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Lecture 2

BIOLOGY 1A03 Lecture 2: Biology Theme 3 Module 4

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Department
Biology
Course
BIOLOGY 1A03
Professor
Alastair Tracey
Semester
Fall

Description
Biology Theme 3 Module 4 Unit 1: Global Gene Expression The relative amounts of a gene product that are produced by an organism can vary according to changing conditions and different signals. As a result, the expression of genes is regulated so that their products are present in the right amount at the right times. Gene expression can be regulated at many different points during the synthesis of a functional protein. This can include at transcription initiation, RNA processing, the overall stability of the RNA molecule, protein synthesis, protein modifications and transport, and finally protein degradation. This kind of multi- level regulation enables a cell to rapidly alter the levels of active proteins in response to internal and external signals. To best understand how cells work, it is important to know when and where specific genes or a group of genes are expressed within an organism. One of the most straightforward ways in which scientists can examine patterns of gene expression of specific genes is to identify the mRNA products that are produced. Consider for example if we suspect that a specific gene plays an important role in the embryonic development of the fruit fly Drosophila. It is possible to identify which embryonic cells or tissues express the gene by undertaking an analysis as to where the corresponding mRNA that is transcribed from that gene can be found during development in the intact organism (or in situ). The mRNA can bedetected with a complementary probe that has a fluorescent tag attached to it. In this case, the complementary probe to the mRNA of interest would be a fluorescently labelled short single-stranded segment of DNA or RNA, and the probe will bind or hybridize in a complementary fashion to the target mRNA molecule in the organism. This process of in situ hybridization can be utilized to study the expression of one or a few genes of interest and can often lead to beautiful results as seen in the image of thisDrosophila embryo. As can be seen, there are spatial differences in the relative expression of these investigated 5 gene products throughout the body of the embryo. These types of in situ hybridizations can be carried out and compared throughout the different stages of development to attain a better understanding of temporal differences that may exist. While in situ hybridization is useful to assess the gene expression levels of a few genes of interest, the development of DNA microarray techniques in the mid 1990’s made it possible to examine the expression of thousands of genes at once. DNA microarrays are largely based on the base-pair interactions and binding of complementary strands of nucleic acids. This is merely an in vitro adaptation of the in situ hybridization technique. Since entire genomes of various organisms are now known, it is possible to set up glass slides that have tiny spots containing a known DNA sequence or gene. These DNA molecules act as probes to detect gene expression (sometimes also referred to as the transcriptome or the set of mRNA transcripts that are expressed by various genes). These slides are the DNA chips or gene chips that are utilized during microarray analysis. Researchers can use genomic DNA sequences as probes to investigate whether specific genes of interest are transcribed and can also look at groups of genes to determine whether they are expressed in any specific coordinated manner. As a result, it is possible to gain insight into possible interacting gene networks within a genome. Currently, it is possible to manufacture DNA microarrays containing up to 100, 000 oligonucleotides (or short fragments of nucleic acids) each representing a different gene. These are the basic strategies that encompass global (or genome-wide) expression studies. Although all cells in our body contain identical genetic material, this does not mean that all of our genes are active at once. There are differences that exist. DNA microarrays are largely useful to visualize variation in gene expression during different stages of development, across different cell types and even in response to different signals. Investigations into which genes are active or not in different cells can provide information as to how these cells can function normally, and what changes when gene expression is altered. DNA microarrays can also be utilized as a great tool to identify differences in gene expression levels between normal and cancerous cells. Whereas many different types of cancer cells were once classified solely based on the organs in which tumours develop, it is now possible, with microarray analysis, to be able to differentiate between the patterns of gene activity between normal and cancerous cells. Consider for example a comparison that can be made between normal breast epithelial cells and cancerous breast carcinoma cells. How do we know what genes are involved in transformation of a normal cell to a tumour cell? An answer to this question can be largely attained by growing both cell types in culture, isolating the gene products or mRNA that is transcribed by these two cell types and then undertaking a DNA microarray analysis. Once the mRNA has been isolated from the cells, these molecules can serve as templates for making complementary cDNA molecules to the mRNA utilizing a specific reverse transcriptase enzyme. During this reverse transcription process, fluorescent nucleotides are utilized and become part of the newly synthesized cDNA molecule. It is important to label the cDNA isolated from both cell types with different colouredluorescent dyes so that they can be easily identified during the microarray analysis. A DNA microarray chip consists of a large number of single-stranded DNA fragments (that represent different genes) which are fixed to a glass slide in a tightly spaced array. The DNA that is fixed to the chip represents all genes known to be in a cell, but as we have discussed there can be marked differences in gene expression at any given time. If we consider the fluorescently labelled cDNA molecules that have been prepared from the normal epithelial cells (which will fluoresce in green) and the breast carcinoma cells (which will fluoresce in red), these can be combined in equal amounts and tested for hybridization with the single-stranded DNA molecules that are present on the microarray chip. Relative differences in fluorescence colour and intensity can then be measured with a special scanner at each spot on the microarray slide. The key to interpreting the results of a DNA microarray is to keep in mind that if a particular gene is active, then it will produce many molecules of mRNA, and as a result, we will have more labeled cDNA molecules available after reverse transcription which will be able to hybridize to the DNA on the microarray chip. This allows for the observation of many bright spots. When an equal mixture of the labeled cDNA of both the normal epithelial cells and breast carcinoma cells are applied to a microarray chip, they will compete for the synthetic complementary DNA fragments that are distributed across all the spots of the microarray chip. What this mean
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