The Study of Developmental Genes in Drosophila

Small, cute looking fruit flies known as Drosophila, fly around the leftover foods, fruits, vegetables, and juicy substances. Never thought about these flies helping the scientists tremendously in the genetics research. Drosophila, one of the most important model organisms, is used worldwide in various experiments. Drosophila is one such genus of the flies that contains more than 900 described species. Hence, it is the most extensively studied organism in the fields of cytology and genetics. Most of the genetic information available today involves data extracted from Drosophila melanogaster studies. Thousands of mutants are available with this insect. Many of the mutants affect the developmental processes. These genes provide a rich array of information through developmental genetics. The mutants helped us in understanding sex determination and many other traits.

                                                                  Image 1: Drosophila (fruit fly)

 The specialty of using fruit flies as model organisms lies in their ability to reproduce on any media. Obtaining Drosophila cultures is not a tough job. It grows on almost all the foodstuffs. Just take a container having holes for the air to pass inside. Put a piece of banana or any juicy fruit inside it and keep it in your window. After some time, these tiny fruit flies get attracted to the fruit inside the container. Another important specialty of Drosophila lies in the presence of a polytene chromosome, widely used in gene studies. The eukaryotic development completely depends on the precise regulation of a group of genes. The genetic regulation of the development in Drosophila progresses significantly. An important point to be noted regarding the developmental genes in Drosophila is that these flies have counterparts in all the organisms, including humans. The body structure of Drosophila involves many segments. Homeotic genes determine the developmental identity of these segments. Studying the genes accelerates the developmental analysis. Thus, in layman terms, the developmental genetics involves the study of mutations deciding the developmental processes gaining the information of the way normal genes get control over the growth, form, behavior, and reproduction. A well-differentiated organism arises from a single cell. Zygote forms due to the fusion of sperm and egg. The cell exhibits totipotency or a potential to develop into any cell type. The genetic programming determines the fate of the cell. This process is known as cell determination. It, later on, involves differentiation, meaning, the determined cells undergo developmental programs and synthesize specific types of cells. Then morphogenesis comes into the picture.

Developmental stages in Drosophila:

A well-ordered sequence of developmentally programmed events follows a strict genetic control. Particular molecular gradients get established before fertilization. There is a region known as polar cytoplasm at the posterior end. The fertilized egg consists of two parental nuclei that fuse to produce a diploid zygote nucleus. It undergoes nine divisions in the cytoplasm and gives rise to multi-nucleate syncytium. The syncytial blastoderm nuclei further migrate and divide, thereby producing a layer at the egg periphery. The nuclei then divide four times resulting in instructions making other important cellular structures such as membranes. After these events, the initial steps in the embryonic development include axes formed in a tight genetic control.

The segment pattern of the embryo involves an adult segment organization. The development of the body structure includes two main processes. The four axes studied in a Drosophila egg include anterior, posterior, dorsal and ventral axes. Along these axes, a molecular gradient occurs. Expression of genes depends on the position of a nucleus involving the intersecting gradient specific region in the adult body. Study of genes involves a cellular blastoderm. Hence, they are known as Para segments. After ten hours or so, these Para segments further look clear and appear like segments. The maternal genes controlling the development of anterior, posterior and dorsoventral axes determine them. Before fertilization, the genes get expressed outside the egg of the mother fly. A wide number of genes get expressed such as Bicoid, Swallo, Oskar, Torso, and Cauda.  The genes express themselves by coding for transcription factors.

The products of these genes, when carried to the egg, establish gradients consisting of RNA and proteins distributed differentially. 

The following table describes the genes and their functions:

Name of the gene	The function of the gene
Bicoid	Involved in axial patterning
Swallow	Plays an important role in bicoid message localization
Oskar	The gene defines a posterior pole (early embryogenesis)
Torso	Determining the anterior and posterior terminal structures.
Caudal	Segmentation of the embryo
Snake (snk)	Extracellular signaling component
Easter	Required for the development of all lateral and ventral pattern elements.

Table 1: Developmental genes and their functions.

Image 2: Development of segments in Drosophila

1.   Formation of embryo axes:

As discussed, there are anteroposterior and dorsoventral axes. The anteroposterior axis requires regulation of Bicoid, Nanos, Acron, Telson, and Torso. Bicoid gene determines the anterior end. It encodes for a mRNA translating to a protein consisting of a helix-turn-helix protein. Mutation in this gene leads to the formation of an embryo lacking a head and the thorax. The Nanos class of genes determines the posterior end of the abdominal segments of the embryo. Further classification of the anterior and posterior structures involves most anterior and most posterior structures respectively. A separate set of genes regulate them.

The dorsal gene involves a product forming ventral to the dorsal gradient in the syncytial blastoderm. The bicoid gene, a key maternal effect gene involves a product forming anteroposterior axes. The bicoid gene encodes a protein known as a morphogen which controls the development. The bicoid gene affects the expression of the caudal gene. After the translation of the bicoid mRNA, the formation of the caudal protein gradient occurs. The protein is lowest at the anterior end and highest at the posterior end. The behavior of the caudal protein antagonizes the behavior of the bicoid protein. The segmentation phase involves the caudal protein. One more maternal effect gene known as Nanos gene helps to form posterior structures. The Drosophila with null mutations in the Nanos genes exhibits phenotype with no abdomen. A hunchback gene expresses a hunchback protein. This protein correctly carries out the developmental processes. It decreases from the anterior to the posterior.  The Nanos gene product increases from anterior to posterior. It is highest at the posterior end.

Antero-posterior structure
Anterior end	Posterior end
Caudal protein: It is lowest at the anterior end and highest at the posterior end.	Hunchback protein: It is highest at the anterior end and lowest at the posterior end.
Morphogen protein It is highest at the anterior end and lowest at the posterior end.	Nanos protein: It is lowest at the anterior end and highest at the posterior end.

Table 2: Proteins playing a crucial role in the development of the anterior and the posterior end.

2.   Segmentation genes:

These genes determine the embryonic and adult segments. Mutations in these genes alter the number. Three main genes include gap genes, pair-rule genes, and segment polarity genes. The homeotic genes specify the identity of the genes. Gap genes include Kruppel, Hunchback, Giant, and Tailless. All these genes encode transcription factors. Pair-rule genes include Hairy, Even-skipped, Runt and Fushi Tarazu genes encoding transcription factors. Engrailed gene is an example of the segment polarity gene. The gap genes divide the embryo into broad regions. The pair-rule gene divides the embryo into seven segments along the craniocaudal axis. The segment polarity genes divide the embryo into fourteen segments.

3.   Determination of the regional characteristics:

The regional characterization of the individual segments in the embryo involves an important group of genes known as homeotic genes. These genes determine whether the embryonic segment bears antennae, legs or wings. Chromosome 3 consists of a total of eight homeotic genes arranged in Antennapedia Bithorax groups. They encode 60 amino acids. Other genes involved in the regional characterization include Antennapedia complex, Bithorax complex and Ultrabithorax complex. Hence, the study of the developmental stages involves a wide variety of mutants. The genes control the development in a temporal regulatory cascade. Hence, studying the developmental genes in Drosophila helps in comparison with the higher organisms.

References:

[1] Genetics, Daniel Hartl, Maryellen Ruvolo

[2] Medical Genetics, G.P. Pal

[3] Drosophila embryogenesis, Wikipedia

[4] Drosophila and the Molecular Genetics of Pattern Formation: Genesis of the Body Plan, NCBI

[5] Homology, Genes, and Evolutionary Innovation, Günter P. Wagner
[6] IGenetics, Peter Russell, second edition

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Top 10 Model Organisms

Conducting trials directly on humans is forbidden on humanitarian grounds. Since the human body is very complex direct involvement of humans as subjects of experiments may not always be possible. Therefore it is ideal to use prokaryotes, eukaryotes, and organisms other than humans. These helper organisms are known as model organisms. They contribute to scientific and industrial research. These organisms include bacteria, fungi, viruses, algae, nematodes, worms, and higher animals. Model organisms are selected based on criteria such as their ease of growing, short life cycle, well-defined cellular structures, easily analyzable properties, safe and ethically sound. Using hazardous toxin-producing bacteria or organisms for conducting experiments is forbidden and is accessed only in rare and unavoidable cases. Only trained, well-qualified professionals handle these organisms. Model organisms are involved in genome analysis, recombination experiments and sequence similarity studies. It requires a lot of care and caution to handle these organisms. Recombinant vaccines, peptides, and biomolecules are produced using model organisms. Biosatellites and spaceships use model organisms in their first launch.

1.   Yeast:

Yeast is a very useful eukaryote with beneficial properties. Yeast is very easy to culture because it can easily grow on fruits, sugary and salty foods. It can easily proliferate from 12⁰C to 40⁰C. The environment of acidic to alkaline is suitable for its growth. Beer and wine industry widely make use of yeast. Preparation of bakery products also involves yeast. Division of yeast occurs due to a process is known as budding. Yeast cells are easy to study since yeast is a single-celled organism. In addition to studying yeast cells with ease, these cells are comparatively safe to handle.

Most of the experiments in genetic engineering and genome research involve the use of yeast as a model organism. It is possible to study cell cycle, DNA replication, protein and cell wall synthesis using yeast cells. Gene cloning can be achieved using vectors. Yeast artificial chromosomes (YACs) are yeast cloning vectors. Typical features of YACs include telomere (TEL), centromere (CEN), selectable markers on each arm, an origin of replication, and restriction sites or multiple cloning sites. YAC vectors can accommodate DNA fragments of several base pairs. Protein-protein interaction studies make use of the yeast two-hybrid system. Cancer genetic studies also involve yeast as model organisms.

2.   Fruit fly:

Most of the genetics experiments involve the use of the fruit fly. It is scientifically known as Drosophila melanogaster. Drosophila is easy to grow since it does not require any specific conditions to grow. It can grow on a wide range of foodstuffs right from a piece of banana to a culture medium. There is a wide range of mutants observed in the fruit fly. Studying its genes is simpler as compared to other organisms. Drosophila is used to study developmental genes, mutant genes, and chromosomes. Morgan was the first scientist to use this model organism. The generation time of fruit fly is rapid as it takes less than two weeks to grow. Transgenic fruit flies are those that consist of foreign gene(s) inserted into their genome.

Drosophila is one such genus of the fruit flies that contains over 900 described species. The genic balance theory was used to identify the concept of sex determination. This theory worked out primarily in Drosophila. According to this theory, sex determination is the ratio of sex chromosomes and polytene chromosomes. A polytene chromosome is a giant chromosome in Drosophila produced by the endomitotic process. It consists of many chromatids. DNA replication and heterochromatin studies are carried out using Drosophila polytene chromosomes. They are also involved in gene mapping and linkage studies. The fruit fly is a model system for understanding human biology and disease. Findings suggest that this model organism has homologs for 177 out of 289 genes involved in human cancers.

Image: Model organisms

3.   E.coli:

Escherichia coli are most widely used bacteria in the biotechnology industry. Genetic engineering principles make very high use of this bacteria for cloning and other experiments. The life cycle of E. coli is simple to understand. Hence they are easy to grow and safe to handle. These bacteria consist of extrachromosomal material known as plasmids. Genetic modifications are possible with this model organism. E.coli can be engineered to produce the desired product. For example, gene modification helps in synthesizing insulin. Plasmid cloning vectors include pUC 19 vectors that contain multiple cloning sites. Important applications in model organisms are as follows:

a.    Gene cloning experiments: Pure samples of cloned DNA are obtained using plasmid vectors. Some of the plasmids with multiple cloning sites enable different types of restriction enzymes to act on them.

b.   Bacteriophage studies: The viruses that infect the bacteria are known as bacteriophages. They follow lytic as well as the lysogenic cycle of infection.

c.     Gene mapping: E.coli bacteria undergo a process of conjugation for transferring genes from one bacterium to another. Thus, gene mapping is possible with the help of conjugation experiments.

d.    Study of DNA replication: Prokaryotic DNA replication study involves E. coli as a model organism.

e.     Identification of mutant phages: E. coli are used to identify mutant phages in site-directed mutagenesis.

f.       Bacterial Artificial Chromosomes (BACs): They are useful for cloning DNA fragments in E. coli. BACs consists of an origin of replication, multiple cloning sites, a selectable marker, and other features.

4.   Nematode C. elegans:

Caenorhabditis elegans is the widely used nematode in zoology. This nematode exhibits anatomical simplicity. It is ideal to carry out gene diversity and cell cycle studies with this organism. The life cycle of this nematode is just three days. Early embryonic genes are clear to study with this organism. It is capable of learning simple tasks. C elegans is used to study the genetic and molecular aspects of embryonic development, morphogenesis, nerve systems, aging, and behavior.

5.   Arabidopsis thaliana:

It is a small plant popular for studying genetic analysis. A complete genome sequence of A. thaliana is available. It is useful to understand processes including nutrient transport and flower development. CRISPR/Cas 9 gene editing also utilizes this plant.

6.   Mus musculus:

The gene content in mice is similar to that of humans. The genome sequence of mice is known to us. Mouse models are used to study organ and immune systems. Mice are just like humans in developing diseases such as cancer, diabetes, atherosclerosis, hypertension and Alzheimer disease. Thus mouse models are far better to work. The mice help in studying various gene mutations, biochemical pathways, metabolism, pharmacokinetics, and pharmacodynamics. Thus, they are ideal for clinical trials and research.

7.   Neurospora crassa:

It is a haploid fungus widely used in cellular processes. It is possible to grow this strain and use it for tetrad analysis. Typical findings using this model organism involve centromere distance, crossing over and poky mutants. The recessive traits easily show up in the offspring. Thus it is simple to study genetic analysis.

8.   Danio rerio:

The common name for Danio rerio is zebrafish. It is a model organism in stem cell biology and developmental genetics. Its embryos are transparent and help in clearly identifying stages of development. It is easy to feed zebrafish by making a genetic cross.

9.   Lambda bacteriophage:

It is a phage that infects E. coli cells. It replicates using bacterial machinery. A bacteriophage is an important tool for genetic analysis. It is a model system for studying genetic recombination, complementation, and cloning experiments. The lambda genome is capable of insertion into the bacterial chromosome.

10.                      Cavia porcellus:

The common name for this model organism is the guinea pig. However, this organism is not a pig but a rodent. Germ theory was established using guinea pigs as model organisms. They are used to study infectious diseases such as cholera, typhus, and brucellosis.

References:

[1] Biotechnology, David P. Clark, Nanette J. Pazdernik

[2] Introduction to Genetic Analysis, Anthony J.F. Griffiths, Susan R. Wessler, Richard C. Lewontin, Sean B. Carroll

[3] Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes, Philip Mark Meneely

[4] Encyclopedia of Genetics, Eric C.R. Reeve

[5] Neurospora: Contributions of a Model Organism, Rowland H. Davis
[6] Model organisms- Wikipedia

© Copyright, 2018  All Rights Reserved

Gene mapping in eukaryotes

Mapping the genes in a correct order helps to know the location of the genes on the chromosomes. Various techniques achieved success in mapping the genes. Mapping techniques achieved success with eukaryotic organisms such as Drosophila and plants before humans. Gene mapping not only helps us in knowing the exact location of the gene but also helps in conducting various other experiments based on the gene location. Thomas Morgan worked with Drosophila strains and found out recombination mechanisms. An experiment sometimes gives an idea of other hidden strategies. For example, gene mapping studies revealed the mechanism of recombination. The studies revealed that the progeny obtained by crossing some strains of the eukaryotic organisms also showed phenotype differing from the parental phenotype.

Morgan’s experiment:

Thomas Morgan and his colleagues worked with Drosophila strains. They cultured Drosophila with a particular X-linked phenotype. The experimenters selected certain strains of fruit flies. The female flies had two X chromosomes with linked genes. The males had one X and one Y chromosome. The female flies had a phenotype of white eyes and miniature wings. The male flies were wild-type flies. The cross between these two types of flies gave rise to an F1 generation having wild-type females, and white-eyed, miniature winged male flies. Interbreeding of the F1 progeny gave different kinds of flies. There were total 2241 flies in the F2 generation. Out of these, total 900 flies had a non-parental phenotypic combination of white eyes and normal wings. Other types of non-parental strains included red-eyed, miniature winged flies. The non-parental ones are known as recombinants. The recombinants arise due to the crossing over between the homologous chromosomes. The theory involves two key concepts. The first one is the site of physical exchange. It is known as the chiasma. The second one involves the genetic recombination between the linked genes. It is known as crossing over. It also involves a reciprocal exchange of chromosome segments.

Image 2: Morgan's experiment

Stern’s Experiment:

Stern worked with X-linked gene loci in Drosophila. The experimenters conducted a cross between the wild-type bar eyed females and carnation type round-eyed males. The female flies had two X chromosomes. One of them additionally had a detached piece of X chromosome. The other X chromosome had an additional attachment of a piece of the Y chromosome. The chromosomes in the males flies had no extra pieces attached. The interbreeding of the F1 progeny gave rise to different types of flies. Four main types of progeny observed included carnation bar, red round, carnation round, and red bar eyed males and females respectively. The results of the experiment revealed the genetic recombination and exchange of identifiable segments. 

Barbara McClintock’s corn experiment:

The corn species selected for the experiment consisted of heterozygotes for the two genes on the 9^th chromosome. One of the genes gave a phenotype of colored versus colorless. The other type of genes resulted in the phenotypes such as standard type starch with amylose and amylopectin versus waxy plants having the only amylopectin. The chromosomes had genes cWx giving normal phenotype. The homologs of the chromosomes having genes cWx had the genotype of Cwx. These homologs had a large double stained knob and a piece of 8^th chromosome attached near the wx gene. It was a translocated segment. These features are known as the cytological markers. Hence, the corn experiments revealed the process of genetic recombination associated with the physical exchange between the parts of the homologous chromosomes.

Linkage studies using testcross:

A cross involving a normal individual with an individual who is homozygous recessive for all the genes is known as a testcross.

·        Two point test cross

Consider the autosomal recessive individuals. Suppose there involves a cross between the double heterozygotes with a genotype of a⁺b⁺/ a⁺b⁺ and double homozygous recessives with a genotype of ab/ab. The F1 generation revealed progeny with a wild-type a⁺b⁺/ab genotype. Upon conducting a testcross with double homozygous recessives, the progeny had 50% parental non-recombinants and 50% recombinant progeny. The formula for the recombination frequency involves (Number of recombinants/ Number of testcross progeny) x 100. The recombination frequency cannot exceed 50%.

Image 2: Two-point test cross

·        Three-point test cross:

Consider a cross between the triple heterozygotes with a genotype of a⁺b⁺c⁺/abc and triple homozygous recessives with a genotype of abc/abc. These crosses reveal the genetic recombination. Consider another example of flowering plants having three linked genes controlling the fruit phenotype. The recessive p allele gives a purple phenotype versus the wild-type yellow phenotype. The recessive r allele gives a round shape versus the wild-type elongated one. The recessive j allele gives juicy phenotype versus the wild-type dry fruit. The order of genes gets determined through a three-point test cross. Two parentals and six recombinants arise due to crossing over. The frequency of the double crossovers was found less than the frequency of the single crossovers.

Gene-centromere distance studies in Neurospora crassa:

The products of meiosis get a specialized arrangement depicting the four chromatids of each of the homologous pair of chromosomes. It usually reflects during the metaphase I. Neurospora consists of ordered tetrads. Meiotic and the mitotic divisions in the tetrads help in studying the process of recombination. It becomes easy to map the distance between the gene and the centromere using the ordered tetrads. The first division segregation tetrad consists of a parental type occupying half the ordered tetrad and another parental type in the other half of the tetrad. It occurs when there is no crossover. A single crossover between the gene and the centromere gives different types of tetrad segregation patterns (the second division segregation). The percentage of the second division tetrads divided by 2. It is known as the gene-centromere map distance. Tetrad analysis also helps in mapping two linked genes.

Mitotic recombination:

Crossing over is also known as genetic recombination between the linked genes or the reciprocal exchange of chromosome segments. It occurs during the mitosis as well as meiosis. The mitotic crossing over is also known as mitotic recombination. It leads to the production of the progeny cells having a combination of genes differing from the diploid parental cell entering the mitotic cycle. A classic example of the mitotic recombination includes fungus Aspergillus nidulans. It has a parasexual cycle of genetic systems. The genetic recombination in Aspergillus occurs through the processes other than regular alteration of meiosis and fertilization. The heterokaryon forms due to the mycelial fusion and the fusion of the two haploid nuclei. It gives rise to a diploid nucleus. The parasexual cycle also consists of mitotic crossing over within the diploid nucleus or haploidization of the diploid nuclei without meiosis. It becomes easy to calculate the gene order and the map distances.

Human gene mapping:

Physical mapping techniques help in mapping human genes. This technique mainly involves large genomes. It is not possible to set up a testcross for human genes since the human genome is vast. We obtain the recombination data from the pedigree analysis in humans. Gene mapping involves the use of gene markers and DNA markers.

                       
References:
[1] Genetics: Analysis of Genes and Genomes, Daniel L. Hartl, Elizabeth W. Jones
[2] Biology, Raven
[3] Biology, Pages 172-180, Neil A. Campbell, Jane B Reece
            

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