Question

In this chapter, we focused on how DNA is organized at the chromosomal level. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, propose answers to the following fundamental questions: (a) How do we know that viral and bacterial chromosomes most often consist of circular DNA molecules devoid of protein? (b) What is the experimental basis for concluding that puffs in polytene chromosomes and loops in lampbrush chromosomes are areas of intense transcription of RNA? (c) How did we learn that eukaryotic chromatin exists in the form of repeating nucleosomes, each consisting of about 200 base pairs and an octamer of histones? (d) How do we know that satellite DNA consists of repetitive sequences and has been derived from regions of the centromere?

Short answer

Question: Explain how we know that viral and bacterial chromosomes most often consist of circular DNA molecules devoid of protein, and how puffs in polytene chromosomes and loops in lampbrush chromosomes are areas of intense transcription of RNA. Additionally, discuss the discovery that eukaryotic chromatin exists in the form of repeating nucleosomes and how satellite DNA consists of repetitive sequences and derives from regions of the centromere. Answer: Viral and bacterial chromosomes were found to consist of circular DNA molecules devoid of protein through electron microscopy, density gradient centrifugation, and biochemical analysis. Puffs in polytene chromosomes and loops in lampbrush chromosomes were discovered to be areas of intense RNA transcription through autoradiography and electron microscopy. The discovery that eukaryotic chromatin exists in the form of repeating nucleosomes came from experiments involving nuclease digestion, transmission electron microscopy, and biochemical studies. Finally, the understanding that satellite DNA consists of repetitive sequences and derives from regions of the centromere emerged through methods such as density gradient centrifugation, sequence analysis, molecular hybridization, and fluorescence in situ hybridization (FISH).

Step-by-step solution

Question (a) Viral and Bacterial Chromosomes

To explain how we know that viral and bacterial chromosomes most often consist of circular DNA molecules devoid of protein, let's discuss the experiments and methods that lead to these discoveries. Key evidence comes from the following: 1. Electron microscopy: Visualization of circular DNA molecules from some viruses and bacteria provided strong evidence of their circular structure. 2. Density gradient centrifugation: This technique allowed scientists to isolate the DNA in viruses and bacteria, showing that indeed, the isolated DNA is devoid of proteins. 3. Biochemical analysis: Studies on the composition and proportions of DNA and protein in viruses and bacteria further supported that their genome is circular and free of proteins.

Question (b) Puffs in Polytene Chromosomes and Loops in Lampbrush Chromosomes

The experimental basis for concluding that puffs in polytene chromosomes and loops in lampbrush chromosomes are areas of intense transcription of RNA was discovered through: 1. Autoradiography: By incubating the cells containing polytene or lampbrush chromosomes with radioactive labeled precursors of RNA, and then visualizing the radioactivity on an autoradiogram, researchers found that the radioactive precursors were primarily incorporated into puffs or loops. 2. Electron microscopy: These techniques allowed for a closer observation of puff and loop structures and revealed that they contained RNA and were associated with RNA polymerase – the enzyme responsible for transcribing RNA from DNA.

Question (c) Eukaryotic Chromatin and Nucleosomes

The discovery that eukaryotic chromatin exists in the form of repeating nucleosomes came from these experiments: 1. Nuclease digestion: Researchers found that chromatin treated with micrococcal nuclease exhibited a pattern of digestion with a regular spacing of around 200 base pairs. This was seen as a "ladder" pattern on an agarose gel, which indicated a repeating unit in the chromatin structure. 2. Transmission electron microscopy: Visualization of chromatin revealed the presence of bead-like structures with DNA wrapped around them, which were named nucleosomes. 3. Biochemical studies: Experiments showed that the core of the nucleosome was composed of an octamer of histones, small proteins rich in lysine and arginine that facilitate DNA packaging.

Question (d) Satellite DNA and Repetitive Sequences

The understanding that satellite DNA consists of repetitive sequences and derives from regions of the centromere emerged through these methods: 1. Density gradient centrifugation: Satellite DNA can be isolated from other types of DNA due to its specific density, which is different from the main genomic DNA. Centrifugation allowed for the separation of these densities, which lead to the discovery of satellite DNA as a distinct entity. 2. Sequence analysis and molecular hybridization: Once isolated, satellite DNA was characterized by analyzing its sequence and found to be highly repetitive, tandemly arranged, and present in large proportion in the centromeric region of the chromosome. 3. Fluorescence in situ hybridization (FISH): By using labeled probes that bind specifically to satellite DNA, researchers were able to visualize and localize it to the centromeric regions of the chromosomes, further confirming its origin and composition.