Download Guide to 2nd Drosophila discussion

Guide to 2nd Drosophila discussion
Pat O’Farrell
Paper for discussion: Hartl TA, Smith HF, Bosco G. (2008) Chromosome alignment
and transvection are antagonized by condensin II. Science 322(5906):1384-7
Although this paper is not heavy on genetic techniques, it will expose you to some
interesting aspects of biology with very strong Drosophila genetics connections —
polytene chromosomes and transvection. We discussed polytene chromosomes in class
and the mysterious interactions that align the many copies of the genome. This
analysis, while not solving the mystery, succeeds in disrupting the interactions in a way
that suggests connections to fundamental interactions in chromatin in the normal cell
cycle and in other aspects of biology. It also uncovers a relationship to an interaction
that allows communication between homologous chromosomes via a pairing interaction.
This communication between chromosomes (without diffusible intermediate) was
discovered and characterized by E.B. Lewis. It was defined by the ability of
chromosomal rearrangements to alter the phenotype of a heteroallelic combination, even
when the rearrangement breakpoints do not influence the function of the alleles. For
decades, the observations stood as an enigma that defied normal views of how genes
functioned. It is now commonly thought that close alignment of the homologous
chromosomes in interphase allows the “cis” acting regulatory sequences to act in trans
on the promoter of the homolog. Thus, mutations in “cis” acting regulatory regions are
thought to complemented by an allele with normal “cis” acting sequences but a defective
coding sequence. While this interpretation is well supported in some cases, a
generalization that all transvection (pairing dependent interactions between homologous
alleles) is due to this one phenomenon is not justified at this time.
The following abstract gives a synopisis of transvection (see the complete review for and
authoritative summary).
Duncan IW. (2002) Transvection effects in Drosophila. Annu Rev Genet. 236:521-56
Abstract
An unusual feature of the Diptera is that homologous chromosomes are intimately
synapsed in somatic cells. At a number of loci in Drosophila, this pairing can significantly
influence gene expression. Such influences were first detected within the bithorax
complex (BX-C) by E.B. Lewis, who coined the term transvection to describe them. Most
cases of transvection involve the action of enhancers in trans. At several loci deletion of
the promoter greatly increases this action in trans, suggesting that enhancers are
normally tethered in cis by the promoter region. Transvection can also occur by the
action of silencers in trans or by the spreading of position effect variegation from
rearrangements having heterochromatic breakpoints to paired unrearranged
chromosomes. Although not demonstrated, other cases of transvection may involve the
production of joint RNAs by trans-splicing. Several cases of transvection require Zeste, a
DNA-binding protein that is thought to facilitate homolog interactions by self-aggregation.
Genes showing transvection can differ greatly in their response to pairing disruption. In
several cases, transvection appears to require intimate synapsis of homologs. However,
in at least one case (transvection of the iab-5,6,7 region of the BX-C), transvection is
independent of synapsis within and surrounding the interacting gene. The latter example
suggests that transvection could well occur in organisms that lack somatic pairing. In
support of this, transvection-like phenomena have been described in a number of
different organisms, including plants, fungi, and mammals.
A couple of background things will help for technical or conceptual context.
— Where did the mutations come from? See supplement, which refers to a paper (abs
below) that describes a collection of lines with mutagenized chromosomes. This
collection was screened to identify the founding mutation and then other alleles where
pulled out of the gene knock out collection or other curated stock collections as detailed
in the supplement. Note that this collection is unusual mostly in that is kept and
screened by many people, whereas fly geneticists usually make a transient collection of
mutagenized chromosomes every time they screen – they just throw away the ones they
don’t want.
Koundakjian EJ, Cowan DM, Hardy RW, Becker AH. (2004) The Zuker collection: a
resource for the analysis of autosomal gene function in Drosophila melanogaster.
Genetics. 2004 May;167(1):203-6.
Abstract
The majority of genes of multicellular organisms encode proteins with functions that are
not required for viability but contribute to important physiological functions such as
behavior and reproduction. It is estimated that 75% of the genes of Drosophila
melanogaster are nonessential. Here we report on a strategy used to establish a large
collection of stocks that is suitable for the recovery of mutations in such genes. From
approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl
methanesulfonate (EMS), approximately 12,000 lines in which the treated second or
third chromosome survived in homozygous condition were selected. The dose of EMS
induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six
nonessential gene mutations per chromosome and seven to nine alleles per locus in the
samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic
mutations induced in the initial exposure to the mutagen, many of the lines are
segregating or are now fixed for lethal mutations on the mutagenized chromosome. The
features of this collection, known as the Zuker collection, make it a valuable resource for
forward and reverse genetic screens for mutations affecting a wide array of biological
functions.
— The biological context of the findings in the discusison paper are likely to be a bit
murky to you (or actually anyone at this point), but there are some interesting
connections. Here is a bit of background discussing roles of cohesin and condensin in
chromatid pairing and chromatid resolution.
Chromosome duplication and segregation are among the most fundamental events that
must be regulated faithfully to maintain genome integrity in eukaryotic cells. Errors in
these processes during mitosis or meiosis increase the probability of genome instability,
potentially leading to cancer, developmental disorders, or birth defects. Extensive
studies during the past decade have revealed that two structurally related protein
complexes, cohesin and condensin, play central roles in controlling a series of events
that makes duplicated chromosomes proficient for faithful segregation (Losada and
Hirano 2005; Nasmyth and Haering 2005). Cohesin participates in holding newly
duplicated chromatids together during S phase, a process known as sister chromatid
cohesion (Lee and Orr-Weaver 2001; Onn et al. 2008). Condensin, on the other hand,
associates with chromatin to initiate chromosome condensation during the early stage of
mitosis (i.e., prophase). At the same time, the bulk of cohesin dissociates from
chromosome arms, leading to the formation of metaphase chromosomes in which sister
chromatids become microscopically discernible as two rod-shaped structures apposed
along their lengths. Although the physiological significance of this process, often referred
to as sister chromatid resolution, is not fully understood, one possibility frequently
discussed is that the resolution process is a prerequisite for rapid and synchronous
separation of sister chromatids in anaphase (Losada et al. 2002). Despite its
fundamental importance, we are still largely ignorant of the molecular mechanisms
behind this process. (paragraph taken from Shintomi and Hirano, 2009 – for more
details see review by the same authors 2010 Chromosoma. 119(5):459-67)
Just one note about reading this paper: at least in my opinion the effort to produce
simple summaries of the transvection data in Fig 3 fail. The figure is really useful to
learn what was scored in terms of phenotype, but I found the diagrams not very helpful
and pictures deceptive because they don’t give the range of the phenotype. I found the
tables of the data in supplement more clear and I recommend that you look at these (at
least tables S1 and S2).