Exercise practice: Molecular Basis of Inheritance

6.1 The last two topics and the exercise section

The Human Genome Project and DNA fingerprinting close the chapter, and the exercise questions test both. The project began in 1990 and was completed in 2003. Its public consortium included the United States Department of Energy and the National Institutes of Health, and the Wellcome Trust became a major partner, with contributions from Japan, France, Germany and China. Two strategies were used: expressed sequence tags, which start from the genes that are expressed as RNA, and sequence annotation, which sequences whole genomes and assigns functions later. DNA was cut into fragments, cloned in bacteria or yeast using BAC and YAC vectors, sequenced with automated sequencers based on the Sanger method, and the sequences were assembled with specialised computer programs.

The main findings are worth memorising as a list. The assembled sequence totals about 3,164.7 million base pairs. An average gene spans around 3,000 bases, while dystrophin, the largest known gene, spans 2.4 million bases. The gene count came out at roughly 30,000, much lower than the earlier estimates of 80,000 to 140,000. Human beings are almost identical at the sequence level, with the usual figure being 99.9 per cent similarity. Protein-coding regions are a small minority, under two per cent of the total. A major share of the genome is made of repeated sequences. Among the chromosomes, the first carries the largest gene count at 2,968 and the Y the smallest at 231. The project also catalogued about 1.4 million positions where a single base differs between individuals, the SNPs.

DNA fingerprinting was developed by Alec Jeffreys. The method compares regions of DNA where short sequences are repeated a varying number of times. The number of repeats differs between individuals, so cutting the DNA with restriction enzymes and separating the fragments by gel electrophoresis produces a pattern of bands, a profile, that is characteristic of an individual. The technique has four steps: isolating the DNA, cutting it with restriction enzymes, separating the fragments by electrophoresis, and detecting specific fragments by hybridisation with a labelled probe followed by autoradiography. It is used in forensic identification, in paternity testing and in population genetics.

6.2 Exercise answers

1. Why is DNA a better genetic material than RNA? DNA is more stable because its deoxyribose lacks the reactive 2 prime hydroxyl group, and the double-stranded structure protects the bases. RNA is chemically more reactive and degrades more easily, though RNA is a better catalyst. The stability and copying ability of DNA make it the genetic material in most organisms.

2. In Griffith's experiment, why did heat kill the bacteria but not the transforming principle? Heat kills the bacterial cells but does not destroy the DNA. The DNA survives heating and can still enter the rough cells and transform them.

3. Why did Avery, MacLeod and McCarty use enzymes? They used proteases and RNases to show that destroying proteins or RNA did not stop transformation, while DNase did. This isolated DNA as the transforming principle.

4. How did Hershey and Chase differentiate DNA from protein? They labelled DNA with radioactive phosphorus and protein with radioactive sulphur, and followed which label entered the bacteria.

5. Why is RNA more labile than DNA? RNA has a 2 prime hydroxyl group that can attack the phosphodiester bond, and its single-stranded form exposes the bases. DNA lacks that hydroxyl and is double-stranded.

6. What are the two functions of the 3 prime and 5 prime ends? The 5 prime end carries the phosphate and the 3 prime end carries the hydroxyl; polymerisation adds new nucleotides at the 3 prime end, and the ends define the direction of the strand.

7. Differentiate repetitive DNA and satellite DNA. Repetitive DNA is any sequence present in many copies, while satellite DNA is a class of highly repeated sequences that form distinct bands in density gradients. Both belong to the non-coding part of the genome.

8. Differentiate mRNA and tRNA. mRNA carries the message from DNA to the ribosome, while tRNA is the adapter that brings amino acids to the ribosome. mRNA is processed from hnRNA, while tRNA is made by RNA polymerase III and folded into a clover shape.

9. Differentiate the template strand and the coding strand. The template strand is read by RNA polymerase and is complementary to the RNA, while the coding strand has the same sequence as the RNA except for uracil in place of thymine.

10. List two roles of the ribosome in translation. The ribosome holds the mRNA and the charged tRNAs in position, and its peptidyl transferase activity forms the peptide bonds between amino acids.

11. Why does the lac operon shut down after lactose is exhausted? The repressor returns to the operator once the inducer is gone, so RNA polymerase is blocked again.

12. Explain the functions of the promoter, tRNA and exons. The promoter is the binding site for RNA polymerase, tRNA carries amino acids to the ribosome, and exons are the coding stretches that remain in the processed mRNA.

13. Give two reasons why the Human Genome Project is counted among the mega projects of biology. It ran for about thirteen years, joined many laboratories across several countries, cost billions, and needed large-scale automation and computing to read roughly three billion base pairs.

14. What is DNA fingerprinting and where is it used? It is the comparison of repeated DNA sequences to produce an individual-specific band pattern, used in forensic identification, paternity testing and population studies.