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    Alternative processing raises many fundamental issues, perhaps none more important than the very concept of the gene itself. Prior to the discovery of alternative promoters, splicing, and poly (A) sites, a eukaryotic gene could be defined as a hereditary unit that codes for a specific polypeptide. However, this one gene-one polypeptide hypothesis does not work for alternatively processed transcription units that can code for two or more protein isoforms. We must therefore find some other way to define a gene. Although an exon has the minimal amount of information that is expressed as a discrete unit, we cannot define a gene as an exon because exons do not contain all of the information that we normally associate with genes. Perhaps the best definition that we can devise at this time is that a gene is a linear collection of exons that are incorporated into a specific mRNA. However, the term gene continues to be used in a more general sense when referring to a transcription unit or protein coding region. Alternative processing also presents challenges for genetic engineering. Some alternatively processed transcription units are known to produce isoforms that have antagonistic effects. Therefore, transforming a cell with an alternatively spliced gene may result in the production of a harmful rather than a beneficial protein.




    Although it might seem that only a few specific endonucleases and a ligase might suffice to catalyze splicing reactions, eukaryotic cells use a very sophisticated piece of machinery to carry out the steps involved in splicing. As often happens in science, the first clue to the nature of this machinery came from studies in a seemingly unrelated field. People suffering from autoimmune diseases known as mixed connective tissue disease and systemic lupus erythematosus make aberrant antibodies that attack components of their own cells. One such antibody, called the anti-Smith or anti-Sm antibody, binds to small ribonucleoprotein particles in the cell nucleus. Joan Steitz and coworkers suspected that these small ribonucleoproteins might help to process pre-mRNA and set about isolating them so that they could study their function(s). The isolation method they devised was to first tag the ribonucleoprotein particles in a human cell extract with antibodies from the serum of a lupus patient and then pull the tagged ribonucleoproteins out of the extract with an insoluble Stapbylococcus aureus cell wall preparation that binds to antibodies. When Steitz and coworkers analyzed RNA molecules that had been extracted from the ribonucleoprotein particles by polyacrylamide gel electrophoresis, they observed discrete bands corresponding to RNA molecules with chain lengths between 100 and 200 nucleotides long. Because each of the small nuclear RNA molecules is uridine-rich, they are called U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, and U6 snRNA. The reason that there is no U3 snRNA is that the lupus antibodies do not bind to the ribonucleoprotein that contains this RNA. U3 snRNA is involved with rRNA rather than mRNA formation. The U1, U2, U4, and U5 snRNAs each has a 2, 2, 7 trimethyl guanosine cap at its 5'-end while U6 has a ?-methyl phosphate cap.
    Each snRNA is present in its own small nuclear ribonucleoprotein particle (snRNP; or 'snurp' f......


    Splicing may occur so that (1) each and every exon in a pre-mRNA is in-corporated into one mature mRNA through the joining of all successive exons, or (2) one combination of exons in a pre-mRNA is incorporated in one pre-mRNA while other combinations are incorporated in other mRNAs. At present it appears that about 42% of total human transcription units use the latter form of splicing, to produce mRNA. However, this estimate is probably low because the ESTs used to detect alternative splicing come from a limited number of tissues or developmental states, and cover only part of each mRNA. More thorough studies of chromosome 22 show that 59% of its genes are alternatively spliced and even this number may be low.
    Exons are either constitutive or regulated. A constitutive exon is included in all mRNAs formed. A regulated exon, also called a cassette exon, is an exon that is included in some mRNAs and excluded from other mRNAs. Many alternate spiicing patterns are possible for a typical multi-exon pre-mRNA. A cassette exon located between two constitutive exons may either be included or skipped when a pre-mRNA is spliced to form mRNA. Inclusion of one cassette exon may prevent the inclusion of other cassette exons. Mutually exclusive splicing occurs when an array of cassette exons is located between two constitutive exons and only one of the cassette exons can be incorporated into mature mRNA. An intron between two constitutive exons may be included in some mRNAs but not in others. Thus, a single DNA sequence may act as either an intron (when it is excised) or an exon (when it is incorporated). Some exons have alternative splice sites at their 3'-end or their 5'-end. When mRNAs formed by alternative splicing of a given pre-mRNA are translated, the proteins formed usually share common functions but differ within one or more domains. Sometimes, alternative splicing introduces a termination codon in mRNA, causing a premature end......

Messenger RNA and hnRNA both have polv(A) tails

Posted by star on 2018-12-25 21:56:29
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    The next major advance in studying eukaryotic mRNA synthesis took place in the early 1970s as a result of efforts to characterize HeLa cell mRNA by digesting it with ribonucleases. The mRNA was extracted from cytoplasmic complexes known as polyribosomes or polysomes, each consisting of two or more ribosomes translating a single mRNA. Two different endonucleases, RNase TI (a product of the fungus Aspergillus oryzae) and pancreatic RNase, were used to digest the mRNA. RNase TI and pancreatic RNase cleave phosphodiester bonds on the 3’-sides of guanylate residues and pyrimidine nucleotide distribution within HeLa cell mRNA, one would expect to find the digests would contain a mixture of short oligonucleotides along with some mononucleotides. Although digesting HeLa cell mRNA with either T1 RNase or pancreatic RNase did indeed produce the expected digestion products, it also produced an entirely unexpected polynucleotide containing 150 to 200 adenylate groups. Further analysis showed that these poly (A) segments are attached to the 3’-end of mRNA. With the one notable exception of histone mRNA, all eukaryotic mRNAs have such 3'-poly (A) tails. Yeast poly (A) tails are usually between 50 and 70 nucleotides long, considerabiy shorter than mammalian poly (A) tails. Subsequent studies showed that a large proportion of the rapidly labeled hnRNA molecules also have poly (A) tails, supporting the idea that these hnRNA molecules are converted to mRNA.
    Poly (A) tails have a practical laboratory application; their baes paring properties can be uesd to separate mRNA and pre-mRNA from other kinds of RNA. This separation is accomplished by passing a mixture of RNA molecules, dissolved in a buffer solution containing a high salt concentration, through a column packed with cellulose fibers linked to oligo (dT). Messenger RNA molecules stick to the column because of base pairing between their poly (A) tails and oligo (dT). The s......


    Although a great deal is now known about structure-function relationships in DNA-binding domains from a wide variety of transcription activator proteins, much less is known about the activation domains (ADs) in these same transcription activator proteins. This disparity in knowledge reflects difficulties in studying activation domains. In contrast to DNA binding domains, which usually fold to form specific three-dimensional structures such as helix-turn-helix motifs or zinc fingers, activation domains tend to be random coils but may assume a defined structure when they interact with other components of the transcription machinery. Furthermore, activation domains do not appear to share common structural features such as conserved amino acid sequences. Activation domain function has been just as difficult to study as activation domain structure. Although activation domains work by binding to specific proteins in the transcription machinery, it is quite difficult to determine the physiological target. In fact, many activation domains appear to have several possible targets and it is quite possible that many of these targets are physiologically important.
    DNA-bound transcriptional activator proteins may work by one or both of the following mechanisms:
    1.Activation by recruitment model. The activation domain interacts with one or more components of the transcription machinery and stabilizes the binding of the component(s) to the template DNA.
    2.Activation by conformational change model. Tne activation domain somehow induces a conformational change in one or more components of the transcription machinery that are bound to it and thereby stimulates RNA polymerase Il to initiate transcription.



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