In general, the best preparation for the exam is to review your homework assignments and lecture notes. You should review my lecture notes posted on this website, paying special attention to topics highlighted below.
The exam covers the following lectures:
Here are some important highlights from these lectures.
We discussed three mechanisms for moving DNA from one bacterium to another:
We didn't spend much time on transformation.
The F factor carries genes that cause F+ cells to be capable of mating. F+ cells have pili and a special system for replicative transfer of the F plasmid. During transfer, the F plasmid is nicked in a specific location and transfers to the recipient cell by rolling circle replication, which transfers a single-stranded copy of the F plasmid to the recipient. The recipient cell completes replication of the F factor by synthesizing the other strand and circularizing the linearized plasmid.
An F factor can integrate into the donor chromosome to create an Hfr cell. The Hfr will initiate transfer of the entire donor chromosome to the host cell. It takes about 90 minutes to transfer the entire chromosome, so most matings do not go to completion. The first part of the F factor transfers first. The last sequence to be transferred, after the entire donor chromosome, is the other part of the F factor. For this reason, matings with Hfr strains typically do not transfer fertility. Large regions of the donor chromosome are transferred in Hfr matings. These undergo recombination with the recipient chromosome to result in exconjugants that have donor genes.
In generalized transduction, we grow a generalized transducing phage on a donor strain. A small fraction (1/10,000) of phage particles are defective, containing only bacterial DNA but no phage DNA. When we infect a recipient strain at a low multiplicity of infection, so that most cells get only one phage particle, some of the recipient cells get a piece of donor DNA instead of a phage genome. We can recover bacterial cells that have picked up selectable markers from the donor genome.
We studied two different operons in E. coli to understand prokaryotic gene regulation: lac and trp. In both of these operons, multiple genes are transcribed as a single transcript.
The lac operon has three genes under coordinate control, but we concentrated on two of them, lacZ, which encodes β-galactosidase, the enzyme that cleaves lactose to galactose and glucose, and lacY, a permease that transports lactose into the cell. The lac operon is regulated by two proteins:
The lac operon therefore offers an example of both negative regulation, where a protein bound to DNA blocks transcription (the lac repressor), and positive regulation, where a protein bound to DNA increases transcription (CAP protein bound to cAMP).
It is very important to be able to work problems in which you are given the genotype of a bacterial cell, which may be a partial diploid, and asked to determine whether particular enzymes are inducible (expressed when inducer is present but not expressed in the absence of inducer), constitutive (expressed both in the presence and absence of inducer), or uninducible (not expressed in the presence or absence of the inducer). Please see the practice problems.
In order to work these problems, you need to understand all the players:
lacI. This gene encodes the lac repressor, which operates in trans, meaning that a functional lacI gene on the F' plasmid can regulate a normal lac operon on the bacterial chromosome. There are three kinds of alleles of lacI that matter:
lacP. This is the promoter for the lac operon, the site of binding of RNA polymerase. For our purposes, there are two alleles, P+, which works normally, and P-, which doesn't work at all. All genes downstream of a broken promoter (P-) will not be transcribed under any circumstances, because RNA polymerase cannot bind. Promoter mutations act only in cis.
lacO. This is the operator for the lac operon, the site of binding of the lac repressor. There are two kinds of alleles: O+, which works normally, and OC, the operator-constitutive alleles. The OC alleles fail to bind repressor, even superrepressor, so all genes downstream of an OC allele will be expressed constitutively. Operator mutations act only in cis.
lacZ. This is the structural gene for β-galactosidase. There are two kinds of alleles: lacZ+, which makes a normal enzyme, and lacZ-, which makes a protein product with no enzyme activity.
lacY. This is the structural gene for lactose permease. There are two kinds of alleles: lacY+, which makes a normal protein, and lacY-, which makes a protein product with no permease activity.
The trp operon encodes five enzymes needed for tryptophan biosynthesis. There is a trp repressor, actually an aporepressor, that negatively regulates the trp operon by binding to an operator sequence. The trp aporepressor binds the operator when it is bound to tryptophan, so in the presence of high concentrations of tryptophan, the operon is repressed. When tryptophan concentrations are low, the repressor does not bind, and the trp operon is expressed.
In addition, the E. coli trp operon is regulated by another mechanism called attenuation. Attenuation is a mechanism that causes the termination of transcription of the trp operon before the coding sequence of the first biosynthetic enzyme is reached. Downstream of the promoter and operator is a leader sequence that encodes a short peptide containing two tryptophan codons. Downstream of the leader is a spacer that contains sequences that can form a stem-loop structure to terminate transcription. Formation of the transcription-terminating stem-loop is inhibited when the concentration of charged tryptophan tRNA (tRNATrp) is low. During translation of the leader mRNA, if the concentration of tryptophan tRNA is low, the ribosome will stall. The presence of the stalled ribosome will cause an alternative stem-loop structure to form, blocking the formation of the transcription-terminating stem-loop and allowing transcription of the operon. Please see the explanation with figures in the lecture notes.
We began our study of eukaryotic gene regulation by noting that eukaryotic DNA is packed into chromatin that is not accessible to transcription in the absence of positive regulation. We can broadly define two classes of chromatin:
Most eukaryotic chromosomes have large segments of heterochromatin near the centromere. In addition, it is possible for portions of the genome that are ordinarily euchromatic to become heterochromatic, for example, one of the mammalian X chromosomes in female mammals, or parts of euchromatin that are undergoing position-effect variegation in Drosophila.
The basic unit of chromatin is the nucleosome. A nucleosome consists of an octamer of two molecules of each of four histones: H2A, H2B, H3, and H4. The histone octamer has two turns of DNA wound around the outside, and is associated with a single molecule of histone H1.
Some Drosophila cells, particularly those of the larval salivary gland, have polytene chromosomes. Polytene chromosomes form by repeated rounds of replication of chromosomal DNA, until the euchromatin of each chromosome arm is replicated to about 1,000 copies. The heterochromatin does not replicate to the same extent. The centric heterochromatin of all of the chromosomes forms an aggregate called the chromocenter from which the chromosome arms emerge.
Polytene chromosomes are useful for studying the dosage compensation system in Drosophila. The basis of dosage compensation is hypertranscription of the X chromosome in males. The genes required for dosage compensation were discovered through the identification of male-specific recessive lethals, mutations that kill males but not females. The protein products of five genes form a complex that associates with the male X chromosome, acetylating a histone at a particular position to make the X chromosome more accessible to transcription. Immunofluorescent staining of polytene chromosomes in male larvae using antibodies to these proteins shows the association of the dosage compensation complex with the male X chromosome.
There are also mutations that transform the somatic sex of Drosophila. Recessive loss-of-function alleles of tra and tra2 transform XX females to sterile males. Sexually transformed XX males do not activate the male-specific dosage compensation system, so dosage compensation and somatic sex determination are at least partially separate processes.
We can discover mutations that activate the dosage compensation system in females by looking for female-specific recessive lethals, mutations that kill females but not males. Recessive alleles of Sxl, like Sxlf1, kill homozygous females but have no effect on males. The Sxl gene encodes an RNA-binding protein that affects splicing of three pre-mRNAs: Sxl, msl-2 (one of the male-specific lethals that is a component of the dosage compensation system), and tra. The SXL protein is present in females but not males. The effect of SXL protein on msl-2 RNA is to prevent its proper splicing and to interfere with its translation, so msl-2 is not expressed in females. None of the other four proteins that make up the dosage compensation system is regulated, but because the proteins work as a complex, regulation that prevents expression of a single component keeps dosage compensation off in females. The effect of SXL protein on tra is to promote a female-specific splice that makes an active gene product. There is no functional TRA protein in males. TRA protein is also an RNA-binding protein with two targets, dsx and fru, both transcription factors that function in somatic sex determination.
Cancer is a disease in which cells accumulate mutations in genes required for the proper control of cell division. When they have accumulated mutations in the right collection of genes to be relieved of normal growth control, they are tumor cells.
We can divide genes that contribute to cancer into two categories, tumor-suppressor genes and oncogenes. Cancer-causing alleles of tumor-suppressor genes are loss-of-function alleles, recessive at the cellular level. Cancer-causing alleles of oncogenes are gain-of-function alleles, contributing to tumor formation even in the presence of a normal copy of the gene.
There are over fifty different hereditary predispositions to cancer. Most of these behave in pedigrees as dominant risk alleles even though they are loss-of-function alleles of tumor suppressor genes. Examples include Hereditary Breast and Ovarian Cancer (BRCA1 and BRCA2), familial retinoblastoma (RB1), and Li-Fraumeni Syndrome (TP53).
Gain-of-function alleles of oncogenes occur by somatic mutation; mutations in specific oncogenes are common across many different tumor types. Activating mutations in RAS, a small GTPase, are common among many tumors.
There are many genes that function to carry out the processes of development in animals that have no role in the adult animal. Because the genetic analysis of development in Drosophila has been so extensive, we take most of our examples from Drosophila.
Much of early embryonic development in Drosophila is influenced by molecular gradients built into the egg by the mother. Genes expressed in the mother that are required for the proper development of the embryo are called maternal-effect genes. Females carrying mutations in these genes lay eggs that cannot develop normally regardless of the genotype of the zygote. These are also called coordinate genes because they establish the coordinates of the anterior-posterior and dorsal-ventral axes in Drosophila.
There are a number of genes that control development through their expression in the embryo. These zygotic genes interact with the initial asymmetry of the egg, and with each other, to produce more refined patterns.
Once the intial gradient of the egg has been refined into a pattern of segments, another set of genes determines segmental identity. Mutations in these genes produce striking transformations of one body part into another, and are called homeotic genes.