As such, it seems that this survey of molecular biology techniques and concepts will be found useful by those performing molecular diagnostics in a medical laboratory setting, as it should provide a finer understanding of the methodologies and suggest rational approaches to troubleshooting. It should also be noted that this book may be of use to students in molecular biology and to senior faculty who have been somewhat removed from the bench of late.
It may suggest possible uses for methodologies not yet tried by the laboratory or provide insights into protocols that you have been using for years. It will, for example, explain why lower concentrations of standard saline citrate SSC or addition of formamide increases the stringency of DNA or RNA hybridizations, and perhaps introduce you to the concept of plasmids as DNA parasites or to the actual definition of yeast. In fact, it might be wise to have such a book around either a medical or molecular biology laboratory as a means of supporting some minimum level of understanding of molecular biology techniques among laboratory members.
Overall, the book is an enjoyable read, and it is likely to remain useful for some time as a reference book. It contains Web addresses of databases and software useful for DNA and protein sequence analyses as well as a glossary of molecular biology terms that many will find useful. On the downside, some definitions in the glossary are somewhat less than precise, whereas some statements in the text are a bit too sweeping and may be a bit misleading to some because of oversimplications.
However, the book is overall recommended for the list of reasons cited above. Skip to main content. Daniel S. DOI: Noted for its outstanding balance between clarity of coverage and level of detail, this book provides an excellent introduction to the fast moving world of molecular genetics. Get A Copy. Paperback , pages. Published December 12th by Wiley first published October 8th More Details Original Title. Other Editions Friend Reviews. To see what your friends thought of this book, please sign up.
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All Languages. More filters. Sort order. Selin rated it it was amazing May 28, Maria Dimitrova rated it it was amazing Jan 04, Kedra rated it it was amazing Jun 10, There is relatively little DNA to which we can ascribe no likely function, compared to eukaryotic cells, and especially animal and plant cells with much larger genomes, where there is a much higher proportion of non-coding DNA.
Increasingly, much of it is recognized as having important functions within the cell. These include enabling the DNA to be folded correctly and in ensuring that the coding regions are available for expression under the appropriate conditions, as well as coding for small non-translated RNA molecules that play a major role in modulating gene expression. However, this is rarely a serious problem. We just have to be careful how we use it depending on whether we are discussing only the coding region ORF , or the length of sequence that is transcribed into mRNA including untranslated regions , or whether we wish to include DNA regions with regulatory functions as well as coding sequences.
In this context, we will also encounter the words allele and locus.
An allele is one version of that locus. So variation of a genetic characteristic between individuals would be due to different alleles at one locus or several loci. The basic dogma Figure 1. Further processes are required before its proper activity can be manifested: these include the folding of the polypeptide, possibly in association with other subunits to form a multisubunit protein, and in some cases modification, for example by glycosylation or phosphorylation.
It should be remembered that in some cases, RNA rather than protein is the final product of a gene e. RNA polymerase recognizes and binds to a specific sequence the promoter , and initiates the synthesis of mRNA from an adjacent position. A typical bacterial promoter carries two consensus sequences i.
It is important to understand the nature of a consensus: few bacterial promoters have exactly the sequences shown, but if you line up a large number of promoters you will see that at any one position a large number of them have the same base Box 1. The RNA polymerase has higher affinity for some promoters than others — depending not only on the exact nature of the two consensus sequences but also, to a lesser extent, on the sequence of a longer region of DNA.
The nature and regulation of bacterial promoters, including the existence of alternative types of promoters, is considered further in Chapter 5.
In eukaryotes, by contrast, the promoter is a considerably larger area around the transcription start site, where a number of trans-acting transcription factors i. The need for this added complexity can easily be imagined; if cells carrying the same genome are differentiated into a multitude of cell types fulfilling very different functions, a very sophisticated control system is needed to provide each cell type with its specific repertoire of proteins, and to fine-tune the degree of expression for each one of them. Nonetheless, the promoter region, however simple or complex, gives rise to different levels of transcription of various genes.
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Spaces are inserted to optimise the alignment. Note that the consensus is derived from a much larger collection of characterized promoters. Position 1 is the transcription start site. By definition, enhancers are position and orientation independent, and are often remote from the actual start site of transcription by several thousand base pairs.
Eukaryotes have three different RNA polymerases. Only one of these, RNA polymerase II, is involved in the transcription of protein-coding transcripts, plus the transcription of a group of small non-coding RNAs called micro-RNAs, which we will encounter in Chapter It is very short-lived as such, being rapidly processed in a number of steps called maturation.
This transcript contains intervening sequences introns between the exons that carry the coding information see below. The final step is the process of splicing, by which the introns are removed and the exons are joined together. This process is quite complex; some introns are removed by specific proteins known as splicing factors, whereas other introns are removed independently through autocatalysis. To complicate things further, in some cases the transcript is edited, leading, for example, to the introduction of a tissue-specific earlier stop codon that is not encoded in the corresponding DNA.
In bacteria, the processes of transcription and translation take place in the same compartment and simultaneously. In eukaryotes, by contrast, the mature mRNA molecule is transported out of the nucleus to the cytoplasm, where translation takes place. The resulting level of protein production is dependent on the amount of the specific mRNA available, rather than just the rate of production.
The level of an mRNA species will be affected by its rate of degradation as well as by its rate of synthesis.
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In bacteria, most mRNA molecules are degraded quite quickly with a half-life of only a few minutes , although some are much more stable. The instability of the majority of bacterial mRNA molecules means that bacteria can rapidly alter their profile of gene expression by changing the transcription of specific genes.
This greater persistence of eukaryote mRNA molecules is, again, a reflection of the fact that an organism or a cell that is able to control its own environment to a substantial extent is subjected to less radical environmental changes. Consequently, mRNA molecules tend to be more stable in multicellular organisms than in, for example, yeast.
Nonetheless, the principle remains — the level of an mRNA in a cell is a function of its production and degradation rates. We will discuss how to study and disentangle these parameters in Chapter The precise sequence of this site and its distance from the start codon affect the efficiency of translation, although in nature this is less important than transcriptional efficiency in determining the level of gene expression. Translation efficiency will also depend on the codon usage, i.
This concept is explored more fully in Chapter 7. In bacterial systems, where transcription and translation occur in the same compartment of the cell, ribosomes will bind to the mRNA, a process known as initiation, as soon as the RBS has been synthesized. Thus, there will be a procession of ribosomes following close behind the RNA polymerase, translating the mRNA in the process of elongation as and when it is being produced. So, although the mRNA may be very short-lived, the bacteria are capable of producing substantial amounts of the corresponding polypeptide in a short time.
Translation stops when a termination codon is reached. In eukaryotes, the mechanism is much more complicated. The sequence AUG may be encountered on the way without initiation, because the surrounding sequence is also important to define the start of protein synthesis.
Sometimes, translation may be initiated at an internal ribosome entry site IRES ; the best studied of these occur in some viruses, but they also occur in some transcripts encoded naturally by the cell. If we are considering protein-coding genes, the transcription product, messenger RNA mRNA , is then translated into a number of separate polypeptides. This can occur by the ribosomes reaching the stop codon at the end of one polypeptide-coding sequence, terminating translation and releasing the product before reinitiating without dissociation from the mRNA.
Alternatively, the ribosomes may attach independently to internal ribosome binding sites within the mRNA sequence. Generally, the genes involved are responsible for different steps in the same pathway, and this arrangement facilitates the coordinate regulation of those genes, i. In eukaryotes, by contrast, the way in which ribosomes initiate translation is different, which means that they cannot usually produce separate proteins from a single mRNA in this way.
Although there are some examples where polygenic transcripts analogous to bacterial operons are produced, these are very much the exception rather than the rule. Generally, when a single mRNA gives rise to different proteins, this is due to alternative processing of the mRNA see below or by producing one long polyprotein or precursor, which is then cleaved into different proteins as occurs in some viruses.
We will consider this in more detail in Chapter This is usually not true for eukaryotic cells, where the initial transcript is many times longer than that needed for translation into the final protein. This pre-mRNA contains blocks of sequence introns that are removed by processing to generate the final mature mRNA for translation Figure 1.
Introns do occur in bacteria, but quite infrequently. This is partly due to the need for economy in a bacterial cell. A further factor arises from the nature of transcription and translation in a bacterial cell. Since the ribosomes translate the mRNA while it is being made, there is less opportunity for sections of the RNA to be removed before translation. These properties are manifested by transcription of the DNA into RNA, which in eukaryotes is processed by removal of introns to produce a messenger RNA that is then translated into protein.
We now know that several aspects of this model are inadequate. Firstly, there can be differences between cells that have identical DNA sequences, and these differences can be passed on from one cell to its progeny.