Archive for the ‘DNA-protein interactions’ Category

I learnt about epistasis in twelfth year of schooling. It was first used by Bateson to describe the masking of one allele(variant of a gene) at a locus masking another allele at a different locus. But today I see this word used in different contexts and I was really confused about its meaning, so looked on the net to find this paper describe wonderfully what I was looking for.
Cordell, H.J. (2002). Epistasis: what it means, what it doesn’t mean, and statistical methods to detect it in humans. Human Molecular Genetics, 11(20), 2463-2468. DOI: 10.1093/hmg/11.20.2463


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Last week I blogged on the talk by Peter Fraser where he showed evidence for the existence of transcription factories. He showed pictures as follows to demonstrate the theory of transcription factory. transcription factory

Fig. 1. Transcription factories are concentrated foci of active RNA polymerase. Immuno-detection of the hyper-phosphorylated form of RNAPII reveals their focal existence in a limiting number of transcription factories. Shown is a deconvoluted, single optical section of a mouse E12.5 fetal liver nucleus. Scale bar, 5 μm. Image courtesy of L. Chakalova. (taken from Seminars in Cell & Developmental Biology).

A new paper in molecular cell has appeared where the authors show evidence for the classical view that Pol II can be recruited to gene loci for activated transcription in living cells. They show this in polytene nuclei in salivary glands of Drosophila. Chromosomal DNA at loci containing the HS(heat shock) protein genes locally decondenses upon HS to form “puffs,” as can be readily observed in fixed, spread polytene chromosomes. Accompanying this decondensation is the strong recruitment of Pol II molecules that become densely packed along the activated transcription units. This is clearly demonstrated by this movie in live cells. Here is a picture which shows the transcription in the polytene chromosomes. The green color shows the presence of active transcription units where RNA pol II(which is ligated to enhaced Green flourscent protein) has been recruited.

polytene chromosome

Figure 1. Recruitment of Pol II to Major HS Puffs at 87A and 87C Observed in Living Cells

(A and B) Two-photon optical sections of a polytene nucleus expressing Rpb3-EGFP under NHS and HS conditions. (A) NHS: yellow arrows indicate Pol II-enriched sites that are transcriptionally active during normal development. (B) HS: red arrows show newly formed Pol II-concentrated sites upon HS.

(C) Recruitment of Pol II to 87A and 87C after HS. Times after HS are shown in minutes.

(D–F) Three sections of the same polytene nucleus under HS show distinct Pol II enriched sites (green). Chromosomes are stained with Hoechst33342 (red). 87A and 87C sites are indicated by the white arrows in (F) (Yao et al., 2006).

(G) A maximum intensity projection image (shown in pseudocolor) of all optical section series of this nucleus expressing EGFP-Rpb3 under HS (D–F). Scale bars, 10 μm.

Next the authors also have performed the dual-color FISH analyses on HS genes(figure 3). Labeled bacterial artificial chromosomes (BACs) containing HS genes were used to probe the interphase, diploid nuclei in larval imaginal disc tissues (Figure 3A). It is well known that HS genes are robustly activated in all larval tissues after HS, and therefore DNA-FISH signal during HS can represent active gene loci. The FISH signal indicates that HS genes occupy spatially distinct domains in most cases. Two Hsp70 loci 87A and 87C that are cytogenetically very close (separated by one subdivision not, vert, similar400 kb) also show distinct FISH signal. From these results, the authors conclude that, in diploid cells, HS genes colocalize at a very low frequency, and therefore, these coregulated genes are not generally cotranscribed within shared transcription factories during activation.no colocalization

Figure 3. Intranuclear Positions of HS Gene Loci in Drosophila Imaginal Disc Diploid Nuclei

(A) A FISH image of imaginal disc nuclei (red, small hsp locus 67B; green, Hsp70 locus 87A). Scale bar, 5 μm.

(B) FISH images on HS genes before and after HS (87C, Hsp70 locus; 63B, hsp83 locus).

(C) Summary of FISH analyses on HS gene pairs before and after HS.

(D) Positions of 87A, 67B, and 63B loci relative to the nuclear periphery or interior regions.

It is real exciting and waiting game to see which of the two theories are going to fail: Is transcription factory a distinct sub-nuclear compartment or the classical view of RNA pol II recruited to the gene loci !!


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Today we had a very fascinating story by Peter Fraser (from the Babraham Institute, Cambridge), suggesting that genes migrate to specialized sites called transcription factories in the nucleus for transcription. These transcription factories are nothing but discrete foci in the nucleus containing high concentrations of RNA polII(the eukaryotic polymerase responsible for transcription of protein-coding genes). This is very much opposed to the well known text book theory that the RNA polymerase is recruited to promoters and RNA are transcribed from de novo transcription start sites.

The number of discrete factories visible in the nucleus is less than the number of expressed genes, which means that multiple genes are transcribed from one factory. This is also evident from the fact that the hbb(hemoglobin beta chain- a component of oxygen carrying hemoglobin protein in blood) gene is  transcribed in the same factory as that other similarly expressed genes in the erythroid cell. These genes are seperated by more than 50 megabases in cis(present of the same chromosome). Also unlinked genes in trans(present on different chromosomes) co-localized in the same the transcription factory but the frequency of such co-localization was low. (ex hbb & hba genes). The most interesting fact was that the quiet allele of active genes are located away from the transcription factories.

He also discussed more about the nuclear co-localization and regulatory functional implication of such localizations, described in detail in ” Nuclear organization of the genome and the potential for gene regulation Nature. 2007 May 24;447(7143):413-7

The interacting cis and trans genes completely dissociated when RNA initiation was inhibited by heat-shocking cells at 45 degrees, while the remained associated when only the RNA elongation was inhibited using a chemical DRB. Futhermore the transcription factories persisted even in the absence of transcription Which he presented as a compelling evidence that these transcription factories are distinct nuclear sub-compartments and not just local concentrated foci of RNA pol II. ref:” Transcription factories are nuclear subcompartments that remain in the absence of transcription Mitchell JA, Fraser P 2008 Jan 1;22(1):20-5

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Today I went to a wonderful seminar by Sebastian J Maerkl. He gave a talk on the a high-throughput microfludic platform that measures the binding affinity of transcription factors.

Transcription factors are protein molecules that bind to the DNA molecule and help to copy the information(genes) in the DNA to RNA and proteins. These transcription factors interact with very specific DNA base sequences generally present upstream of genes. The transcription factors bind to these sequences very strongly and the affinity between a DNA sequence and a protein molecule is due to the chemical complimentarity between them. This affinity is measured in terms of a constant called association constant or the reverse of it called the dissociation constant.

Special techniques have been described to measure this affinity at very low concentrations. Some techniques are filter binding assay, gel shift assay, surface plasmon resonance etc. These techniques are all low throughput techniques. The protein binding microarrays is a high throughput method that was used to measure the affinity, but actually the PBMs measure only the off-rate of the DNA-protein interaction because of the strigent wash that is involved in the protocol. The wash also causes loss of weakly bound DNA-protein complexes.

The method described by Maerkl et al, measures the binding affinities of a large number DNA molecules in high throughput manner by trapping the bound DNA-protein complexes mechanically and allows one to measure the the equilibrium concentrations of the molecular interactions. The experiments have shown a very high reproducibilty in measuring the affinty values(only 20% error on the golbal measurement).

It has been published in Science 12 January 2007: Vol. 315. no. 5809, pp. 233 – 237. Although yeilding a lot of useful information, a major limitation of this approach is that it requires knowledge of the likely consensus site to be used as the starting sequence, a requirement that is not inherent in PBM or SELEX-SAGE techniques. But the another major advantage of this method is it allows for the designing oligonucloetides with more than one single base substitutions(which is has been traditionally done) and hence allows us to examine the inter nucleotide dependence.

It would be great to have such microfludic platforms to study long promoter sequences which will enable us to study the binding specificities and affinities for all transcription factors in several model organisms, including yeast, Drosophila and mouse, which span a range of genome sizes and modes of transcriptional regulation.

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