Analysis of Gene Clusters, Crossings Over, and Tandem Repeats

1. Chapter 7Clusters and Repeats

2. 7.1 Introduction • gene family – A set of genes within a genome that encode related or identical proteins or RNAs. • The members were derived by duplication of an ancestral gene followed by accumulation of changes in sequence between the copies. • Most often the members are related but not identical.

3. 7.1 Introduction • pseudogenes – Inactive but stable components of the genome derived by mutation of an ancestral active gene. • Usually they are inactive because of mutations that block transcription or translation or both. • gene cluster – A group of adjacent genes that are identical or related.

4. 7.1 Introduction Figure 07.01: Chiasma formation can result in the generation of recombinants. Figure 07.02: Recombination involves pairing between complementary strands of the two parental duplex DNAs.

5. 7.1 Introduction • unequal crossing over (nonreciprocal recombination) – Unequal crossing over results from an error in pairing and crossing over in which nonequivalent sites are involved in a recombination event. • It produces one recombinant with a deletion of material and one with a duplication. Figure 07.03: Unequal crossing-over results from pairing between nonequivalent repeats in regions of DNA consisting of repeating units.

6. 7.1 Introduction • satellite DNA – DNA that consists of many tandem repeats (identical or related) of a short basic repeating unit.

7. 7.1 Introduction • minisatellite – DNAs consisting of tandemly repeated copies of a short repeating sequence, with more repeat copies than a microsatellite but fewer than a satellite. • The length of the repeating unit is measured in tens of base pairs. • The number of repeats varies between individual genomes.

8. 7.2 Unequal Crossing Over Rearranges Gene Clusters • When a genome contains a cluster of genes with related sequences, mispairing between nonallelic loci can cause unequal crossing over. • This produces a deletion in one recombinant chromosome and a corresponding duplication in the other. Figure 07.04: Gene number can be changed by unequal crossing-over.

9. 7.2 Unequal Crossing Over Rearranges Gene Clusters • Different thalassemias are caused by various deletions that eliminate α- or -globin genes. • The severity of the disease depends on the individual deletion. Figure 07.06: Deletions in the β-globin gene cluster cause several types of thalassemia. Figure 07.05: α-thalassemias result from various deletions in the α-globin gene cluster.

10. 7.2 Unequal Crossing Over Rearranges Gene Clusters • HbH disease – A condition in which there is a disproportionate amount of the abnormal tetramer β4 relative to the amount of normal hemoglobin (α2β2). • hydrops fetalis – A fatal disease resulting from the absence of the hemoglobin α gene.

11. 7.2 Unequal Crossing Over Rearranges Gene Clusters • Hb Lepore – An unusual globin protein that results from unequal crossing over between the β and δ genes. • The genes become fused together to produce a single β-like chain that consists of the N-terminal sequence of δ joined to the C-terminal sequence of β.

12. 7.2 Unequal Crossing Over Rearranges Gene Clusters • Hb anti-Lepore – A fusion gene produced by unequal crossing over that has the N-terminal part of β globin and the C-terminal part of δ globin. • Hb Kenya – A fusion gene produced by unequal crossing over between the Aγ- and β-globin genes.

13. 7.3 Genes for rRNA Form Tandem Repeats, Including an Invariant Transcription Unit • Ribosomal RNA is encoded by a large number of identical genes that are tandemly repeated to form one or more clusters. • Each rDNA cluster is organized so that transcription units giving a joint precursor to the major rRNAs alternate with nontranscribed spacers. • The genes in an rDNA cluster all have an identical sequence.

14. 7.3 Genes for rRNA Form Tandem Repeats, Including an Invariant Transcription Unit • The nontranscribed spacers consist of shorter repeating units whose number varies so that the lengths of individual spacers are different. Figure 07.07: A tandem gene cluster has an alternation of transcription unit and nontranscribed spacer and generates a circular restriction map.

15. 7.3 Genes for rRNA Form Tandem Repeats, Including an Invariant Transcription Unit • nucleolus – A discrete region of the nucleus where ribosomes are produced. • nucleolar organizer – The region of a chromosome carrying genes encoding rRNA.

16. Figure 07.08: The nucleolar core identifies rDNA under transcription, and the surrounding granular cortex consists of assembling ribosomal subunits. Photo courtesy of Oscar Miller

17. Figure 07.09: Transcription of rDNA clusters generates a series of matrices, each corresponding to one transcription unit and separated from the next by the nontranscribed spacer. Photo courtesy of Oscar Miller

18. 7.3 Genes for rRNA Form Tandem Repeats, Including an Invariant Transcription Unit • Bam islands – A series of short, repeated sequences found in the nontranscribed spacer of Xenopus rDNA genes. Figure 07.10: The nontranscribed spacer of X. laevis rDNA has an internally repetitious structure that is responsible for its variation in length.

19. 7.4 Crossover Fixation Could Maintain Identical Repeats • Unequal crossing over changes the size of a cluster of tandem repeats. • Individual repeating units can be eliminated or can spread through the cluster. • concerted evolution (coincidental evolution) – The ability of two or more related genes to evolve together as though constituting a single locus.

20. 7.4 Crossover Fixation Could Maintain Identical Repeats • gene conversion – The alteration of one strand of a heteroduplex DNA to make it complementary with the other strand at any position(s) where there were mispaired bases. • crossover fixation – A possible consequence of unequal crossing-over that allows a mutation in one member of a tandem cluster to spread through the whole cluster (or to be eliminated). Figure 07.11: Unequal recombination allows one particular repeating unit to occupy the entire cluster. The numbers indicate the length of the repeating unit at each stage.

21. 7.5 Satellite DNAs Often Lie in Heterochromatin • Highly repetitive DNA (or satellite DNA) has a very short repeating sequence and no coding function. • simple sequence DNA – Short repeating units of DNA sequence. • Satellite DNA occurs in large blocks that can have distinct physical properties. Figure 07.12: Mouse DNA is separated into a main band and a satellite by centrifugation through a density gradient of CsCl.

22. 7.5 Satellite DNAs Often Lie in Heterochromatin • cryptic satellite – A satellite DNA sequence not identified as such by a separate peak on a density gradient. • It remains present in main-band DNA.

23. 7.5 Satellite DNAs Often Lie in Heterochromatin • in situ hybridization – Hybridization performed by denaturing the DNA of cells squashed on a microscope slide so that reaction is possible with an added single-stranded RNA or DNA. • The added preparation is radioactively labeled and its hybridization is followed by autoradiography. Figure 07.13: Cytological hybridization shows that mouse satellite DNA is located at the centromeres. Photo courtesy of Mary Lou Pardue and Joseph G. Gall, Carnegie Institution. Courtesy of Mary Lou Pardue and Joseph G. Gall, Carnegie Institution

24. 7.5 Satellite DNAs Often Lie in Heterochromatin • Satellite DNA is often the major constituent of centromeric heterochromatin. • euchromatin – Regions that comprise most of the genome in the interphase nucleus, are less tightly coiled than heterochromatin, and contain most of the active or potentially active single copy genes.

25. 7.6 Arthropod Satellites Have Very Short Identical Repeats • The repeating units of arthropod satellite DNAs are only a few nucleotides long. • Most of the copies of the sequence are identical. Figure 07.14: Satellite DNAs of D. virilis are related. More than 95% of each satellite consists of a tandem repetition of the predominant sequence.

26. 7.7 Mammalian Satellites Consist of Hierarchical Repeats • Mouse satellite DNA has evolved by duplication and mutation of a short repeating unit to give a basic repeating unit of 234 bp in which the original half-, quarter-, and eighth-repeats can be recognized. Figure 07.15: The repeating unit of mouse satellite DNA contains two half-repeats, which are aligned to show the identities (in blue).

27. 7.7 Mammalian Satellites Consist of Hierarchical Repeats Figure 07.16: The alignment of quarter-repeats identifies homologies between the first and second half of each half-repeat. Figure 07.17: The alignment of eighth-repeats shows that each quarter-repeat consists of an α half and a β half.

28. 7.7 Mammalian Satellites Consist of Hierarchical Repeats Figure 07.18: The existence of an overall consensus sequence is shown by writing the satellite sequence in terms of a 9 bp repeat.

29. 7.8 Minisatellites Are Useful for Genetic Mapping • The variation between microsatellites or minisatellites in individual genomes can be used to identify heredity unequivocally by showing that 50% of the bands in an individual are derived from a particular parent. • variable number tandem repeat (VNTR) – Very short repeated sequences, including microsatellites and minisatellites. Figure 07.20: Alleles may differ in the number of repeats at a minisatellite locus, so that cleavage on either side generates restriction fragments that differ in length.

30. 7.8 Minisatellites Are Useful for Genetic Mapping • DNA fingerprinting – Analysis of the differences between individuals of restriction fragments that contain short repeated sequences, or by PCR. • The lengths of the repeated regions are unique to every individual, so the presence of a particular subset in any two individuals shows their common inheritance (e.g., a parent–child relationship).

31. 7.8 Minisatellites Are Useful for Genetic Mapping Figure 07.21: Replication slippage occurs when the daughter strand slips back one repeating unit in pairing with the template strand.