We are studying the transcriptional regulatory mechanisms that control stress response from a variety of eukaryotic model systems.

Transcription, the process of copying RNA from DNA, is one of the primary steps for control of gene expression as it ensures least wastage of resources in synthesis of macromolecules.  Starting with bacterio-phages, increasingly complex organisms have been used as model systems to study transcriptional regulatory mechanisms with an aim to understand regulation of complex phenomena such as response to environment, differentiation, and development.  The study of a given phenomenon in a variety of related and unrelated model systems allows one to understand the underlying theme.  Yeast, S. cerevisiae, makes a good eukaryotic model system owing to the fact that on one hand it is very simple to handle and manipulate while on the other it exhibits, albeit rudimentary in some cases, many phenomena akin to those observed in higher organisms.

One such phenomenon is stress response. It involves probably one of the most highly conserved family of proteins, called the heat shock proteins.  Originally characterised in response to heat shock, these proteins include regulators of the response as well as the mediators required for coping up with the stress.  These are common to a variety of apparently unrelated stressful conditions.  The regulators generally are not part of the basal transcriptional machinery.  Interestingly, observations few years ago suggested that the yeast RNA polymerase II may have components that may contribute specifically to stress response related gene expression and indeed, many components of the core polymerase are turning out to be modulators of specific gene transcription.

We discovered that a two-protein subcomplex within the yeast RNA polymerase II affects many stress responses in an interesting fashion, previously not encountered in eukaryotes.  One of the components of this subcomplex, Rpb7, is essential for viability of yeast and is very highly conserved from archaebacteria to humans.  The high degree of conservation suggests an essential role played by this protein.  The other component of the subcomplex Rpb4 on the other hand is dispensable for growth under normal conditions and in fact is not that well conserved among even the eukaryotic systems.  We have initiated the characterisation of the involvement of this subcomplex in regulating the stress response in yeast.  As mentioned earlier, we are also pursuing the idea that the counterparts of this subcomplex in other eukaryotic model systems may also function similarly.  If the above assumption were true, then we would be able to identify the components that act downstream of the subcomplex in regulating the stress response in a given organism.  Since the transcriptional machinery in yeast is probably one of the most well characterised system, we initiated our inquiry working with S. cerevisiae.  The specific ongoing projects include: a) Characterisation of the domains of the two proteins, involved in protein-protein interaction, using molecular genetic screens designed in the lab as well as biochemical techniques. b) Using the state of the art microarray technology for whole genome level transcriptional analysis, study the effects of stress and the subunit dependent transcription.  We have already identified novel leads towards understanding the role of these subunits in stress regulation. c) Identify the specific regulators that control various stress responses such as sporulation, heat shock response using two hybrid system as well as other genetic screens. d) Solve the structure of the two subunits and compare the structure function relationship among their eukaryotic homolgs.

We are interested in using two other eukaryotic model systems described below to study functional conservation of the stress response regulators.  Each of the system chosen adds its own flavour to the question asked.

Pathogenicity determinants of Candida albicans: C. albicans is an organism of impor-tance as it is the causative agent of one of the most prevalent secondary infections in patients with immunodeficiency such as in AIDS.  Interestingly, virulent form of C. albicans exhibits altered morphology essential for pathogenicity.  This alteration in morphology is very similar to the one shown by S. cerevisiae in response to starvation.  We are studying the effect of expression of the homologs of the above mentioned RNA polymerase II subunits. Using the morphological alteration as a marker, we expect to discover regulators that control the virulence of this pathogen.

Study of the transcriptional regulators of stress dependent differentiation in Dictyostelium discoideum.  D. discoideum is an interesting organism with very well characterised cascade of multicellular developmental stages accompanied by cellular differentiation in response to starvation stress. We have cloned the RPB7 homolog and seen that it is differentially expressed.  In this system, we are trying to use the antisense technology to regulate the expression of the homologs from the system.

SELECTED PUBLICATIONS:

 

1:	Singh V, Sinha I, Sadhale PP.
	Global analysis of altered gene expression during morphogenesis of Candida albicans in vitro. Biochem Biophys Res Commun. 2005 Sep 9;334(4):1149-58.
2:	Sampath V, Sadhale P.  Rpb4 and Rpb7: a sub-complex integral to multi-subunit RNA polymerases performs a multitude of functions. IUBMB Life. 2005 Feb;57(2):93-102. Review.
3:	Singh SR, Rekha N, Pillai B, Singh V, Naorem A, Sampath V, Srinivasan N, Sadhale PP.
	Domainal organization of the lower eukaryotic homologs of the yeast RNA polymerase II core subunit Rpb7 reflects functional conservation.
4:	Maya Rani Jaiprakash, Beena Pillai, Purna Venkatesh, Subramanian N, Vilas P. Sinkar and Parag P. Sadhale
	RNA Isolation From High-Phenolic Freeze-Dried Tea (Camellia sinensis) Leaves  (Published by Abstract) Plant Molecular Biology Reporter, Volume 21, Number 4, December 2003, 465a-465g
5:	Parag Sadhale
	“A stress regulatory role of a core RNA polymerase subunit: insights from Genetic and Genome wide transcription profiling studies” (2004) p185-187  in ‘Deep roots, Open skies: new Biology in India’ eds., SK Basu, J K Batra and DM. Salunke.  Narosa publishin house New Delhi India..
6:	Sampath V, Nambudiry R, Srinivasan N, Sadhale PP.
	The conserved and non-conserved regions of Rpb4 are involved in multiple phenotypes in Saccharomyces cerevisiae. J Biol Chem. 2003 Oct 6 [Epub ahead of print]
7:	Pillai B, Verma J, Abraham A, Francis P, Kumar Y, Tatu U, Brahmachari SK, Sadhale PP.
	Whole genome expression profiles of yeast RNA polymerase II core subunit, Rpb4, in stress and nonstress conditions. J Biol Chem. 2003 Jan 31;278(5):3339-46. Epub 2002 Nov 11
8:	Pillai B, Sampath V, Sharma N, Sadhale P.
	Rpb4, a non-essential subunit of core RNA polymerase II of Saccharomyces cerevisiae is important for activated transcription of a subset of genes. J Biol Chem. 2001 Aug 17;276(33):30641-7. Epub 2001 May 29.
9:	Beena Pillai, Samir K. Brahmachari and Parag P. Sadhale		PDF
	Genome-wide expression profile of RNA polymerase II subunit mutant of yeast using microarray technology. CURRENT SCIENCE, VOL. 81, NO. 5, 10 SEPTEMBER 2001
10:	Tan Q, Li X, Sadhale PP, Miyao T, Woychik NA.
	Multiple mechanisms of suppression circumvent transcription defects in an RNA polymerase mutant. Mol Cell Biol. 2000 Nov;20(21):8124-33.
11:	Sharma, N. and Sadhale, P.P. (1999)
	Over-expression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotype associated with absence of the largest, non essential subunit Rpb4. J. Genet. 78, 149-156
12:	Parag Sadhale, Nimisha Sharma, P Beena, Aparna Katoch, Narottam Acharya and Sanjay K Singh		PDF
	Modulation of polymerase II composition: A possible mode of transcriptional regulation of stress response in eukaryotes. J. Biosci. 23. No. 4. October 1998, pp 331-335. (C) Indian Academy of Science