Squash preparation of polytene chromosome from Drosophila larvae PDF

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this document gives the experimental practical of squash preparation and the observations....

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Squash preparation of polytene chromosome from Drosophila larvae. INTRODUCTION Polytene chromosomes are well known from their use in a variety of genetical, cytogenetical and molecular studies. These chromosomes remain in permanent interphase but due to repeated cycles of endoreduplication and tight lateral association of all chromatids, each arm chromosome becomes thicker and distinctly visible as cable -like structure with alternating light and dark regions, the bands and interbands respectively. Polytene chromosomes are most commonly found in dipteran insects. In addition, they are also seen in certain other insects, macronucleus of some ciliates and in certain plant tissues. Drosophila has been very widely used for studies on polytene chromosomes. Salivary glands of late third stage instar larvae provided cytologically excellent polytene chromosome preparations due to high level of polyteny achieved by several cells in this tissue. With increasing larval age, the levels of polyteny of chromosome in the cells of salivary glands also increases. Each salivary gland has about 120 cells. Of these the more posterior cells endoreplication more often than the anterior ones so that posterior cells provide better chromosome preparations. By late third instar stage, many of these cells have completed 8 or 9 rounds of replication. All the resulting chromatids maintain their lateral association in such a way that their differentially coiled regions remain in tight register, this results in the characteristic banding patterns consisting of more dense band regions alternating with light stained interbands. The chromatin is more densely packed in the bands while it is less coiled in the interband. Transcriptionally active regions can be easily identified under light microscope as “puffs”. The chromatin fibrils are more loosely arranged in a puff and the newly synthesized RNA also accumulates here. These events results in the enlarged diameter and lighter staining of puff regions compared to the transcriptionally inactive regions (bands). The pericentromeric heterochromatin regions of different chromosomes remain in close association with each other and together they form the chromo center. The euchromatic arms of different chromosomes appear to radiate from this common chromocenter (in some dipterans, like Chironomus, chromocenter formation does not occur in polytene cells.) . A major part of the DNA in chromocenter region does not participate in endo-replication i.e., it remains under- replicated. Drosophila melanogaster has an acrocentric X-chromosome, two pairs of metacentric chromosome (chromosome 2 & 3) and a pair of very small dot like 4th chromosome. Females have 2 X chromosomes while males have only one X chromosome and a large submetacentric Y- chromosome. Y chromosome like the other heterochromatic region, remains buried within the chromo center mass. As in other somatic cells of Drosophila and many other diptera, the homologous chromosomes in polytene cells also remained tightly synapsed. As a result, a polytene nucleus of D. Melanogaster shows a common chromo center (formed by centromeric and pericentromeric heterochromatic regions of all chromosomes) from which 5 long and a very short euchromatic banded chromosome arms radiate out. The 5 long arms represent the X chromosome, left and right arms of chromosome 2 and 3 respectively, while the very short arm is formed by the 4th chromosome. Each chromosome arm has a characteristic banding pattern due to which each region of every chromosome can be very easily distinguished and identified. Every band is given a specific number identity: for D. Melanogaster, the polytene chromosome prepared by C.B bridges in 1930s are followed to identify each of the approximate 5000 bands seen in the salivary glands polytene nucleus. As the salivary glands are histolysed after pupation, polytene chromosome preparations can be prepared from salivary glands of larvae only. Salivary glands of adult flies do not contain polytene cells. For cytological studies, polytene chromosome preparations are made by the classical squashing technique following a brief fixation and staining these preparation could be temporary or permanent, depending upon how they are made.

Materials required: Healthy late third instar stage larvae, blotting paper, dissecting needles and fine forceps, cavity slides, clean glass slides, cover glasses, 37 C incubator, stereo binocular microscope, cloth for cleaning slides, diamond markers, marker pen, slide box, razor blade, dipping jars, slide tray, microscope for examination of preparation. Solutions required:  IX PBS for dissection  50% acetic acid  Aceto- methanol (1:3 freshly prepared)  Aceto- orcein (2%)  For making aceto- orcein , dissolve 2g orcein in 50% acetic acid by keeping on stirring hot plate at 40C for 20 min. Filter when cool. It is strongly desirable to filter the stain every time before use.  Aceto-carmine (2%)- same as aceto-orcein  DPX mountant  Absolute alcohol

Cleaning of slides and cover glasses:

To obtain good squash preparation, it is absolutely essential that the slides and cover glasses are totally free of any dustparticles, fibers and greasy material. A simple way to achieve this is to store the fresh (or soap-cleaned, if desired) slides and cover glass in 90% ethanol in suitable containers and wipe them dry, immediately before use, with a clean soft cloth. These are stored covered till use.

PROCEDURE: Method one (temporary preparations)  Take wandering late third instar larvae from a healthy culture. Wash them free of adhering food particles. This can be done by transferring the larvae on a filter paper soaked in water and placed in a petri dish.  Using fine forceps and/or dissecting needles, pull forward the mouth parts of larvae to rupture larval skin. This forces out internal organs. Salivary glands are seen as a pair of whitish translucent elongated structures connected at their anterior ends with a common salivary duct.  Using tips of the dissecting needles transfer the cleaned salivary glands to a drop of IX PBS solution on a clean slide. Drain out the excess PBS solution (do not let the gland dry). Keeping the slide in a slanting position, add drops of freshly prepared fixative. Wipe out excess fixative with a piece of filter paper (the total duration of fixation should not exceed 1 min. Because longer fixation makes chromosomes brittle and difficult to spread). Add a few drops of acetoorcein stain and leave the slide covered with a watch glass for a few minutes. ( A better staining is obtained by a mix of aceto-orcein and aceto carmine stains: add 2 drops of aceto carmine and 1 drop of aceto orcein and keep covered).  Drain out the stain and add a few drops of 50% acetic acid to remove excess stain. Finally place a drop of 50% acetic acid, cover with a clean cover glass.  For squashing, put the slide with its cover glass between folds of filter paper lightly tap the cover glass with a needle (tapping breaks the cell and nuclear membrane and releases chromosome free in cytoplasm). Too strong tapping would break chromosomes in pieces.hold the cover glass in position with fingers of one hand placed over the filter paper such that they press on two diagonal corners of cover glass. Apply firm pressure of thumb of the other hand on the cover glass. This act of squashing spreads the polytene chromosome arms of a nucleus and makes them flat in one plane. Any lateral movement of cover glass relative to the slide at this stage is likely to cause “rolling” of chromosomes, making them totally unsuitable for study. Too strong a thumb pressure may cause the chromosome unduly stretched.  After squashing, seal the cover glass with DPX (to prevent evaporation of acetic acid and drying of the slide and observe under microscope.

OBSERVATIONS:

A good squash preparation reveals many polytene nuclei with well spread polytene chromosome arms connected to a common chromocenter. The chromocenter is an irregular mass of densely attained chromatin giving a granular appearance. This granular and irregularly arranged chromatin is termed as beta-heterochromatin. A small very densely stained compact region, the alphaheterochromatin can often be seen with this mass. Five long (corresponding to the X, left and right arms of chromosomes 2 and 3 (2L, 2R, 3R and 3L respectively) and one short chromosome 4 arms radiate from the chromocenter. Each chromosome arm displays a typical pattern of dark stained bands and light inter bands: this banding pattern allows identification of not only each chromosome arm but also specific region since each band has been assigned a specific number. Certain specific regions, the puffs appear swollen (greater diameter) and light stained. Specific region that are puffed and the size of each puff (the puffing pattern) are characteristic of the developmental stages of the larva.

PRECAUTIONS:  

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Getting good preparation is an art and requires some practice, following are the some common encountered problems: Excessive tapping may lead to breaking of individual chromosome arm into pieces; likewise, excessive pressure during squashing may cause over-stretching of some chromosome region. In a well-spread preparation all chromosome region must remain in focus at one plane. Any lateral movement of the cover glass relative to the slide during squasing results in chromosome being “rolled” and fragmented: such chromosomes appear as small “rolls” of homogeneously stained material. Salivary glands from the larvae that have not reached the late third instar stage or that are weak due to overcrowding or growth at high temperature, do not provide well spread, thick and distinctly branded chromosomes due to their reduced levels of polytene. Any traces of grease on slides and cover glass hamper good spreading of chromosomes. The slides and cover glass must be kept in 90% alcohol for some time and should be wiped dry before use with fresh clean and soft clothexamination of the slide and cover glass under reflected light from a lamp will reveal if traces of oil or fibres etc are present on their surface....