Genetics Ch 8

Cotransformation

Genes that are close enough to each other to be cotransformed are linked.

Genetists believe

that transfer of the F factor during conjugation involves separation of the two strands of its double helix and movement of one of the two strands into the recipient cell. Both strands, one moving across the conjugation tube and one remaining in the donor cell, are replicated. The result is that both the donor AND the recipient cells become F+. See page 164.

prototroph

A bacterium that can completely grow in a minimal medium without supplements.

lysogenic

A bacterium that harbors a prophage is said to be lysogenic, capable of being lysed.

F+ cells

F for fertility. When cells server as donors of parts of their chromosomes, they are designated as F+ cells

Genetic Recombination

For prokaryotes: leads to the replacement of one or more genes present in one strain with those from a genetically distinct strain. For eukaryotes: the term describes crossing over that results in reciprocal exchange of events from one chromosome to another, resulting in an altered genotype.

auxotroph

If a bacterium loses, through mutation, the ability to synthesize one or more organic components, it is called this. If a bacterium loses the ability to make histidine, then this amino acid must be added as a supplement to the minimal medium for growth to occur. The resulting bacterium is designated as a his- auxotroph, in contrast to its prototrophic his+ counterpart.

Later evidence with L and T

Later experiments by Leaderberg and Tatum and by Hayes and Cavalli-Sforza show taht certain conditions eliminate the F factor in otherwise fertile cells. However, if these "infertile" cells are then grown with fertile donor cells, the F factor is regained.

F- cells

Recipient bacteria receive the donor chromosome material (now known to be DNA), and recombine it with part of their own chromosome. They are designated as F- cells.

Conjugation

The genetic information from one bacterium is transferred to and recombined with that of another bacterium. Conjugation may be initiated by a bacterium housing a plasmid called the F factor in its cytoplasm, making it a donor (F+) cell. Following conjugation, the reciepient (F-) cells receives a copy of the F factor and is converted to the F+ status.

prophage

The viral DNA that integrates into the bacterial chromosome

temperate phages

Viruses that lyse the cell or behave as phrophages

virulent phages

Viruses that only lyse the cell

R plasmid

consists of two components: the resistance transfer factor (RTF) and one or more r-deteminants. The RTF encodes gen. info. essential to transferring the plasmid btwn bacteria, and the r-deteminants are genes that confer resistance to antibiotics or mercury. Sometimes several r-determinants occur in a single plasmid, conferring multiple resistance to several antibiotics.

Interrupted mating technique

demonstrated that specific genes of a given Hfr strain were transferred and recombined sooner than others. See Figure 8.6 page 164.

Col plasmid (dervied from E. coli - ColE1)

encodes one more more proteins that are highly toxic to bacterial strains that do not harbor the same plasmid. These proteins are called colicins, and can kill neighboring bacteria. Not usually transferable to other cells.

F factor

is an example of a plasmid

Leaderberg-Zinder experiment

led to the discovery of phage transduction in bacteria. After placing 2 auxotrophic strains on opposite sides of a Davis U-tube, Leaderberg and Zinder recovered prototrophs from the side with the LA-22 strain, but not from the side containing the LA-2 strain. Fig 8.16

lysogeny

occurs when: 1. the phage DNA integrates into the bacterial chromosome 2. its replicated along with the chromosome 3. its passed to daughter cells

Wollman and Jacob

postulated that the E. coli chromosome was circular. They demonstrated an ordered transfer of genes that correlated with the length of time conjugation proceeded.

adaptation hypothesis

this hypothesis put forth to explain this type of observation, implies that the interaction of the phage and bacterium is essential to the acquisition of immunity. The phage "induces" resistance in the bacteria.

Bacteriophages

viruses that use bacteria as their host

The F' State and Merozygotes -Adelberg Fig 8.10

Adelberg discovered that the F factor could lose its integrated status, causing the cell to revert to the F+ state (1). When this occurs, the F factor frequently carries several adjacent bacterial genes along with it (2). He labeled this condition as F' to distinguish it from F+ and Hfr. F' like Hfr, is thus another special case of F+, but this conversion is from Hfr to F'. The presence of bacterial genes within a cytoplasmic F factor creates an interesting situation. An F' bacterium behaves like an F+ cell by initiating conjugation with F- cells (3). When this occurs, the F factor, containing chromosomal genes, is transferred to the F- cell (4). As a result, whatever chromosomal genes are part of the F factor are now present as duplicates in the recipient cell (5) because the recipient still has a complete chromosome. This crates a partially diploid cells, merzoygote. Pure cultures of F' merzoygotes can be established. They have been extremely useful in the study of bacterial genetics, esp genetic regulation.

To summarize

An E. coli cell may or may not contain the F factor. When this factor is present, the cell is able to form a sex pilus and potentially serve as a donor of genetic information. During conjugation, a copy of the F factor is almost always transferred from the F+ cell to the F- recipient, converting the recipient to the F+ state.

Davis U-tube experiment

At the base of the tube is a sintered glass filter with a pore size that allows passage of the liquid medium but that is too small to allow the passage of bacteria. The F+ cells are placed on one side of the filter, and the F- cells on the other side. The medium is moved back and forth across the filter so that the cells share a common medium during bacteria incubation. When Davis plated the samples from both sides of the tube on minimal medium, no prototrophs were found, and he logically concluded that physical contact between cells of the two strains is essential to genetic recombination. We now know that this physical interaction is the initial step in the process of conjugation established by a structure called the F pilus (or sex pilus, pl. pili). Bacteria often have many pili, which are tubular extensions of the cell. After contact is initiated between mating pairs, chromosome transfer is then possible.

minimal medium

If the nutrient components of the growth medium are simple and consist only of an organic carbon source (such as glucose or lactose) and a variety of ions, including NA+, K+, Mg2+, Ca2+, and NH4+, present as inorganic salts, it is called this. Bacteria are grown in a liquid culture medium or in a petri dish on a semisolid agar surface. To grow on such a medium, a bacterium must be able to synthesize all essential organic compounds (e.g., amino acids, purines, pyrimidines, sugars, vitamins, and fatty acids). A bacterium that can accomplish this remarkable biosynthetic feat is a prototroph. It is said to be wild type for all growth requirements.

Transformation (another mechanism for recombination)

In bacteria, which does not require cell to cell contact, involves exogenous DNA that enters a recipient bacterium and recombines with the host's chromosome. Linkage mapping of closely aligned genes is possible during the analysis of transformation. In transformation, small pieces of extracellular DNA are taken up by a living bacterial cell and are integrated stably into the chromosome. The recombinant region contains one host strand (present originally) and one donor strand. These strands are from different sources, so this helical region is referred to as a heteroduplex. The two strands of DNA are not perfectly complementary in this region.

Leaderberg and Tatum's initial experiment

Initial experiments were performed with 2 multiple auxotrophic strains of E. coli K12. Strain A required methionine and biotin in order to grow, whereas strain B required threonine, leucine, and thiamine. Therefore, neither strain would grow on minimal medium. The 2 strains were first grown separetely in supplemented media, and cells from both were mixed and grown together for several more generations and then plated on minimal medium. Any bacterial cells that grew on minimal medium were prototrophs. It is highly improbable that any of the cells that contained 2 or 3 mutant genes underwent spontaneous mutation simultaneously at 2 or 3 independent locations, leading to wild-type cells. Therefore, the researchers assumed that any prototrophs recovered arose as a result of some form of genetic exchange and recombination between the 2 mutant strains. Leaderberg and Tatum proposed that genetic exchange had occured.

transduction and mapping

Like transformation, generalized transduction can be used in linkage and chromosomal mapping of the bacterial chromosome. Two closely aligned (linked) genes can be simultaneously transduced (cotransduction). - The closer linked genes are to each other, the greater the frequency of transduction. The precise order of genes can be determined.

Example: E. coli

Strains of E. coli are known to be sensitive to infection by the bacteriophage T1. Infection by this bacteriophage leads to the virus reproducing at the expense of the bacterial cell, from which new phages are released as the host cell is disrupted, or lysed. If a plate of E. coli is uniformly sprayed with T1, almost all cells are lysed. Rare E. coli cells are isolated and established in pure culture, all of the descendants are resistant to T1 infection.

Phage T4: Structure and Life Cycle (Fig. 8.13)

The life cycle is initiated when the virus binds by adsorption to the bacterial host cell. The tail contracts, the central core penetrates the cell wall, and the DNA moves into the host cytoplasm.- The bacterial DNA, RNA, and protein synthesis in host is inhibited. Host DNA degradation is initiated. Phage DNA replication occurs, then components of the head, tail, and tail fibers are synthesized. When approximately 200 new viruses have been constructed, the bacterial cell is ruptured by the enzyme lysozyme (a phage gene product) and mature phages are released from the host cell

Plaque assay

The number of phages produced following the infection of bacteria can be determined by the plaque assay. This technique entails performing serial dilutions of virally infected bacteria, which are then poured onto agar plates. By counting the number of plaques (areas clear of bacteria) on the plates, the number of phages in the original culture can be determined.

Transudction

Viral transfer of genetic material between bacteria (virions packaged with bacterial DNA) is the process by which DNA is transferred from one bacterium to another by a virus.[1] It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNase resistant (transformation is susceptible to DNase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome. Bacteriophages, which can themselves undergo genetic recombination, can be involved in a mode of bacterial genetic recombination called transduction.

specialized transduction

a small piece of bacterial DNA is package along with the phage DNA, and transfer is only strain-specific (not random). This (not diagrammed) involves excision of lysogenized phage from the bacterial genome. Sometimes, it excises in a faulty manner, taking a little bit of the adjacent bacterial chromosome with it, so any strain, if it does this, always carries ONLY the bacterial DNA adjacent to the phage insertion site in the bacterial genome (not random fragments of bacterial chromosome)

RecBCD protein

an enzyme consisting of polypeptide subunits encoded by 3 other rec genes. Is important when double-stranded DNA serves as the source of genetic recombination. RecBCD unwinds the helix, facilitating recombination that involves RecA. These discoveries extended our knowledge on the process of recombination, value of isolating mutations, establishing phenotypes, determining normal, wild type genes.

Plasmids

are double-stranded closed circle of DNA (dsDNA). Contain one or more genes and often, quite a few. Their replication depends on the same enzymes that replicate the chromosome of the host cell, and they are distributed to daughter cells along with the host chromosome during cell division. Exist in multiple copies in the cytoplasm. Replicate independently of the bacterial chromosome. Classified according to the genetic information specified by their DNA.

Prokaryotes

bacteria

The Nature of Transduction

bacterial DNA is randomly package in a phage particle and transferred to a recipient host.

Abortive transduction

bacterial DNA is transmitted to one progeny cell following each division

complete transduction

bacterial DNA recombines with its homologous region of the bacterial chromosome AND passes to all daughter cells.

High-frequency recombination (Hfr)

behave as donors, they're a special class of F+ cells. HFr strains allow genetic mapping. F+ x F- = F+ (low rate of recombination) Hfr x F- = F- (higher rate of recombination)

F factor plasmid

confers fertility and contains genes essential for sex pilus formation, on which genetic recombinatino depends

single strand displacement

is a common form of recombination in many bacterial species. when double-stranded DNA enters a recipient cell, one strand is often degraded, leaving the complementary strand as the only source of recombination. this strand must find its homolgous region along the host chromosome, and once it does, RecA facilitates recombination.

The conclusion of the F factor

is a mobile element is further supported by the observation that, following conjugation and genetic recombination, recipient cells always become F+. Thus, in addition to the rare cases of gene transfer from the bacterial chromosome (genetic recombination), the F factor itself is passed to all recipient cells. On this basis, the initial cross of Leaderberg and Tatum is as follows: Strain A Strain B F+ x F- Donor Recipient

Merozygotes

is a state when a cell, usually bacterial, is temporarily partial diploid as result of DNA transfer processes like conjugation. One example of how merozygotes can be formed is the transfer of genetic material of an Hfr cell to an F- cell through conjugation.

episome

is a viral DNA that can be integrated into the host DNA or replicated in the bacterial cytoplasm.

Rec Proteins Are Essential to Bacterial Recombination

researchers looked for and subsequently isolated several functional gene products present in normal cells but missing in rec mutant cells and showed that they played a role in gen. recomb.

Genetic map

shows that the chromosome of the Hfr bacterium was transferred linearly and that the gene order and distance between genes, as measured in minutes, could be predicted from such experiments, see Fig. 8.7. This info served as the basis for the first genetic map of the E. coli chromosome. Minutes in bacterial mapping are equivalent to map units in eukaryotes.

RecA protein

this protein plays in an important role involving either a single-stranded DNA molecule or the linear end of a double-stranded DNA molecule that has unwound.

lysed

undergo or cause to undergo lysis

point of origin (O)

varies from strain to strain.