Polytene Chromosomes in the Salivary Glands of Drosophila Renren Barroga, Ma. Rica Paulene Marquez Department of Biology - University of t he Philippines Baguio
ABSTRACT Polytene chromosomes are result of repeated DNA synthesis without division into daughter chromosomes. This experiment aims to aims to orient students with the proper technique in isolation of polytene chromosomes from D. melanogaster salivary glands and to explain basic concepts about the chromosome. Polytene chromosome was observed in this experiment by extracting salivary glands from third instars of Drosophila melanogaster. Salivary glands were stained with aceto-orcein and observed under a light microscope.
INTRODUCTION
In certain cells and tissues of some insects and amphibians, chromosomal structures can be observed that relate to their function. Polytene (“many threads”) chromosomes are formed as a result of repeated DNA synthesis without cell division, have a distinct pattern of chromosome banding readily visible under the light microscope. These chromosomes are first observed by E.G. Balbiani in cells of salivary glands of Drosophila melanogaster and Chironomus. (Passarger, 2007)
salivary glands were stained with aceto-orcein for ten minutes. A cover slip was then placed in top of the specimen. Enough pressure was then applied over the specimen to crush the cells and spread the chromosomes. The excess stain was blotted off. After which, the specimen was observed under a light microscope. Two slides of clearly discernable polytene chromosomes were prepared in this exper iment.
RESULTS AND DISCUSSION
These polytene chromosomes are a result of ten cycles of replication without division into daughter chromosomes. Thus, there are about 1024 (2 10) identical chromatid strands, which lie strictly side by side. (Passarge, 2007) Generally, polytene chromosomes aare much larger than metaphase chromosomes both because of the multiple chromatids and because of lower compaction (Goldstein and Fyrberg, 1994). The Drosophila genome contains about 5000 bands (Passarge, 2007). This experiment aims to aims to orient students with the proper technique in isolation of polytene chromosomes from D. melanogaster salivary glands and to explain basic concepts about the chromosome.
METHODOLOGY
Healthy-looking third instar larvae were obtained from stock of wild fruitflies. The food around the larvae was rinsed off in a petri dish. The larvae were then transferred to moistened glass slide after it has been rinsed. Using two dissecting needles, the mouth hooks were pulled together with the attached structures from the larval body. The salivary glands were then separated from the other tissues. Once the unwanted tissues were removed from the slide, the
Drosophila melanogaster is a holometabolous insect with four main stages to its lifecycle: embryo, larva, pupa, and adult. As a larva, the organism is primarily concerned with obtaining food for the rapid increase in size. During this time, salivary glands must be large and well developed to sufficiently supply salivary enzymes which will be used for food digestion. These salivary glands achieve their growth through an increase in cell mass and volume rather than an increase in cell number (Clark, 2015). After an initial population of salivary gland cells is established during early larval development, cell division ceases (Clark, 2015). Since the salivary cells are not dividing, the nuclei are not undergoing mitosis. Thus, the chromosomes are in an extended interphase of the cell cycle and are stretched out to their full length. The exact reason for this unusual process, increase in cell mass rather than cell number, is still unknown; however, it is apparently a more efficient way of producing salivary enzymes need for rapid larval development (Clark, 2015). Because polytene chromosomes are extended and consist of so much DNA, they are easily visible under the light mic roscope. Different methods were carefully done to prepare the polytene chromosome. The success of the experiment is
determined to a large extent by the developmental stage of the larvae (Clark, 2015). If they are too small, their salivary glands will not be sufficiently developed to yield a good chromosome. If they are too large and approaching pupation, the salivary glands begin to degenerate and are no longer suitable for chromosome preparation. Larvae that are approaching pupation are visibly darker than the rest due t o the darkening of the cuticle (Clark, 2015).
mitosis, thus, salivary cells increase in cell mass rather than in cell number. These chromosomes are called polytene chromosomes. A pair of salivary glands was isolated from appropriate larvae (third instar larvae). From these salivary glands, the polytene with distinct banding was observed. Problems aroused during the exercise. The aceto orcein used was either diluted or expired which caused the incomplete staining of the chromosomes, thus, banding is not distinctly visible. It was also observed applying pressure could squash the salivary glands, thus, making them flattened and distorted.
QUESTIONS FOR RESEARCH
Figure 1 Salivary Glands
After obtaining the appropriate larva, the salivary glands were obtained by piercing the head and pulling it away from the body (Figure 1). The salivary glands are paired and each is identical in size and shape, and has a glistening, translucent appearance (Cai, et. al., 2010). The salivary glands were then stained using aceto orcein. Aceto orcein was used to label chromosomal loci (Cai, et. al., 2010). A cover slip was then placed in top of the specimen. Enough pressure was then applied over the specimen. This pressure forces the salivary gland cells to burst open and the chromosomes to spread out (Cai, et. al., 2010). Figure 2 shows the prepared polytene chromosome.
CONCLUSION
Salivary chromosomes of Drosophila melanogaster in the larval stage does not undergo
the
Chromosomes are coiled strands of DNA that appear inside the nucleus during cell division. There are various ways of classifying chromosomes. Chromosomes can be classified according to position of centromere: acrocentric chromosomes (those with centromere is present near one end), metacentric (centromere is situated at the center), submetacentric (centromere is situated between the midpoint and at one end of the chromosome), and telocentric (centromere is situated at the end having only one arm). Another way of classifying chromosomes is by the Denner-London System classification, according to this system chromosomes are classified in different groups according to their length. And lastly, chromosomes can also be classified using the Paris conference (1972) classification, wherein chromosomes are classified based on banding pattern. This system of classification provides more accuracy to the identification of parts of each chromosomes.
Figure 2 Polytene Chromosomes
What are chromosomes? Enumerate criteria used in classifying chromosomes.
Explain the different methods of staining chromosomes.
Chromosome banding methods are either based on staining chromosomes with a dye or on assaying for a particular function. The most common methods of dye based chromosome banding are G(Giemsa), R-(reverse), C-(centromere) and Q(quinacrine) banding. Bands that show strong staining are referred to as positive bands; weakly staining bands are negative bands. However the staining patterns are not black and white, different bands stain to different intensities (Francke, 1994). G-positive bands are usually just called G-bands and likewise for Rpositive (R-) bands.
Positive C-bands contain constitutive heterochromatin. Q-bands are considered equivalent to Gbands. The most widely used function-based banding method is replication banding and is based on the fact that different bands replicate theirDNAat different times during S phase of the cell cycle. Generally, R-band DNA is replicated earlier than G-bands (Dutrillaux et al., 1976). G-bands also correspond to the condensed chromomeres of meiotic chromosomes and R-bands to the interchromomeric regions.
Give the importance of chromosome number.
Chromosome number is important in the context of species perpetuation. Parents should have the same amount of chromosomes to pass on to their offspring. These chromosomes must match up (successful homologous pairing) in order for the offspring to become fertile. Therefore, if a chromosome number was not maintained in a species, the species would not be able to mate. Differing chromosome numbers alter the genetic code of the organism. Any change in an organism's genetic code is called a mutation. All mutations are random. Although a small percentage can be potentially beneficial, many of them are deleterious (meaning they damage or cause harm to the organism). Occasionally, the correct numbers of chromosomes are not passed onto offspring for various reasons. One well-known disease caused by a chromosomal disorder is Down Syndrome. This disorder is caused by an extra copy of chromosome 21.
REFERENCES
(1994). Drosophila melanogaster: Practical uses in cell and molecular biology. In L. Goldstein & E. A. Fyrberg (Eds.), Retrieved from https://books.google.com.ph/books?id=HBBblg ZvOA4C&pg=PA335&dq=polytene+chromosom es&hl=en&sa=X&ei=8csXVZ7ZNofh8gXYrICgAg& redir_esc=y#v=onepage&q=polytene%20chrom osomes&f=false Cai, Weili, et. al. (2010). Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling. Data Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PM C2818712/ on March 30, 2015 Clark, Jonathan. (2015). Polytene Chromosomes in Drosophila. Data Retrieved from http://faculty.weber.edu/jclark1/CellBiology.ht ml on March 30, 2015 Dutrillaux B, Couturier J, Richer C-L and ViegasPe´guinot E (1976) Sequence of DNA replication in 277 R-and Q-bands of human chromosomes using a BrdU treatment. Chromosoma 58: 51 – 61. Francke U (1994) Digitized and differentially shaded human chromosome ideograms for genomic applications. Cytogenetics and Cell Genetics 65: 206 –219. Passarge, E. (2007). Color atlas of Genetis :Third Edition. Retrieved from https://books.google.com.ph/books?id=W0NjjP 51MJIC&pg=PA178&dq=polytene+chromosome s&hl=en&sa=X&ei=8csXVZ7ZNofh8gXYrICgAg&r edir_esc=y#v=onepage&q=polytene%20chromo somes&f=fals
