Gene Expression and Regulation

How does a gene, which consists of a string of DNA hidden in a cell's nucleus, know when it should express itself? How does this gene cause the production of a string of amino acids called a protein? How do different types of cells know which types of proteins they must manufacture? The answers to such questions lie in the study of gene expression. Thus, this topic room begins by showing how a quiet, well-guarded string of DNA is expressed to make RNA, and how the messenger RNA is translated from nucleic acid coding to protein coding to form a protein. Along the way, the room also examines the nature of the genetic code, how the elements of code were predicted, and how the actual codons were determined.

Next, this topic room turns to the regulation of genes. Genes can't control an organism on their own; rather, they must interact with and respond to the organism's environment. Some genes are constitutive, or always "on," regardless of environmental conditions. Such genes are among the most important elements of a cell's genome, and they control the ability of DNA to replicate, express itself, and repair itself. These genes also control protein synthesis and much of an organism's central metabolism. In contrast, regulated genes are needed only occasionally—but how do these genes get turned "on" and "off"? What specific molecules control when they are expressed?

It turns out that the regulation of such genes differs between prokaryotes and eukaryotes. For prokaryotes, most regulatory proteins are negative and therefore turn genes off. Here, the cells rely on protein–small molecule binding, in which a ligand or small molecule signals the state of the cell and whether gene expression is needed. The repressor or activator protein binds near its regulatory target: the gene. Some regulatory proteins must have a ligand attached to them to be able to bind, whereas others are unable to bind when attached to a ligand. In prokaryotes, most regulatory proteins are specific to one gene, although there are a few proteins that act more widely. For instance, some repressors bind near the start of mRNA production for an entire operon, or cluster of coregulated genes. Furthermore, some repressors have a fine-tuning system known as attenuation, which uses mRNA structure to stop both transcription and translation depending on the concentration of an operon's end-product enzymes. (In eukaryotes, there is no exact equivalent of attenuation, because transcription occurs in the nucleus and translation occurs in the cytoplasm, making this sort of coordinated effect impossible.) Yet another layer of prokaryotic regulation affects the structure of RNA polymerase, which turns on large groups of genes. Here, the sigma factor of RNA polymerase changes several times to produce heat- and desiccation-resistant spores. Within this topic room, the articles on prokaryotic regulation delve into each of these topics, leading to primary literature in many cases.

For eukaryotes, cell-cell differences are determined by expression of different sets of genes. For instance, an undifferentiated fertilized egg looks and acts quite different from a skin cell, a neuron, or a muscle cell because of differences in the genes each cell expresses. A cancer cell acts different from a normal cell for the same reason: It expresses different genes. (Using microarray analysis, scientists can use such differences to assist in diagnosis and selection of appropriate cancer treatment.) Interestingly, in eukaryotes, the default state of gene expression is "off" rather than "on," as in prokaryotes. Why is this the case? The secret lies in chromatin, or the complex of DNA and histone proteins found within the cellular nucleus. The histones are among the most evolutionarily conserved proteins known; they are vital for the well-being of eukaryotes and brook little change. When a specific gene is tightly bound with histone, that gene is "off." But how, then, do eukaryotic genes manage to escape this silencing? This is where the histone code comes into play. This code includes modifications of the histones' positively charged amino acids to create some domains in which DNA is more open and others in which it is very tightly bound up. DNA methylation is one mechanism that appears to be coordinated with histone modifications, particularly those that lead to silencing of gene expression. Small noncoding RNAs such as RNAi can also be involved in the regulatory processes that form "silent" chromatin. On the other hand, when the tails of histone molecules are acetylated at specific locations, these molecules have less interaction with DNA, thereby leaving it more open. The regulation of the opening of such domains is a hot topic in research. For instance, researchers now know that complexes of proteins called chromatin remodeling complexes use ATP to repackage DNA in more open configurations. Scientists have also determined that it is possible for cells to maintain the same histone code and DNA methylation patterns through many cell divisions. This persistence without reliance on base pairing is called epigenetics, and there is abundant evidence that epigenetic changes cause many human diseases.

In order for transcription to occur, the area around a prospective transcription zone needs to be unwound. This is a complex process requiring the coordination of histone modifications, transcription factor binding and other chromatin remodeling activities. Once the DNA is open, specific DNA sequences are then accessible for specific proteins to bind. Many of these proteins are activators, while others are repressors; in eukaryotes, all such proteins are often called transcription factors (TFs). Each TF has a specific DNA binding domain that recognizes a 6-10 base-pair motif in the DNA, as well as an effector domain. In the test tube, scientists can find a footprint of a TF if that protein binds to its matching motif in a piece of DNA. They can also see whether TF binding slows the migration of DNA in gel electrophoresis.

For an activating TF, the effector domain recruits RNA polymerase II, the eukaryotic mRNA-producing polymerase, to begin transcription of the corresponding gene. Some activating TFs even turn on multiple genes at once. All TFs bind at the promoters just upstream of eukaryotic genes, similar to bacterial regulatory proteins. However, they also bind at regions called enhancers, which can be oriented forward or backwards and located upstream or downstream or even in the introns of a gene, and still activate gene expression. Because many genes are coregulated, studying gene expression across the whole genome via microarrays or massively parallel sequencing allows investigators to see which groups of genes are coregulated during differentiation, cancer, and other states and processes.

Most eukaryotes also make use of small noncoding RNAs to regulate gene expression. For example, the enzyme Dicer finds double-stranded regions of RNA and cuts out short pieces that can serve in a regulatory role. Argonaute is another enzyme that is important in regulation of small noncoding RNA–dependent systems. This topic room contains an introductory article on these RNAs, but more content is needed; please contact the topic room editor if you are interested in contributing.

Imprinting is yet another process involved in eukaryotic gene regulation; this process involves the silencing of one of the two alleles of a gene for a cell's entire life span. Imprinting affects a minority of genes, but several important growth regulators are included. For some genes, the maternal copy is always silenced, while for different genes, the paternal copy is always silenced. The epigenetic marks placed on these genes during egg or sperm formation are faithfully copied into each subsequent cell, thereby affecting these genes throughout the life of the organism.

Still another mechanism that causes some genes to be silenced for an organism's entire lifetime is X inactivation. In female mammals, for instance, one of the two copies of the X chromosome is shut off and compacted greatly. This shutoff process requires transcription, the participation of two noncoding RNAs (one of which coats the inactive X chromosome), and the participation of a DNA-binding protein called CTCF. As the possible role of regulatory noncoding RNAs in this process is investigated, more information regarding X inactivation will continue to be discovered, and further additions to this topic room will therefore be warranted.

Protein biosynthesis

Protein biosynthesis (Synthesis) is the process in which cells build proteins.

Protein synthesis is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA which is then used as input to translation.

The cistron DNA is transcribed into a variety of RNA intermediates. The last version is used as a template in synthesis of a polypeptide chain. Proteins can often be synthesized directly from genes by translating mRNA. When a protein is harmful and needs to be available on short notice or in large quantities, a protein precursor is produced. A proprotein is an inactive protein containing one or more inhibitory peptides that can be activated when the inhibitory sequence is removed by proteolysis during posttranslational modification. A preprotein is a form that contains a signal sequence (an N-terminal signal peptide) that specifies its insertion into or through membranes; i.e., targets them for secretion. The signal peptide is cleaved off in the endoplasmic reticulum. Preproproteins have both sequences (inhibitory and signal) still present.

For synthesis of protein, a succession of tRNA molecules charged with appropriate amino acids have to be brought together with an mRNA molecule and matched up by base-pairing through their anti-codons with each of its successive codons. The amino acids then have to be linked together to extend the growing protein chain, and the tRNAs, relieved of their burdens, have to be released. This whole complex of processes is carried out by a giant multimolecular machine, the ribosome, formed of two main chains of RNA, called ribosomal RNA (rRNA), and more than 50 different proteins. This molecular juggernaut latches onto the end of an mRNA molecule and then trundles along it, capturing loaded tRNA molecules and stitching together the amino acids they carry to form a new protein chain.

Protein biosynthesis, although very similar, is different for prokaryotes and eukaryotes.

Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL–induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect—that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

The transformation of complex chromatographic raw data into quantitative and qualitative information

Using the new Chromatography Data System Geminyx III , an in-house software development from Goebel Instrumentelle Analytik, the detector raw data from complete sample sequences can be converted into meaningful quantitative and qualitative information with just a few mouse clicks:

The automatic integration ensures that complete sample sequences are automatically and correctly integrated without time-consuming parameter selection. This can be checked by simply paging through the individual samples.

The user can zoom in on details and, if required, use the graphical editor to move peak limits, reset the baseline or separate a peak shoulder using simply mouse clicks. Once all runs have been checked the sequence can be printed as a complete summary report.

All calibration functions usually used in the chromatography laboratory are supported in Geminyx III. Outliers in standard runs are automatically recognized using tightly defined limits, marked as outliers and are not then used for the calibration calculations.

The integrated report designer permits the professional design of the sample report. The method defines which results are shown in the integration report. Using a report template you define the positioning and formatting of the graphics and sample data and can also add the company logo to the template. The analysis results are thus printed using a professional layout and can be saved in all usual electronic formats or be sent as e-mail.

Alongside the quantitative calculations from the chromatograms Geminyx III also, in conjunction with a diode-array detector, supplies valuable qualitative information with respect to peak purity and component identity. The peak purity is displayed both numerically and graphically using comparisons made between high-resolution normalized spectra obtained across the peak. Using automatic solvent correction the system compensates for the changes in solvent spectrum during a gradient run.

The spectrum search, using self-created or commercially available spectral libraries, is a reliable indicator as to whether the detected component is in reality the substance expected at this retention time.

Naturally Geminyx III executes the quantitative and qualitative calculations defined in the method fully automatically so that checking only requires paging through the individual samples before the complete report of a sample sequence is generated with a single mouse click.

Goebel Instrumentelle Analytik offers complete HPLC solutions from a single source, alongside the configurable modules of the Celeno HPLC range we can also adapt our internally developed Geminyx Software to suit your needs. Further we also offer you reliable and cost-effective service support designed to preserve the value of your system and improve the quality of your analysis results!

Human Microbiome Project

Microbes occupy a number of niches on the surface of and inside human body. In fact, in an adult human being, microbe cells outnumber human cells 10 to 1. It makes us a supra-organism or a composite of species. The gut microbiome, for example, consists of at least many thousands of bacteria and archea species many of which can not be cultured. Arrival of fast and economic sequencing techniques e.g. 454 and SOLEXA has made the molecular (16S rRNA based) identification or microbes within the reach of labs. It appears that there is a lot of diversity in a specific niche across humans and other mammals.

NIH Human Microbiome Project is an intitiative to understand this composite of species. Specifically, the aim is to understand whether there is a core group of microbes that all humans share. The second one is to understand whether the composition of microbiota in a specific niche relates to health. For example there is strong evidence to suggest that gut microbiota of obese individuals is different from lean ones. Transfer of gut microbiota from a obese human to a germ free mouse (a humanized mouse) can induce adipocity which can again be transferred to other mice, yet again. Such a finding has far reaching consequences for a population dealing with calorie-rich diet and obesity.

Stem cells without virus infection

Recently there has been a lot of new studies showing how to reprogram adult cells into stem cells. Just a few months ago researchers showed that only four genes (c-Myc, Klf4, Oct4, and Sox2) were needed to obtain stem cells, and a few weeks ago Oct4 was shown to be the only gene needed to reprogram cells into stem cells.
All these studies required the use of viruses to reprogram cells into stem cells. The typical experiment was basically as follows: introduce your genes (Oct4, etc) into a retrovirus and then infect a cell with it. The retrovirus integrates into the cell's genome, expresses its genes (Oct4, etc) and the cell becomes a stem cell.
Now two studies, both published in the latest issue of Nature, have shown how to get around using a virus. One approach cloned the now called Yamanaka factors (c-Myc, Klf4, Oct4, and Sox2) into a vector and introduced it into cells. After the cells were reprogrammed the vector was digested using a restriction enzyme, only leaving a small trace of DNA. The other approached used a transposable element called piggyBac (from the cabbage looper moth). As with the vector, all four Yamanaka factors were cloned into piggyBac and then introduced into cells, which lead to reprograming into stem cells. The beauty of this new method is that this transposable element can be removed entirely with the use of a transposase (an enzyme that makes the transposable element completely excise itself from DNA, not leaving any trace of its existence). The transposase experiments remain to be performed, as well as a complete characterization of the pluripotent nature of these reprogrammed stem cells (there are a lot of experiments that need to be performed to fully characterize a cells as a stem cell).
These new techniques have great potential for genetic therapy. Up until now in order to introduce genetic modifications into cells to turn them into stem cell you had to infect them with a retrovirus (not the nicest thing and potentially dangerous). By getting rid of viruses altogether, these new techniques made genetic therapy and stem cell research significantly safer and easier.

IISER-Kolkatta recruits @ BT2020- JRF cum PhD position in Marine Microbiology/ Marine Molecular ecology

Dr. Punyasloke Bhadury, Assistant Professor, Indian Institute of Science Education and Research (IISER)- Kolkata,West Bengal would like to recruit suitable candidate for the following position.

(with PhD opportunity)

Marine Microbiology/Marine Molecular ecology

M.Sc. Life Science / Microbiology / Marine Biology / Ecology (First Class);
CSIR-UGC NET qualifion or NET-LS qualification.
Participation in international oceanographic cruises will be required.

03 years

Rs 12,000 + HRA pm/-

12 June 2009

To apply:
Mail your resumes with the subject line "IISER" to,