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Anjali Singh Author
Anjali Singh is a freelance writer. Following her passion for science and research she did her Master’s in Plant Biology and Biotechnology from the University of Hyderabad, India. She has a strong research background in Plant Sciences with expertise in Molecular techniques, Tissue culture, and Biochemical Assays. In her free time outside work, she likes to read fictional books, sketch, or write poems. In the future, she aspires to pursue a doctorate in Cancer Biology while continuing her excellence as a scientific writer.
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Anjali Singh Author
Anjali Singh is a freelance writer. Following her passion for science and research she did her Master’s in Plant Biology and Biotechnology from the University of Hyderabad, India. She has a strong research background in Plant Sciences with expertise in Molecular techniques, Tissue culture, and Biochemical Assays. In her free time outside work, she likes to read fictional books, sketch, or write poems. In the future, she aspires to pursue a doctorate in Cancer Biology while continuing her excellence as a scientific writer.
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Introduction

A Chromosome is an organized package of DNA, located inside the nucleus. The structure of a chromosome involves six essential parts, that include: Pellicle and matrix, chromonemata, chromomere, centromere, secondary constriction, and satellite body.

So, chromonemata are the spiral structures that are embedded in the matrix of the chromosome and are the focus of this study.

The first study on the spiral structure of chromosomes was reported in 1880, by Baranetzsky in pollen mother cells of Tradescantia zebrina.

However, the study remained short of information until the discovery of cell division processes, and its significance was made around 1894[1].  Later, an efficient method was developed to study the spiral nature of chromosomes, which you will discover later in this article.

In the beginning, the spiral structure of chromosomes was only studied in the multiple species of three genres, to define the structure. It included Tradescantia, Trillium, and Osmunda[1]. Some other plants included later were, Lilium, Vicia, and Lathyrus. Furthermore, the structure was also observed in a few animals (especially in higher groups), like flagellate protozoa and grasshopper (family Acrididae– in which it was first reported).

The studies were performed by observing the chromosomes in different phases (especially prophase, metaphase, and anaphase are preferred) of meiosis and mitosis.

This article will give an overview of the spiral structure of chromosomes and what methods can be used to study them in plants, as well as animals.

Figure: The schematic diagram of the structure of the chromosome, showing chromonema embedded in the matrix[4].

Source:https://www.biologydiscussion.com/chromosome/chromosome-its-parts-functions-and-types-1934-words-biology/486

Structure of Spiral Chromosomes

What are spiral structures?

Spiral structures are the condensed, thin, coiled, long, thread-like structures present in the matrix of a chromosome. They are also called chromonemata (plural) or chromonema. The term was given by Vejdovsky, in 1912[1].

1. Diameter and number of Gyres

The chromonema in the meiotic-I phase has a wide diameter and few gyres. This type of spiral is known as a “major spiral”. Whereas, in meiotic-II or mitotic phases, it has a narrow diameter and more number of gyres. This type of spiral is called a “minor spiral”[1].

The presence of major and minor spiral differs in different species, and on this basis, they are divided into three categories:

  1. Type I: The major spiral is present at both the meiotic divisions and the minor spiral is only observed in the mitotic stage. Example: Trillium, Vicia, and Sagittaria[1].
  2. Type II: The major spiral is only present in meiosis I, whereas, the minor spiral is observed during meiosis II and Mitosis, with similar spiral structures. Example: Tradescantia[1].
  3. Type III: the major spiral is only observed in the meiotic phase, however, the minor spiral is demonstrated in both, meiotic II and mitotic phase. In this case, the structure of the minor spiral is not the same in both cases[1]. The minor spiral in the meiotic II stage has fewer gyres than at the mitotic stage.

2. The geometrical form of the spiral

The interlocking (major and minor spirals interlock with each other or spiralize individually) of the two chromatids has a strong correlation with the geometrical properties of the spirals.

The chromonema has fibrils whose numbers differ depending on the type of species. The number of fibrils also varies depending on the phase of the division. It may be one at one phase and 2-4 at another phase[4]. The fibrils are coiled with each other and the geometry of coiling is categorized into two forms: plectonemic and paranemic.

  1. Plectonemic: When the two fibrils of chromonema are intertwined with each other in a manner that makes it difficult to separate them into two individual fibrils. When the two strands are pulled, they will be twisted around each other as many times as there will be gyres of the spiral[1].
  2. Paranemic: In this form, the chromonemal fibrils are held together without rotation of their ends. When they are separated by pulling up the ends, they will become perfectly straight and not twisted around each other[1].

Figure: The diagram shows the two forms of coiling of the chromonemata: (A) Plectonemic coil, and (B) Paranemic Coil.

Source: Manton, I. (1950). The Spiral Structure Of Chromosomes. Biological Reviews, 25(4), 486–508. Doi:10.1111/J.1469-185x.1950.Tb00770.X

3. Direction of coiling

The direction of coiling is random in the chromonemata, it can be right-handed or left-handed, on both sides of the centromere or in both the chromatids. In some chromosomes, the symmetrical coiling (one chromatid is right-handed while the other is left-handed) is also observed[3].

Figure: It shows (a) Microscopic image, and (b) Schematic illustration, of chromosome 2 of humans, which shows the symmetrical gyre direction[3].

L=left-handed, R=right handed

Source: Ohnuki, Y. (1968). Structure of chromosomes. Chromosoma, 25(4), 402–428. DOI:10.1007/bf02327721

4. Centromere

Normally, in the centromeric region of the chromosome, the chromonemata are present in uncoiled thread-like structures. However, the centromere has three different appearances[3]:

  1. Simple uncoiled-thread like form (most common).
  2. Appears in the ellipsoidal forms: This is observed when the chromosome is contracted. So, it is a temporary modified form of chromonemata.
  3. Appear as two separate chromonemata: This is observed in ordinary non-spiralized chromosome preparations[3].

5. Telomere structure

Based on the different termination pattern of chromonemata, the telomere is categorized into two groups[3]:

  1.  Pointed-end termination: In this case, the chromonemata terminate at a point where it is “uncoiled”, and that results in a point-end, called pointed-end termination[3].
  2. Rounded-end termination: In this case, the chromonemata “coils” terminate at the end of the chromosomes, resulting in a rounded-shaped end, called rounded-end termination[3].

Method to visualize the spiral nature of human chromosome

Human leukocyte is a favorite material for scientists to study the spiralization of the chromosome at mitotic phases, as demonstrated in the following steps[3].

Procedure

  1. Culture the leucocyte using Eagle’s balanced salt solution supplemented with 10% fetal calf serum at 37 ℃ for 3-5 days.
  2. After 3 days treat the culture with 0.06 µg/ml colchicine for 1 hour at 37 ℃.
  3. Suspend the cell cultures in T-30 or T-60 flasks by shaking.
  4. Harvest and transfer the culture into a 15 ml conical centrifuge tube.
  5. Centrifuge the tube for 5 minutes at 1000 rpm.
  6. Discard the supernatant and provide the pellet a hypotonic treatment. This causes the swelling of the cell and makes it easy to release the chromosome for the demonstration of the spiral structure.
  7. Resuspend the cell in a centrifuge tube containing 2-3 ml of a 4:2:0.8 mixture of equimolar solution (0.055 M and pH 6.5-7.0) of KCl, NaNO3, and CH3COONa.
  8. Keep the cell in the solution for 90 minutes at room temperature (21-23 ℃). During this period resuspend cells for 2-3 times by gentle pipetting.
  9. Add 10 ml of freshly prepared chilled fixative in 1:1 mixture of ethanol or methanol and glacial acetic acid.
  10. Make three changes of fixative at every 10 minutes of interval.
  11. Put one or two drops of cell suspension on a clean slide and allow it to dry without flaming[3].
  12. Dilute the Giemsa staining solution 70 times with distilled water.
  13. Stain the slides with the diluted Giemsa stain for 30 minutes.
  14. Wash the slides under running water and allow it to stand at room temperature for complete dehydration.
  15. Drip the slides in xylene for 5 minutes and then mount it in Permount.
  16. Observe the slide in a phase-contrast microscope or brightfield microscope and analyze the spiral structure of the chromosome[3].

Figure: A microscopic image of the human chromosome showing spirals at the metaphase stage of mitotic division, in cultured leucocytes[3].

Source: Ohnuki, Y. (1968). Structure of chromosomes. Chromosoma, 25(4), 402–428. DOI:10.1007/bf02327721

Method to visualize the spiral nature of plant chromosome

Here’s a procedure of demonstration of the spiral chromosome in Lilium longiflorum[2]. However, demonstration methods need a bit of modification depending on the species of the plant. So, the method explained below may not work for the visualization of the spiral chromosome in every other plant.

Procedure

  1. Isolate the pollen mother cells (PMCs) of the Lilium longiflorum.
  2. Take a clean slide and suspend PMCs in a small drop of 3 % saccharose solution on the slide.
  3. Invert the slides onto a Petri dish containing 10 ml of 20 % alcohol.
  4. Add  8 drops of 25% ammonia solution in the Petri dish. This is done to loosen the tightly packed coiling structure[2].
  5. Stain the cells with 0.5% acetocarmine and prepare the squash of the cells on the slide.
  6. Observe the cells under a microscope and analyze the spiral structure of the chromosome.
  7. The length of the chromonema (spiral structure) can be determined using the formula [) ], where n= number of gyres, d=spiral diameter, and p=pitch[2].

Figure: the microscopic image of the chromosome of the pollen mother cell of Lilium longiflorum, at the anaphase stage[2].

Source: Nokkala. S. And Nokkala. C. 1985. Spiral structures of meiotic chromosomes in plants. ~ Hereditas 103: 187-194. Lund. Sweden. ISSN 0018-0661

Conclusion

So far, researchers have studied the two forms (major and minor spiral) of the spiral structure, in the meiotic and mitotic phases of the chromosome, especially in prophase, metaphase, and anaphase. They observed that the number of gyres is lesser in the prophase, clearly visible in metaphase, and condensed or more in the anaphase.

The scientists then concluded that the spiral structure shows variations depending on the division (meiotic or mitotic), phases of the division, and the species which is being used for the study.

Colchicine and ammonia treatments have been a popular method to study the structure. The chemicals de-condense the spiral structure inside the chromosome to study its different properties. For example, diameter, gyre number, and coiling in different regions of the chromosome, such as centromere and telomere. However, the structure of chromosomes still needs a deep-dive to understand the processes that involve its functioning.

References

  1. Manton, I. (1950). The Spiral Structure Of Chromosomes. Biological Reviews, 25(4), 486–508. DOI:10.1111/j.1469-185x.1950.tb00770.x
  2. Nokkala. S. And Nokkala. C. 1985. Spiral structures of meiotic chromosomes in plants. ~ Hereditas 103: 187-194. Lund. Sweden. ISSN 0018-0661
  3. Ohnuki, Y. (1968). Structure of chromosomes. Chromosoma, 25(4), 402–428. DOI:10.1007/bf02327721.
  4. https://www.biologydiscussion.com/chromosome/chromosome-its-parts-functions-and-types-1934-words-biology/486.

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