Quadrapedal locomotion in Humans.

In their paper entitled “Mutations in the very low-density lipoprotein receptor VLDLR cause cerebellar hypoplasia and quadrupedal locomotion in humans”, which appeared in March 18^th issue of PNAS, Ozcelik et al. shows that a mutation in very low-density lipoprotein receptor VLDLR lead to cerebellar hypoplasia and quadrupedal locomotion in humans.

Fig. Phenotypic (A) and cranial radiologic (B) presentation of quadrupedal gait in families A and D. (A) Affected brothers VI:20 and VI:18 and cousin VI:25 in family A (Upper) and the proband II:2 in family D (Lower) display palmigrate walking. This is different from quadrupedal knuckle-walking of the great apes (2). The hands make contact with the ground at the ulnar palm, and consequently this area is heavily callused as exemplified by VI:20. Strabismus was observed in all affected individuals. (B) Coronal and midsagittal MRI sections of VI:20, demonstrating vermial hypoplasia, with the inferior vermial portion being completely absent. Inferior cerebellar hypoplasia and a moderate simplification of the cerebral cortical gyri are noted. The brainstem and the pons are particularly small (Left and Center). Similar findings are observed for II:2 (Right).

Ref
1. Ozcelik T, et al. (2008) Mutations in the very low-density lipoprotein receptor VLDLR cause cerebellar hypoplasia and quadrupedal locomotion in humans. Proc Natl Acad Sci USA 105:4232–4236.[Abstract/Free Full Text]

What is ChIP on Chip?

What is ChIP on Chip?

It is a deadly combination of Chromatin Immuno Precipitation with Microarray to provide a powerful technique for identifying the functional DNA elements within a genome. Functional elements can be promoters, silencers, enhancers, Chromatin domain boundaries, heterochromatin proteins, and histones etc, because these elements are normally bound with specific proteins like transcription factors. This can provide a large quantum of data that is unbiased and can provide information on a whole genome basis.

Steps involved:-

Formaldehyde cross linking of proteins.

Shearing of DNA to smaller fragments.

Immuno pull down using desirable antibody

Reverse cross linking

Amplifying and labeling of pulled down fragments

Hybridization on to a Microarray (Chip)

Data analysis.

For example if one is looking for the genome wide association of certain histone variants, use an antibody against the variant of interest. After the hybridization on to the Chip you can make out to which regions of the genome this particular variant is associated (like centromere, telomere etc).

What is PMSF?

PMSF or phenylmethanesulphonylfluoride or phenylmethylsulphonyl fluoride is serine protease inhibitor. It is regular ingredient of most of the protein preparations.

Preparation of PMSF solution

PMSF is normally unstable in aqueous solutions. But highly stable and soluble in non-polar organic solvents like isopropanol, methanol, ethanol etc.

PMSF is soluble (>= 1M) in polar organic solvents such as isopropanol, methanol, ethanol, and 1,2 propanediol. A stock solution of PMSF in 100% isopropanol is stable at least 9 months at 25degC. Stability and solubility in aqueous solution: PMSF is sparingly soluble (slightly greater than 1mM, 25degC) in aqueous buffers. It is also very_ unstable. Aqueous solutions of PMSF are inactivated rapidly (hydrolyzed), an effect accelerated by rising pHG and temperature. The half-life of 100uM PMSF at 25degC (measured as ability to inhibit chymotrypsin) is 110 min. at pH 7.0 (phosphate buffer), 55min. at pH 7.5 (Hepes), and 35 minutes at pH 8.0 (Tris). At 4degC, 100uM PMSF is completely inactivated in 30h at pH 7.6 (Hepes), 22h at pH 8.0 (Tris), and 10h at pH 8.6 (Tris). Presence of EDTA and other metal chelators, DTT or 2-mercaptoethanol does not interfere with the action of PMSF.

The white crystal is fairly stable at room temperature.

PMSF (10mg/ml in isopropanol), working concentration 17-174ug/ml (100-100uM). Stable at 25C for 9 months.

References

1) Fahrney and Gold, J Am Chem Soc 85: 997-100 (1963)

2) Gold and Fahrney, Biochemistry 3: 783-791 (1964)

3) Turini, et al., J Pharmacol Exp Ther 167: 98-104 (1969)

4) Gold, Methods Enzymol 11: 706-711 (1967)

5) James. Anal Biochem 86: 574-579 (1978)