1. In order to better understand how the Trp operon functions you do a series of experiments that involve introducing a copy of the Trp operon into an E. coli cell that already contains a Trp operon in its own DNA. In each of the following scenarios, explain how the normal control of the Trp operon will be affected (i.e. will genes be turned on and off in the same way? will they be on all the time? off all the time? etc.), and then explain what effect the addition of the extrachromosomal version of the gene will have. (4 points each answer)
If there is no functional repressor the operon will always be on - independent of Trp levels. When the extra operon is added there will be normal regulation because there will be a functional repressor again.
Since there is an operator mutation as described, the operon will always be on because repressor can never bind. Adding a normal operon will not have any affect on the regulation of the "genomic" operon because the problem lies in the operator DNA sequence, which is incapable of binding with a repressor. Thus, the genomic operon will always be on.
This mutation will prevent the synthesis of the TrpC product. Therefore, this cell will be incapable of making tryptophan and no repressor-Trp complex can be formed to turn the operon off - it will always be on. Addition of the extrachromosomal operon will restore normal behavior because normal TrpC can be made and therefore normal control will be possible.
Any two of the following is OK (other answers may be possible):
1) In T7 phage, switching from early to late genes is accomplished by synthesizing a new T7 RNA polymerase that recognizes a new promoter.
2) In T4 phage, the switch from early to middle and from middle to late operons is brought about by synthesizing new "sigma" factors that provide the core RNA polymerase with the ability to recognize new promoter sequences.
3) In lambda phage, the switch from immediate early to delayed early is made by interfering with the rho dependent termination site... thereby allowing genes downstream of the rho-dependent termination site to be transcribed.
3. Name the three known eukaryotic RNA polymerases and describe what genes they each are responsible for transcribing. ( 6 points )
RNA polymerase I - responsible for synthesizing ribosomal RNAs
RNA polymerase II - responsible for synthesizing mRNAs (pre-mRNAs)
RNA polymerase III - responsible for synthesizing tRNAs
4. RNA polymerase II does not seem to recognize a specific termination signal, yet the RNA that it produces from a given gene is always the same length. Explain how this is accomplished. ( 3 points )
Even though the RNA polymerase continues to synthesize RNA, the RNA itself is recognized by enzymes that will cut the RNA at a specific location. This will produce an RNA of a defined length.
Enhancers can bind transcription factors (TF). The enhancer-TF complex can then "bend" over to contact TFs that are bound to a promoter sequence. This larger complex of TFs and DNA acts as a strong binding signal to RNA polymerase, thereby stimulating the rate of transcription initiation (i.e. - the level of transcription is higher)
6. After an RNA is made from a eukaryotic gene in there are three major post-synthesis processing steps that must occur. Describe each of these steps. ( 6 points )
capping - places a unique "cap" structure on the 5' end of the RNA
splicing - removes introns from the precursor RNA to produce the final "product" RNA
polyadenylation - places a string of adenosines at the 3' end of the RNA - a poly(A) tail
7. How can a mutation in an intron affect the level of product that is made from that gene? ( 3 points )
If the mutation is at a splice junction sequence (or at a branch point) then the pre-mRNA will be spliced incorrectly and not code for or produce a normal protein.
8. Explain one theory of how multi-exon genes are thought to have evolved from simpler single exon "mini-genes". ( 5 points )
The primitive genes coded for small proteins, each of which performed a specific job, such as binding DNA. When these primitive cells developed the ability to splice RNA, large transcripts that spanned more than one of these genes could be spliced to produce a mRNA that contained more than one function and could be of benefit to the cell. Thus, if a gene were duplicated and translocated to another region of the DNA, it might be able to combine with a neighboring gene to produce a novel product. This rearrangement could be approximate because the splicing would remove the intervening RNA. On the other hand, if rearrangement at the DNA level was required to produce the "new" gene, then the DNA translocation would have to be exact (same reading frame). If it was not beneficial, the cell would have lost the ability to use that translocated sequence. By "experimenting" at the RNA level with splicing, the genome is not being permanently altered/damaged. [NOTE: this much detail is not needed]
This family of genes consists of many genes that are of similar sequence. By comparing the organization of these genes in different species, we can piece together how they might have evolved. Initially, in primitive organisms, there was a single globin gene. This single gene might have been similar to the myoglobin gene we now have in muscle tissue. During evolution, this gene was duplicated and the two copies diverged (perhaps forming alpha and beta globin genes). Later, these two genes were duplicated to form clusters of beta-like and alpha-like genes. It is not clear how these animal genes are related to the leghemoglobin gene in plants which has an extra intron, but the two forms (animal and plant) must have diverged early on.
10. Explain how a transducing phage (like Lambda) can carry host DNA from one host to another. ( 4 points )
The phage can integrate into the host genome. When it is excised at a later time, it might accidently take some of the neighboring DNA and package it into the phage. This new phage then can attack a different cell and deliver the original host DNA into the new target cell.
A phage plaque is the result of placing a single phage on a lawn of bacteria. The phage will kill a bacterial cell to which it is attached and release new progeny phage. These new phage will attack and lyse nearby bacteria. The process will continue and will result in a clear area in the bacterial lawn called a plaque.
12. Explain how restriction enzymes enable the assembly of DNA from different sources into a new recombinant DNAs. ( 4 points )
When a restriction enzyme cuts a DNA it can leave an overhanging cut - with one strand being longer than the other at the cut end of the DNA. Since the enzyme will always leave the same overhang (it recognizes a specific site), these "sticky" ends will always be complementary no matter what the source of the DNA. Therefore, the ends will be capable of base-pairing and reassembling into a new recombinant DNA molecule.
A genomic library contains sequences derived directly from the genome - introns, exons, upstream sequences and sequences between genes. A cDNA library represents a mRNA population from a specific cell population. It contains only those sequences that are actually found in mRNAs. It does not contain any upstream sequences, intron sequences, or sequences from between genes.
An expression vector is a vector which contains a promoter sequence upstream of the insertion point for foreign DNA. This enables the inserted DNA to be transcribed when the promoter is turned on. Expression vectors are used to make products from cDNAs that are inserted into the vector. The products can be used for commercial purposes or for screening cDNA libraries by looking for protein products.
15. a. Explain what is mean by chromosome walking. ( 4 points )
Chromosome walking involves using a piece of genomic DNA of interest to screen a phage library for adjacent fragments of DNA. This is accomplished by using a fragment of the probe DNA (e.g. the 3' end) to screen the library for overlapping phage clones. These new clones are then used in the same manner to screen the library for additional adjacent fragments of DNA.
b. In some genomes, there are sequences that are repeated many times throughout the DNA. How would the presence of all these repeated sequence impact the process of chromosome walking? ( 4 points )
These repeated sequences could mess up chromosome walking because if the library were probed with a repeated sequence (say from the 3' end of a phage insert), that sequence would not only hybridize to the appropriate adjacent sequence, but would bind to other locations in the genome that contained that sequence as well.
RFLP markers represent specific locations in the genome, although they might not reside in genes. A certain RFLP marker was found to always be associated with patients having cystic fibrosis, and was therefore nearby on the chromosome. By chromosome walking from the RFLP location in both directions and screening the adjacent DNA, it was possible to identify a specific gene that was always identified with cystic fibrosis when it contained a mutation.
A reporter gene is used to measure the level of activity from a specific promoter. Reporter genes make some easily measurable product, such as Green Fluorescent Protein - GFP, that is placed downstream of the promoter being tested. The level of transcription and subsequent translation of the mRNA (as measured by GFP in this case) will represent the level of activity of the promoter driving the synthesis of this RNA.
To study the level of tissue specific expression, the reporter gene with its promoter being tested is injected into an egg cell. As the organism develops, the reporter gene is replicated in every cell. Thus, by looking for fluorescence during development or even in the mature organism, one could identify the tissues in which that promoter is active.
Oncogenes are genes that will cause cancer by being inappropriately activated. This is a dominant effect so that just turning on the single gene copy can cause cancer. Tumor suppressor genes are genes that normally make a product that inhibits (suppresses) the activation of oncogenes. Tumor suppressor genes can be mutated and can result in the expression of the product they were inhibiting. However, since blocking a single suppressor gene will not block its corresponding `sister" gene this mutation is usually a recessive mutation that necessitates two independent hits in order for the effect to be observed.
18 b. Why are tumor suppressor genes more likely (than oncogenes) to be involved in inherited predispositions to cancer? ( 2 points )
Oncogenes by their very definition are dominant. Therefore, anyone carrying a dominant and expressed oncogene will get cancer and perhaps not survive. Tumor suppressor genes are recessive and therefore can be inherited from an otherwise healthy parent.