The Boston barberry is an imaginary plant with a diploid...

The Boston barberry is an imaginary plant with a diploid chromosome number of $4,$ and Boston barberry cells are easily grown in suspended cell cultures. $^{3}$ H-Thymidine was added to the culture medium in which a $G_{1}$ -stage cell of this plant was growing. After one cell generation of growth in 'H-thymidine-containing medium, colchicine was added to the culture medium. The medium now contained both 'H-thymidine and colchicine. After two "generations" of growth in $^{3} \mathrm{H}$ -thymidine-containing medium (the second seneration" occurring in the presence of colchicine as well), the two progeny cells (each now containing eight chromosomes) were transferred to culture medium containing nonradioactive thymidine ('H-thymidine) plus colchicine. Note that a "generation" in the presence of colchicine consists of a normal cell cycle's chromosomal duplication but no cell division. The two progeny cells were allowed to continue to grow, proceeding through the "cell cycle," until each cell contained a set of metaphase chromosomes that looked like the following. If autoradiography were carried out on these metaphase chromosomes (four large plus four small), what pattern of radioactivity (as indicated by silver grains on the autoradiograph) would be expected? (Assume no recombination between DNA molecules.)

Key Concepts

Semiconservative DNA Replication

In semiconservative DNA replication, each daughter double helix contains one original (template) strand and one newly synthesized strand. This principle is crucial when using labeled nucleotides (such as 3H?thymidine) to trace the incorporation of new DNA, because one can distinguish between the originally labeled strands and those synthesized later, allowing researchers to follow the distribution of radioactive label over successive rounds of replication.

Thymidine Labeling

Thymidine labeling involves supplying cells with thymidine molecules that are radioactively tagged (in this case with tritium, or 3H) during S-phase. When the cell’s DNA replicates, the radioactive thymidine is incorporated into new DNA strands. This method provides a marker that can be visualized later by autoradiography, distinguishing between DNA synthesized in the presence of the label versus DNA synthesized with nonradioactive nucleotides.

Autoradiography

Autoradiography is a technique used to visualize the distribution of radioactive substances within a sample. When applied to chromosomes, silver grains deposited on a photographic emulsion reveal where the radioactive label is present. This allows researchers to determine which parts of the chromosomes were synthesized during times when the radioactive nucleotide was available, and to study the pattern of replication and segregation.

Cell Cycle Manipulation Using Colchicine

Colchicine is a chemical that disrupts microtubule polymerization, thereby blocking cell division (cytokinesis) while allowing DNA replication to continue. This manipulation creates cells with duplicated but undivided chromosomes, such as polyploid cells or cells arrested in metaphase. It is used experimentally to accumulate cells in a particular stage and to generate distinctive chromosomal structures for analysis.

Chromosome Structure at Metaphase

Metaphase chromosomes are highly condensed and visible under a microscope, typically displaying paired sister chromatids joined at the centromere. In experiments utilizing differential thymidine labeling, such as combining cycles of radioactive and nonradioactive thymidine incorporation, the differences become visible in the final metaphase spreads. Autoradiography can reveal one chromatid per chromosome labeled with silver grains, reflecting the history of DNA replication in different labeling conditions.

Transcript

00:01 Dna replication is a semi -conservative mechanism, meaning that every time the dna molecule replicates, a parent strand remains with a newly formed daughter strand.

00:18 So if we take dna from this made -up boston barbary plant, it has a diploid number of four.

00:28 And so we expect to maintain that diploid number of four through cell division.

00:33 So if we look at a first generation where the dna is allowed to do semi -conservative replication with the radioactive thimidine, we would expect all four molecules of dna or chromosomes to be with a 3h femudine strand.

00:58 So all four of them.

01:00 So we have two large and two small chromosomes, all containing three h.

01:06 H -thymidine.

01:08 When we allow this newly formed molecule of dna with a radioactive label in it undergo a second generation, but we swap out the 3h thimidine for a non -radioactive 1h themadine, we still have a 2n equals 4 number because the cell division is allowed to proceed forward.

01:36 So we still have four chromosomes in each cell.

01:41 So technically it made eight chromosomes in two cells.

01:45 And so that would be four chromosomes in each cell, each with one chromosome that contains the 3h thimidine parental molecule.

01:57 And the newly formed daughter cells will all have 1h thimidine.

02:04 I'm going to draw them as x's.

02:07 So here's one age.

02:08 H, 1h, 1h for each of the daughter's cells...