. The genetic code is made of four interchangeable parts of DNA molecules, called "bases": adenine, cytosine, thymine (uracil in RNA), and guanine and is redundant, meaning multiple combinations of these base pairs (which are read in triplicate) produce the same amino acid. Proteomics and genomics are fields in biology that come out of the study of molecular genetics and the central dogma.

Structure of DNA

An organism's genome is made up by its entire set of DNA and is responsible for its genetic traits, function and development. The composition of DNA itself is an essential component to the field of molecular genetics; it is the basis of how DNA is able to store genetic information, pass it on, and be in a format that can be read and translated.

DNA is a double stranded molecule, with each strand oriented in an antiparallel fashion. Nucleotides are the building blocks of DNA, each composed of a sugar molecule, a phosphate group and one of four nitrogenous bases: adenine, guanine, cytosine, and thymine. A single strand of DNA is held together by covalent bonds, while the two antiparallel strands are held together by hydrogen bonds between the nucleotide bases. Adenine binds with thymine and cytosine binds with guanine. It is these four base sequences that form the genetic code for all biological life and contains the information for all the proteins the organism will be able to synthesize.

Its unique structure allows DNA to store and pass on biological information across generations during cell division. At cell division, cells must be able to copy its genome and pass it on to daughter cells. This is possible due to the double-stranded structure of DNA because one strand is complementary to its partner strand, and therefore each of these strands can act as a template strand for the formation of a new complementary strand. This is why the process of DNA replication is known as a semiconservative process.

Techniques

Forward genetics

Forward genetics is a molecular genetics technique used to identify genes or genetic mutations that produce a certain phenotype. In a genetic screen, random mutations are generated with mutagens (chemicals or radiation) or transposons and individuals are screened for the specific phenotype. Often, a secondary assay in the form of a selection may follow mutagenesis where the desired phenotype is difficult to observe, for example in bacteria or cell cultures. The cells may be transformed using a gene for antibiotic resistance or a fluorescent reporter so that the mutants with the desired phenotype are selected from the non-mutants.

Mutants exhibiting the phenotype of interest are isolated and a complementation test may be performed to determine if the phenotype results from more than one gene. The mutant genes are then characterized as dominant (resulting in a gain of function), recessive (showing a loss of function), or epistatic (the mutant gene masks the phenotype of another gene). Finally, the location and specific nature of the mutation is mapped via sequencing. Forward genetics is an unbiased approach and often leads to many unanticipated discoveries, but may be costly and time consuming. Model organisms like the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the zebrafish Danio rerio have been used successfully to study phenotypes resulting from gene mutations.

An example of forward genetics in C. elegans (a nematode) using mutagenesis

Reverse genetics

Diagram illustrating the development process of avian flu vaccine by reverse genetics techniques

Reverse genetics is the term for molecular genetics techniques used to determine the phenotype resulting from an intentional mutation in a gene of interest. The phenotype is used to deduce the function of the un-mutated version of the gene. Mutations may be random or intentional changes to the gene of interest. Mutations may be a missense mutation caused by nucleotide substitution, a nucleotide addition or deletion to induce a frameshift mutation, or a complete addition/deletion of a gene or gene segment. The deletion of a particular gene creates a gene knockout where the gene is not expressed and a loss of function results (e.g. knockout mice). Missense mutations may cause total loss of function or result in partial loss of function, known as a knockdown. Knockdown may also be achieved by RNA interference (RNAi). Alternatively, genes may be substituted into an organism's genome (also known as a transgene) to create a gene knock-in and result in a gain of function by the host. Although these techniques have some inherent bias regarding the decision to link a phenotype to a particular function, it is much faster in terms of production than forward genetics because the gene of interest is already known.

Molecular genetic tools

Molecular genetics is a scientific approach that utilizes the fundamentals of genetics as a tool to better understand the molecular basis of a disease and biological processes in organisms. Below are some tools readily employed by researchers in the field.

Microsatellites

Microsatellites or single sequence repeats (SSRS) are short repeating segment of DNA composed to 6 nucleotides at a particular location on the genome that are used as genetic marker. Researchers can analyze these microsatellites in techniques such DNA fingerprinting and paternity testing since these repeats are highly unique to individuals/families. a can also be used in constructing genetic maps and to studying genetic linkage to locate the gene or mutation responsible for specific trait or disease. Microsatellites can also be applied to population genetics to study comparisons between groups.

Genome-wide association studies

Genome-wide association studies (GWAS) are a technique that relies on single nucleotide polymorphisms (SNPs) to study genetic variations in populations that can be associated with a particular disease. The Human Genome Project mapped the entire human genome and has made this approach more readily available and cost effective for researchers to implement. In order to conduct a GWAS researchers use two groups, one group that has the disease researchers are studying and another that acts as the control that does not have that particular disease. DNA samples are obtained from participants and their genome can then be derived through lab machinery and quickly surveyed to compare participants and look for SNPs that can potentially be associated with the disease. This technique allows researchers to pinpoint genes and locations of interest in the human genome that they can then further study to identify that cause of the disease.

Karyotyping

Karyotyping allows researchers to analyze chromosomes during metaphase of mitosis, when they are in a condensed state. Chromosomes are stained and visualized through a microscope to look for any chromosomal abnormalities. This technique can be used to detect congenital genetic disorder such as Down syndrome, identify gender in embryos, and diagnose some cancers that are caused by chromosome mutations such as translocations.

Modern applications

Genetic engineering

Genetic engineering is an emerging field of science, and researcher are able to leverage molecular genetic technology to modify the DNA of organisms and create genetically modified and enhanced organisms for industrial, agricultural and medical purposes. This can be done through genome editing techniques, which can involve modifying base pairings in a DNA sequence, or adding and deleting certain regions of DNA.

Gene editing

Gene editing allows scientists to alter/edit an organism's DNA. One way to due this is through the technique Crispr/Cas9, which was adapted from the genome immune defense that is naturally occurring in bacteria. This technique relies on the protein Cas9 which allows scientists to make a cut in strands of DNA at a specific location, and it uses a specialized RNA guide sequence to ensure the cut is made in the proper location in the genome. Then scientists use DNAs repair pathways to induce changes in the genome; this technique has wide implications for disease treatment.

Personalized medicine

Molecular genetics has wide implications in medical advancement and understanding the molecular basis of a disease allows the opportunity for more effective diagnostic and therapies. One of the goals of the field is personalized medicine, where an individual's genetics can help determine the cause and tailor the cure for a disease they are afflicted with and potentially allow for more individualized treatment approaches which could be more effective. For example, certain genetic variations in individuals could make them more receptive to a particular drug while other could have a higher risk of adverse reaction to treatments. So this information would allow researchers and clinicals to make the most informed decisions about treatment efficacy for patients rather than the standard trial and error approach.

Forensic genetics

Forensic genetics plays an essential role for criminal investigations through that use of various molecular genetic techniques. One common technique is DNA fingerprinting which is done using a combination of molecular genetic techniques like polymerase chain reaction (PCR) and gel electrophoresis. PCR is a technique that allows a target DNA sequence to be amplified, meaning even a tiny quantity of DNA from a crime scene can be extracted and replicated many times to provide a sufficient amount of material for analysis. Gel electrophoresis allows the DNA sequence to be separated based on size, and the pattern that is derived is known as DNA fingerprinting and is unique to each individual. This combination of molecular genetic techniques allows a simple DNA sequence to be extracted, amplified, analyzed and compared with others and is a standard technique used in forensics.