GENETICS: PART I

Lecture 1: GENETICS TRANSMISSION

Genetics

It is the science that seeks to understand, explain and ultimately exploit the

phenomenon of heredity (i.e. transmission of biological characteristics, its

mechanism and its variation).

Genetic material

The genetic material of a cell can be a gene, a part of a gene, a group of genes,

a DNA molecule, a fragment of DNA, a group of DNA molecules, or the

entire genome of an organism or even RNA in certain viruses. They are found in

the cytoplasm (prokaryotes), nucleus, mitochondria and plastids (for algae and plant)

of eukaryotes, which play a fundamental role in determining the structure and nature

of cell substances, and capable of self-propagating and variation.

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Heredity – the passing on of characteristics from parents to offspring.

Genetic transmission (genetic transfer, GT) is almost synonymous with heredity,

which study of how genetic information from genes are transmitted from cell to cell

and generation to generation (from parent to offspring), how they recombine and

segregate, with the goal of explaining the numerical proportions of the progeny.

There are two types of genetic transmission:

1. Vertical gene transmission (VGT) is the transmission of genes from one

generation of species (the parental or ancestor generation) to the next generation

of the same species (offspring or new generation) via sexual or asexual

reproduction (figure below).

DNA encodes all the information necessary to make an organism.

DNA → RNA → Protein

Every organism's DNA is made of the same basic parts, arranged in different

orders.

There are two ways for vertical gene transfer:

Sexual reproduction is a biological process by which organisms create

descendants that have a combination of genetic material contributed from two

(usually) different members of the species using meiosis. Sexually reproducing

organisms have different sets of genes (2 or more) for every character (trait) obtained

from a combination of the parents' genes. Most animals (including humans)

and plants reproduce sexually. Some microorganisms also do it.

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Asexually reproducing organisms creates descendants that have a copy of the

same genetic material using mitosis. Bacteria and Euglina divide asexually via

binary fission; Hydras (invertebrates of the order Hydroidea) and yeasts are

able to reproduce by budding. Other ways of asexual reproduction include

fragmentation (as Spirogyra) and spore formation (as in Fungi).

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Budding in Hydra

Fragmentation in Spirogyra

Budding in yeast

Binary fusion in

Euglina

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Growth and development in Human

Both these mechanisms (sexual and asexual rep.) involve duplication of DNA,

which then gets passed to offspring. RNA is a key component in the duplication of

DNA.

Spore formation in Fungi

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2. Horizontal gene transmission (HGT) refers to the transfer of genes between

organisms, from one species to another species, in a manner other than

traditional reproduction. It is also termed lateral gene transfer.

HGT has been shown to be an important mechanism that drives evolution of

many organisms due to the incorporation of genetic material from an organism

to another one without being the offspring of that organism.

There are three main mechanisms for horizontal gene transfer:

a. Transformation, the genetic alteration of a cell resulting from the

introduction, uptake and expression of foreign genetic material in form of

fragment (DNA or RNA) or plasmids either naturally or artificially (figure

below).

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Application:

Agrobacterium tumefaciens is a widespread naturally occurring Gram-negative soil

bacterium that causes crown gall, and has the ability to transfer a portion of DNA

(transferred DNA or T-DNA) from its tumor-inducing (Ti) plasmid to plant cells. The

T-DNA contains the genes for inducing tumor formation and opine biosynthesis.

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A. rhizogenous is a Gram-negative soil bacterium that produces hairy root

disease in dicotyledonous plants. It is able to transform plant genomes and induces

the formation of proliferative multiple-branched adventitious roots at the site of

infection, so-called hairy roots.

In the laboratory, Agrobacterium bacterial T-DNA genes have been replaced by

genes of interest. Agrobacterium is capable of transforming a range of

dicotyledonous plant species; however, monocots are less responsive towards this

method. The principle advantage of Agrobacterium-mediated gene transfer is the

high occurrence of single-copy T-DNA integration with a relatively stable high level

of transgene expression.

In methods based on biolistics (or microprojectile bombardment), transformation is

achieved by coating gold or tungsten microparticles with the desired DNA, and

accelerating them using high pressure gas such that they are able to penetrate the cell

(wall and membrane) and enter the plant nucleus. This method is routinely used for

the transformation of monocots, cereals, legumes, and microalgae; species that are

typically recalcitrant to Agrobacterium-mediated transformation Bombardment offers

a relatively high efficiency, the ability of delivering DNA without vector backbone,

and also the opportunity to deliver DNA directly to organelle (nucleus or plastids)

genomes. However, there can be substantial variation between experiments (due to

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differences in bombardment conditions), integrations at multiple loci and substantial

damage to bombarded tissue.

Artificial transformation happens in laboratories to insert novel genes into

microorganisms, plant, animal, stem cell, insect, … for research experiments or for

industrial or medical applications through molecular biology and biotechnology.

Transformation of plants (as crops or medicinal plants) with Agrobacterium

tumefaciens or by gene gun (biolistic) for fungal, bacterial, salt or drought tolerance.

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Artificial transformation of some crops and medicinal plants with Agrobacterium

rhizogenes or by gene gun (biolistic) is performed to increasing important

metabolites in the ameliorated root system.

At The New York Times.com (Tues. 4 Nov. 2014)

Biotech potato tubers were developed by the J. R. Simplot Company, an

initial supplier of frozen French fries to McDonald’s since the 1960s has been

approved for commercial planting, by the Department of Agriculture of USA

on Friday. They were genetically engineered to reduce the amounts of a

potentially harmful ingredient (acrylamide) in French fries and potato chips,

which is suspected of causing cancer in people, when the potato is fried.

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b. Conjugation, a process in which a cell transfers genetic material to another

cell by cell-to-cell contact or by a bridge-like connection between two cells

(figures below). During conjugation the donor cell provides a conjugative or

mobilizable genetic element that is most often a plasmid . Most conjugative

plasmids have systems ensuring that the recipient cell does not already contain

a similar element. This process is common in bacteria.

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Conjugation is the process by which one bacterium transfers genetic material to

another through direct contact. During conjugation, one bacterium serves as the

donor of the genetic material, and the other serves as the recipient. The donor

bacterium carries a DNA sequence (plasmid) called the fertility factor, or F-factor.

The F-factor allows the donor to produce a thin, tube-like structure called a pilus,

which the donor uses to contact the recipient. The pilus then draws the two bacteria

together, at which time the donor bacterium transfers genetic material to the recipient

bacterium. Typically, the genetic material is in the form of a plasmid, or a small,

circular piece of DNA. The genetic material transferred during conjugation often

provides the recipient bacterium with some sort of genetic advantage. For instance, in

many cases, conjugation serves to transfer plasmids that carry antibiotic resistance

genes.

Bacterial resistance to antimicrobial drugs is an increasing health and economic

problem. Bacteria may be innate resistant or acquire resistance to one or few classes

of antimicrobial agents. After a bacterium gains resistance genes to protect itself

from various antimicrobial agents, bacteria can use several biochemical types of

resistance mechanisms: antibiotic inactivation (interference with cell wall synthesis,

e.g., β-lactams and glycopeptide), target modification (inhibition of protein synthesis,

e.g., macrolides and tetracyclines; interference with nucleic acid synthesis, e.g.,

fluoroquinolones and rifampin), altered permeability (changes in outer membrane,

e.g., aminoglycosides; new membrane transporters, e.g., chloramphenicol), and

"bypass" metabolic pathway (inhibition of metabolic pathway, e.g., trimethoprim-

sulfamethoxazole).

From the human perspective, one of the significant consequences of a

bacterium's ability to pass genetic information along to other cells via conjugation is

its link to the widespread incidence of antibiotic resistance. The genes that encode for

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resistance to a variety of antibiotics like penicillin and tetracycline are commonly

found on plasmids. When a population of susceptible bacteria is exposed to a given

antibiotic, most of them will be killed. However, if the population contains cells with

conjugative plasmids bearing the genes for resistance, they can rapidly spread the

trait throughout the population. These plasmids are large and are often promiscuous,

so that transfer of antibiotic resistance genes need not be restricted to cells of like

species. In some cases, this has resulted in disease-causing bacteria that are resistant

to almost every antibiotic available. For instance, antibiotic resistant tuberculosis

bacteria are a significant public health threat in some metropolitan areas.

Application: The genetic information transferred is often beneficial to the

recipient. Benefits may include antibiotic resistance, xenobiotic tolerance or the

ability to use new metabolites (figure below).

DNA from one bacterium is transferred to another bacterium. The recipient bacterial strain subsequently develops

phenotypic characteristics of the donor strain with its own ones.

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A problem that has plagued antibiotic therapy from the earliest days is the

resistance that bacteria can develop to the drugs. The end result is bacterial infections

in humans that are untreatable by one or even several of the antibiotics customarily

effective in such cases. The indiscriminate and inexact use of antibiotics encourages

the spread of such bacterial resistance.

At Healthline news.com (Frid. 18 Oct. 2013)

Though the human body contains trillions of bacteria and (usually) remains

healthy, many bacteria continue to evolve to outsmart (resist) current antibiotic

medications. Tom Frieden, director of the U.S. Centers for Disease and Control

and Prevention (CDC), warns that we’re buying time on the biological clock and

that current medications soon will not be able to cure people of life-threatening

infections.

c. Transduction is a method in which bacterial DNA is moved from one

bacterium to another by a virus. When the process whereby foreign DNA is

introduced into another cell by bacteriophage is called Generalized

transduction (natural), while the process used a viral vector (prophage) is

known as Specialized transduction (artificial). Transduction does not require

physical contact between the cell donating the DNA and the cell receiving the

DNA (which occurs in conjugation). Phages also inject their DNA into host

DNA so it’s copied and passed on (figure below).

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Generalized transduction

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Specialized transduction

Transduction does not require physical contact between the cell donating the

DNA and the cell receiving the DNA (which occurs in conjugation), and it

is DNase resistant. Transduction is a common tool used by molecular biologists to

stably introduce a foreign gene into a host cell's genome (both bacterial and

mammalian cells). When viruses, including bacteriophages (viruses that infect

bacteria), infect bacterial cells, their normal mode of reproduction is to harness

the replicational, transcriptional, and translation machinery of the host bacterial cell

to make numerous virions, or complete viral particles, including the viral DNA or

RNA and the protein coat. Viral packaging mechanisms may incorporate bacterial

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genetic material into the new virion. The new virus capsule now loaded with part

bacterial DNA continues to infect another bacterial cell.

Application: Transduction is a common tool used by molecular biologists to

stably introduce a foreign gene into a host cell's genome. Transduction is especially

important because it explains:

- mechanism by which antibiotic drugs become ineffective due to the transfer of

antibiotic-resistance genes between bacteria, which is becoming a medical challenge

to deal with. This is the most critical reason that antibiotics must not be consumed

and administered to patients without appropriate prescription from a medical

physician.

- the evolution of bacteria that can degrade novel compounds such as human-created

pesticides, and in the evolution, maintenance, and transmission of virulence.

- Correcting genetic diseases by direct modification of genetic errors:

Mammalian cell transduction with viral vectors

Transduction with viral vectors can be used to insert or modify genes in mammalian

cells. It is often used as a tool in basic research and is actively researched as a

potential means for gene therapy. In these cases, a plasmid is constructed in which

the genes to be transferred are flanked by viral sequences that are used by viral

proteins to recognize and package the viral genome into viral particles. This plasmid

is inserted (usually by transfection) into a producer cell together with

other plasmids (DNA constructs) that carry the viral genes required for formation of

infectious virions. In these producer cells, the viral proteins expressed by these

packaging constructs bind the sequences on the DNA/RNA (depending on the type of

viral vector) to be transferred and insert it into viral particles. For safety, none of the

plasmids used contains all the sequences required for virus formation, so that

simultaneous transfection of multiple plasmids is required to get infectious virions.

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Moreover, only the plasmid carrying the sequences to be transferred contains

signals that allow the genetic materials to be packaged in virions, so that none of the

genes encoding viral proteins are packaged. Viruses collected from these cells are

then applied to the cells to be altered. The initial stages of these infections mimic

infection with natural viruses and lead to expression of the genes transferred and (in

the case of lentivirus/retrovirus vectors) insertion of the DNA to be transferred into

the cellular genome. However, since the transferred genetic material does not encode

any of the viral genes, these infections do not generate new viruses (the viruses are

"replication-deficient".

Example:

Fusion of a lentiviral vector with the cell membrane of mammalian cells, here

rLV-LifeAct.

Recombinant lentiviral vectors have been shown to be powerful tools for stable

gene transfer to both dividing and non-dividing cells in vitro and in vivo as they

integrate into the host genome. They have a broad host cell range that also includes

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cell types such as neurons, lymphocytes, and macrophages. Moreover, lentiviral

vectors have also proven to be effective in transducing brain, liver, muscle, and retina

in vivo without toxicity or immune responses. The ibidi LifeAct-Lentiviral Vectors

mediate efficient transduction, integration, and long-term expression of LifeAct into

these cell types.

At Reuters.com (Wed. 26 Nov. 2014)

The Western world's first gene therapy drug (Glybera) from Dutch biotech firm

UniQure and its unlisted Italian marketing partner Chiesi is set to go on sale in

Germany. It fights an ultra-rare genetic disease called lipoprotein lipase deficiency

(LPLD) that clogs the blood with fat. The drug consists of a harmless modified virus

that carries a corrective gene into the body's cells.

NOTE: Most thinking in genetics has focused upon vertical transfer, but there is

a growing awareness that horizontal gene transfer is a highly significant phenomenon

and among single-celled organisms perhaps the dominant form of genetic transfer.