This is old Lecture 13
Textbook Assignment: Chapter 6, pages 168-179; Plus additional information in these notes that is not included in the textbook.
Major concepts
Introduction: This lecture and the one that follows mark the transition in our course from classical Mendelian genetics to modern molecular genetics. Studies on the genetic fine structure of bacteriophage (this lecture) provided a detailed understanding of the phenomenon of complementation and the linear nature of genetic information, and studies on biochemical complementation in Neurospora (next lecture) provided much of our early understanding of the "one gene-one enzyme" theory, which has become a fundamental part of our current understanding the nature of genetic information. The subject material for these two lectures has been pulled together from two different parts of the textbook in an attempt to provide a smoother transition between classical and molecular genetics.
Growth of bacteriophage : Bacteriophages are bacterial viruses. The bacteriophage particle consists of a hollow head packed with DNA, a tail structure, which is a hollow tube used to inject the DNA into the bacterial cell, and tail fibers, which are involved in attachment to the bacterial cell (Fig. 6.15). Bacteriophage inject their DNA into bacteria such as E. coli. Host transcriptional and translational machinery is used to produce new bacteriophage proteins (often with changes in transcriptional specificity for expression of "late" viral genes). The bacteriophage DNA is replicated to 100 fold or more. New bacteriophage particles form inside the bacterial cell, and the products of late-acting genes lyse the host cell, giving rise to a burst of new bacteriophage particles. When single bacteriophage are plated on a lawn of bacterial cells on an agar plate, the first burst initiates a second round of infection, leading to more bursts and a growing circle of bacterial lysis, which is known as a plaque.
Bacteriophage genetics: The size and shape of plaques and the strains of bacteria that will support multiplication of particular types of bacteriophage can be used as markers for genetic studies. Through use of a high multiplicity of infection (far more bacteirophage than there are bacteria), it is possible to infect each bacterial cell with several bacteriophage particles. Interaction of the bacteriophage genomes within the individual host cells makes it possible to undertake a variety of genetic studies, including recombination and complementation.
Fine structure mapping: After the discovery by several investigators in the late 1940's that recombination could occur when two different strains of bacteriophage invaded the same bacterial cell, Seymour Benzer undertook a series of studies designed to push recombination over short genetic distances to its limit. For these studies, he employed a mutant strain of bacteriophage T4 designated rII, which could form plaques on E. coli strain B, but not on strain K12. His goal was to look for recombination within a single gene, taking advantage of the ability to detect as few as one recombinant phage in 100 million.
Complementation: When certain pairs of rII strains were coinfected into bacteria, they were found to form plaques readily without the need for rare recombinational events. Benzer proposed that each strain was providing a function that the other lacked, such that by working together they could replicate under conditions where neither could replicate alone. He called this phenomenon complementation. His research provided evidence for the existence of two separate genetic functions in the rII region, which came to be known as rIIA and rIIB. Subsequent studies have shown that rIIA and rIIB are in fact two separate, but closely-linked genetic loci, each coding for a different protein product. A functional copy of each coding sequence is needed to support full function. There are also some special cases in which two defects in the same protein can complement each other by interacting to form a functional dimer. These relatively rare cases will be discussed later in this lecture.
Complementation in other organisms: The basic concept of complementation or complementary gene action has been known for a long time, and occurs in highly diverse organisms. At this point, these notes depart from the textbook to explore some of these other cases before returning to complementation in bacteriophage. Mutations are said to be complementary if the genomes (or partial genomes) that contain them support normal function when both are introduced into the same cell. The two separate genomes can be introduced in many different ways, including the maternal and paternal components of a heterozygous diploid genome, or a full haploid genome plus a partial genome in a bacterial merozygote (also sometimes called a merodiploid), or two different types of nuclei in a common cytoplasm in heterokaryons of Neurospora, or two different bacteriophage strains infecting the same bacterial cell or any other combination that allows the undamaged part of each genome to compensate for the damage that has occurred in the other one. The critical requirement is that the two mutations must not destroy precisely the same genetic function and that when combined in a diploid or diploid-like situation, each of the damaged genomes must be able to compensate for the losses suffered by the other.
Restricted to one phenotypic variable: If the definitions given above are taken literally, any two recessive mutations in a trans- relationship can be called complementary. However, in practice, the term "complementation" is usually only used to describe the interaction between mutations that influence the same general phenotypic variable. One example of this is the complementary gene action we saw earlier in the semester, in which knocking out either of two enzymes that acted sequentially in the synthesis of purple pigment in sweet peas resulted in a white flower. However, the F1 hybrid between these two mutant strains produced purple flowers, because each of the mutant strains introduced a wild type allele of the gene coding for the other enzyme. This is the essence of complementation: each mutant strain must be deficient in a sufficiently different function so that the two together can achieve full function without the need for genetic recombination to occur.
Limitations on complementation tests: There are several additional limitations to complementation tests. The mutant phenotypes that are being studied need to be recessive to wild type. A dominant gene will exert its effects irrespective of the overall genetic background it is in. In addition, the extent of recombination must be substantially less than the level of complementation that is observed so that it will be clear that the restoration of function occurred because the two mutations destroyed two different units of function, and not because recombination created a normal gene and a doubly-defective gene. Also, the gene products that are involved must be diffusible, such that they can move enough within the cytoplasm to interact in a way that will allow normal function. Thus, strictly cis-acting mutations (those whose effect is on the function of the DNA molecule that they are part of), such as mutations in promoters or operators, would not be expected to be capable of complementation.
Cis-trans test: As used here, the term cis refers to genetic changes that are on the same DNA molecule in simple organisms or in the same haploid genome in cases where there are mutiple chromosomes. The term trans refers to genetic changes that are carried by different genomes that have been introduced into the same cell (in any of the possible ways discussed above). The cis-trans test asks whether two mutations that occur in different genomes have altered the same unit of fuction (usually the coding sequence for a single protein). It is expected that when the two mutations are put together in the same genome (cis) that they will not support normal function, even if they occur in two separate functional units. Such a test is straightforward in haploid organisms. However, to see cis lack of function in a diploid organism, it is necessary to make the organism homozygous for the cis genome. Otherwise there will be a wild type genome opposite it that will support normal function. In most cases, the cis part of the cis-trans is an implied control that could be run, rather than an important part of the actual experimental study.
The trans test, where complementation is expected to occur if two different coding sequences are involved, requires the presence of two complete genomes, or at least two copies of the portions of the genomes where the mutations occur, so that a good copy of each coding unit will be present (in cases where the mutations involve two different coding units). Thus, in haploid organisms, it becomes necessary to find a way to introduce a second genome (merodiploids in bacteria, optional diploid phase in yeast, heterokaryons in Neurospora, mixed infection of bacteriophage into bacterial cells, etc.).
Complementation in diploid organisms: As discussed above, use of the term "complementation" is usually reserved for cases in which the two mutations whose interactions are being studied have a similar phenotypic effect. Numerous examples of such effects can be cited, including the ebony and black mutations in Drosophila, the A and B color loci for corn kernels, or the C and P color loci in sweet peas. In every case, it is necessary to have at least one wild-type allele at both genetic loci in order to obtain a wild-type phenotype (normal body color in Drosophila, purple kernels of corn, purple flowers on the sweet peas). Such systems typically yield a 9:7 phenotypic ratio in the F2 (Table 4.8 and discussion on page 93).
Cistron: Returning now to the studies of genetic fine structure undertaken by Seymour Benzer, he introduced the term cistron to describe the genetic units that were identified in his cis-trans tests as the basic units of complementation in bacteriophage T4. When the original work was done in the 1950's, genes could only be identified as units of heredity that were known to have a 1:1 relationship to enzymes and other protein products in some cases. (At that time, the genetic code was not yet known and practical methods for sequencing DNA had not been developed). The cis-trans test allowed the rII "gene" in the bacteriophage T4 genome to be subdivided by complementation testing into two subunits, rIIA and rIIB, which he described as separate cistrons.
One cistron-one polypeptide: With the rapid advances in molecular biology that were occurring at the time, it soon became evident that a cistron was, in fact, the coding unit for one polypeptide chain (as opposed to one enzyme, which could be composed of several subunits). For many years, cistron was the preferred term for identifying the coding sequence for a single protein chain. However, as older vague usages have slowly faded away, the word "gene" has gradually regained popularity as the appropriate word to describe a single coding sequence. Nevertheless, use of the term "cistron" has persisted in certain cases, such as the description of the transcripts from operons as "polycistronic" (meaning that they contain the coding sequences for several different proteins in a single large RNA molecule).
Intracistronic complementation: In certain rare cases, complementation can occur within a single coding sequence. This is commonly referred to as intracistronic complementation. The simplest way to visualize this is to think of subunits of a dimeric enzyme. In certain cases identical subunits carrying the same defect may not be able to form a dimer, whereas those that have complementary defects can. Thus, for example, a hydrophobic interaction between two different regions in the monomer might be lost because a charged amino acid has replaced a hydrophobic amino acid. However, if a second mutation places the opposite charge at the interacting site, the positive and negative might interact and allow a stable dimer to be formed, even though neither type of monomer could interact with itself.
Beta-galactosidase as an example: A real life example cited in a previous textbook involves two different mutations in the beta-galactosidase (lacZ) gene in E. coli that can complement each other. In this case, the functional enzyme is a homotetramer. There are two known mutations that each individually prevent formation of an active tetramer. However, the mutations involve different parts of the protein monomer, such that a mixture of the two types of proteins can still form a heterotetramer (containing two copies of each of the mutant proteins) that is catalytically active.
Deletion mapping: Benzer accumulated some 20,000 rII mutations, which were distributed about equally between rIIA and rIIB. Detailed recombinaitonal analysis was greatly facilitated by the use of a number of deletion mutations. Since each deletion mutant had lost a chromosomal region, it could not restore function by recombining with any point mutations that fell within the region covered by the deletion. This allowed point mutations to be localized to restricted areas within one of the cistrons before precise recombinational mapping with nearby loci was undertaken (fig. 6.25).
Linear arrays of mutations: Benzer's work demonstrated that the smallest functional genetic unit, the cistron, was, in fact, a linear array of genetic information that was entirely consistent with the growing belief that the sequence of nucleotides in a linear DNA molecule was the carrier of genetic information (fig. 6.26). Other concepts also emerged, such as mutational hot spots, which will be explored later in the semester.
Metabolic pathways: In the next lecture, we will examine the one gene:ene enzyme concept in greater detail, with particular emphasis on complementation among mutations that affect sequential enzymatic steps in biosynthetic pathways.