when it doesn’t work
I think most people who work in an experimental research lab recognise that not all experiments yield good results. And so today, I found out that the RTS 100 synthesis I did yesterday was of no good at all. What I saw in my gels were all alike, including the GFP vector.
Instead of sulking, I think I shall make myself happy by having a pint of ice cream after a sumptuous dinner. Have a nice evening to you all. I leave you with a clever sandwich:
Twiggy and Ageing
I cannot avoid mentioning that Twiggy, the so called “The Original Supermodel” is turning 60. And it is without doubt that she still remains in the limelight and continues to be a darling of the press. She recently published a book on ageing, for women above 40. I reckon this book is partly autobiographical too. What is inside the book intrigues me. Check her out.
vintage and the hamburger bed
If you have not heard of it yet, do not be alarmed, you are only missing a bed. Yes, it a real bed, a real hamburger bed. It went on sale as of July 16 on Ebay. Apparently, it has been featured in seven magazines and over 100 blogs. What a remarkable, world-famous, handmade hamburger bed. Should you decide to purchase it, you will not only own the bed, but the official Hamburger Bed website and its Facebook fan page with 12,000 fans too. It does look like a good bargain. You never run out of that lovely burger as you get old. Now I’m off to a vintage fair and will try my luck on any vintage hamburger bed paraphernalia.
for the sake of time
Looks like we’re going to give up the benzonase mystery soon. We are a bit constrained by time. Solving the benzonase mystery seems to be an exciting prospect. However, we will have to carry on with our purification efforts.
the benzonase mystery
Recently, we have been intrigued by the association of benzonase and our molecule of interest. And so I thought it might be handy to dig up information on benzonase. Benzonase is the commerical version of the extracellular endonuclease from the pathogenic enteric Gram-negative bacterium Serratia marcescens (1-4). It is composed of a single polypeptide chain (245 residues, Mr 26,700). It is capable of digesting DNA and RNA, both single- and double-stranded. Similar to DNase I, it is magnesium-dependent and catalyzes the cleavage of the 3′ O-P bond (4). In addition to its broad specificity, it retains catalytic activity even in extreme conditions such incubation with 4 M urea (7). Now, the mystery involves the formation of precipitates upon incubation of our clear lysate with benzonase. SDS-PAGE of the supernatant and the precipitate indicated that our molecule of interest is is in the precipitate, and not in the supernatant. Upon solubilisation of that precipitate (or pelle) with another round of lysis buffer, followed by SDS-PAGE, resulted into the similar gel profile of our molecule of interest showing up in the precipitate and not in the supernatant. What is going on?
References:
1) Product Specifications for Benzonase, The first industrial endonuclease. (Benzon Pharma A/S, Helseholmen 1, PO box 1185, DK-2650 Hvidovre Denmark. 1993.
2) Saito H, Elting L, Bodey GP, Berkey P. (1989) Rev Infect Dis. 11: 912-920
3) Acar JF. (1986) Infect Control. 7: 273-278
4) Ball TK, Saurugger PN, Benedik MJ (1987) Gene. 57: 183-192
5) Eaves GN, Jeffries CD. (1963) J Bact. 85: 273-278
6) Nestle M, Roberts WK. (1969) J Biol Chem. 244: 5219-5225
7) Filimonova MN, Balaban NP, Sharipova FR, Leshchinskaya IB. (1980) Biokhimiya. 45: 2096-2103
Miller MD, Tanner J, Alpaugh M, Benedik M, Krause KL. (1994) Nat Struc Biol 1: 461-468
moments of not knowing
We are still struggling up to this moment in purifying the molecule that we are interested in – DmWRNexo. It appears to behave in some “mysterious” ways. The peaks that come out after chromatography seems to vary. Last Friday, we got three peaks (three fractions). Under the electron microscope, I saw this morning how the individual particles from the first fraction look like. Quite nice, they remind me of tiny doughnuts (with a hole in the middle). But there weren’t enough of them in a single field of view, so we did not take a photo. Tomorrow, we shall see. We have a new sample today (completed at 1700), but the chromatography profile was so different, it had only one peak! In the end, Ivan and I exclaimed, “What is going on? We don’t know!”
rDNA theory of aging
The ribosomes are the most abundant protein complexes in the cell. They are synthesised from ribosomal DNA (rDNA) genes within nucleoli. In most eukaryotic cells, rDNA exists in tandem arrays, which are repeating units of similar sequences. Oftentimes the rDNA provide sites for recombination, and the copy number seems to fluctuate. In this regard, the rDNA is hypothesised to be involved in genome stability and cellular senescence.
There was a paper written by Takehiko Kobayashi last year, 2008, in the journal BioEssays, wherein he proposed the rDNA theory of aging, stating that the rDNA may be the region most sensitive to DNA damage. He highlighted two key molecular entities that would link rDNA to aging: FOB1 and SIR2. These two are well known aging genes.
“… in yeast Saccharomyces cerevisiae … lifespan expansion in a fob1 mutant is associated with increased rDNA stability (no copy number variation), while the lifespan reduction in a sir2 mutant is associated with decreased rDNA stability (high copy number variation) …”
“rDNA is more unstable and induces checkpoint control faster than any other part of genome, and that the nucleolus may be more sensitive to protein damage because it is protein-dense …”
He stated further that rDNA may serve as ‘‘sensor’’ for DNA damage, and also as ‘‘shock absorber’’ that protects the genome from damage. I think these are broad statements as of this time.
He and his colleagues have observed that fob1 mutants show better growth than wild type, indicating checkpoint induction is reduced and lifespan is extended. More specifically, he mentioned that rDNA copy number gradually decreases in a fob1 mutant by a FOB1-independent form of homologous recombination. This implies termination of growth, presumbaly because of a shortage of rRNA and ribosomes. In this manner, lifespan extends due growth delay or termination. And yet, the number of abnormal cells is likely to increase.
In contrast, he described that sir2 mutants show increased rDNA instability. He stated that this leads to greater checkpoint control and shorter lifespan. I shall look further into this as it seems unclear to me.
Below is one of the images he presented in the paper:
A new role of the rDNA and nucleolus in the nucleus – rDNA instability maintains genome integrity
Takehiko Kobayashi
National Institute of Genetics and The Graduate University for Advanced Studies, SOKENDAI, 1111 Yata, Mishima 411-8540 Japan
email: Takehiko Kobayashi (takobaya@lab.nig.ac.jp)
BioEssays (2008)
Volume 30 Issue 3, Pages 267 – 272
Williams–Beuren syndrome transcription factor and the gamma-H2A.X
Andrew Xiao and colleagues reported their findings (Nature 457, 57-62 ) recently (1 January 2009) about factors that are directly involved in regulating H2A.X. In mammals, H2A.X Ser 139 phosphorylation (
-H2A.X) occurs to reorganize chromatin in case of DNA double-strand breakage. Because the regulation of -H2A.X phosphorylation during the repair process remains unclear, they studied a new mechanism involving WSTF (Williams–Beuren syndrome transcription factor, also known as BAZ1B) – a component of the WICH complex (WSTF–ISWI ATP-dependent chromatin-remodelling complex). They showed that WSTF has intrinsic tyrosine kinase activity by means of a domain that shares no sequence homology to any known kinase fold. Furthermore, they demonstrated that WSTF phosphorylates Tyr 142 of H2A.X.
maternally inherited piRNAs
In hybrid dysgenesis (Drosophila melanogaster), the progeny of intercrosses between wild males and laboratory-strain females are sterile because of defects in gamete formation. This has something to do with the mobilization of transposons. Depending on the parent of transposon origin, there exist cytoplasmically inherited determinants of the outcome phenotype. These are often transmitted through the maternal germ line.
Networks of small RNAs act through RNA-interference (RNAi) pathways to restrain the spread of selfish genetic elements, regulate gene expression, and many other functions. Like microRNAs and small interfering RNAs, such small species guide Argonaute proteins to silencing targets. The control of mobile elements in germ cells depends on a system composed of Piwi-family proteins (Piwi, Aubergine, and AGO3) and piRNAs. Piwi and Aubergine (Aub) transfer maternal piRNAs into the germ line.
Do maternally deposited small RNAs affect transposon suppression in a heritable fashion? Are piRNAs the maternal suppressor of hybrid dysgenesis?
Julius Brennecke and colleagues recently reported an epigenetic role for maternally inherited piRNAs in transpososon silencing. They found that small RNAs themselves serve as vectors for epigenetic information. “In both P- and I-element–mediated hybrid dysgenesis models, daughters show a markedly different content of Piwi-interacting RNAs (piRNAs) targeting each element, depending on their parents of origin. Such differences persist from fertilization through adulthood. This indicates that maternally deposited piRNAs are important for mounting an effective silencing response and that a lack of maternal piRNA inheritance underlies hybrid dysgenesis.”
Science 28 November 2008:
Vol. 322. no. 5906, pp. 1387 – 1392
DOI: 10.1126/science.116517
Fig. 2. I-R hybrid dysgenesis correlates with maternal piRNA inheritance. (A) Normalized piRNA counts for Repbase transposons are plotted for w1118 inducer and wK reactive ovaries. (B) (Left) Fold differences in piRNA counts comparing w1118 and wK mothers are shown (red line indicates a 1:1 ratio). (Right) Transposon piRNA ratios for mothers, embryos, and F1 progeny (SF: RSF ratio) are shown as a heat map. (C) Scatter plots indicating transposon piRNA correlations between w1118 and wK mothers (top) and their respective intercross progeny (bottom).
