We have transformed cells with an aberrant PCR product which happens to be a circular piece of DNA (plasmid) but we are not sure if the promotor for that gene was included in that circular product. Yet, our gene seems to have been expressed by the successfully transformed cells. Is this possible, if no promoters were on the circular PCR product? Thank you. ANSWER ASAP, pls.
138792
You're trying to say you have a synthetic chromosome. I would check all the experiments for contamination, and check the cells for endogenous expression before jumping to that conclusion. There are a lot of hurdles to leap, as mentioned in the other posts.
295140
Yes, you can use it for over expression.  Make sure your plasmid has the proper self cloning sequences, one plasmid per cell won't do you much good.  \n\nPrecautions?  \n\nMake sure your gene is pure, as in not having anything that might ruin your experiment. Test culture your target bacterium first to weed out the non functionals and then cultivate the good ones, the ones that produce your targe protein.  You might need to assay for this stuff though, unless there's one of the specialized agars that will detect your target protein.
56304
http://universe-review.ca/I11-21-rRNA1.jpg\nhttp://dna01.bse.kyutech.ac.jp/jouhou/image/dna-protein/all/small_N1qa6.gif\nhttp://dna01.bse.kyutech.ac.jp/jouhou/image/dna-protein/all/small_N1i6u.png
173090
i dont think you'll be able to access this without logging in to my school, so i'll paste it below in case. try publications at your school as well, pubmed is always good, as ia google.\n\nTransgenics\nThe production of transgenic mice is a gain-of-function approach to manipulation of the mouse genome (Clarke, 2002; Nagy et al., 2003; Hedrich, 2004). Transgenic mice can be designed for numerous purposes including: (1) identification and characterization of specific regulatory sequences; (2) ubiquitous or tissue-specific overexpression of a transgene; and (3) cell lineage ablation achieved by coupling a tissue-specific promoter to a sequence encoding a toxin which kills cells that express it. Transgenic mice are typically generated by microinjection of the transgene construct into the male pronucleus of fertilized oocytes, which are then reimplanted into the oviducts of foster females. Expression of the transgene is dependent on its site of integration, with individual insertion sites typically containing multiple copies of the transgene. It is important to keep in mind that transgene copy number and expression levels of the transgene do not necessarily correlate. When chromosomal integration occurs in the fertilized oocyte, the developing animal will be fully transgenic, since all nucleated cells in the animal will contain the transgene; however, if integration occurs later, at a postzygotic stage, the animal will be a mosaic, with some cells containing the transgene and others lacking it. Founders are identified through analysis of tail biopsies, and copy number and transgene integrity are then evaluated. To establish a transgenic line, a founder is bred to mice of a defined genetic background and offspring are characterized for expression of the transgene. Generally at least two founders should be maintained for experiments. Due to the nature of random integration, no two transgenic lines will be identical. Details on the identification and analysis of founders and the establishment of transgenic lines can be found in UNIT 23.9.\n\nTransgenics carrying large segments flanking a gene of interest isolated as bacterial artificial chromosomes (BACs) have been used to minimize the abovementioned variation due to the effects of different integration sites (UNIT 23.1). The versatility of this strategy has been significantly increased by the development of homologous recombination–based strategies to mutagenize and manipulate the BAC constructs (UNITS 1.16 & 23.11) and the use of regulated transgenics (e.g., with tetracycline; UNIT 23.12). In the GENSAT project (http://www.ncbi.nlm.nih.gov/projects/gensat; Gong et al., 2003), for example, an atlas of gene expression in the mouse central nervous system is based on large numbers of BAC transgenics in which the protein-coding sequences of genes expressed in the brain are replaced by an EGFP reporter.
658984
tetracyclin inhibits translation, i don't think it's able to penetrate eukaryotic cells, because it would inhibit translation in them as well.
195113
One way that transformation can be helpful for bacteria is that this is a main way for the bacterium to gain genes for antibiotic resistance, environmental stress resistance (like heat or pH), or an advantage against the host's immune system.  Think of it as a big "fruit basket turnover" where one bacterium's DNA (along with its advantages) can be trasferred to another bacterium simply by the cells making contact.\n\nAnother example is that scientists use transformation as a tool in molecular biology.  We can put our DNA of interest into a bacterial cell (usually E. coli) and basically make it do whatever we want it to do.  It's a pretty handy trick, actually.
484966
Yes. DNA is a polynucleotide, and the bases referred to are actually the paired up ATCG. I think the person who answered this question thought that you were referring to the sides of the molecule, i.e. the ribose-sugar backbone, also comonly called the 'rails'.
524972
The DNA has to be wrapped up very tightly to fit all of it into the nucleus.  This is a complex process involving chromatin (DNA and associated proteins).  For example, histones are groups of protein molecules that the DNA strand wraps around to help compact its shape.  For a gene to be expressed (transcribed) a section of the the DNA has to become 'unwrapped' and 'exposed' to the transcription machinery.  Methylation and Acetylation of histones play significant roles in this process.\n\nAny good, recent, biochemistry textbook will explain these issues.  If you go to pubmed, they have an online book section.  Click on any of the biochemistry books and enter into the search field "DNA, chromatin", or "DNA, histones", or "gene expression, chromatin" and you will find lots of info. and probably pictures too.\n\nBest wishes and good luck.
392802
I'm almost 100% sure that the answer is "It ensures that the DNA will be replicated for new cells"\n\nWhat the question is talking about is the way in which the bases pair together.  I'm sure you've been taught that the bases pair as follows:  A-T and C-G.  When DNA is replicated, it is "unzipped" leaving two strands of DNA that each have bases that aren't paired.  In order to make 2 new complete strands, the correct bases must be added to each of these half-complete strands.  This is where the question comes into play.  If the bases could pair in any way possible the new strands would not be identical to the original.  This is why it is critical that A only pairs with T and C only pairs with G.\n\nI hope this helps.  Questions like this really test your knowledge of the material.  It is one thing to know A-T and C-G.  It is another to know why these combinations are critical in the process of replicating DNA.
445314
To be honest with you i don't quite understand your question, it is often that restriction enzymes are used to create genomic library for which you look into the overlapping sequence and then be able to determine which fragment is next to which. If that is what you have done, you should have used the fragmented DNA sequence to determine the orginall DNA sequence that you digested it with the enzyme, to confirm the sequence you ahve found is correct, you can use PCR, you got the hypothetical sequence, thus by making primers from the start and the end of the sequence (doesn't matter which way round) you should have amplify the same sequence which you started with, you got the amplified DNA (same as the orginal DNA because you used primers that complemented the very start and the very end of the sequence which you found out after the restruction enzyme mapping) run it on gel electrophoresis with a DNA size marker that will include the size of DNA you have sequenced, if the amplified DNA size is the same as the sequence you have found from restriction mapping, that means your primers did complemented from the start and the end, that means the sequence you thought is actually the correct sequnece.\nI am sorry, i don't think i am making this clear enough, but anyway, that's the best i can think of.
145930
This is dependent on the type of organism, the stage in the lifecycle of the organism, and a wide variety of environmental factors such as temperature, availability of nutrients, presence of toxic compounds, and organism-specific biological signalling compounds which play a role in regulating gene expression.\n\nTo take a simple organism such as a bacterium, it is a reasonable assertion that levels of gene expression are highest under conditions that are optimal for reproduction (optimal temperature and moisture conditions, high availability of nutrients, low concentrations of toxic compounds).  This is because during asexual reproduction, proteins are required both as building materials for replication (e.g. cell wall constituents, structural proteins, enzymes required for metabolism etc.) and for the actual process of replication (proteins involved in DNA replication and protein synthesis etc.).\n\nOf course, without knowing the specific organism you are referring to it is not possible to answer your question except in broad terms.  You might try looking at a text book such "Biological Science" by R.Soper (Cambridge University Press).
192119
Each half of the double-helix sort-of "unzips" during replication.  Since each of the bases is kind of like a jigsaw-puzzle piece, the two strands can then wind back up, if supplied with the appropriate free bases, to form two identical copies of the original DNA strand.\n\nNeat, huh?
276821
I gave a lecture on this recently and have had first hand experience with this technology in a lab, so let me give you a brief and easy summary to take with you about this topic about Recombinant DNA Technology:\n\nIn order to clone you need 2 things: Bacterial DNA (the circular form called a "plasmid"), and the DNA you want to clone (be it human, mouse, plant, other species etc)\n\nThen you will need to cut the Bacterial DNA with enzymes called "endonucleases" that work like scissors. (So imagine a circle and I take a pair of scissors and cut the circle at some point in it, b/c I want to insert the foreign DNA (non native DNA) where I had cut.) I will have to do the same with the foreign DNA and have endonucleases cut OUT the particular piece i want cloned. \n\nThen we can put the two pieces together, the cut circular plasmid DNA from the bacteria, and the CUT out piece of foreign DNA. \n\nWhen we put the two pieces together it is called "ligation", and it is done as a reaction in a tube with enzymes inside that "glue" the "sticky ends" of the bacteria DNA to the foreign DNA. \n\nAfter the reaction normally done in a 16 deg. Celsius water bath, we now will have "circular" plasmids with our Foreign DNA inserted into the bacterial DNA (the circle DNA)\n\nThen we would re-insert this DNA back into the bacterial host (that the circle DNA first came out of) in a process called "transformation", this is where the broth from the water bath we just discussed is grown in a growth medium at 37 deg. celsius so that the bacteria can grow and produce MORE of those circle plasmids with our foreign DNA inserted into it. \n\nThen we would "plate" these onto agar plates, and let them grow in incubators at 37 deg. Celsius over night until we get small colonies of bacteria on the plate. \n\nEach "mound" of bacteria (small like white dots on the plate) contains millions of copies of our gene (inserted in the circular plasmid DNA of the bacteria). \n\nNow we can take these small colonies, and harvest them further, by putting them in large Erlenmeyer flasks to be grown in the same "growth broth" over night on a shaker for 37 deg. celsius. \n\nAfter of which we can harvest, and purify that culture so that only the circular plasmids remain (this is a long and convuluted process so we won't go into it in detail here), and from there you can store those in a 4 deg. Celsius fridge and keep it in stock for further research purposes. \n\nHope that helps!
180168
I think you miscopied the third item.  You have copied one of the first helf cells, not given a reaction.
458335
I don't know exactly what are you asking for, but if you asked it two days ago anf anybody answers you I'm not the only person that don't know at all what we've to answer.\n\nI'm going to try, and it will be difficult to me because I'm from Barcelona and my english isn't very well at all.\n\nPolymerase does the works to incorporate ribonucleotides in the new RNA. The direction of the transciption is 5'->3' . Primase is the protein that begins the replication synthesing RNA primer, but in the transcription is different. RNA polymerase knows the beginnig of the transcription because of the promoter. In the DNA there is a promoter and when the RNA polymerase recognise the promoter (thanks of its sigma factor) it begins the transcription.\n\n1) formation of a closed-promoter complex.\n2) formation of a open-promoter complex.\n3) initiation of mRNA synthesis, almost always with a purine\n4) elongation of mRNA by about 8 more nucleotides\n5) release of sigma as polymerase proceeds down the template.\n\nThe polymerase ends the transcription when the rho factor recognise the ending of the transcription.\n\nIt is the answer of your question??\n\nsorry for my english!!
271058
I think this is done by alternative sigma factors. Sigma factors from a complex with RNA polymerase. Different sigma factors recognize different promoter sequences, so regulating a particular sigma factor will regulate all of the genes that are controlled by that sigma factor.
623281
Your question isn't easy to read, but i think i understand it enough. There is one mole of 1kbp DNA strand with an EcoR1 restriction site in the middle. The amount of 5'ends produced when you addd EcoR1 to the DNA will be double because you will be effectively cutting the DNA in half. I hope this is what you were asking