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, what answers would you propose 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

Polytene chromosomes display puffs and lampbrush chromosomes have loops in regions of intense transcription; these areas show the presence of nascent RNA and increased concentration of RNA polymerase. 3) How was the nucleosome structure in eukaryotic chromatin discovered? Roger Kornberg discovered the nucleosome structure using nuclease digestion and gel electrophoresis, which revealed the repeating structure of chromatin with a 200 base pair DNA segment wrapped around a histone core. 4) What is satellite DNA and how is it related to the centromere regions of chromosomes? Satellite DNA consists of repetitive sequences derived from the centromere regions of chromosomes. It was identified through density gradient ultracentrifugation and cytological studies that localized it to the centromere regions.

Step-by-step solution

a) Viral and bacterial chromosomes: circular DNA molecules devoid of protein

Viral and bacterial chromosomes are shown to be circular DNA molecules without protein through a series of experiments. One of the key experiments that provided strong evidence for this is cesium chloride density gradient centrifugation which separates molecules based on their density. By using this method, researchers found that viral and bacterial DNA was separated from the protein material, proving that their chromosomes consist primarily of circular DNA molecules without associated proteins. Additionally, electron microscopy has shown the circular nature of bacterial and viral DNA.

b) Polytene chromosomes: puffs and loops indicate intense transcription of RNA

The experimental basis for concluding that puffs in polytene chromosomes and loops in lampbrush chromosomes are areas of intense RNA transcription comes from observing the presence of nascent RNA and an increased concentration of RNA polymerase in these regions. Pioneering studies by Joseph Gall and early autoradiography experiments, which used radioactive labeling to track RNA synthesis, provided evidence that these areas had high levels of RNA synthesis activity. Radioactive RNA precursors were incorporated into the chromosomal regions displaying puffs and loops, confirming their involvement in active transcription of RNA.

c) Eukaryotic chromatin: nucleosomes with 200 base pairs and octamer of histones

The discovery of the nucleosome structure and its repeating nature in eukaryotic chromatin is attributed to experiments conducted by Roger Kornberg in 1974. By using nuclease digestion (micrococcal nuclease) and gel electrophoresis, Kornberg found that chromatin was digested into regular fragments containing 200 base pairs of DNA. This established the repeating unit of chromatin and indicated that the structure was composed of DNA wrapped around a protein core. The octamer of histones in nucleosomes was identified through protein analysis, with researchers determining that histones H2A, H2B, H3, and H4 are present in the core in pairs.

d) Satellite DNA: repetitive sequences from centromere regions

Satellite DNA was found to consist of repetitive sequences and be derived from the centromere regions due to several lines of experimental evidence. Firstly, the discovery of satellite DNA was made by density gradient ultracentrifugation, which separated the main bulk of the DNA from the repetitive sequences. These repetitive sequences were dubbed "satellite" DNA because they were found to be satellite bands in the density gradient. Further characterization of satellite DNA showed that it was highly repetitive, and cytological studies determined its localization to the centromere regions of chromosomes. These findings solidified the understanding that satellite DNA is composed of repetitive sequences deriving from centromere regions.