Giant Chromosome

They are also known as polytene chromosomes. They commonly occur in the dipteran species of the flies such as Drosophila. First, they look like normal chromosomes. However, later on, these regions undergo repeated rounds of DNA replication. They look longer than the normal chromosomes. Hence, they are known as the giant chromosomes. The process of repeated DNA replication without the cell division is known as endoreplication. However, the centromere does not undergo endoreplication. The reason remains unclear. The process of replication requires the removal of the internal eliminated sequences (IES). The non-coding genes get removed or excised. The polytene chromosomes become larger and appear banded. A rare event occurs in the cells with the polytene chromosome. The centromeres come together and bundle up. The structure so formed is known as a chromocenter. Dissecting the Drosophila larvae and observing their cells under the microscope reveals polytene chromosomes. They commonly occur in the larvae as compared with the adult flies. Balbiani first reported these chromosomes in the year 1881. Thousands of DNA strands occupy the polytene chromosomes. They play a crucial role in the salivary glands. A polytene band represents the vertical stripes on the polytene chromosomes. It results from the specified association between the homologous chromomeres. A somatically paired bundle of chromosomes consists of these chromomeres at the same level. The polytene chromosome is also known as the Balbianin chromosome due to the presence of large puffs known as Balbiani rings. The RNA puffs rarely exist.

The large size of the polytene chromosomes occurs due to the presence of chromonemata (longitudinal strands). They form after repeated cytoplasmic divisions known as endomitosis. The polytene chromosomes consist of two main types of bands. The dark bands involve a dark stain. The light bands stain light. They are known as interbands. The dark bands possess more DNA and less RNA. The light bands contain more of RNA.

Image: Giant Chromosome or Polytene Chromosome

Functions of giant chromosomes:

The polytene bands get enlarged and form swellings. These structures are known as puffs. They consist of active genes. The chromonemata uncoil in the puffs. Thus, it opens up and forms many loops. The individual chromomeres also uncoil. These puffs indicate RNA synthesis. The loops appear like the ring structures. The scientist Balbiani discovered the rings. Hence, they are known as Balbiani rings. They consist of nucleic acids and proteins. These sites are known as transcriptionally active sites. Thus, they have RNA polymerases and ribonucleoproteins actively involved in the transcription process. The protozoan puffs consist of only DNA. Thus, they do not undergo transcription in the puffs. Multiple gene copies in the polytene chromosome permit a very high level of gene expression. Many DNA replication rounds resulting in the endoreplication in the cells of the salivary glands secrete a mucoprotein. It possesses a sticky glue-like characteristic useful for the pupae stage. The regions near the Centromeres possess polytene bands. These bands undergo a tandem duplication. Mainly, they occur near the centromere of the X chromosome. The flies showing tandem duplication in this region possess a phenotype of bar eyes with kidney shape. The interband get involved in the interaction with the important structures. They include active chromatin proteins, nucleosome remodeling complexes, and origin recognition complexes. They help in initiating the process of replication. The interphase replication involved in producing giant chromosome with many stranded cables (polytenes) is known as polyteny.

The polytene chromosomes have a larger size in comparison with the metaphase chromosomes. Both light and electron microscopy reveal the structure of the polytene bands. The tight association also takes place between the chromatids of one single chromosome. Ectopic pairing also involves the polytene chromosome. The pairing occurs between the nonhomologous chromosomes in the ectopic pairing. It occurs between the single chromosome bands. However, it is a site-specific feature. 

Role of chromocenter in polytenization:

It is a diffuse region. It consists of fused pericentric regions of all the chromosomes. It mainly consists of the heterochromatic region. The heterochromatic region surrounds the mitotic chromosome. The chromocenter mainly contains two types of heterochromatin such as the alpha and the beta-heterochromatin. The former one does not participate in the polytenization. The latter on, the beta-heterochromatin gets involved in the polytenization. It contains sequences capable of cross-hybridization with many mobile genetic elements. The telomeric heterochromatin also plays a crucial role in polytenization. The DNA rich banded regions of the polytene chromosomes consist of histone proteins. These proteins help in various hybridization assays. Immunostaining technique helps in identifying the proteins present abundantly for the polytene chromosomes.

Control of genes present in the polytene chromosomes:

The insect hormone known as ecdysone plays a crucial role in controlling the genes in the polytene chromosome. The larval development involves a periodic rise and fall of the ecdysone hormone. The larva requires certain proteins for each of its molt and pupation stage. The levels of the ecdysone hormone control the expression of the genes involved in producing these proteins. The activation and deactivation of the transcriptional events lead to the receding of the old puffs. Also, the new puffs arise. It happens when the organism switches from one developmental stage to the other. The polytene chromosomes roughly contain about 5000 genes.

Polytene chromosomes in mammals:

Mammalian trophoblast cells also contain polytene chromosomes. They show a ploidy between the 32C and 2048C. However, these polytene chromosomes do not have a banded structure like that of the Drosophila polytene chromosomes. These chromosomes have a transitory existence. Later on, they break down into smaller fragments.

References:

[1] Biology: A Functional Approach, M. B. V. Roberts, Giant chromosome, Page 241

[2] Laboratory Manual for Biotechnology, Verma, Ashish S./ Das Surajit & Singh Anchal, Page 190-192

[3] Advanced Topics In Zoology, H V Kashyap, Page 16-17

[4] Polytene Chromosome –Wikipedia

Copyright, 2019. All Rights Reserved

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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