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Chapter 4, The transmission of DNA at cell division

Specific Reading Assignment. pp 91-112. In mitosis and meiosis concentrate on chromosome replication and movement rather than the details of all the nuclear changes.  Crucial Figures are 3, 4, 5, 6, 7,8, 9, 14, 15, 16, 17, 18, 20, Figures in p. 96. Important Figures are 19, 21, 22, 24, mitosis figure in p. 91. Skip Figures: 10, 11, 12, 13, 23

DNA REPLICATION (pp. 93-99)

Objectives. To learn: 1) The concept of semi-conservative replication. 2) The experiment of Meselson and Stahl. 3) The concept of replication fork. 4) The main steps involved in the process of DNA synthesis: continuous and discontinuous synthesis, unwinding the DNA, priming, elongation, ligation.

1a) Make a diagram in the style of Fig. 4-3. Invent a sequence and indicate old and newly synthesized strands with different colors.

1b) Meselson and Stahl=s experiment (p. 96): Why is DNA in generation 0 or parental generation heavier than DNA of later generations? What isotope of nitrogen was used to grow the first generation? The DNA produced in this first generation could be described as Ahybrid@, why? What would have been the result of the M&S experiment (in generations 1 and 2) if replication occurred by a conservative rather than a semi-conservative mechanism?

2) The polymerization process.

2a) In Fig. 4-4, the replication fork advances from right to left; why does the top newly synthesized polynucleotide strand (light blue) grow Abackwards@, from left to right?

2b) Immagine that in the next step a stretch of nucleotides will be denatured at the left end of the figure. How will replication start to copy the new section of the top template strand?

2c) Why are RNA primers needed?

2d) Review your understanding of: replication fork, RNA primer, leading and lagging strands, Okasaki fragments. What is the function of each of the following enzymes: helicase and gyrase, primase, DNA polymerase, ligase?

2e) What is wrong with the position of primase in Fig. 4-7, relative to the primer?

3a) Is there a single site where the replication of DNA of a eukaryotic chromosome start?

3b) How many replication forks are produced at each origin of replication?

3c) How far does the replication fork of one origin of replication travel?

3d) In Fig. 4-5a, which is the leading strand, the top one or the bottom one?

Answers to DNA REPLICATION

1a) See Fig. 4-3.

1b) Bacteria had been grown in the heavy isotope ¹⁵N for many generations; this made its DNA Aheavy@, denser than DNA from cells grown in the normal ¹⁴N. This is the beginning of the experiment, called Generation 0. Then cells are transferred to medium with ¹⁴N (and here the first generation grew). We could call it Ahybrid@ because one strand is made up entirely of nucleotides with the heavy N and the other with nucleotides with the light (normal) isotope. If a conservative mechanism were in place in the first generation the two heavy strands would stay together in one molecule and a new molecule would have two light strands. [Diagram]

2a) The top strand is the discontinuous one in this figure. A polynucleotide strand always has to grow at its 3' end; it will extend to the right until it encounters the primer of the previous Okasaki fragment (Fig. 4-6b, c, and d).

2b) By laying down an RNA primer first, the primer will then be elongated with deoxynucleotides. Note that in Fig. 4-4 the primer from which the top strand is elongated should have been indicated.

2c) Because RNA polymerase is able to start a new chain without a primer, but DNA polymerases cannot; they can only elongate.

2e) If primase is at all associated with the primer, it should be at its 3' end, where it is elongating it.

3a) In eukaryotic chromosomes replication starts at many specific sites more or less simultaneously (At the beginning of the S or synthesis phase of the cell cycle). (In prokaryotes, with much smaller chromosomes, there is a single origin of replication.)

3b) Two replication forks, traveling in opposite directions, are produced at each origin of replication.

3c) Until they encounter a replication fork from a neighboring origin traveling in the opposite direction. At that point the two fuse, and replication is complete in that region of the chromosome (Fig. 4-8b).

3d) In the fork that goes to the left, the top is the leading strand; in the fork that travels to the right, the bottom is the leading strand.

CELL DIVISION (pp. 100-112)

Objectives. To learn 1). The behavior of chromosomes in mitosis and meiosis. 2). The "rationale" for that behavior. 3). The nomenclature of the main stages in mitosis and meiosis (It is not necessary to memorize the names of all the sub-stages of meiotic prophase, but it is important to know the main events in that prophase). 4). The main stages in the life cycle of sexual organisms.

4) Cell division that conserves the genetic material (Mitosis).

4a) In what tissues and stages of development is asexual cell division (mitosis) found in 1) humans, 2) flowering plants, 3) fungi? What is the process by which the nucleus divides in asexual cell division?

4b) The mitotic process occurs, with few variations, in most eukaryotes; i.e., it evolved a long time ago, and has been maintained by natural selection pretty much unchanged. Why? What is the outcome insured by mitosis that is so important?

4c) Compare the middle diagram (mitosis in diploids) in Fig. 4-20 to the photographs on p. 91. Which drawings in Fig. 4-20 represent the lower left, upper right and lower right photos.

4d) In Fig. 4-20 the authors use the ADNA representation@ way of drawing chromosomes; this is to remind you that there is a linear, double-stranded DNA molecule in each chromatid. The second circle from the top shows how, after DNA synthesis, each chromosome is made up of two chromatids, each with its own DNA molecule. Draw these top two stages (before and after DNA synthesis) using different colors to indicate the Aold@ and Anew@ DNA strands.

4e) Draw by yourself a schematic diagram of mitosis in a diploid cell using as a guideline Fig. 4-20, but instead of the ADNA representation@ use the ASolid representation@, only that instead of drawing the chromosomes as little sausages, represent them as a line, with a little circle to indicate the centromere (As in Table 2-4; check your answer to Guide and Orientation Question 7f) in the Chapter 2 assignment). We will call this the ALine representation@ of chromosomes. Label the stages of mitosis that you draw.

4f) Draw mitosis as in question 4e), but instead of a diploid cell with two chromosomes represent a diploid cell with four chromosomes, two metacentric, and two acrocentric. Indicate genes in both pairs of chromosomes.

4g) The drawings you made correspond to what stages in Fig. 4-21?

5a) In what organs and/or stages of development does meiosis occur in animals, plants and molds?

5b) Similar to question 3b), what is the Apoint@ of meiosis?

5c) Is there a process that to some extent Areverses@ what is accomplished in meiosis?

5d) Draw meiosis (instead of mitosis) as in question 4f).

5e) How many times would you have to draw meiosis of the same cell (as in 5d)) to show all possible outcomes?

5f) Why would you say in your answer to 4c) that the process is not fully reversed? i.e., that you do not get exactly the cell you had before meiosis?

5g) The drawings you made correspond to what stages in Fig. 4-22?

Answers to CELL DIVISION

4a) Asexual cell division is found in all tissues and stages of development in humans (all diploid cells) and flowering plants (mostly diploid cells), except for those divisions in the germ line that give rise to haploid cells. In fungi, mitosis is found in the growth of the vegetative tissue (haploid). The process by which the nucleus divides in asexual cell division is mitosis (Fig. 4-16).

4b) Because mitosis insures that the two daughters cells produced during cell division have all the genetic information of the mother cell (or insures that each daughter cell receives a full chromosomal complement).

4c) Bottom left is a cell in metaphase, third drawing from the top, top right is anaphase, fourth drawing from the top, and bottom right is telophase, two bottom drawings.

4d)

4e) Note that what is important, for our purpose in this genetics course, is not so much everything that happens in mitosis, but only the behavior of the chromosomes.

4f)

4g) Genetics in Process: a) (assuming it is before synthesis, G1) - b) (after synthesis, G2 or ptophase) - d) metaphase - e) anaphase - f) telophase and daughter cells not shown in the book.

5a) Meiosis occurs in the gonads of animals, in cells that will produce the gametes, in the flowers of plants in cells that will produce the female gametophyte and male pollen grains and in the zygote in fungi, immediately after fusion of the gametes (Fig. 4-16).

5b) The Amain point@of meiosis is to produce daughter cells with half the number of chromosomes, but this is not just a question of numbers. Just as in mitosis each daughter cell receives a copy of each chromosome, in meiosis each meiotic product receives one of each Akind@ of chromosome (there being two of each kind of chromosome in the original diploid cell). We go from one diploid meiocyte (cell undergoing meiosis), with one round of DNA synthesis, to four haploid cells.

5c) Fertilization, the joining of two gametes reverses the meiotic process in terms of numbers of chromosomes. We go from two haploid cells to one diploid zygote.

5d) 

5e) Twice for a cell with two pairs of chromosomes: once showing that the chromosome with A goes with the chromosome carrying B (and a with b), and a second time showing A going with b and a with B. Note that the outcome depends on the alignment of chromosomes in the first metaphase plate, and that this alignment occurs at random. [Note that there are so many genes in each chromosome that, in practice, no two members of a pair are identical; i.e. there will be many genes for which the chromosomes will have different alleles); note also that the number of possible outcomes goes up exponentially with the number of pairs of chromosomes; thus, for 2n = 6 the number of possibilities is 4 (2²); for 2n = 46 (as in humans) the total number is 2²².]