Question

Contrast the concepts of 'housekeeping' genes (and proteins) from the cell-specific genes (and proteins) that characterize and define a differentiated cell type. Propose an experiment that would distinguish the tissue-specific gene expression from housekeeping gene expression in muscle cells

Answer 1

Contrast between housekeeping and tissue specific genes

House keeping genes are also called constitutive genes (also proteins since these genes will be translated into proteins) which are necessary for maintaining basic cellular functions and are expressed throughout every cell type of an organism in normal as well as patho-physiological conditions. These genes are absolutely vital for the survival of an organism and basically form a huge portion of the active part of the genome.

Housekeeping genes are usually fine tuned according to the need of the tissues and thus their expression levels may differ from tissue to tissue but they will be present in every tissue and expressed at some level. Examples :- GAPDH, MYC, eEF-2

Tissues in animals are distinguished and differentiated on the basis of specific gene expression patterns. These genes have specific transcription factors as well as specific promoters which are controlled by the factors either by binding or removal of bound factors which initiates their transcription and translation.

These are controlled and regulated by the network of regulatory genes and controlling to how much extent a gene is expressed.

Tissue environment plays a huge role in meditating the transcription of genes and their expression in a particular cell type.Tissue specific proteins can be either enzymes, constitutive proteins of those tissues, carrier proteins or cell surface receptors.

Examples of tissue/cell specific genes :-

Male sex organ: Tubulin alpha 3

Female sex organ: Glycam1

Skin: Keratin, lymphocyte antigen 6 complex locus G6C

Experiment to distinguish tissue specific gene from housekeeping gene expression.

For this experiment we will use eukaryotic elongation factor 2 as housekeeping gene since it is constitutively expressed throughout the body almost equally.

For muscle specific gene, we will be taking Tropomyosin 3 gamm (Tpm3) gene which is exclusively found in the skeletal muscles.

For control, we can take any tissue type other than muscles. This will ideally express eukaryotic elongation factor 2 similar to muscle cells while there will be no expression of Tpm3 in it.

First experimental step will involve RT-qPCR which can be done using SYBR green dye method and will give us exact amount of mRNA present in the cell.

Secondly it will be followed by Western blotting method. In this, proteins first will be subjected to SDS-PAGE followed with their transfer to nitrocellulose membrane. Presence of a particular protein then can be determined with the help of specific antibodies. Which further can be visualized with the fluorescent tagged secondary antibody. If the target protein for primary antibodies is present, they can be visualized and will give a positive result.

Lastly, the expression level can also be determined with the help of micro arrays.

Thank you