1. COMPARATIVE GENOMICS II Variation in Chromosome Structure GENE 330

2. Genetic Variation in Natural Pop. Cont... Without variation, populations evolve. Soon after Mendel’s principles were rediscovered, biologists began to document genetic variation in natural population Initially, these efforts focused on conspicuous features of the phenotype- pigmentation, size and so forth. Later, they emphasized characteristics that are more directly related to chromosomes and genes VARIATION IN PHENOTYPES There are phenotypic variation within many species, e.g., land snails have different colored bands on their shells, squirrels and other small mammals have different patterns In plant kingdom, phenotypic variation maybe manifested by different kinds of flowers All these phenotypic differences may have a genetic basis. However, to evaluate the underlying genetic factors, it is necessary to bring the organisms into the laboratory and cross them with others. Unfortunately, for many species this approach is not feasible. Thus, geneticists have tendered to focus their investigations of naturally occurring phenotypic variation on organisms that can be reared and bred in the laboratory

3. Yellow Tiger Swallowtail (Papilio glaucuas) Black Tiger Swallowtail

4. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Phenotypic variation can be reflection of the underlying genetic variability Flies captured in the wild can be captured in the laboratory and bred to produce larvae, which can be examined for alterations in the banding patterns of their polytene chromosomes The four common types of chromosomal mutations leading to changes in structure are: deletions, duplications, inversions and translocations. All result from breaks in chromosomes

5. KARYOTYPING: THE BEGINNING OF COMPARATIVE GENETICS The term “karyotype” refers to a description or depiction (karyogram) of the set of all chromosomes in organism. It is customary to depict the autosomal (nonsex) chromosomes arranged in homologous pairs in a standard order, usually from largest to smallest, with the shorter arm of each one oriented toward the top of the picture. The sex chromosomes typically are placed last. Each eukaryotic chromosome is linear with a constriction somewhere along its length called the centromere, and is capped by condensed regions called telomeres. Each chromosome looks more like the letter X, with each arm in two replicates (sister chromatids) emanating from the centromere. The shorter arm is designated as the “p” arm (petit- little); the longer arm is labeled the “q” arm (queue-tail).

6. KARYOTYPING: THE BEGINNING OF COMPARATIVE GENOMICS Different banding techniques provide cytogeneticists with the means to analyze fine details of chromosome structure. Until the late 1960s and early 1970s, chromosome spreads were stained with Feulgen’s reagent. More detailed studies now employ dyes that stain chromosomes differentially along their lengths. Several types of banding can be produced by different dyes and treatments. Chromosomes they have been stained with quinacrine or giemsa create a reproducible pattern of bands on each chromosome. Today are used a different staining procedures called ; - Q banding (Quinacrine stain) - G banding (Gienmsa stain) - R banding (reverse patterns-dark bands that corresponds to light G bands) - C banding (stain the region around centromere) Using these bands, cytogeneticists can identify particular chromosomes in a cell, and they can analyze these chromosomes for structural abnormalities.

7. KARYOTYPING: THE BEGINNING OF COMPARATIVE GENOMICS The correct chromosome count for humans was not established until 1956. A great deal of variation among karyotypes of different organisms was apparent. In some organisms, the chromosomes all have the same morphology (e.g. all mouse chromosomes are acrocentric- centromere near the end), other organisms, such as humans, have a mixture of different chromosome morphological types. Chromosomes vary considerably in size and numbers of chromosomes. Even closely related and phenotypically similar species can differ greatly in chromosome number.

8. Preparation of cells for cytological analysis.

9. Q bands – the band appear only when the chromosomes are exposed to UV light.

10. G banding

11. Polytene chromosomesPolytene chromosomes are a giant chromosome containing non- separated multiple copies of replicated DNA

12. Polytene chromosomes in Drosophila

13. Banding pattern of the polytene X chromosome. Chromosome is divided into 20 numbered sections.

14. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE D. pseudoobscura and D. persimilis are subling species – morphologically similar and able to be interbred in the laboratory. However, hybrid males from these crosses are sterile and hybrid females have severely reduced fertility. D. mirand is less closely related to other two species. It rarely produce hybrids in crosses with neither D. pseudoobscura nor D. persimilis, and when hybrids are produces, they are always sterile There are many different arrangements of the banding patterns in the polytene chromosomes of the species Each arrangement consists of one or more inversions of the most common banding pattern. e.g. in the third chromosome of D. pseudoobscura, there 17 different arrangements in natural population The standard banding pattern (ST) is most frequent in populations along the coast of California (48%) and in Northern Mexico (58%) Different arrangements predominates in other areas, e.g., 88% Arrowhead (AR) arrangement in Arizon, Nevad and Atah. 71% Pike’s Peak (PP) arrangement in Taxas The changes in arrangement frequencies are both seasonally and long-term

15. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Chromosomes can undergo several types of changes which fall into two classes. The first type of change involves changes in chromosome number and is referred to as aneuploidy and euploidy The chromosomal changes alter the linear order of the chromosome and occur because of deletions, duplications, inversions, translocations and insertions of chromosomal DNA The analysis of these types of changes to a large part has been performed in genetic stocks of the fruit fly, Drosophila melanogaster The chromosomes of this species are of particular interest, are those found in the salivary glands of larvae These tissues grow not by cell division but by enlargement. During this enlargement the chromosomes also undergo replication but this replication is different than in other tissues because: 1.the homologous chromosomes remain synapsed, that is paired throughout the duplication; and 2.the chromosomes undergo about ten rounds of replication to produce 12 10 (1024) double-stranded DNA molecules. 3. homologous chromosomes are paired-2048 molecules in total

16. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE The banding patterns of these chromosomes are distinct from metaphase chromosomes and have been associated with specific genes Metaphase chromosomes also have distinct bands, but these are thought to be associated with genetically inactive heterochromatin DNA The term used to describe these chromosomes and similar chromosomes in other diptera species is polytene chromosomes and these chromosomes have primarily been used to: 1.to locate genes; and 2.to analyze structural changes in chromosomes

17. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Duplications and Deletions A mechanisms that could generate a duplication is multiple replication of a looped region of DNA by DNA polymerase That individual would have one chromosome with an extra copy of the sequence whereas the homologous chromosome would still retain a single copy of the sequence A second proposed molecular mechanism that can generate a duplication of a chromosome by its very nature will also generate a deletion This mechanism is unequal crossing-over. This occurs when pairing occurs between homologous chromosomes in regions that are out of register This pairing can be mediated by sequences that are repeated on the same chromosome Following recombination (or crossing over) one chromosome will lose DNA sequence whereas the homologous chromosome will gain the sequences lost by the first The presence, and extent of a deletion, can be detected quite readily with polytene chromosomes

18. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Duplications and Deletions A loop will appear on one paired chromosome, and those sequences found in the looped region mark the length of the deletion Analysis of heterozygote chromosomes during pairing will also reveal a loop structure, but without the band markers that are available with polytene chromosomes, it is difficult to define the region It is easier to detect the larger deletions, but with good preparation and patience, the deletion of a single band can be detected, and thus the chromosomal location of a specific phenotype can be made Evolution and Unequal Crossing Over One possible result of unequal crossing-over is the creation of a multigene family. These are defined as a tandem array of genes that have similar function The best studied family is that which encodes the β-chain of human hemoglobin. Six β-globin genes are found within 50 kb on chromosome 11

19. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Evolution and Unequal Crossing Over The one of β genes is a pseudogene that has a mutation which prevents its expression. The sequences of these six genes are quite similar which suggests they occurred by duplication of an ancestral β-globin gene Once a gene duplication event has occurred evolutionary constraints working upon these gene presumably lessen First, another unequal crossing-over event could generate a third copy of the gene, further expanding the family. Other similar events will further spread the family As the family expands, previous harmful mutations can now be tolerated because functional copies will still exist Duplicate genes could now diversify and take on new or more specialized functions. Thus, over evolutionary time the sequences of the family members can diverge, and their role in phenotypic expression of the individual could change Deletions and duplications involve an actual gain or loss of genetic material by the genome of the specific individual. If these changes are maintained and prove to beneficial then eventually these changes might become stabilized in the species.

20. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Duplications of the Bar locus of Drosophila One phenotype that has been analyzed in Drosophila with respect to duplications is bar eye. The eye of the fly is normally an elongated oval shape whereas the bar eye phenotype is much thinner When the chromosomes of males with bar eye are analyzed, a duplication in region 16A of the chromosome is detected Another mutant of the eye shape is the double bar eye. These individuals have a second duplication of the same 16A region

21. Effects of duplications for region 16A of the X chromosome on the size of the eyes in Drosophila.

22. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Inversions Another group of changes in chromosome structures are associated with a relocation of the genetic material either within a chromosome or between non- homologous chromosomes. These two types of changes are inversions and translocations Inversions result when a segment of a chromosome is excised, inverted 180o, and reintegrated into the same chromosome. Two specific types of inversions are recognized, and they are classified as to whether or not the centromere is a portion of the inverted segment: 1. Paracentric inversion - an inversion of a segment of the chromosome that does not involve the centromeric region 2.Pericentric inversion - an inversion of a segment of the chromosome that involves the centromeric region In humans, it is difficult to detect inversions. If a large inversion occurs, then a new banding pattern will be seen in the regions that under went the inversion One manner in which inversions can be detected is by a change in the location of the centromere

23. Structure of an inversion. The chromosome has been broken at two points, and the segment between them has been inverted.

25. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Inversions Inversions are not easily detected in homozygotes of other species, unless as with humans, distinct chromosomal banding patterns have been established for normal members of that species Heterozygotes derived from a cross of a normal individual and an individual with an inversion in one chromosome will have specific patterns that occur because of pairing problems In Drosophila, polytene chromosome will contain loops, evidence that one of the synapsed chromosomes contains an inversion The appearance of meiotic chromosomes also will be affected if one of the chromosomes has an inversion. Loops appear and define the region in which the inversion has occurred Furthermore, if recombination occurs in a paracentric inversion region, then one-half of the gametes will be normal and the other half will contain deleted chromosomes If the recombination involves a pericentric inversion then one-half of the gametes will be normal with respect to the initial chromosomal arrangements and the other gametes will contain chromosomes that contain both additions and deletions

26. Genetic Variation in Natural Pop. Cont... VARIATION IN CHROMOSOME STRUCTURE Inversions These abnormalities can generate unbalanced gametes and in general, individuals with pericentric inversions are reproductively inferior In general, inversions do not change the phenotype of the individual unless an excision site of the inversion is within the regulatory or structural region of a gene The primary change that is seen with inversions is a change in linkage relationships. A survey of 50 known paracentric inversions in humans revealed that: 1.14 chromosomes were involved 2.eight involved chromosome 3, 10 involved chromosome 7, and 7 involved chromosome 11 3.34/50 appeared to familial 4. no harmful effects or reduced fertility of the paracentric inversion noted

27. Translocation Translocation occur when a segment from one chromosome is detached and reattached to a different (that is, nonhomologous ) chromosome.

28. Translocation During meiosis these translocated chromosomes would be expect to pair with their untranslocated homo- logues in a cruciform, or crosslike pattern. This pairing configuration is diagnostic of a translocation heterozygote.