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Synthesis of an additional DNA in Pachytene and Zygotene

Synthesis of an additional DNA in Pachytene and Zygotene


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I've read, that in Pachytene and Zygotene additional DNA material is synthesized, about 0,3, 0,1% respectively. Why is it so?


About 0.3% of total DNA complement is synthesized during those 2 stages as a measure of replication repair mechanism.

Reference:

http://www.ncbi.nlm.nih.gov/pubmed/3731943

http://www.nature.com/nature/journal/v219/n5153/abs/219489a0.html


The occurrence and role of DNA synthesis during meiosis in wheat and rye

The incorporation of 3 H-thymidine into the DNA of rye meiocytes at zygotene, pachytene-diplotene and metaphase I to telophase II stages has been studied. Low levels of 3 H were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far.

The DNA synthesis inhibitor 2′-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.

G. W. R. W. is on sabbatical leave from the Department of Genetics, University of Alberta, Edmonton, Alberta, Canada.


Appels R, Bouchard RA, Stern H (1982) cDNA clones from meiotic-specific poly(A)+RNA in Lilium: Homology with sequences in wheat, rye, and maize. Chromosoma 85:591–602

Callan HG (1972) Replication of DNA in the chromosomes of eukaryotes. Proc R Soc Lond B 181:19841

Gonda DK, Radding CM (1983) By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology. Cell 34:647–654

Holm PB (1977) The premeiotic DNA replication of euchromatin and heterochromatin in Lilium longiflorum. Carlsberg Res Commun 42:249–281

Hotta Y, Shepard J (1973) Biochemical aspects of colchicine action on meiotic cells. Mol Gen Genet 122:243–260

Hotta Y, Stern H (1965) Polymerase and kinase activities in relation to RNA synthesis during meiosis. Protoplasma 60:218–232

Hotta Y, Stern H (1971a) Analysis of DNA synthesis during meiotic prophase in Lilium. J Mol Biol 55:337–355

Hotta Y, Stern H (1971b) A DNA-binding protein in meiotic cells of Lilium. Dev Biol 26:87–99

Hotta Y, Stern H (1971c) Meiotic protein in spermatocytes of mammals. Nature 234:83–86

Hotta Y, Stern H (1974) DNA scission and repair during pachytene in Lilium. Chromosoma 46:279–296

Hotta Y, Stern H (1975) Zygotene and pachytene-labeled sequences in the meiotic organization of chromosomes. In: Peacock WJ, Brock RD (eds) The eukaryote chromosome. Australian Nat Univ Press, pp 283–300

Hotta Y, Stern H (1976) Persistent discontinuities in late replicating DNA during meiosis in Lilium. Chromosoma 55:171–182

Hotta Y, Stern H (1981) Small nuclear RNA molecules that regulate nuclease accessibility in specific chromatin regions of meiotic cells. Cell 27:309–319

Ito M, Stern H (1967) Studies of meiosis in vitro. I. In vitro culture of meiotic cells. Dev Biol 16:36–53

Kurata N, Ito M (1978) Electron microscope autoradiography of 3 H-thymidine incorporation during the zygotene stage in microsporocytes of lily. Cell Struct Funct 3:349–356

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Lab, p 545

Morrison A, Cozzarelli NR (1979) Site-specific cleavage of DNA by E. coli DNA gyrase. Cell 17:175–174

Moses MJ, Poorman PA (1981) Synaptonemal complex analysis of mouse chromosomal rearrangements. II. Synaptic adjustment in a tandem duplication. Chromosoma 81:519–536

Ninnemann H, Epel B (1973) Inhibition of cell division by blue light. Exp Cell Res 79:318–326

Rasmussen SW, Holm PB (1980) Mechanics of meiosis. Hereditas 93:187–216

Roth TF, Ito M (1967) DNA dependent formation of the synaptonemal complex at meiotic prophase. J Cell Biol 35:247–255

Stern H, Hotta Y (1967) Chromosome behavior during development of meiotic tissue. In: Goldstein L (ed) The control of nuclear activity. Prentice Hall, pp 47–76

Stern H, Hotta Y (1970) Culture of meiotic cells for biochemical studies. In: Prescott DM (ed) Methods in cell physiology, vol. 4. Academic Press, pp 497–513

Stern H, Hotta Y (1974) Biochemical controls of meiosis. Ann Rev Genet 7:37–66

Stern H, Hotta Y (1977) Biochemistry of meiosis. Phil Trans R Soc Lond B 277:277–293

Stern H, Hotta Y (1980) The organization of DNA metabolism during the recombinational phase of meiosis with special reference to humans. Mol Cell Biochem 29:145–158

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Class 11 Biology chapter 7 Cell Division Textbook solutions

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1. Choose correct option
A. The connecting link between Meiosis-I
and Meiosis-II is .
a. interphase-I
b. interphase-II
c. interkinesis
d. anaphase-I

B. Synapsis is pairing of .
a. any two chromosomes
b. non-homologous chromosomes
c. sister chromatids
d. homologous chromosomes

C. Spindle apparatus is formed during which stage of mitosis?
a. Prophase.
b. Metaphase.
c. Anaphase.
d. Telophase.

D. Chromosome number of a cell is almost doubled up during .
a. G1-phase
b. S-phase
c. G2-phase
d. G0-phase

E. How many meiotic divisions are necessary for formation of 80 sperms?
a. 80
b. 40
c. 20
d. 10

F. How many chromatides are present in anaphase-I of meiosis-I of a diploid cell having 20 chromosomes?
a. 4
b. 6
c. 20
d. 40

G. In which of the following phase of mitosis chromosomes are arranged at equatorial plane?
a. Prophase
b. Metaphase
c. Anaphase
d. Telophase

H. Find incorrect statement -
a. Condensation of chromatin material
occurs in prophase.
b. Daughter chromatids are formed in anaphase.
c. Daughter nuclei are formed at metaphase.
d. Nuclear membrane reappears in telophase.

I. Histone proteins are synthesized during .
a. G1 phase
b. S-phase
c. G2
phase
d. Interphase


2. Answer the following questions
A. While observing a slide, student observed many cells with nuclei. But some of the nuclei were bigger as compared to others but their nuclear membrane was not so clear. Teacher inferred it as one of the
phase in the cell division. Which phase may be inferred by teacher?
Answer : The phase which was inferred by the teacher was 'Prophase'. Because In this phase, the chromosomes starts getting condensed. If this phase occurs in cells of animal then the centrioles starts moving in the opposite ends or poles. Mitotic Spindle starts appearing in this phase.

B. Students prepared a slide of onion root tip. There were many cells seen under microscope. There was a cell with two groups of chromosomes at opposite ends of the cell. This cell is in which phase of mitosis?
Answer : The Anaphase is the mitosis phase revealing two groups of chromosomes at opposite ends of the cell. because of The most commonly used is the root tip to study the mitosis reaction. The onion tip is squashed which allows being flattened on the microscope.

The DNA specific stains are used to view the DNA under the microscope are Fuelgen stains and acetocarmine stain.

C. Students were shown some slides of cancerous cells. Teacher made a comment as if there would have been
a control at one of its cell cycle phase, there wouldn’t have been a condition like this. Which phase the teacher was
referring to?
Answer : This phase was the initial phase, phase I. The phase I can be treated with chemotherapy but cannot be controlled or by any medicinal treatment. Cancer cannot be completely cured but it can be treated, in the initial stage but at the last stage, the treatment is very difficult.

D. Some Mendelian crossing experimental results were shown to the students. Teacher informed that there are two genes located on the same chromosome. He enquired if they will be ever separated from each other?
Answer : When two genes are located on the same chromosome and as much they are near to each other they defy the Mendel's Law of Independent Assortment which says that "alleles coding for separate traits are passed independently of one another" but William Bateson, Edith Rebecca Saunders and Reginald Punnett in 1905 discovered the phenomenon of Genetic linkage, according to which two genes located on the same chromosome and are near to each other are unlikely to get separated and tends to gets inherited together. So, there is a very low probability of the two genes to separate from each other. However, the most important consideration in this matter will be the distance between them.

E. Students were observing a film on
Paramoecium. It underwent a process
of reproduction. Teacher said it is due to cell division. But students objected and said that there was no disappearnce of nuclear membrane and no spindle formation, how can it be cell division? Can you clarify?
Answer : reproduction in paramecium is through binary fission

Explanation: During reproduction, the macronucleus splits by a type of amitosis, and the micronuclei undergo mitosis. The cell then divides transversally, and each new cell obtains a copy of the micronucleus and the macronucleus.

F. Is the meiosis responsible for evolution? Justify your answer
Answer : Yes, Meiosis is responsible for evolution. Explanation: During Meiosis, recombination occurs which leads to variation in the progeny. So, variations due to Meiosis are considerable factors for evolution

G. Why mitosis and meiosis-II are called
as homotypic division?
Answer: Mitosis and Meiosis 2 is called equational or homotypic division because number of chromosomes remain equal before and after division.

H. Write the significance of mitosis.
Answer :

  1. Mitosis results in the production of diploid daughter cells with identical genetic complement usually.
  2. Cell divides by mitosis to restore the nucleo-cytoplasmic ratio.
  3. Helps in cell repair. Mitotic divisions in the meristematic tissues result in a continuous growth of plants throughout their life.

I. Enlist the different stages of prophase-I.
Answer : Prophase I. Prophase I is divided into five phases: leptotene, zygotene, pachytene, diplotene, and diakinesis.

3. Draw labelled digrams and write explanation

A. With the help of suitable diagram,
describe the cell cycle.
Answer : Sequential events occurring in the life of a cell is called cell cycle. There are two phases of cell cycle as interphase and M-phase. During interphase, cell undergoes growth or rest as per the need. During M-phase, the cell undergoes division. Interphase alternates with the period of division.

Interphase : Interphase is the stage between two successive cell divisions. It is the longest phase of cell cycle during which the cell is highly active and prepares itself for cell division. The interphase is divisible into three sub-phases as G1-phase, S-phase and G2-phase.

G1-phase : This is also known as first gap period or first growth period. It starts immediately after cell division. Cell performs RNA synthesis (mRNA, rRNA and t-RNA), protein synthesis and synthesis of membranes during this phase.

S-phase : It is synthesis phase in which DNA is synthesized or replicated, so that amount of DNA per cell doubles. Histone proteins are also synthesized during this phase.

G2 phase : G2 is the second growth phase,
during which nucleus increases in volume.
Metabolic activities essential for cell division occur during this phase. Various proteins necessary for cell division are synthesized during this phase. Besides, RNA synthesis also occur during this phase. In animal cells, a daughter pair of centrioles appear near the pre-existing pair.

M-phase or period of division : 'M' stands
for mitosis or meiosis. M-phase involves
karyokinesis and cytokinesis. Karyokinesis is the division of nucleus into two daughter nuclei whereas cytokinesis is division of cytoplasm resulting in two daughter cells

B. Distinguish between mitosis and meiosis.
Answer :
1) mitosis division takes place in somatic cells and as a result of this division growth occurs. Meisis takes place usually in reproductive cells and as a result of this character one generation pass into the other.

2) Mitosis Completed in one stage. Meiosis Completed in two stages.

3) Mitosis. Prophase is smaller (as compared to prophase of Meiosis). Meiosis Prophase longer than prophase of mitosis and divided into five substages.

4) In mitosis No crossing over takes place. In Meiosis Crossing over takes place in which exchange of segments of chromatids occurs.

5) Synapsis does not take place at metaphase. In Meiosis Synapsis between homologous chromosomes takes place (bivalent stage).

6) At metaphase, centromere is towards equatorial plate and ends of chromosomes towards poles. Centromere divides. In Meiosis metaphase I, the centromere is towards poles and ends of chromosomes towards equatorial plate. Centromere does not divide.

7) Chromatids are long and thin. In Meiosis Chromatids are shorter and thick

8) In mitosis Cytokinesis follows karyokinesis. In Meiosis Telophase I, cytokinesis does not takes place always (may occur).


C. Draw the diagram of metaphase.
Answer :


Plus One Botany Chapter Wise Questions and Answers Chapter 6 Cell Cycle and Cell Division

Plus One Botany Cell Cycle and Cell Division One Mark Questions and Answers

Question 1.
Meiosis results in
(a) Production of gametes
(b) Reduction in the number of chromosomes
(c) Introduction of variation
(d) all of the above
Answer:
(d) all of the above

Question 2.
At which stage of meiosis does the genetic constitution of gametes is finally decided
(a) Metaphase I
(b) Anaphase II
(c) Metaphase II
(d) Anaphase I
Answer:
(d) Anaphase I

Question 3.
Meiosis occurs in organisms during
(a) Sexual reproduction
(b) Vegetative reproduction
(c) Both sexual and vegetative reproduction
(d) None of the above
Answer:
(a) Sexual reproduction

Question 4.
During anaphase-l of meiosis
(a) Homologous chromosomes separate
(b) Non-homologous autosomes separate
(c) Sister chromatids separate
(d) non-sister chromatids separate
Answer:
(a) Homologous chromosomes separate

Question 5.
Mitosis is characterised by
(a) Reduction division
(b) Equal division
(c) Both reduction and equal division
(d) None of the above
Answer:
(b) Equal division

Question 6.
A bivalent of meiosis-l consists of
(a) Two chromatids and one centromere
(b) Two chromatids and two centromere
(c) Four chromatids and two centromere
(d) Four chromatids and four centromere
Answer:
(c) Four chromatids and two centromere

Question 7.
Cells which are not dividing are likely to be at
(a) G1
(b) G2
(c) Go
(d) S phase
Answer:
(c) Go

Question 8.
Which type cell divisions occur in meristematic cell of root apex?
Answer:
Mitosis

Question 9.
In which stage the actual reduction of chromosome number occurs in meiosis.
Answer:
Anaphase 1

Question 10.
Give the term for the failure of separation of homologous chromosomes.
Answer:
Non-disjunction

Question 11.
Name the cell divisions which help in growth and recombination of genes.
Answer:
Mitosis and meiosis

Question 12.
It is observed that heart cells do not exhibit cell division. Such cells do not divide further and exit ____ phase to enter an inactive stage called ____ of cell cycle. Fill in the blanks.
Answer:
G1 and G0

Question 13.
In the case of plant cells, the formation of new cell wall begins with a simple precursor. What is this precursor?
Answer:
Middle lamella

Question 14.
It is said that the one cycle of cell division in human cells (eukaryotic cells) takes 24 hours. Which phase of the cycle, do you think occupies the maximum part of cell cycle?
Answer:
Interphase.

Question 15.
At what stage of cell cycle does DNA synthesis take place?
Answer:
substage of Interphase

Question 16.
If the failure of division of cytoplasm occurs after nuclear division, What will happen to the cell?
Answer:
Free nucleii are formed

Question 17.
An anther has 1200 pollen grains. How many pollen mother cells must have been there to produce them?
Answer:
300 pollen mother cells

Question 18.
What is the peculiarity of zygotene?
Answer:
The pairing of the homologous chromosome called synapsis

Question 19.
It is the inactive stage of cell division but cell differentiation occurs. Name it.
Answer:
G0 Phase /Quiscent stage.

Plus One Botany Cell Cycle and Cell Division Two Mark Questions and Answers

Question 1.
Meiosis is the type of cell division which maintain the race. Discuss.
Answer:
Reduces the chromosome number to half, so that chromosome number is maintained in the next generation.

Question 2.
Interphase in cell cycle is sometimes referred to as resting phase. Do you consider this statement true? Substantiate your answer.
Answer:
No.

Question 3.
A diagram of typical cell cycle of a higher plant is shown here. Identify each stage of the cycle and explain what happens during these stages.

Answer:

  • G1 – pre mitotic gap -synthesis of RNA &Proteins
  • S – phase of synthesis – DNA replication
  • G2 – Post mitotic Gap phase synthesis of RNA &Proteins continues
  • M – Mitotic phase
  • G0 – Inactive phase

Question 4.
Cytokinesis differs in plant and animal cell. Substantiate this statement.
Answer:

Question 5.
Analyse column A and B arrange the matter is an appropriate order.

Question 6.
Identify the stages of Mitosis in which the following events take place:

Question 7.
Different stages of Prophase I of Meiosis are given in column A, arrange them in correct order and match them with the events in column B.

A B
i) Diplotene a) Chromosomes become gradually visible under the light microscope
ii) Pachytene b) The pairing of Homologous chromosomes
iii) Leptotene c) The appearance of recombination nodules and crossing over takes place
iv) Zygotene d) Dissolution of synaptonemal complex and separation of bivalents.
e) Terminalisation of Chiasmata

Question 8.
The pairing of homologous chromosomes is called synapsis.

  1. Name each pair of homologous chromosomes.
  2. Name the stage of prophase at which it takes place.

Question 9.
Specific chromosome number of each species is conserved across generations in sexually reproducing organisms. What is the reason for this? Write the different steps of this process.
Answer:

Question 10.
The life cycle of a cell is called cell cycle. It consists of four stages such as Gv S, G2, and M.

  1. Construct a pie diagram showing the different stages indicated above.
  2. State the major events occurring in G, S, and G2 phases.

Answer:
1. Pie diagram of cell cycle.

2. G1 Phase -1, Cell grows in size and prepares the machinery needed for the DNA replication. RNA and proteins are synthesized. S phase – DNA replication. G2 phase – Synthesis of RNA and proteins.

Question 11.
Arrange the following stages of cell cycle in correct sequence S, G2, G1, M.
Answer:
S, M, G1, S, G2

Question 12.
X’ shaped structure called ‘chiasmata occurs during a particular stage of cell division.

Question 13.
Given below are the five phases of prophase I of Meiosis I. Arrange them in correct order.
Zygotene, diakinesis, diplotene, leptotene, pachytene
Answer:
Leptotene, Zygotene, Pachytene, diplotene and diakinesis

Question 14.
Give the scientific term of the following.

  1. Interchange of genetic material between non-sister chromatids of the homologues chromosomes
  2. The plane of alignment of the chromosomes at metaphase

Question 15.
Identify the diagram and label a, b,c and write the events during this.

Answer:
(a) G1
(b) S
(c) G2

  • G1 – Interval between mitosis and initiation of DNA replication.
  • S – DNA synthesis or replication of DNA occurs.
  • G2 – In this phase proteins are synthesised for mitosis.

Question 16.
You have supplied I set of glass slides showing gametogenesis or garnets formation in an animal In one of the slide you observed the following features. Four cells with haploid number of chromosomes. Your friend told you that this is a meiotic division. (Hint: The diploid number of chromosome is 16.)
Are you agree with this statement. Justify your answer.
Answer:
Yes. Meiosis takes place in diploid cell or meiocyte to form four haploid cells. These cells contain 8 chromosomes each.

Question 17.
Differentiate reduction division from equational division.
Answer:
1. Reduction division:
It occurs in diploid cells to form 4 haploid cells, ie, the Chromosome number reduced to half in Meiosis I and results 2 daughter cells, which again divides to form 4 daughter cells. All cells formed in meiosis are haploid.

2. Equational division:
It is the mitotic division results 2 daughter cells carrying same set of chromosomes as that of parent cell. No genetic variation occurs.

Question 18.
Can there be mitosis without DNA replication in S phase?
Answer:
There cannot be mitosis without DNA replication in 5 phase of interphase because the trigger for mitosis is disturbance of nucleocytoplasmic ratio caused by DNA replication in S phase. Mitosis restores the quantity of genetic material to the species-specific level.

Question 19.
How does anaphase of mitosis and anaphase I of meiosis differ from each other?
Answer:
In anaphase of mitosis chromatids separate while in anaphase 1 of meiosis homologous chromosomes separate.

Question 20.
How does cytokinesis in plant cells differ from that in animal cells?
Answer:
1. In an animal cell a furrow in the plasma membrane.lt joins in the centre and dividing the cell cytoplasm into two.

2. In-plant cells, wall formation starts in the centre of the cell and grows outward to meet the existing lateral walls. Then Cell division occurs.

Question 21.
How cytokinesis is different in an animal and a plant cell?
Answer:
In-plant cell cytokinesis occurs by cell plate formation while in the animal cell it occurs by cell furrow formation.

Question 22.
Why mitosis is called equational division ? Give the occurrence of mitosis.
Answer:
It keeps the chromosome number constant. It occurs in somatic cells.

Question 23.
What is the feature of a metacentric chromosome?
Answer:
The metacentric chromosome has a centromere in the middle region with two equal arms of the chromosome.

Question 24.
What is kinetochore? Give its function.
Answer:
It is a disc-like area in each chromatid and is site of attachment of spindle microtubule.

Question 25.
Why is meiosis essentially in sexually reproducing organisms?
Answer:
Meiosis reduces the chromosome number to half as it is followed by fertilization which restores diploidy.

Question 26.
Name the stage of cells cycle at which one of the following events occurs.

  1. Chromosomes are moved to spindle equator
  2. Centromere splits and chromatids separate
  3. Pairing between homologous chromosomes. takes place
  4. Crossing over between homologous chromosomes takes place.

Question 27.
Downs syndrome and Klinefelter’s syndrome occurs due to mistake in cell division. What does it indicate?
Answer:
It is due to the failure of separation of homologous chromosomes during meiosis.

Question 28.
The events occur in prophase and telophase are one opposite to other,

  1. Name the cell structures shown the above events
  2. How many daughter nuclei are formed at the end of mitotic and meiotic prophase?

Question 29.
Mangolism or Trisomy is due to the failure of one event in cell division.

  1. What will be the ploidy level of dyad and tetrad of cells in meiosis?
  2. How is it occurs?
  1. Dyad – haploid, Tetrad – Haploid
  2. It occurs in meiosis due to separation of homologous chromosomes and chromosome number reduced to half.

Plus One Botany Cell Cycle and Cell Division Three Mark Questions and Answers

Question 1.
In which phase of meiosis is the following formed? Choose the answers from hint points given below.

  1. Synaptonemal complex ______
  2. Recombination nodules ______
  3. Appearance/activation of enzyme recombinase _____
  4. Termination of chiasmata _______
  5. Interkinesis ________
  6. Formation of dyad of cells ________
  1. Zygotene
  2. pachytene
  3. pachytene
  4. diakinesis
  5. After Meiosis-I before meiosis II
  6. after first cytokinesis

Question 2.
The interphase stage is significant in mitotic and meiotic cell division

  1. Give one specific event
  2. Name the stage of interphase this event occurs
  3. How will you differentiate interphase from interkinesis?
  1. DNA replication
  2. S – phase
  3. Interphase – cell prepares for cell division Interkinesis – short interval between meiosis I and meiosis II

Question 3.
The following events occur during the various phases of the cell cycle, Write the phase against each of the events.

Question 4.
Life cycle of a cell is called cell cycle. ‘S’ phase is an important phase of cell cycle.

  1. Justify your answer.
  2. Name the stages of cell cycle at which the following events occur.
    • Crossing over of homologous chromosome.
    • Pairingof homologous chromosomes.
    • Chromosomes are arranged at the equatorial plane.
  1. phase of DNA synthesis
  2. stages of the cell cycle
    • Pachytene
    • Zygotene
    • Metaphase

Question 5.
Name the stages of cell division in which the following events occur?

  1. Chromosomes are moved to spindle equator.
  2. Centromere splits and chromatids separate.
  3. Crossing over between homologous chromosomes takes place.

Question 6.
Match the words listed in column I with suitable words from column II.

  1. a) – Gametic meiosis
  2. b) – Nuclear division
  3. c) – Zygotic meiosis
  4. d) – Cytoplasmic division
  5. e) – Meiocytes
  6. f) – Plant cells

Plus One Botany Cell Cycle and Cell Division NCERT Mark Questions and Answers

Question 1.
Distinguish cytokinesis from karyokinesis.
Answer:
The division of cytoplasm is called cytokinesis, while the division of the nucleus is called Karyokinesis.

Question 2.
What is G0 (quiescent phase) of cell cycle?
Answer:
Some cells in the adult animals do not appear to exhibit division (e.g., heart cells and many other cells divide only occasionally, as needed to replace cells that have been lost because of injury or cell death.

These cells do not divide further exit G1 phase to enter an inactive stage called quiescent stage (G0) of the cell cycle. Cells in this stage remain metabolically active but no longer proliferate unless called on to do so depending on the requirement of the organism.

Question 3.
Describe the event taking place during interphase.
Answer:
The interphase is divided into three further phases:
1. G1 phase (Gap 1). G1 phase corresponds to the interval between mitosis initiation of DNA replication. During G, phase the cell is metabolically active and continuously grows but does not replicate its DNA.

2. S phase (Synthesis). S or synthesis phase marks the period during which DNA synthesis or replication takes place.

3. During this time the amount of DNA per cell doubles. If the initial amount of DNA is denoted as 2C then it increases to 4C. However, there is no increase in the chromosome number, if the cell had diploid or 2n number of chromosomes at G1, even after S phase the number of chromosomes remains the same, i.e., 2n.

4. G2 phase (Gap 2). In animal cells, during the S phase, DNA replication begins in the nucleus, and the centriole duplicates in the cytoplasm during the G2 phase, proteins are synthesised in preparation for mitosis White cell growth continues.

Question 4.
Why is mitosis called equational division?
Answer:
Since the number of chromosomes remains same in parent and daughter cells so mitosis is also called a equational division.

Question 5.
Name the stage of cell cycle at which one of the following events occur.

  1. Chromosomes are moved to spindle equator.
  2. Centromere splits and chromatids separate.
  3. Pairing between homologous chromosomes takes place.
  4. Crossing over between homologous chromosomes takes place.

Question 6.
What is the significance of meiosis?
Answer:
Significance of Meiosis:

  1. Maintaining genetic identity by maintaining number of chromosomes.
  2. Bringing variations to ensure better species.
  3. Facilitates sexual reproduction.

Question 7.
Discuss with your teacher about.

  1. haploid insects and lower plants where cell division occurs, and
  2. Some haploid cells in higher plants where cell division does not occur.
  1. Male bees, wasps and ants are haploid organisms because they are produced from unfertilized eggs.
  2. Synergids and antipodal cells in the ovule don’t undergo cell division.

Question 8.
Can there be mitosis without DNA replication in ‘S’ phase?
Answer:
DNA replication is necessary for cell division, and cell division cannot happen without DNA replication.

Question 9.
Can there be DNA replication without cell division?
Answer:
DNA replication takes place in order to prepare for cell division. Cell division is the next logical step after DNA replication.

Question 10.
Analyse the events during every stage of cell cycle and notice how the following two parameters change

  1. Number of chromosomes remains same after mitotic cell division and becomes half after meiotic cell division.
  2. During S phase the DNA content doubles, but number of chromosomes remains the same.

Plus One Botany Cell Cycle and Cell Division Multiple Choice Questions and Answers

Question 1.
Cleavage is a unique form of mitotic cell division in which
(a) there is no growth of cells
(b) the nucleus does not participate
(c) no spindle develops to guide the cells
(d) the plasma membranes of daughter cells do not separate.
Answer:
(a) there is no growth of cells

Question 2.
In animal cells, cytokinesis involves
(a) the separation of sister chromatids
(b) contraction of the contractile ring of microfilament
(c) depolymerisation of kinetochore microtubules
(d) a protein kinase that phosphorylates other enzymes
Answer:
(b) contraction of the contractile ring of microfilament

Question 3.
During mitosis, the number of chromosomes gets
(a) change
(b) no change
(c) maybe change if cell is mature
(d) maybe change if cell is immature
Answer:
(b) no change

Question 4.
A diploid living organism develops from zygote by which type of the following repeated cell divisions?
(a) Meiosis
(b) Amitosis
(c) fragmentation
(d) Mitosis
Answer:
(d) Mitosis

Question 5.
If you are provided with root-tips of onion in your class and are asked to count the chromosomes, which of the following stages can you most conveniently look into?
(a) Metaphase
(b) Telophase
(c) Anaphase
(d) Prophase
Answer:
(a) Metaphase

Question 6.
At which stage of mitosis, chromatids separated and passes to different poles
(a) prophase
(b) Metaphase
(c) anaphase
(d) Telophase
Answer:
(c) anaphase

Question 7.
The two chromatids of a metaphase chromosome represent
(a) replicated chromosomes to be separated at anaphase
(b) homologous chromosomes of a diploid set
(c) non-homologous chromosomes joined at the centromere
(d) maternal and paternal chromosomes joined at the centromere
Answer:
(a) replicated chromosomes to be separated at anaphase

Question 8.
The process of cytokinesis refers to the division of
(a) nucleus
(b) chromosomes
(c) cytoplasm
(d) nucleus and cytoplasm
Answer:
(c) cytoplasm

Question 9.
Which of the following serves as mitotic spindle poison?
(a) Ca2
(b) azide
(c) Tubulin
(d) Colchicine
Answer:
(d) Colchicine

Question 10.
Pairing of homologous chromosomes occurs at which stage?
(a) Zygotene
(b) Leptotene
(c) Metaphase
(d) Pachytene
Answer:
(a) Zygotene

Question 11.
In meiosis, division is
(a) I reductional and II equational
(b) I equational and II reductional
(c) Both reductional
(d) Both equational
Answer:
(a) I reductional and II equational

Question 12.
Which type of chromosomes segregate when a cell undergoes meiosis?
(a) Homologous chromosomes
(b) Non-homologous chromosomes
(c) Both (a) and (b)
(d) centric and acentric chromosomes
Answer:
(a) Homologous chromosomes

Question 13.
Chiasmata are most appropriately observed in meiosis during
(a) diakinesis
(b) diplotene
(c) metaphase-ll
(d) pachytene
Answer:
(b) diplotene

Question 14.
During cell division, sometimes there will be failure of separation of homologous chromosomes. This event is called
(a) interference
(b) complementation
(c) non-disjunction
(d) coincidence
Answer:
(c) non-disjunction

Question 15.
The second meiotic division leads to
(a) separation of sex chromosomes
(b) fresh DNA synthesis
(c) separation of chromatids and centromere
(d) separation of homologous chromosomes.
Answer:
(c) separation of chromatids and centromere

Question 16.
Term meiosis was proposed by
(a) Farmer and Moore
(b) Flemming
(c) Strasburger
(d) Darlington
Answer:
(a) Farmer and Moore

Question 17.
Synapsis occurs in the phase of meiosis.
(a) zygotene
(b) diplotene
(c) pachytene
(d) leptotene
Answer:
(a) zygotene

Question 18.
When the number of chromosomes is already reduced to half in the first reductional division of meiosis, where is the necessity of second meiotic division
(a) The division is required for the formation of four gametes
(b) Division ensures equal distribution of haploid chromosomes
(c) Division ensures equal distribution of genes on chromosomes
(d) Division is required for segregation of replicated chromosomes
Answer:
(d) Division is required for segregation of replicated chromosomes


Mechanisms of recombination

Recombination occurs when a piece of the paternal chromosome is swapped for the homologous piece of DNA on the matching maternal chromosome (or vice versa). Obviously, this kind of a DNA swap must be done carefully and with equivalence, so that the resultant DNA does not gain or lose information. To ensure this precision in recombination, the non-sister homologous chromatids are held together via proteins in a synaptonemal complex (SC) during prophase I. This ladder-like complex begins to form in the zygotene stage of prophase I and completes in pachytene. The complete SC consists of proteinaceous lateral elements (aka axial elements) that run along the length of the chromatids and a short central element composed of fibrous proteins forming the rungs of the ladder perpendicular to the two lateral elements.

Recombination may occur with or without the formation of double-strand breaks, and in fact, can occur without the formation of the synaptonemal complex, although the SC probably enhances the efficiency of recombination. In S. pombe, meiosis occurs without the formation of a synaptonemal complex, but there are small discontinuous structures somewhat similar to parts of the SC. In the fruit fly, Drosophila melanogaster, females undergo meiosis using a synaptonemal complex, but males do not undergo meiotic recombination, and their chromosomes do not form synaptonemal complexes. In most cases, recombination is preceded by the formation of recombination nodules, which are protein complexes that form at potential points for recombination.

The best studied mechanism for meiotic recombination involves a double-stranded break of one of the chromosomes initiated by the meiosis-specific endonuclease, Spo11. The 5&rsquo ends (one in each direction) of this cut are degraded slightly to form 3&rsquo single-stranded overhangs. These unpaired ends lead to the formation of Holliday junctions (named after Robin Holliday) with a strand from another chromatid acting as a template for synthesis of the missing portion of the chromatids, leading to two sister chromatids that are "entangled" by having one strand of DNA paired with a different chromatid. This entanglement may be resolved with or without a crossover. The recombination is initiated in pachytene and completes in diplotene, at which time the synaptonemal complex breaks down. As the chromatids begin to separate, chiasmata (sites where chromatids remain in contact) become apparent at some of the recombination sites. As prophase completes, the chiasmata resolve from the center of the chromosomes to the ends.

Figure (PageIndex<2>). Recombination of homologous chromosomes.

Video (PageIndex<1>): In this animation, explore how a Holliday junction is formed, and how it can subsequently be resolved. (www.youtube.com/watch?v=MvnWxN81Qps)


MATERIALS AND METHODS

Plant Material and Genotyping

The maize (Zea mays) UFMu-07260 mutant line (ZmmtopVIB-1) in the W22 inbred background was obtained from the UniformMu stock center of MaizeGDB (https://maizegdb.org/ McCarty et al., 2013). Another mutant allele, EMS4-0742ae (ZmmtopVIB-2) in the B73 inbred background, was obtained from the Maize EMS Induced Mutant Database (http://www.elabcaas.cn/memd/ Lu et al., 2018). All plants were cultivated and fertilized under normal field conditions during the growing season or in a growth chamber (16 h light at 28ଌ, 8 h dark at 22ଌ, 60% to 70% humidity). Maize genomic DNA was extracted using a method previously described (Li et al., 2013). Primers used for genotyping and sequencing of the two mutant alleles are listed in Supplemental Table S1.

Pollen Viability

Pollen viability was assessed using the Alexander staining method (Alexander, 1969 Johnson-Brousseau and McCormick, 2004). Mature pollen grains were dissected out of anthers from the wild type and ZmmtopVIB mutants during the pollination stage and then stained with 10% Alexander solution. Images of stained pollen grains were taken using a Leica EZ4 HD stereo microscope equipped with a Leica DM2000 LED illumination system.

RT-qPCR Analysis

Total RNA was isolated from root, stem, leaf, developing embryo, endosperm, young tassel, and young ear using a TRNzol-A + Kit Reagent (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was performed using a PrimeScript II first strand cDNA Synthesis Kit (TaKaRa) with Oligo-T primers to obtain cDNA. Quantitative PCR was conducted with a 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR Green Master Mix (TaKaRa). All reactions were performed with three biological replicates and technical repeats. Gene-specific primers and reference gene (Ubiquitin) primers for internal control are listed in Supplemental Table S1.

Meiotic Chromosome Preparation and DAPI Staining

Young tassels were fixed in Carnoy’s solution (ethanol:glacial acetic acid [3:1]) for 1 d at room temperature and then stored in 70% (v/v) ethanol at 4ଌ. Anthers were dissected in 45% (v/v) acetic acid solution. Meiocytes were squeezed from anthers and squashed onto slides using coverslips. Slides were frozen in liquid nitrogen and the coverslips were removed immediately. After serial dehydration in 70%, 90%, and 100% (v/v) ethanol, the air-dried slides were stained and mounted with DAPI in Vectashield antifade medium (Vector Laboratories).

FISH and Chromosome Painting

Chromosome spreads were prepared by the method described previously (Wang et al., 2006). Three repetitive DNA probes were used, including the pTa794 clone containing 5S rDNA repeats (Li and Arumuganathan, 2001), the pAtT4 clone containing telomere-specific repeats (Richards and Ausubel, 1988), and cy5-conjugated 180-bp knob oligonucleotides. The rDNA and telomere probes were labeled by the Nick Translation Kit (Roche). The chromosome 3 painting probe was labeled with ATTO-550 as previously described (Albert et al., 2019). Slides were counterstained using DAPI in antifade mounting medium (Vector Laboratories). Chromosome images were captured under a Ci-S-FL fluorescence microscope (Nikon) equipped with a DS-Qi2 microscopy camera (Nikon) or under a Delta Vision ELITE system (GE Healthcare) equipped with an Olympus IX71 microscope.

Immunofluorescence Assay

Immunofluorescence was performed as described previously (Pawlowski et al., 2003), with minor modifications. After being dissected and permeabilized in 1× buffer A solution with 4% (w/v) paraformaldehyde for 30 min at room temperature, fresh young anthers were washed twice in 1× buffer A at room temperature and then stored in 1× buffer A at 4ଌ. Meiocytes were squeezed from anthers and squashed onto slides. After freezing in liquid nitrogen, coverslips were removed immediately. The meiocytes were incubated in blocking buffer diluted with primary antibodies for 1 h in a 37ଌ humidity chamber, then washed three times in 1× phosphate-buffered saline. Goat anti-rabbit antibodies conjugated with Fluor 555 diluted in blocking buffer were added to the slides. After incubation at 37ଌ for 1 h, the slides were washed three times in 1× phosphate-buffered saline. Finally, cells were counterstained with DAPI in antifade mounting medium (Vector Laboratories). The antibodies against ASY1, ZYP1, and γH2AX were prepared as described previously (Jing et al., 2019a). Antibodies against AFD1, RAD51, and DMC1 were gifts from collaborators. All primary and secondary antibodies were diluted at 1:100. Images of meiocytes were observed and captured using a Ci-S-FL microscope (Nikon) equipped with a DS-Qi2 microscopy camera (Nikon). Two-dimensional projected images were generated using NIS-Elements software. Further image processing was conducted using ImageJ software (https://imagej.nih.gov/ij/index.html).

Accession Numbers

Genes referenced in this article can be found in GenBank/National Center for Biotechnology Information data libraries under accession numbers Zm00001d014728 (ZmMTOPVIB) AT1G60460 (Arabidopsis MTOPVIB) GSBRNA2T00026842001 (Brassica napus MTOPVIB) GLYMA (Glycine max MTOPVIB _01G029900) LOC107872805 (Capsicum annuum MTOPVIB) LOC107787690 (Nicotana tabacum MTOPVIB) BRADI_Ig34717 (Brachypodium distachyon MTOPVIB) Os06g0708200 (O. sativa MTOPVIB) SETIT_008523mg (Setaria italica MTOPVIB) and SORBI_3010G257500 (Sorghum bicolor MTOPVIB).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Phylogenetic analysis of MTOPVIB homologs in different plant species.

Supplemental Figure S2. Multiple sequence alignment analysis of MTOPVIB proteins in maize, Arabidopsis, and rice.

Supplemental Figure S3. Tissue-specific expression patterns of ZmMTOPVIB revealed by RT-qPCR.


Chapter 10 : meiosis

In prophase 1 of meiosis, the DNA coils tighter, and individual chromosomes first become visible under the light microscope as a matrix of fine threads. Because the DNA has already replicated before the onset of meiosis, each of these threads actually consist of two sister chromatids joined at their centromeres. In prophase 1, homologous chromosomes become closely associated in synapsis, exchange segments by crossing over, and then separate.

Prophase 1 is traditionally divided into five sequential stages: leptotene, zygotene, pachytene, diplotene, and diakinesis.

Leptotene: during which each chromosome becomes visible as two fine threads (chromatids)

Zygotene: A lattice of protein is laid down between the homologous chromosomes in the process of synapsis, forming a structure called a synaptonemal complex.

Pachytene: Pachytene begins when synapsis is complete (just after the synaptonemal complex forms and lasts for days. This complex, about 100 nm across, holds the two replicated chromosomes in precise register, keeping each gene directly across from its partner on the homologous chromosome. Within the synaptonemal complex, the DNA duplex unwind at certain sites, and single strands of DNA from base-pairs with complementary strands on the other homologue. The synaptonemal complex thus provides the structural framework that enables crossing over between the homologues chromosomes. As you will see, this has a key impact on how the homologues separate later in meiosis.

Diplotene: the fourth stage of the prophase I of meiosis, following pachytene, during which the paired chromosomes begin to seperate (the synaptonemal complex disassembles) into two pairs of chromatids . Diplotene is a period of intense cell growth. During this period the chromosomes decondense and become very active in transcription

Diakinesis: At the beginning of diakinesis, the transition into metaphase, transcription ceases and the chromosomes recondense.

Synapsis: During prophase, the ends of the chromatids attach to the nuclear envelope at specific sites. The sites the homologous attach to are adjacent, so that the members of each homologous pair of chromosomes are brought close together. They then line up side by side, apparently guided by heterochromatin sequences, in the process called synapsis.

Crossing Over

Within the synaptonemal complex, recombination is thought to be carried out during pachytene by very large protein assemblies called recombination nodules. A nodule’s diameter is about 90 nm, spanning the central element of the synaptonemal complex. The details of the crossing over process are not well understood, but involve a complex series of events in which DNA segments are exchanged between nonsister or sister chromatids. In humans, an average of two or three such crossover events occur per chromosome pair.

When crossing over is complete, the synaptonemal complex breaks down, and the homologous chromosomes are released from the nuclear envelope and begin to move away from each other. At this point, there are four chromatids for each other. At this point, there are four chromatids for each type of chromosome (two homologous chromosomes, each of which consists of two sister chromatids). The four chromatids do not separate completely, however, because they are held together in two ways: (1) the two sister chromatids of each homologue, recently created by DNA replication, are held near by their common centromeres and (2) the paired homologues are held together at the points where crossing over occurred within the synaptonemal complex.

Chiasma Formation

Evidence of crossing over can often be seen under the light microscope as an X-shaped structure known as a chiasma. The presence of a chiasma indicates that two chromatids (one from cach homologue) have exchanged parts. Like small rings moving down two strands of rope, the chiasmata move to the end of the chromosome arm as the homologous chromosomes separate.

Synapsis is the close pairing of homologous chromosomes that takes place early in prophase 1 of meiosis. Crossing over occurs between the paired DNA strands, creating the chromosomal configurations known as chiasmata. The two homologues are locked together by these exchanges and they do not disengage readily.

Metaphase 1

By metaphase 1, the second stage of meiosis 1, the nuclear envelope has dispersed and the microtubules form a spindle, just as in mitosis. During diakinesis of prophase 1, the chiasmata move down the paired chromosomes from their original points of crossing over, eventually reaching the ends of the chromosomes. At this point, they are called terminal chiasmata. Terminal chiasmata hold the homologous chromosomes together in metaphase 1, so that only one side of each centromere faces outward from the complex the other side is turned inward toward the other homologue. Consequently, spindle microtubules are able to attach to kinetochore proteins only on the outside of each centromere, and the centromeres of the two homologues attach to microtubules originating from opposite poles. This one-sided attachment is in marked contrast to the attachment in mitosis, when kinetochores on both sides of a centromere bind to microtubules.

Each joined pair of homologues then lines up on the metaphase plate. The orientation of each pair on the spindle axis is random: either the maternal or the paternal homologue may orient toward a given pole.

Chiasmata play an important role in aligning the chromosomes on the metaphase plate.

Completing Meiosis

After the long duration of prophase and metaphase, which together make up 90% or more of the time meiosis 1 takes, meiosis 1 rapidly concludes. Anaphase 1 and telophase 1 proceed quickly, followed – without an intervening period of DNA synthesis – by the second meiotic division.

In anaphase 1, the microtubules of the spindle fibers begin to shorten. As they shorten, they break the chiasmata and pull the centromeres toward the poles, dragging the chromosomes along with them. Because the microtubules are attached to kinetochores on only one side of each centromere, the individual centromeres are not pulled apart to form two daughter centromeres, as they are in mitosis. Instead, the entire centromere moves to one pole, taking both sister chromatids with it. When the spindle fibers have fully contracted, each pole has a complete haploid set of chromosomes consisting of one member of each homologous pair. Because of the random orientation of homologous chromosomes on the metaphase plate, a pole may receive either the maternal or the paternal homologue from each chromosome pair. As a result, the genes on different chromosomes assort independently that is, meiosis 1 result in the independent assortment of maternal and paternal chromosomes into the gametes.

Telophase 1

By the beginning of telophase 1, the chromosomes have segregated into two clusters, one at each pole of the cell. Now the nuclear membrane re-forms around each daughter nucleus. Because each chromosome within a daughter nucleus replicated before meiosis 1 began, each now contains two sister chromatids attached by a common centromere. Importantly, the sister chromatids are no longer identical, because of the crossing over that occurred in prophase 1. Cytokinesis may or may not occur after telophase 1. The second meiotic division, meiosis 2, occurs after an interval of variable length.

The Second Meiotic Division

After a typically brief interphase, in which no DNA synthesis occurs, the second meiotic division begins.

Meiosis 2 resembles a normal mitotic division. Prophase 2, metaphase 2, anaphase 2, and telophase 2 follow in quick succession.

Prophase 2 . At the two poles of the cell the clusters of chromosomes enter a brief prophase 2, each nuclear envelope breaking down as a new spindle forms.

Metaphase 2. In metaphase 2, spindle fibers bind to both sides of the centromeres.

Anaphase 2. The spindle fibers contract, splitting the centromeres and moving the sister chromatids to opposite poles.

Telophase 2. Finally, the nuclear envelope re-forms around the four sets of daughter chromosomes.

The final result of this division is four cells containing haploid sets of chromosomes. No two are alike, because of the crossing over in prophase 1. Nuclear envelopes then form around each haploid set of chromosomes. The cells that contain these haploid nuclei may develop directly into gametes, as they do in animals. Alternatively, they may themselves divide mitotically, as they do in plants, fungi, and many protists, eventually producing greater numbers of gametes or, as in the case of some plants and insects, adult individuals of varying ploidy.

During meiosis 1, homologous chromosomes move toward opposite poles in anaphase 1, and individual chromosomes cluster at the two poles in telophase 1. At the end of meiosis 2, each of the four haploid calls contains one copy of every chromosome in the set, rather than two. Because of crossing over, no two cells are the same. These haploid cells may develop directly into gametes, as in animals, or they may divide by mitosis, as in plants, fungi, and many protists.


Sorting out meiosis

Funding support by the Natural Sciences and Engineering Research Council of Canada (NSERC).

In his elegant and authoritative volume of work Meiosis, Bernard John aptly quoted the late J. Herbert Taylor, best known for his metabolic labeling studies on nucleic acid synthesis and segregation during the 1950s “Meiosis is still a potential battleground where dead hypotheses litter the field or rest uneasily in shallow graves, ready to emerge and haunt any conscientious scientist who tries to consolidate victory for any particular thesis” 1 . Even today with our ability to bring the combined power of genomics, transcriptomics, proteomics, and systems biology to bear on the problem much of the process of meiosis in mammals remains mysterious and incompletely characterized.

Meiosis is a segment of gametogenesis, the developmental program by which diploid progenitor germ cells reduce their ploidy through meiotic divisions to form haploid gametes that undergo profound genomic and morphological differentiation. The gametes, sperm, and ova in humans, are essential for sexual reproduction. Defects in this differentiation program manifest in phenotypes ranging from infertility and increased incidence of birth defects to cancers 2, 3 . Despite the importance of this process it remains less well understood than the process of mitotic cell division. Many aspects of gametogenesis remain unclear in part owing to our limited ability thus far to completely recapitulate the process in vitro. Unlike most basic processes involved in cell proliferation, gametogenesis only proceeds effectively in vivo with the support of surrounding cells and structures and bathed in the appropriate milieu of hormones and growth factors 4 . Attempts to recapitulate gametogenesis in vitro are making headway but to date neither spermatogenesis nor oogenesis with human germ cells has been fully accomplished in vitro 5, 6 .

The inability to reproduce the complete process of gamete formation in vitro should not preclude analysis of its stages, all that is required is the ability to recover a population of cells undergoing gametogenesis and separate them based on the phase of development. This is the approach that has been taken previously where isolated meiocytes were separated by sedimentation through a bovine serum albumin (BSA) gradient 7 . Although this technique has been successful in isolating populations of prophase, pachytene, and diplotene cells, it is limited by the inability to separate cells in the early stages of prophase I. The problem here is that the leptotene and zygotene phase is relatively short and cells pass through these stages quickly so in comparison with pachytene cells and spermatocytes, cells in the leptotene and zygotene phase are in very low abundance 8 . This limitation is compounded by the fact that cells in the leptotene and zygotene phase have similar physical characteristics (size, volume, and DNA content) and hence it is difficult to separate them based on any of those physical parameters 9 . This is problematic because prophase I is arguably the most important phase of meiosis.

During prophase I the chromosomes that were replicated in the preceding interphase begin to align in leptotene, the chromosomal telomeres cluster and interact with the nuclear envelope, promoting localized movement of the chromosomes to aid in the homology search required for bivalent formation that will occur as the cells enter the zygotene stage 10 . The homology search that allows alignment is an essential precursor to the formation of DNA double strand breaks and meiotic recombination along with formation of the synaptonemal complex and chiasmata. These events are crucial to ensure the integrity of the chromosome divisions that follow. Defects in chromosome alignment, recombination, even reduced levels of recombination can lead to increased rates of nondisjunction at meiosis I resulting in aneuploidy that manifests as reduced fertility or birth defects 2 . Indeed there is some evidence that such aneuploidy can result in cancer.

Progression through prophase I is accompanied by changes in gene expression, chromatin modification state, and chromatin condensation. These changes help to drive progress into meiosis. A comprehensive understanding of these processes in mammalian cells requires the application of genomic and proteomic technologies but these procedures are dependent upon the ability to isolate cell populations enriched for cells in the various stages of meiotic prophase.

In this issue of Cytometry (page 556), Gaysinskaya et al. describe a procedure for the recovery of germ cells from male adult mice and a fluorescence activated cell sorting strategy that effectively separates cells in all the phases of gametogenesis allowing recovery of populations with high purity. It is particularly notable that these investigators have optimized the preparation, staining, and analysis of the cells to allow separation of leptotene and zygotene populations. Sorting methods have been previously described for the separation of meiocytes and have been applied to guinea pigs which have a higher percentage of early prophase I cells and to adult mice 9, 11 , but no previously published protocols have achieved the same degree of separation of leptotene and zygotene stage cells as the protocol presented by Gaysinskaya et al. (page 556). The success of the newly described technique is built on three new approaches to the problem first a more extensive treatment of the seminal vesicles to increase the recovery of meiocytes, second, the use of Hoechst 33342 staining, and third, the application of a series of back-gating procedures to allow clear separation of individual populations of meiotic cells.

The isolation and preparation method used by the authors is similar to the methods used by other investigators but the details can make a big difference. Tissue from mouse testis was treated with collagenase/DNase solution followed by pipetting to disperse the seminiferous tubules and release interstitial cells. This was followed by a further, more rigorous treatment with collagenase/DNase and trypsin to dissociate the tissue and release single cells. The authors specify the times of treatment and concentration of enzymes used, which they titrated to achieve optimal cell preparations. The sample preparation is a critical step in this procedure because early prophase cells are in very low abundance and to isolate sufficient cells for sorting and downstream analysis requires near quantitative recovery from the tissue.

The second critical aspect of the cell preparation for sorting is staining with Hoechst 33342. This nucleic acid binding stain does not require permeablization of the cells and importantly allows the cell preparation to be subsequently stained with propidium iodide to rapidly exclude dead cells from the sort. Although other investigators have used Hoechst for sorting meiotic cells 12 , Gaysinskaya et al. specify that optimal staining is achieved with 6 µg Hoechst/million cells (page 556). The authors indicate that the ratio of stain to cells is critical to achieve good results and provides reproducibility thus allowing for populations from independent preparations to be pooled. This is an important consideration not only to allow reproducibility between experiments but also because collecting sufficient samples for RNA or proteomic analysis from low abundance populations like leptotene cells may require pooling samples from more than one sort.

Flow cytometric analysis of the prepared meiotic cells was initiated with conditions to exclude debris based on setting a size gate with the forward scatter. Although this eliminates much of the debris in the sample preparation, it also has the effect of gating out the abundant small-elongated spermatozoa. This gating reduced the ability to capture and analyze all of the meiotic cell types in a single experiment. However, it significantly reduces the noise in later analysis and the trade off is worthwhile as the elongated spermatozoa population can be examined independently if it is important to collect them for any particular analysis.

One of the benefits of Hoechst 33342 staining is the ability to detect the stain in either the red or blue channel. The authors took advantage of this property by setting a gate on the blue channel based on DNA content to allow exclusion of haploid cells that have completed the meiotic program. This treatment further simplifies the pattern of meiotic cells detected and in the authors hands this allows striking discrimination between leptotene/zygotene and pachytene/diplotene populations however, it is not possible to separate cells in the leptotene and zygotene phases at this point in the analysis. To isolate a high purity population of early prophase cells a back-gating strategy was applied. This strategy bases its initial gating on the fluorescence characteristics of the cells followed by analysis of the physical (forward scatter (FSC) and side scatter (SSC)) characteristics. The authors initially gated leptotene and zygotene cells based on Hoechst 33342 fluorescence but FSC and SSC plots revealed a large degree of overlap in the two populations resulting in extensive contamination. This is not surprising given the similar shape and diameter of cells in the leptotene and zygotene stages. This information allowed the authors to impose more conservative gates on the FSC and SSC profiles to reduce contamination of the populations based on light scattering parameters. This strategy thus creates gates based on both Hoechst 33342 dye fluorescence and FSC/SSC that allows separation of even the leptotene and zygotene populations.

Application of the back-gating approach to murine cells in all stages of spermatogenesis allowed the isolation of high purity populations of cells. Immuno-staining with antibodies directed at phosphorylated histone γH2AX a marker for DNA double strand breaks, and SYCP3, a marker for the synaptonemal complex, as well as simply staining the DNA of chromosome spreads showed that multiple sorts allowed the collection of preleptotene, and pachytene cell populations of 80–90% purity and diplotene cells of greater than 95% purity. Most importantly for this article leptotene cells with purity of up to 85%, and zygotene cell populations of up to 90% purity could be captured. Although not perfect such populations will undoubtedly be sufficient for transcriptomic and proteomic analysis, and this will be an important technique to apply to studies of gametogenesis. This tool will greatly enhance our ability to investigate the transcriptional program and proteomic dynamics that regulate progression through gametogenesis in mammalian cells.