GENE AND DEVELOPMENT: TECHNIQUES AND ETHICAL

ISSUES

By:

Dr. Tess Consulta

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THE DEBATES:

1. Which controls heredity? (nucleus / cytoplasm)

2. Embryology vs. genetics

WHICH CONTROLS HEREDITY?• Late 1800s: Edmund Beecher Wilson and

Thomas Morgan, et al. began to study the mechanism by which fertilized eggs give rise to adult organism : cytoplasm / nucleus?

Theodor Boveri: chromosomes and cytoplasm in heredity and development

What was his contribution?

Boveri’s Work

• Sea Urchin: showed that it was necessary to have all chromosomes present in order for proper embryonic development to take place.– How did he performed

his experiment?

Boveri’s Work

• significant discovery was the centrosome (1888), which he described as the especial organ of cell division.

• also discovered the phenomenon of chromatin diminution during embryonic development of the nematodeParascaris.

Boveri’s Work

• 1902 , he said that a cancerous tumor begins with a single cell in which the makeup of its chromosomes becomes scrambled, causing the cells to divide uncontrollably.

Boveri’s Work

• proposed the existence of cell cycle check points, tumour supressor genes, oncogenes and that aberrant mitoses and uncontrolable growth might be caused by radiation, physical or chemical insults or by microscopic pathogens.

• Thomas Hunt Morgan in 1915 demonstrated that Boveri was correct.

E.B Wilson and Nettie Stevens

• Critical correlation between nuclear chromosomes and organismal development: XO or XY (male and female)

Thomas Morgan

• Mutations correlated with sex and the X chromosomes

• Shows that nuclear chromosomes are responsible for the development of inherited characteristics

Split between embryology and genetics

What were their point of arguments?

• Geneticist believed that embryologists were old-fashioned

• Embryologists regarded the geneticists as uninformed about how organisms actually developed

3 major challenges that genetics must meet

1. Geneticists had to explain how chromosomes—which were thought to be identical in every cell of the organism—produce different and changing types of cell cytoplasms.

2. Geneticists had to provide evidence that genes control the early stages of embryogenesis.

3. Geneticists had to explain phenomena such as sex determination in certain invertebrates in which the environment determines sexual phenotype.

Fig 4.3• Embryologists attempted to

keep genetics from “taking over” their field in the 1930s.

• (A) Frank Lillie ,(B) Hans Spemann (left) and Ross Harrison (right) perfected the transplantation operations used to discover when the body and limb axes are determined. They argued that geneticists had no mechanism for explaining how the same nuclear genes could create different cell types during development.

• (C) Ernest E. Just made critical discoveries on fertilization. He spurned genetics and emphasized the role of the cell membrane in determining the fates of cells.

Early attempts at developmental genetics

Fig 4.4• Two of the founders of developmental genetics. (A)

Salome Gluecksohn-Schoenheimer (1907),(B) Conrad Hal Waddington (1905–1975) did not believe in the distinction between genetics and embryology and sought to find mutations that were active during development.

Salome Gluecksohn-Schoenheimer• showed that mutations in the Brachyury genes of the

mouse caused the aberrant development of the posterior portion of the embryo, and she traced the effects of these mutant genes to the notochord, which would normally have helped induce the dorsal axis

• What is it?

• Brachyury is a protein that in humans is encoded by the T gene. Brachyury is a transcription factor within the T-box complex of genes. It has been found in all bilaterian animals that have been screened, and is also present in the cnidaria.

Waddington (1939)•  isolated several genes that caused wing malformations in

fruit flies (Drosophila). He, too, analyzed these mutations in terms of how the genes might affect the developmental primordia that give rise to these structures. The Drosophila wing, he correctly claimed, “appears favorable for investigations on the developmental action of genes

Evidence for Genomic Equivalence

How could nuclear genes direct development when they are the

same in every cell type?

Evidence for Genomic Equivalence

1. Metaplasia2. Cloning

a. Restriction of nuclear potencyb. Pluripotency of somatic cells

What is Metaplasia?

Metaplasia

• the reversible replacement or change from one type of cell to another .

• the original cells are not robust enough to withstand the new environment, and so they change into another type more suited to the new environment.

• If the stimulus that caused metaplasia is removed or ceases, tissues return to their normal pattern of differentiation.

Experiment on Metaplasia (fig 4.5)

Different types of cloning

1. Embryo cloning2. Somatic cell nuclear transfer3. Therapeutic cloning

Amphibian cloning: The restriction of nuclear potency

• The ultimate test of whether the nucleus of a differentiated cell has undergone any irreversible functional restriction is to have that nucleus generate every other type of differentiated cell in the body.

• If each cell's nucleus is identical to the zygote nucleus, then each cell's nucleus should be totipotent (capable of directing the entire development of the organism) when transplanted into an activated enucleated egg

3 techniquesFig 4.6

1. a method for enucleating host eggs without destroying them;

2. a method for isolating intact donor nuclei; and

3. a method for transferring such nuclei into the host egg without damaging either the nucleus or the oocyte.

What happens when nuclei from more advanced developmental stages are

transferred into activated enucleated oocyte?

Fig 4.7• Percentage of successful nuclear transplants as a function of the

developmental age of the donor nucleus. The abscissa represents the developmental stage at which a donor nucleus (from R. pipiens) was isolated and inserted into an activated enucleated oocyte. The ordinate shows the percentage of those transplants capable of producing blastulae that could then direct development to the swimming tadpole stage.

Amphibian cloning: The pluripotency of somatic cells

Is it possible that some differentiatied cell nuclei differ from others in their

ability to direct development?

Fig 4.8• A clone of Xenopus

laevis frogs. The nuclei for all the members of this clone came from a single individual—a female tailbud-stage tadpole whose parents (upper panel) were both marked by albino genes. The nuclei (containing these defective pigmentation genes) were transferred into activated enucleated eggs from a wild-type female (upper panel). The resulting frogs were all female and albino (lower panel)

Fig 4.9• Procedure used to obtain

mature frogs from the intestinal nuclei of Xenopus tadpoles. The wild-type egg (with two nucleoli per nucleus; 2-nu) is irradiated to destroy the maternal chromosomes, and an intestinal nucleus from a marked (1-nu) tadpole is inserted. In some cases, there is no cell division; in some cases, the embryo is arrested in development; but in other cases, an entire new frog, with a 1-nu genotype, is formed

Cloning Mammals

Fig 4.10

Fig 4.11

TRANSGENIC ORGANISMS

TOBACCO PLANT

• genetic engineering; - 1986

• a glowing transgenic tobacco plant

• bears the luciferase gene of fireflys

GLO FISH

• took a gene from a jellyfish that naturally produced a green fluorescent protein and inserted it into the zebrafish genome.

• This caused the fish to glow brightly under both natural white light and ultraviolet light.

GENETICALLY MODIFIED ORGANISMS

Bt corn• Bacillus thuringiensis  - Gram-positive, soil 

dwelling bacterium of the genus Bacillus. • B. thuringiensis also occurs naturally in the 

caterpillars of some moths and butterflies, as well as on the surface of plants

GOLDEN RICE• a variety of rice produced through 

genetic engineering to biosynthesize the precursors of beta-carotene (pro-vitamin A) in the edible parts of rice. 

• Golden rice was created by transforming rice with two beta-carotene biosynthesis genes:– psy (phytoene synthase) from daffodil

(Narcissus pseudonarcissus) – crt1 from the soil bacterium

Erwinia uredovora

How do we make a GMO/transgenic organisms?

cloning and gene etc, dev bio\DNA\recombinant DNA technology.MPGcloning and gene etc, dev bio\DNA\isolation of DNA plasmid.MPG

Molecular Cloning

Gene of interest

DNA of donor organism

Restriction enzymes

Gene of interest

Molecular cloning

Plasmid DNA

Plasmid DNA

Molecular cloning

Gene of interestPlasmid DNA+ + Ligase

RecombinantDNA

molecule

Making a GM plant

GMO

BIOLISTICS

Plant tissue culture

Bacteria carrying Ti plasmid with gene of interest

Gene Gun or Biolistics

Why clone mammals?

Pros and cons of cloning

Pros1. cloning vital organs of human body can be used as back-up in

case of an organ failure. When a crucial body organ such as kidney or heart fails to perform its normal functions, it can be replaced with a cloned organ substitute.

2. can also provide a viable solution to infertility in human beings. It can help the infertile individuals in producing children. Cloned embryos can be planted into women’s bodies to produce babies.

3. can make it possible to reproduce a certain desirable trait in human beings through the cloned embryo. Not only that, cloning technologies may help to understand the composition of genes and their effect on human traits and behavior in a more comprehensive and elaborate manner.

Pros

4. Can make it possible to alter genetic constituents in cloned humans, so as to simplify their analysis of genes. A wide range of genetic diseases can be averted through cloning.

5. Lastly, Genetic alteration of plants and animals can

also be enabled by cloning. It can also help to replicate animals that can be used for research purposes by scientists. And it can even help in saving endangered species.

Cons1. since cloning creates identical genes and it is a process of

replicating a complete genetic constitution, it can significantly hamper the much needed DNA diversity in human beings. The lessening of genes diversity will weaken our adaptation ability. Similarly, cloning will also severely affect diversity in plants and animals. A cloned species may not know how to react to viruses and other destructive agents as scientists cannot predict such potential developments.

2. by allowing man to interfere with genetics in human beings, cloning raises a concerning probability of deliberate reproduction of undesirable traits in human beings, if so desired. Cloning of body organs opens the possibility of malpractice in the medical fraternity.

Cons

3. technical and economic barriers need a consideration in cloning human organs for transplant. Cloned organs may not be cost-effective for a good part of human society. The benefits of cloning techniques reaching the common man remain a big question mark.

4. moral and ethical front as well, cloning raises several serious questions. It devalues man-kind, as a new birth is a natural process, and seriously undermines the value of human life.

Is cloning bad or Good?

The Exception to the Rule: Immunoglobulin Genes

WHILE THE RULE is that the genome is the same in every cell in the body, some white blood cells that

function as part of the immune system provide exceptions to that rule.

cloning and gene etc, dev bio\antibodies.MPG

cloning and gene etc, dev bio\immunoglobin.MPG

Fig 4.12

Differential Gene expression

If the genome is the same in all somatic cells within an organism how do the cells

become different with one another?

3 postulates of differential gene expression

1. Every cell nucleus contains the complete genome established in fertilized egg. In molecular terms, the DNAs of all differentiated cells are identical.

2. The unused genes in differentiated cell are not destroyed or mutated, and they retain the potential for being expressed.

3. Only a small percentage of the genome is expressed in each cell, and a portion of the RNA synthesized in the cell is specific for that cell type.

Figure 4.13

RNA Localization technique

1. Northern Blottingoning and gene etc, dev bio\DNA\southern blot technique.MPGcloning and gene etc, dev bio\DNA\gel elctrophoresis.MPGcloning and gene etc, dev bio\DNA\gel electrophoresis.MPGcloning and gene etc, dev bio\DNA\microarray experiment.MPG

2. In situ hybridizationcloning and gene etc, dev bio\DNA\in situ hybridization.MPGcloning and gene etc, dev bio\DNA\FISH (hybridization).MPG

Fig 4.14

Fig 4.15

Fig 4.16

What is a PCR?

cloning and gene etc, dev bio\DNA\PCR - ok.MPGcloning and gene etc, dev bio\DNA\PCR.MPG

Fig. 4.17

Determining the Function of Genes during Development

1. Inserting new DNA into a cell2. Chimeric mice3. Gene targeting (knockout) experiments

Fig. 4.18Insertion of new DNA into embryonic cells. Here, DNA (from cloned genes)

is injected into the a pronucleus of a mouse egg

Fig 4.19

Fig 4.20

Fig 4.21

Human Somatic and Germ Line Gene Therapy

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Fig. 4.22

Determining the function of a message: Antisense RNA

Fig 4.23

Identifying the Genes for Human Developmental Anomalies

Fig. 4.24

Fig 4.25