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112-404: Most bacterial cells including E. coli lack introns in their genome. They also lack a nuclear membrane . Hence the gene regulation by lac operon occurs at the transcriptional level, by preventing conversion of DNA into mRNA . Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as

224-418: A complementation test . This test is illustrated in the figure ( lacA is omitted for simplicity). First, certain haploid states are shown (i.e. the cell carries only a single copy of the lac genes). Panel (a) shows repression, (b) shows induction by IPTG, and (c) and (d) show the effect of a mutation to the lacI gene or to the operator, respectively. In panel (e) the complementation test for repressor

336-408: A chemical reaction , or to a surface on which other chemical reactions or microscopy are performed. In the former sense, a reagent is added to the substrate to generate a product through a chemical reaction. The term is used in a similar sense in synthetic and organic chemistry , where the substrate is the chemical of interest that is being modified. In biochemistry , an enzyme substrate

448-400: A cistron . Although introns are sometimes called intervening sequences , the term "intervening sequence" can refer to any of several families of internal nucleic acid sequences that are not present in the final gene product, including inteins , untranslated regions (UTR), and nucleotides removed by RNA editing , in addition to introns. The frequency of introns within different genomes

560-451: A sugar source for the bacterium, the three genes of the lac operon can be expressed and their subsequent proteins translated: lacZ , lacY , and lacA . The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide , into glucose and galactose . lacY encodes β-galactoside permease , a membrane protein which becomes embedded in the Plasma membrane to enable

672-445: A cryptic splice site or mutate a functional site. They can also be somatic cell mutations that affect splicing in a particular tissue or a cell line. When the mutant allele is in a heterozygous state this will result in production of two abundant splice variants; one functional and one non-functional. In the homozygous state the mutant alleles may cause a genetic disease such as the hemophilia found in descendants of Queen Victoria where

784-405: A culture of wild type using phenyl-Gal, as described above, operator mutations are rare compared to repressor mutants because the target-size is so small. But if instead we start with a strain which carries two copies of the whole lac region (that is diploid for lac ), the repressor mutations (which still occur) are not recovered because complementation by the second, wild type lacI gene confers

896-412: A decreased level of expression in the presence of IPTG and even in strains of the bacterium lacking the repressor or operator. The discovery of cAMP in E. coli led to the demonstration that mutants defective the cya gene but not the crp gene could be restored to full activity by the addition of cAMP to the medium. The cya gene encodes adenylate cyclase, which produces cAMP. In a cya mutant,

1008-405: A double mutant defective in both O 2 and O 3 is dramatically de-repressed (by about 70-fold). In the current model, lac repressor is bound simultaneously to both the main operator O 1 and to either O 2 or O 3 . The intervening DNA loops out from the complex. The redundant nature of the two minor operators suggests that it is not a specific looped complex that is important. One idea

1120-400: A fifth family, but little is known about the biochemical apparatus that mediates their splicing. They appear to be related to group II introns, and possibly to spliceosomal introns. Nuclear pre-mRNA introns (spliceosomal introns) are characterized by specific intron sequences located at the boundaries between introns and exons. These sequences are recognized by spliceosomal RNA molecules when

1232-573: A group II intron, and intronization. In theory it should be easiest to deduce the origin of recently gained introns due to the lack of host-induced mutations, yet even introns gained recently did not arise from any of the aforementioned mechanisms. These findings thus raise the question of whether or not the proposed mechanisms of intron gain fail to describe the mechanistic origin of many novel introns because they are not accurate mechanisms of intron gain, or if there are other, yet to be discovered, processes generating novel introns. In intron transposition,

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1344-488: A lactose metabolite called allolactose, made from lactose by the product of the lacZ gene, binds to the repressor, causing an allosteric shift. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins. The second control mechanism is a response to glucose, which uses the catabolite activator protein (CAP) homodimer to greatly increase production of β-galactosidase in

1456-497: A mutation in one of the introns in a blood clotting factor gene creates a cryptic 3' splice site resulting in aberrant splicing. A significant fraction of human deaths by disease may be caused by mutations that interfere with normal splicing; mostly by creating cryptic splice sites. Incorrectly spliced transcripts can easily be detected and their sequences entered into the online databases. They are usually described as "alternatively spliced" transcripts, which can be confusing because

1568-431: A property termed enzyme promiscuity . An enzyme may have many native substrates and broad specificity (e.g. oxidation by cytochrome p450s ) or it may have a single native substrate with a set of similar non-native substrates that it can catalyse at some lower rate. The substrates that a given enzyme may react with in vitro , in a laboratory setting, may not necessarily reflect the physiological, endogenous substrates of

1680-414: A second, functional transmitter. In contrast, he said, consider a bomber with a defective receiver. The behavior of this bomber cannot be changed by introduction of a second, functional aeroplane. To analyze regulatory mutants of the lac operon, Jacob developed a system by which a second copy of the lac genes ( lacI with its promoter, and lacZYA with promoter and operator) could be introduced into

1792-419: A significant error rate even though there are spliceosome accessory factors that suppress the accidental cleavage of cryptic splice sites. Under ideal circumstances, the splicing reaction is likely to be 99.999% accurate (error rate of 10 ) and the correct exons will be joined and the correct intron will be deleted. However, these ideal conditions require very close matches to the best splice site sequences and

1904-404: A single cell. A culture of such bacteria, which are diploid for the lac genes but otherwise normal, is then tested for the regulatory phenotype. In particular, it is determined whether LacZ and LacY are made even in the absence of IPTG (due to the lactose repressor produced by the mutant gene being non-functional). This experiment, in which genes or gene clusters are tested pairwise, is called

2016-443: A single gene and a single precursor mRNA transcript. The control of alternative RNA splicing is performed by a complex network of signaling molecules that respond to a wide range of intracellular and extracellular signals. Introns contain several short sequences that are important for efficient splicing, such as acceptor and donor sites at either end of the intron as well as a branch point site, which are required for proper splicing by

2128-475: A single gene. Furthermore, some introns play essential roles in a wide range of gene expression regulatory functions such as nonsense-mediated decay and mRNA export. After the initial discovery of introns in protein-coding genes of the eukaryotic nucleus, there was significant debate as to whether introns in modern-day organisms were inherited from a common ancient ancestor (termed the introns-early hypothesis), or whether they appeared in genes rather recently in

2240-468: A small difference in efficiency of transport or metabolism of glucose v. lactose makes it advantageous for cells to regulate the lac operon in this way. The lac gene and its derivatives are amenable to use as a reporter gene in a number of bacterial-based selection techniques such as two hybrid analysis, in which the successful binding of a transcriptional activator to a specific promoter sequence must be determined. In LB plates containing X-gal ,

2352-409: A specific location within the anticodon loop of unspliced tRNA precursors, and are removed by a tRNA splicing endonuclease. The exons are then linked together by a second protein, the tRNA splicing ligase. Note that self-splicing introns are also sometimes found within tRNA genes. Group I and group II introns are found in genes encoding proteins ( messenger RNA ), transfer RNA and ribosomal RNA in

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2464-442: A substrate is called 'fluorogenic' if it gives rise to a fluorescent product when acted on by an enzyme. For example, curd formation ( rennet coagulation) is a reaction that occurs upon adding the enzyme rennin to milk. In this reaction, the substrate is a milk protein (e.g., casein ) and the enzyme is rennin. The products are two polypeptides that have been formed by the cleavage of the larger peptide substrate. Another example

2576-457: A tendency towards intron gain in larger species due to their smaller population sizes, and the converse in smaller (particularly unicellular) species. Biological factors also influence which genes in a genome lose or accumulate introns. Alternative splicing of exons within a gene after intron excision acts to introduce greater variability of protein sequences translated from a single gene, allowing multiple related proteins to be generated from

2688-504: A two-part control mechanism to ensure that the cell expends energy producing the enzymes encoded by the lac operon only when necessary. In the absence of lactose, the lac repressor , encoded by lacI, halts production of the enzymes and transport proteins encoded by the lac operon. It does so by blocking the DNA dependent RNA polymerase . This blocking/ halting is not perfect, and a minimal amount of gene expression does take place all

2800-430: A very wide range of living organisms. Following transcription into RNA, group I and group II introns also make extensive internal interactions that allow them to fold into a specific, complex three-dimensional architecture . These complex architectures allow some group I and group II introns to be self-splicing , that is, the intron-containing RNA molecule can rearrange its own covalent structure so as to precisely remove

2912-540: A wide variety of genes throughout organisms, bacteria, and viruses within all of the biological kingdoms. The fact that genes were split or interrupted by introns was discovered independently in 1977 by Phillip Allen Sharp and Richard J. Roberts , for which they shared the Nobel Prize in Physiology or Medicine in 1993, though credit was excluded for the researchers and collaborators in their labs that did

3024-439: A wild type phenotype. In contrast, mutation of one copy of the operator confers a mutant phenotype because it is dominant to the second, wild type copy. Explanation of diauxie depended on the characterization of additional mutations affecting the lac genes other than those explained by the classical model. Two other genes, cya and crp , subsequently were identified that mapped far from lac , and that, when mutated, result in

3136-434: Is lacI , encoding the lactose repressor—"I" stands for inducibility . One may distinguish between structural genes encoding enzymes, and regulatory genes encoding proteins that affect gene expression. Current usage expands the phenotypic nomenclature to apply to proteins: thus, LacZ is the protein product of the lacZ gene, β-galactosidase. Various short sequences that are not genes also affect gene expression, including

3248-425: Is a DNA sequence with inverted repeat symmetry. The two DNA half-sites of the operator together bind to two of the subunits of the repressor. Although the other two subunits of repressor are not doing anything in this model, this property was not understood for many years. Eventually it was discovered that two additional operators are involved in lac regulation. One (O 3 ) lies about −90 bp upstream of O 1 in

3360-401: Is adjacent to the mutant operator is expressed without IPTG. We say that the operator mutation is cis-dominant , it is dominant to wild type but affects only the copy of the operon which is immediately adjacent to it. This explanation is misleading in an important sense, because it proceeds from a description of the experiment and then explains the results in terms of a model. But in fact, it

3472-671: Is because the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and EIIA shuts down lactose permease to prevent transport of lactose into the cell. This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxie . Only lacZ and lacY appear to be necessary for lactose catabolic pathway . By numbers, lacI has 1100 bps, lacZ has 3000 bps, lacY has 800 bps, lacA has 800 bps, with 3 bps corresponding to 1 amino acid. Three-letter abbreviations are used to describe phenotypes in bacteria including E. coli . Examples include: In

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3584-438: Is indeed the case. While the catalytic reaction may be accurate enough for effective processing most of the time, the overall error rate may be partly limited by the fidelity of transcription because transcription errors will introduce mutations that create cryptic splice sites. In addition, the transcription error rate of 10 – 10 is high enough that one in every 25,000 transcribed exons will have an incorporation error in one of

3696-401: Is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the lac operator . The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels. When cells are grown in the presence of lactose, however,

3808-493: Is no less than 0.1% per intron. This relatively high level of splicing errors explains why most splice variants are rapidly degraded by nonsense-mediated decay. The presence of sloppy binding sites within genes causes splicing errors and it may seem strange that these sites haven't been eliminated by natural selection. The argument for their persistence is similar to the argument for junk DNA. Although mutations which create or disrupt binding sites may be slightly deleterious,

3920-751: Is not an endogenous, in vivo substrate for FAAH. In another example, the N -acyl taurines (NATs) are observed to increase dramatically in FAAH-disrupted animals, but are actually poor in vitro FAAH substrates. Sensitive substrates also known as sensitive index substrates are drugs that demonstrate an increase in AUC of ≥5-fold with strong index inhibitors of a given metabolic pathway in clinical drug-drug interaction (DDI) studies. Moderate sensitive substrates are drugs that demonstrate an increase in AUC of ≥2 to <5-fold with strong index inhibitors of

4032-437: Is observed to vary widely across the spectrum of biological organisms. For example, introns are extremely common within the nuclear genome of jawed vertebrates (e.g. humans, mice, and pufferfish (fugu)), where protein-coding genes almost always contain multiple introns, while introns are rare within the nuclear genes of some eukaryotic microorganisms, for example baker's/brewer's yeast ( Saccharomyces cerevisiae ). In contrast,

4144-449: Is often true that the model comes first, and an experiment is fashioned specifically to test the model. Jacob and Monod first imagined that there must be a site in DNA with the properties of the operator, and then designed their complementation tests to show this. The dominance of operator mutants also suggests a procedure to select them specifically. If regulatory mutants are selected from

4256-400: Is related not to intracellular glucose concentration but to the rate of glucose transport, which influences the activity of adenylate cyclase. (In addition, glucose transport also leads to direct inhibition of the lactose permease.) As to why E. coli works this way, one can only speculate. All enteric bacteria ferment glucose, which suggests they encounter it frequently. It is possible that

4368-421: Is shown. If one copy of the lac genes carries a mutation in lacI , but the second copy is wild type for lacI , the resulting phenotype is normal—but lacZ is expressed when exposed to inducer IPTG. Mutations affecting repressor are said to be recessive to wild type (and that wild type is dominant ), and this is explained by the fact that repressor is a small protein which can diffuse in the cell. The copy of

4480-402: Is that the system works through tethering; if bound repressor releases from O 1 momentarily, binding to a minor operator keeps it in the vicinity, so that it may rebind quickly. This would increase the affinity of repressor for O 1 . The repressor is an allosteric protein , i.e. it can assume either one of two slightly different shapes, which are in equilibrium with each other. In one form

4592-427: Is the chemical decomposition of hydrogen peroxide carried out by the enzyme catalase . As enzymes are catalysts , they are not changed by the reactions they carry out. The substrate(s), however, is/are converted to product(s). Here, hydrogen peroxide is converted to water and oxygen gas. While the first (binding) and third (unbinding) steps are, in general, reversible , the middle step may be irreversible (as in

lac operon - Misplaced Pages Continue

4704-405: Is the material upon which an enzyme acts. When referring to Le Chatelier's principle , the substrate is the reagent whose concentration is changed. In the latter sense, it may refer to a surface on which other chemical reactions are performed or play a supporting role in a variety of spectroscopic and microscopic techniques, as discussed in the first few subsections below. In three of

4816-455: Is the process by which mutations create novel introns from formerly exonic sequence. Thus, unlike other proposed mechanisms of intron gain, this mechanism does not require the insertion or generation of DNA to create a novel intron. The only hypothesized mechanism of recent intron gain lacking any direct evidence is that of group II intron insertion, which when demonstrated in vivo, abolishes gene expression. Group II introns are therefore likely

4928-467: Is transferred via a phosphorylation cascade consisting of the general PTS (phosphotransferase system) proteins HPr and EIA and the glucose-specific PTS proteins EIIA and EIIB, the cytoplasmic domain of the EII glucose transporter. Transport of glucose is accompanied by its phosphorylation by EIIB, draining the phosphate group from the other PTS proteins, including EIIA. The unphosphorylated form of EIIA binds to

5040-424: The cellular transport of lactose into the cell. Finally, lacA encodes β-galactoside transacetylase . [REDACTED] Note that the number of base pairs in diagram given above are not for scale. There are in fact over 5300 base pairs in the lac operon. It would be wasteful to produce enzymes when no lactose is available or if a preferable energy source such as glucose were available. The lac operon uses

5152-403: The lac operon adjacent to the defective lacI gene is effectively shut off by protein produced from the second copy of lacI . If the same experiment is carried out using an operator mutation, a different result is obtained (panel (f)). The phenotype of a cell carrying one mutant and one wild type operator site is that LacZ and LacY are produced even in the absence of the inducer IPTG; because

5264-411: The lac permease and prevents it from bringing lactose into the cell. Therefore, if both glucose and lactose are present, the transport of glucose blocks the transport of the inducer of the lac operon. The lac repressor is a four-part protein, a tetramer, with identical subunits. Each subunit contains a helix-turn-helix (HTH) motif capable of binding to DNA. The operator site where repressor binds

5376-470: The lac promoter, lac p , and the lac operator, lac o . Although it is not strictly standard usage, mutations affecting lac o are referred to as lac o , for historical reasons. Specific control of the lac genes depends on the availability of the substrate lactose to the bacterium. The proteins are not produced by the bacterium when lactose is unavailable as a carbon source. The lac genes are organized into an operon ; that is, they are oriented in

5488-399: The lacI genes are available from GenBank (view) . The first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called the lactose repressor to hinder production of β-galactosidase in the absence of lactose. The lacI gene coding for the repressor lies nearby the lac operon and is always expressed ( constitutive ). If lactose

5600-558: The lacZ gene are thus suited to X-gal plates or ONPG liquid broths. Intron An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word intron is derived from the term intr agenic regi on , i.e., a region inside a gene. The term intron refers to both the DNA sequence within a gene and the corresponding RNA sequence in RNA transcripts . The non-intron sequences that become joined by this RNA processing to form

5712-479: The mitochondrial genomes of vertebrates are entirely devoid of introns, while those of eukaryotic microorganisms may contain many introns. A particularly extreme case is the Drosophila dhc7 gene containing a ≥3.6 megabase (Mb) intron, which takes roughly three days to transcribe. On the other extreme, a 2015 study suggests that the shortest known metazoan intron length is 30 base pairs (bp) belonging to

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5824-705: The spliceosome . Some introns are known to enhance the expression of the gene that they are contained in by a process known as intron-mediated enhancement (IME). Actively transcribed regions of DNA frequently form R-loops that are vulnerable to DNA damage . In highly expressed yeast genes, introns inhibit R-loop formation and the occurrence of DNA damage. Genome-wide analysis in both yeast and humans revealed that intron-containing genes have decreased R-loop levels and decreased DNA damage compared to intronless genes of similar expression. Insertion of an intron within an R-loop prone gene can also suppress R-loop formation and recombination . Bonnet et al. (2017) speculated that

5936-450: The CAP regulatory protein has to assemble on the lac promoter, resulting in an increase in the production of lac mRNA . More available copies of the lac mRNA results in the production (see translation ) of significantly more copies of LacZ (β-galactosidase, for lactose metabolism) and LacY (lactose permease to transport lactose into the cell). After a delay needed to increase the level of

6048-540: The DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase, enabling the cell to hydrolyse lactose and release galactose and glucose. More recently inducer exclusion was shown to block expression of the lac operon when glucose is present. Glucose is transported into the cell by the PEP-dependent phosphotransferase system . The phosphate group of phosphoenolpyruvate

6160-573: The Lac-operon. The specific binding site for the Lac-repressor protein is the operator. The non-specific interaction is mediated mainly by charge-charge interactions while binding to the operator is reinforced by hydrophobic interactions. Additionally, there is an abundance of non-specific DNA sequences to which the repressor can bind. Essentially, any sequence that is not the operator, is considered non-specific. Studies have shown, that without

6272-399: The absence of any competing cryptic splice site sequences within the introns and those conditions are rarely met in large eukaryotic genes that may cover more than 40 kilobase pairs. Recent studies have shown that the actual error rate can be considerably higher than 10 and may be as high as 2% or 3% errors (error rate of 2 or 3 x 10 ) per gene. Additional studies suggest that the error rate

6384-462: The absence of cAMP makes the expression of the lacZYA genes more than ten times lower than normal. Addition of cAMP corrects the low Lac expression characteristic of cya mutants. The second gene, crp , encodes a protein called catabolite activator protein (CAP) or cAMP receptor protein (CRP). However the lactose metabolism enzymes are made in small quantities in the presence of both glucose and lactose (sometimes called leaky expression) due to

6496-453: The absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below, about 60 bp upstream of the transcription start site), which assists the RNAP in binding to

6608-444: The amount of available repressor in the cell. This in turn reduces the amount of inducer required to unrepress the system. A number of lactose derivatives or analogs have been described that are useful for work with the lac operon. These compounds are mainly substituted galactosides, where the glucose moiety of lactose is replaced by another chemical group. The experimental microorganism used by François Jacob and Jacques Monod

6720-413: The bacterial endosymbiont invaded the host genome. In the beginning these self-splicing introns excised themselves from the mRNA precursor but over time some of them lost that ability and their excision had to be aided in trans by other group II introns. Eventually a number of specific trans-acting introns evolved and these became the precursors to the snRNAs of the spliceosome. The efficiency of splicing

6832-436: The cAMP receptor protein). However, the lacI gene (regulatory gene for lac operon) produces a protein that blocks RNAP from binding to the operator of the operon. This protein can only be removed when allolactose binds to it, and inactivates it. The protein that is formed by the lacI gene is known as the lac repressor. The type of regulation that the lac operon undergoes is referred to as negative inducible, meaning that

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6944-436: The case of Lac, wild type cells are Lac and are able to use lactose as a carbon and energy source, while Lac mutant derivatives cannot use lactose. The same three letters are typically used (lower-case, italicized) to label the genes involved in a particular phenotype, where each different gene is additionally distinguished by an extra letter. The lac genes encoding enzymes are lacZ , lacY , and lacA . The fourth lac gene

7056-410: The case of more than one substrate, these may bind in a particular order to the active site, before reacting together to produce products. A substrate is called 'chromogenic' if it gives rise to a coloured product when acted on by an enzyme. In histological enzyme localization studies, the colored product of enzyme action can be viewed under a microscope, in thin sections of biological tissues. Similarly,

7168-433: The cell using pre-existing transport protein encoded by lacY. This lactose then combines with the repressor and inactivates it, hence allowing the lac operon to be expressed. Then more β-galactoside permease is synthesized allowing even more lactose to enter and the enzymes encoded by lacZ and lacA can digest it. However, in the presence of glucose, regardless of the presence of lactose, the operon will be repressed. This

7280-432: The claim of function must be accompanied by convincing evidence that multiple functional products are produced from the same gene. While introns do not encode protein products, they are integral to gene expression regulation. Some introns themselves encode functional RNAs through further processing after splicing to generate noncoding RNA molecules. Alternative splicing is widely used to generate multiple proteins from

7392-485: The colour change from white colonies to a shade of blue corresponds to about 20–100 β-galactosidase units, while tetrazolium lactose and MacConkey lactose media have a range of 100–1000 units, being most sensitive in the high and low parts of this range respectively. Since MacConkey lactose and tetrazolium lactose media both rely on the products of lactose breakdown, they require the presence of both lacZ and lacY genes. The many lac fusion techniques which include only

7504-454: The damaged operator site, does not permit binding of the repressor to inhibit transcription of the structural genes. The operator mutation is dominant. When the operator site where repressor must bind is damaged by mutation, the presence of a second functional site in the same cell makes no difference to expression of genes controlled by the mutant site. A more sophisticated version of this experiment uses marked operons to distinguish between

7616-437: The duplication of this sequence on each side of the transposon. Such an insertion could intronize the transposon without disrupting the coding sequence when a transposon inserts into the sequence AGGT or encodes the splice sites within the transposon sequence. Where intron-generating transposons do not create target site duplications, elements include both splice sites GT (5') and AG (3') thereby splicing precisely without affecting

7728-579: The emergence of eukaryotes, or the initial stages of eukaryotic evolution, involved an intron invasion. Two definitive mechanisms of intron loss, reverse transcriptase-mediated intron loss (RTMIL) and genomic deletions, have been identified, and are known to occur. The definitive mechanisms of intron gain, however, remain elusive and controversial. At least seven mechanisms of intron gain have been reported thus far: intron transposition, transposon insertion, tandem genomic duplication, intron transfer, intron gain during double-strand break repair (DSBR), insertion of

7840-401: The end of the lacI gene, and the other (O 2 ) is about +410 bp downstream of O 1 in the early part of lacZ . These two sites were not found in the early work because they have redundant functions and individual mutations do not affect repression very much. Single mutations to either O 2 or O 3 have only 2 to 3-fold effects. However, their importance is demonstrated by the fact that

7952-432: The enzyme's reactions in vivo . That is to say that enzymes do not necessarily perform all the reactions in the body that may be possible in the laboratory. For example, while fatty acid amide hydrolase (FAAH) can hydrolyze the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide at comparable rates in vitro , genetic or pharmacological disruption of FAAH elevates anandamide but not 2-AG, suggesting that 2-AG

8064-474: The evolutionary process (termed the introns-late hypothesis). Another theory is that the spliceosome and the intron-exon structure of genes is a relic of the RNA world (the introns-first hypothesis). There is still considerable debate about the extent to which of these hypotheses is most correct but the popular consensus at the moment is that following the formation of the first eukaryotic cell, group II introns from

8176-554: The experiments resulting in the discovery, Susan Berget and Louise Chow . The term intron was introduced by American biochemist Walter Gilbert : "The notion of the cistron [i.e., gene] ... must be replaced by that of a transcription unit containing regions which will be lost from the mature messenger – which I suggest we call introns (for intragenic regions) – alternating with regions which will be expressed – exons." (Gilbert 1978) The term intron also refers to intracistron , i.e., an additional piece of DNA that arises within

8288-404: The fact that splicing of RNA molecules containing group II introns generates branched introns (like those of spliceosomal RNAs), while group I introns use a non-encoded guanosine nucleotide (typically GTP) to initiate splicing, adding it on to the 5'-end of the excised intron. The spliceosome is a very complex structure containing up to one hundred proteins and five different RNAs. The substrate of

8400-414: The fact that the RNAP can still sometimes bind and initiate transcription even in the absence of CAP. Leaky expression is necessary in order to allow for metabolism of some lactose after the glucose source is expended, but before lac expression is fully activated. In summary: The delay between growth phases reflects the time needed to produce sufficient quantities of lactose-metabolizing enzymes. First,

8512-565: The function of introns in maintaining genetic stability may explain their evolutionary maintenance at certain locations, particularly in highly expressed genes. The physical presence of introns promotes cellular resistance to starvation via intron enhanced repression of ribosomal protein genes of nutrient-sensing pathways. Introns may be lost or gained over evolutionary time, as shown by many comparative studies of orthologous genes. Subsequent analyses have identified thousands of examples of intron loss and gain events, and it has been proposed that

8624-410: The gene is turned off by the regulatory factor ( lac repressor) unless some molecule (lactose) is added. Once the repressor is removed, RNAP then proceeds to transcribe all three genes ( lacZYA ) into mRNA. Each of the three genes on the mRNA strand has its own Shine-Dalgarno sequence , so the genes are independently translated. The DNA sequence of the E. coli lac operon , the lacZYA mRNA , and

8736-573: The human MST1L gene. The shortest known introns belong to the heterotrich ciliates, such as Stentor coeruleus , in which most (> 95%) introns are 15 or 16 bp long. Splicing of all intron-containing RNA molecules is superficially similar, as described above. However, different types of introns were identified through the examination of intron structure by DNA sequence analysis, together with genetic and biochemical analysis of RNA splicing reactions. At least four distinct classes of introns have been identified: Group III introns are proposed to be

8848-402: The human genome contains an average of 8.4 introns/gene (139,418 in the genome), the unicellular fungus Encephalitozoon cuniculi contains only 0.0075 introns/gene (15 introns in the genome). Since eukaryotes arose from a common ancestor ( common descent ), there must have been extensive gain or loss of introns during evolutionary time. This process is thought to be subject to selection, with

8960-422: The idea that tandem genomic duplication is a prevalent mechanism for intron gain. The testing of other proposed mechanisms in vivo, particularly intron gain during DSBR, intron transfer, and intronization, is possible, although these mechanisms must be demonstrated in vivo to solidify them as actual mechanisms of intron gain. Further genomic analyses, especially when executed at the population level, may then quantify

9072-406: The intron and link the exons together in the correct order. In some cases, particular intron-binding proteins are involved in splicing, acting in such a way that they assist the intron in folding into the three-dimensional structure that is necessary for self-splicing activity. Group I and group II introns are distinguished by different sets of internal conserved sequences and folded structures, and by

9184-447: The lactose metabolizing enzymes, the bacteria enter into a new rapid phase of cell growth . Two puzzles of catabolite repression relate to how cAMP levels are coupled to the presence of glucose, and secondly, why the cells should even bother. After lactose is cleaved it actually forms glucose and galactose (easily converted to glucose). In metabolic terms, lactose is just as good a carbon and energy source as glucose. The cAMP level

9296-436: The large number of possible such mutations makes it inevitable that some will reach fixation in a population. This is particularly relevant in species, such as humans, with relatively small long-term effective population sizes. It is plausible, then, that the human genome carries a substantial load of suboptimal sequences which cause the generation of aberrant transcript isoforms. In this study, we present direct evidence that this

9408-659: The mature RNA are called exons . Introns are found in the genes of most eukaryotes and many eukaryotic viruses and they can be located in both protein-coding genes and genes that function as RNA ( noncoding genes ). There are four main types of introns: tRNA introns, group I introns, group II introns, and spliceosomal introns (see below). Introns are rare in Bacteria and Archaea (prokaryotes). Introns were first discovered in protein-coding genes of adenovirus , and were subsequently identified in genes encoding transfer RNA and ribosomal RNA genes. Introns are now known to occur within

9520-553: The microscopy data. Samples are deposited onto the substrate in fine layers where it can act as a solid support of reliable thickness and malleability. Smoothness of the substrate is especially important for these types of microscopy because they are sensitive to very small changes in sample height. Various other substrates are used in specific cases to accommodate a wide variety of samples. Thermally-insulating substrates are required for AFM of graphite flakes for instance, and conductive substrates are required for TEM. In some contexts,

9632-408: The most common nano-scale microscopy techniques, atomic force microscopy (AFM), scanning tunneling microscopy (STM), and transmission electron microscopy (TEM), a substrate is required for sample mounting. Substrates are often thin and relatively free of chemical features or defects. Typically silver, gold, or silicon wafers are used due to their ease of manufacturing and lack of interference in

9744-526: The most commonly purported intron gain mechanism, a spliced intron is thought to reverse splice into either its own mRNA or another mRNA at a previously intron-less position. This intron-containing mRNA is then reverse transcribed and the resulting intron-containing cDNA may then cause intron gain via complete or partial recombination with its original genomic locus. Transposon insertions have been shown to generate thousands of new introns across diverse eukaryotic species. Transposon insertions sometimes result in

9856-473: The number of conserved introns flanked by repeats in other organisms, though, for statistical relevance. For group II intron insertion, the retrohoming of a group II intron into a nuclear gene was proposed to cause recent spliceosomal intron gain. Intron transfer has been hypothesized to result in intron gain when a paralog or pseudogene gains an intron and then transfers this intron via recombination to an intron-absent location in its sister paralog. Intronization

9968-444: The presence of non-specific binding, induction (or unrepression) of the Lac-operon could not occur even with saturated levels of inducer. It had been demonstrated that, without non-specific binding, the basal level of induction is ten thousand times smaller than observed normally. This is because the non-specific DNA acts as sort of a "sink" for the repressor proteins, distracting them from the operator. The non-specific sequences decrease

10080-458: The presumed ancestors of spliceosomal introns, acting as site-specific retroelements, and are no longer responsible for intron gain. Tandem genomic duplication is the only proposed mechanism with supporting in vivo experimental evidence: a short intragenic tandem duplication can insert a novel intron into a protein-coding gene, leaving the corresponding peptide sequence unchanged. This mechanism also has extensive indirect evidence lending support to

10192-432: The protein-coding sequence. It is not yet understood why these elements are spliced, whether by chance, or by some preferential action by the transposon. In tandem genomic duplication, due to the similarity between consensus donor and acceptor splice sites, which both closely resemble AGGT, the tandem genomic duplication of an exonic segment harboring an AGGT sequence generates two potential splice sites. When recognized by

10304-408: The reaction is a long RNA molecule and the transesterification reactions catalyzed by the spliceosome require the bringing together of sites that may be thousands of nucleotides apart. All biochemical reactions are associated with known error rates and the more complicated the reaction the higher the error rate. Therefore, it is not surprising that the splicing reaction catalyzed by the spliceosome has

10416-413: The reaction of interest, but they frequently bind the reagents with some affinity to allow sticking to the substrate. The substrate is exposed to different reagents sequentially and washed in between to remove excess. A substrate is critical in this technique because the first layer needs a place to bind to such that it is not lost when exposed to the second or third set of reagents. In biochemistry ,

10528-409: The relative contribution of each mechanism, possibly identifying species-specific biases that may shed light on varied rates of intron gain amongst different species. Structure: Splicing: Function Others: Substrate (biochemistry) In chemistry , the term substrate is highly context-dependent. Broadly speaking, it can refer either to a chemical species being observed in

10640-476: The rennin and catalase reactions just mentioned) or reversible (e.g. many reactions in the glycolysis metabolic pathway). By increasing the substrate concentration, the rate of reaction will increase due to the likelihood that the number of enzyme-substrate complexes will increase; this occurs until the enzyme concentration becomes the limiting factor . Although enzymes are typically highly specific, some are able to perform catalysis on more than one substrate,

10752-421: The repressor will bind to the operator DNA with high specificity, and in the other form it has lost its specificity. According to the classical model of induction, binding of the inducer, either allolactose or IPTG, to the repressor affects the distribution of repressor between the two shapes. Thus, repressor with inducer bound is stabilized in the non-DNA-binding conformation. However, this simple model cannot be

10864-604: The resulting data collection. Silicon substrates are also commonly used because of their cost-effective nature and relatively little data interference in X-ray collection. Single-crystal substrates are useful in powder diffraction because they are distinguishable from the sample of interest in diffraction patterns by differentiating by phase. In atomic layer deposition , the substrate acts as an initial surface on which reagents can combine to precisely build up chemical structures. A wide variety of substrates are used depending on

10976-460: The same direction immediately adjacent on the chromosome and are co-transcribed into a single polycistronic mRNA molecule. Transcription of all genes starts with the binding of the enzyme RNA polymerase (RNAP), a DNA-binding protein , which binds to a specific DNA binding site, the promoter , immediately upstream of the genes. Binding of RNA polymerase to the promoter is aided by the cAMP -bound catabolite activator protein (CAP, also known as

11088-413: The splice sites leading to a skipped intron or a skipped exon. Almost all multi-exon genes will produce incorrectly spliced transcripts but the frequency of this background noise will depend on the size of the genes, the number of introns, and the quality of the splice site sequences. In some cases, splice variants will be produced by mutations in the gene (DNA). These can be SNP polymorphisms that create

11200-474: The spliceosome, the sequence between the original and duplicated AGGT will be spliced, resulting in the creation of an intron without alteration of the coding sequence of the gene. Double-stranded break repair via non-homologous end joining was recently identified as a source of intron gain when researchers identified short direct repeats flanking 43% of gained introns in Daphnia. These numbers must be compared to

11312-526: The splicing reactions are initiated. In addition, they contain a branch point, a particular nucleotide sequence near the 3' end of the intron that becomes covalently linked to the 5' end of the intron during the splicing process, generating a branched ( lariat ) intron. Apart from these three short conserved elements, nuclear pre-mRNA intron sequences are highly variable. Nuclear pre-mRNA introns are often much longer than their surrounding exons. Transfer RNA introns that depend upon proteins for removal occur at

11424-431: The substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate(s). In the case of a single substrate, the substrate bonds with the enzyme active site , and an enzyme-substrate complex is formed. The substrate is transformed into one or more products , which are then released from the active site. The active site is then free to accept another substrate molecule. In

11536-460: The term does not distinguish between real, biologically relevant, alternative splicing and processing noise due to splicing errors. One of the central issues in the field of alternative splicing is working out the differences between these two possibilities. Many scientists have argued that the null hypothesis should be splicing noise, putting the burden of proof on those who claim biologically relevant alternative splicing. According to those scientists,

11648-446: The time. The repressor protein is always expressed, but the lac operon (i.e. enzymes and transport proteins) are almost completely repressed, allowing for a small level of background expression. If this weren't the case, there would be no lacY transporter protein in the cellular membrane; consequently, the lac operon would not be able to detect the presence of lactose. When lactose is available but not glucose, then some lactose enters

11760-460: The two copies of the lac genes and show that the unregulated structural gene(s) is(are) the one(s) next to the mutant operator (panel (g). For example, suppose that one copy is marked by a mutation inactivating lacZ so that it can only produce the LacY protein, while the second copy carries a mutation affecting lacY and can only produce LacZ. In this version, only the copy of the lac operon that

11872-407: The whole story, because repressor is bound quite stably to DNA, yet it is released rapidly by addition of inducer. Therefore, it seems clear that an inducer can also bind to the repressor when the repressor is already bound to DNA. It is still not entirely known what the exact mechanism of binding is. Non-specific binding of the repressor to DNA plays a crucial role in the repression and induction of

11984-436: The word substrate can be used to refer to the sample itself, rather than the solid support on which it is placed. Various spectroscopic techniques also require samples to be mounted on substrates, such as powder diffraction . This type of diffraction, which involves directing high-powered X-rays at powder samples to deduce crystal structures, is often performed with an amorphous substrate such that it does not interfere with

12096-448: Was improved by association with stabilizing proteins to form the primitive spliceosome. Early studies of genomic DNA sequences from a wide range of organisms show that the intron-exon structure of homologous genes in different organisms can vary widely. More recent studies of entire eukaryotic genomes have now shown that the lengths and density (introns/gene) of introns varies considerably between related species. For example, while

12208-401: Was not metabolized during the first part of the diauxic growth curve because β-galactosidase was not made when both glucose and lactose were present in the medium. Monod named this phenomenon diauxie . Monod then focused his attention on the induction of β-galactosidase formation that occurred when lactose was the sole sugar in the culture medium. A conceptual breakthrough of Jacob and Monod

12320-472: Was testing the effects of combinations of sugars as nutrient sources for E. coli and B. subtilis . Monod was following up on similar studies that had been conducted by other scientists with bacteria and yeast. He found that bacteria grown with two different sugars often displayed two phases of growth. For example, if glucose and lactose were both provided, glucose was metabolized first (growth phase I, see Figure 2) and then lactose (growth phase II). Lactose

12432-583: Was the common laboratory bacterium, E. coli , but many of the basic regulatory concepts that were discovered by Jacob and Monod are fundamental to cellular regulation in all organisms. The key idea is that proteins are not synthesized when they are not needed— E. coli conserves cellular resources and energy by not making the three Lac proteins when there is no need to metabolize lactose, such as when other sugars like glucose are available. The following section discusses how E. coli controls certain genes in response to metabolic needs. During World War II , Monod

12544-449: Was to recognize the distinction between regulatory substances and sites where they act to change gene expression. A former soldier, Jacob used the analogy of a bomber that would release its lethal cargo upon receipt of a special radio transmission or signal. A working system requires both a ground transmitter and a receiver in the airplane. Now, suppose that the usual transmitter is broken. This system can be made to work by introduction of

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