A. Bhutkar, S. W. Schaeffer, S. M. Russo, M. Xu, T. F. Smith, W. M. Gelbart (2008). Chromosomal Rearrangement Inferred From Comparisons of 12 Drosophila Genomes Genetics, 179 (3), 1657-1680 DOI: 10.1534/genetics.107.086108

Back when I was a carefree postdoc, one of the projects I worked on was the assembly of a molecular physical map of the Drosophila melanogaster genome. Of course, Drosophila researchers had for years been using a physical map, the polytene chromosome map, and indeed we used this as the framework on which we assembled our molecular map using cosmid clones. These papers take the genome sequences of 11 Drosophila species (plus the sequence of Drosophila melanogaster, determined back in 2000), fit them to the polytene chromosome maps, and examine chromosome rearrangments seen in inter-species comparisons.  It seems to me there isn’t anything hugely sexy in this work, but there is a huge amount of work that sets the evolutionary relationships between these Drosopholids in context.  It’s also an opportunity to expound on chromosomes in Drosophila!

Polytene chromosomes are unusual structures – originally found by Balbiani in the 19th century in Chironomus, they are in fact highly endroreduplicated chromosomes.  More than that, all the chromosome copies are in an interphase state and precisely aligned, resulting long rope-like structures with highly reproducible banding patterns (these bands are the basis on which polytene chromosome maps are devised).

Polytene chromosomeDrosophila polytene chromosomess are found in a variety of tissues in the true flies (Diptera), and perhaps reach there greatest lab utility in Drosophila melanogaster.  The picture on the left shows a complete set of D. melanogaster polytene chromosomes – note that each centromere and the pericentromeric heterochromatin (which is not amplified) are clustered to form the chromocentre, from which each of the chromosome arms radiate (though the animal is diploid, both homologues are tightly paired – so for example both 2L arms form one structure.  This summarised in the diagram below.

structure of polytene chromosomes Panel (a) shows the female mitotic karyotype, with the arms identifiable by labels and colours.  The male karyotype has a single X chromosome paired with a heterochromatic Y chromosome.  Panel (b) sows how these arms relate to the polytene chromosomes (no to scale).  Notice that despite the animal being diploid, each pair of chromosome arms is represented by a single arm in the polytene configuration. In the mal, the Y chromosome remains part of the chromocentre, along with the centromeres and pericentromeric heterochromatin.

 Thomas Painter originally used polytene chromosomes to map genetic variants, but it was Calvin Bridges who devised  the first usable map: each of the five chromosome arms were divided into 20 divisions, numbered 1 to 102 (the tiny fourth chromosome has only two divisions), and each division was subdivided into 6 subdivisions, indicated by the letters A-B.  In later revisions, Bridges numbered each individual band of each subdivisions.  Considering this work was conducted by light microscopy, it is remarkable that the map can still be used today, and indeed has been supported by electron microscopy.  It represents a real tour de force of cytogenetic mapping, and is the prototype map for a variety of insect species.

The image above shows a couple of divisions (i.e. about 2%) of Bridges’ polytene map.  The polytene map permitted detailed mapping of chromosome rearrangements such as deficiencies, inversions, etc; and from the early days of moleclar genetics direct localisation of cloned genes by in situ hybridisation.  And, of course, they played their part in evolutionary studies, particularly in other Drosophila species.

The D. melanogaster polytene map is in many ways the "gold standard" map: the most highly used, and indeed the most detailed.  For other Drosophila species, maps are not necessarily constructed in the same way in terms of nomenclature.

Muller’s elements

Muller proposed that the chromosomes of Drosophila species correspond to a set of "elements"  – essentially representing syntenic blocks.  Muller named these elements A-F: in D. melanogaster element A corresponds to the X chromosome, element B to the left arm of chromosome 2, B to the right arm of chromosome 2 and so forth.   

The diagram below (click for a larger version) illustrates the correspondence between Muller’s elements and the karyotypes of the species featured in these papers.Muller's elements

A large scale effort to sequence the genomes of several Drosophila species now means that in addition to D. melanogaster, the genomes of a further 11 species have been determined.  All species in the phylogeny above have been sequenced.

So, on to the first paper, Polytene Chromosomal Maps of 11 Drosophila Species: The Order of Genomic Scaffolds Inferred From Genetic and Physical Maps.  This paper aligns the species scaffolds against the polytene chromosome maps (and in some cases updating the polytene chromosome maps), with the aim of contributing to investigating five questions:

What is the molecular basis of differences between these species?; what is the mechanistic basis of distinct  sex chromosomes?; How do new inversions originate?; What is the basis of gene arrangement polymorphisms within a species?; and finally, why in some species do gene arrangement polymorphisms occur only on some of the chromosome arms?

< p>By using both syntenic analysis and physical mapping, the sequence scaffold blocks for each of the 11 species studied have been aligned to their respective polytene chromosome maps.  This represents a lot of work – there are many supplementary documents (which I confess I’ve not read).  The genome sizes of these species vary widely: from 236.6 Mb for D. willistoni to 137.8 Mb in D. simulans.  These differences are principally due to differing amounts of heterochromatic or other unassigned DNA located around centromeres.

The second paper,  Chromosomal Rearrangement Inferred From Comparisons of 12 Drosophila Genomes, describes the patterns ofchromosome rearrangements in a phylogenetic context.  They also address structural genome features associated with chromosome inversion breakpoints, in particular in those species such as D. pseudoobscura which have well characterised intraspecific inversion polymorphisms.

The figure below is a schematic showing how chromosome inversions and other rearrangents map out on Muller’s elements. You can click on the image for a bigger version (may need subscription).

polytene rearrangements

Each element is composed of a set of hues, and inverted segments are linked by lines.  In fact single genes are represented by single lines, andare coloured in blocks of a hundered or so genes in D. grimshawi.  It’s a neat graphic that cleverly shows that the vast majority of rearrangementshave occurred within single elements: a notable exception is the translocation between D and A in D. psuedoobscura (and D. persimilis).

One of the things I was interested to glean from these studies was the nature of inversion breakpoints.  Some years ago I worked on the malaria mosquito Anopheles gambiae – this species isactually a species complex of 6 species pretty well indistinguishable on morphological criteria, but which were clearly reproductively isolated (at least in the wild) on the basis of the distribution of chromosome inversions between the species.  Many of these inversions share breakpoints, at least at the level of polytene cytology.  One inference was that these breakpoints were seen multiple times because there was a hotspot, possibly due to some feature in the DNA – possibly a transposable element or a segment of heterochromatin.  The same situation is, I think, seen in the polymorphic inversions of D. pseudoobscura, which have been studied in considerable detail, as they seem to vary in frequency depending on environmantal factors such as altitude, temperature and season.  What do Bhutkar et al say that is relevant to this?

Of course, as with the rest of the paper, deep understanding (at least for me) is made difficult by the fact that much of the terminology is from a mathematical or computing biology source.  However, as far as I understand it, they’ve analysed patterns of breakpoint reuse, as judged by mapping them to boundaries between syntenic blocks.  I presume that direct comparison between species’ sequences is difficult due to sequence divergence.  It follows then that apparent beakpoint reuse may only reflect the distance between syntenic blocks.  The prospect that is also raised is that some regions may lack breakpoints because they would split coordinately expressed blocks of genes (previous workers had identified long domains of genes with similar or identical expression patterns, and the inference was that they were co-ordinately expressed.

Bhutkar et al conclude on the basis of both synteny analysis and computational modelling that breakpoint reuse does occur.  This in turn implies that some feature of the chromosome is responsible.  One hypothesis that was attractive was that transposable elements would turn out to be responsible for the formation of inversions- however, many studies in a variety of systems havefailed to support this, though in many cases, reptitive sequences do localise to inversion breakpoints.  The evidence presented here does not support elevation of repeat sequences at breakpoints, though the authors do note that the present situation need not reflect the situation at the time an inversion arose.

A second hypothesis for breakpoint formation was proposed by Novitski (1946). Novitski proposed that synapsis of inverted and standard arrangement chromosomes would lead to elevated frequencies of double strand breaks in the vicinity of breakpoints – consistent with observed patterns of breakpoints within and between species.  the sequence of events would use progressively more distal breakpoints.  Unfortnately the data to date are not clear enough on the sequential nature of inversions to provide support (or not) for Novitski’s hypothesis.

I don’t know where the studies of these genome sequences are going, but it would be of interest if some level of analyses could be applied to D. pseudoobscura and its intraspecific inversion polymorphisms.  Richards et al (2005), in the publication describing the D. pseudoobscura genome sequence look at inversions in this species and frequently find repetitive DNA at inversion breakpoints.  In one case, the intraspecific polymorphic Arrowhead inversion (in Muller element C), and molecular evidence implicates short inverted repeat sequences in the formation of the inversion.