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1: Fundamental Properties of Genes - Biology

1: Fundamental Properties of Genes - Biology


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Genetic dissection by complementation

Genes are the hereditary units that when altered change a phenotype; genes are classically defined by their effects on phenotype. But in many cases more than one gene affects a phenotype. Metabolic pathways, such as synthesis of DNA, repair of DNA, synthesis of leucine, or breakdown of starch occur in multiple steps catalyzed by enzymes. Each subunit of each enzyme is encoded in a gene, and all those genes are needed for the efficient running of the pathway. Multiple genes also determine complex traits, such as susceptibility to substance abuse, diabetes, and other diseases, and probably less pressing concerns, such as retaining a healthy head of hair after you are 40.

Many pathways have been elucidated by finding many mutants that are defective in that process, hopefully enough to sample every gene in the organism (saturation mutagenesis), and grouping them according to the gene that is mutated. All the mutations in the same gene fall into the same complementation group. Two mutants complement each other if they restore the normal phenotype when together in a diploid. This occurs when the mutants have mutations in different genes. If one is examining mutants with a similar phenotype (e.g. inability to grow on leucine or inability to make DNA), then tests of all pairwise combinations of the mutants will place them into complementation group, which complement between groups but not within groups. The complementation groups then define the genes in the process under study. This is a powerful method of genetic dissection of a pathway. Complementation and its functions are discussed in more detail here.

Genetic methods in microorganisms

The genetic systems found in bacteria and fungi are particularly powerful. The small size of the genome(all the genetic material in an organism), the ability to examine both haploid and diploid forms, and the ease of large-scale screens have made them the method of choice for many investigations. Some of the key features will be summarized in this section.

Microorganisms such as bacteria and fungi have several advantages for genetic analysis. They have a haploid genome, thus an investigator can detect recessive phenotypes easily and rapidly. In the haploid (1N) state, only one allele is present for each gene, and thus its phenotype is the one observed in the organism.

Bacteria can carry plasmids and can be infected with viruses, each of which are capable of carrying copies of bacterial genes. Thus bacteria can be partially diploid, or merodiploid, for some genes. This allows one to test whether alleles are dominant or recessive.

Bacteria are capable of sexual transfer of genetic information, during which time homologous chromosomes can recombine. Thus one can use recombination frequencyto map genes, analogous to the process in diploid sexual organisms. Indeed, a high frequency of recombination was essential in investigations of the fine structure of genes.

Bacteria grow, or increase in cell number, very rapidly. Generation times can be as short as 20 to 30 minutes. Thus many generations can be examined in a short time.

An investigator can obtain large quantities of mutant organisms for biochemical fractionation.

Bacterial genomes are small, ranging from about 0.580 (Mycoplasma genitalium) to 4.639 million base pairs (E. coli), with about 500 to 4300 genes, respectively. Compared to organisms with genomes 100 to 1000 times larger, this makes it easier to saturate the genome with mutations that disrupt some physiological process. Also, the smaller genome size, plus the availability of transducing phage, made it possible to isolate bacterial genes for intensive study.

Genomes of several bacteria are now completely sequenced, so all the genes, and their DNA sequences are known.

Yeast, such as Saccharomyces cerevisiae, are eukaryotic microorganisms that have both a haploid and a diploid phase to their life cycle, and thus have these same advantages as bacteria. Although its genome is larger (12 million base pairs), and it has 16 chromosomes, it is a powerful model organism for genetic and biochemical investigation of many aspects of molecular and cell biology. The genome of Saccharomyces cerevisiaeis completely sequenced, revealing about 6100 genes.

One can use mutagens to increase the number of mutations, e.g. to modify bases, intercalate, etc. Specific mutagens will be considered in Part Two of the course.

Replica plating allows one to test colonies under different growth conditions. This is illustrated in Figure 1.8 for finding mutant with new growth factor requirements. Replica plating can be used to compare growth of cells on complete medium, minimal medium, and minimal medium supplemented with a specific growth factor, e.g. an amino acid like Arg (the abbreviation for arginine). Cells that grow on minimal medium supplemented with Arg, but not on minimal medium are Arg auxotrophs. The word auxotrophmeans "increased growth requirements". These are cells that require some additional nutrient (growth factor) to grow. Prototrophs(usually the wild type cells) do not have the need for the additional factor and grow on minimal medium. In this case, they still make their own Arg.

Figure 1.8. Replica plating of microorganisms. Panel A shows the technique of replica plating to screen for drug sensitivity. Panel B illustrates its application to finding mutants with growth factor requirements.

Sometimes the trait one is selecting for is lethal to the organism. In this situation, one can screen for conditional mutants. These are mutants that grow under one condition and not under another condition. Conditional mutants that grow at a low temperature but not at a high temperature are are called "temperature sensitive" or ts mutants. Conditional mutants are not necessarily associated with lethality. The dark ear tips, nose and feet of a Siamese cat are the phenotype of a temperature sensitive mutation in the clocus (determining fur color). The enzyme encoded is not functional at higher temperatures, but is functional at lower temperatures, such as the extremities of the cat. Hence the fur on these parts of the Siamese cat’s body is pigmented.

Figure 1.9. Coat color in Siamese cats is determined by a temperature sensitive mutation in an enzyme needed for pigment formation. Siamese are homozygous chch, which encodes an enzyme that is active at low temperature (in the extremities of the cat) but inactive elsewhere.

Conjugation in bacteria

The ability to plate out large numbers of haploid bacteria or fungi on a Petri dish, and to examine a single colony (or clone) under a variety of conditions (with an without a growth factor, with and without a drug, or at high and low temperature), makes it relatively easy to screen through many individuals to find mutants with a particular phenotype. However, in order to carry out a complementation analysis, one needs to be able to combine the two haploid mutants in one cell. Many fungi, such as yeast, do this thorough a natural meiotic sporulation and mating process. Figure 1.6 illustrates the use of fungal matings in complementation.

Bacteria can also, although not by meiosis and fertilization, and only a part of the genome of one bacterium is transferred to another. The sexual transfer of information in E. coliuses plasmids called F (fertility) factors or Hfr strains. Male E. coli cells have a large plasmid, the F or fertility factor. A plasmidis a circular, extrachromosomal DNA molecule that is not essential to the bacterium. The F plasmid can transfer DNA from the male cell to an F- or female cell, in a process called conjugation (Figure 1.10). The male and female cells are brought close together by attachments at pili, the cells join and DNA is synthesized from the F plasmid and transferred into the recipient cells. This converts the female cell to a male cell, in response to conjugation via pili.

In some strains of E. coli the F factor is integrated. In this case, the DNA transfer starts in F region of the chromosome, but it also transfers adjacent chromosomal DNA. These are called hfrstrains, for their high frequency of recombination. The transferred DNA recombines with the DNA in the recipient cell.

Some F-related plasmids are a hybrid of F DNA and host bacterial DNA. These F’plasmids appear to be derived from F factors but they have replaced some of the F DNA with bacterial DNA. Thus they are convenient carriers of parts of the E. coli genome.

This conjugal transfer can be used to create partial diploids, also called merodiploids, in E. coli. For some time after conjugation, a portion of two different copies of the chromosome is present in the same cells. Another method is to introduce F’ factors, carrying bacterial DNA, into another strain. These are two ways to do complementation analysis in E. coli.

Figure 1.10. F-factor mediated conjugal transfer of DNA in bacteria.

Gene mapping by conjugal transfer

Conjugal transfer can also be used for genetic mapping. By using many different hfr strains, each with the F factor integrated at a different part of the E. colichromosome, the positions of many genes were mapped. These studies showed that the genetic map of the E. coli chromosome is circular. During conjugal transfer, genes closer to the site of F integration are transferred first. By disrupting the mating at different times, one can determine which genes are closer to the integration site. Thus on the E. colichromosome, genes are mapped in terms of minutes (i.e., the time it takes to transfer to recipient).

For example, for an hfr strain with the F factor integrated at 0 min on the E. colimap, conjugal transfer to a female recipient would transfer

  • leuACBD at 1.7 min
  • pyrH at 4.6 min
  • proAB at 5.9 min
  • bioABFCD at 17.5 min.

Use of hfr strains with different sites of integration (initiation of transfer) allows the entire circular genome to be mapped (Figure 1.11). 0/100 is thrABC.

Figure 1.11.Circular genetic map of E. coli.

Bacteriophage

Bacteriophageare viruses that infect bacteria. Because of their very large number of progeny and ability to recombine in mixed infections (more than one strain of bacteria in an infection), they have been used extensively in high-resolution definition of genes. Much of what we know about genetic fine structure, prior to the advent of techniques for isolating and sequencing genes, derive form studies in bacteriophage.

Bacteriophage have been a powerful model genetic system, because they have small genomes, have a short life cycle, and produce many progeny from an infected cell. They provide a very efficient means for transfer of DNA into or between cells. The large number of progeny makes it possible to measure very rare recombination events.

Lytic bacteriophage form plaqueson lawns of bacteria; these are regions of clearing where infected bacteria have lysed. Early work focused on mutants with different plaque morphology, e.g. T2 r, which shows rapid lysis and generates larger plaques, or on mutants with different host range, e.g. T2 h, which will kill both host strains B and B/2.

A cis-trans complementation test defines a cistron, which is a gene

Seymour Benzer used the rIIlocus of phage T4 to define genes by virtue of their behavior in a complementation test, and also to provide fundamental insight into the structure of genes (in particular, the arrangement of mutable sites - see the next section). The difference in plaque morphology between rand r+phage is easy to see (large versus small, respectively), and Benzer isolated many r mutants of phage T4. The wild type, but not any rIImutants, will grow on E. colistrain K12(l), whereas both wild type and mutant phage grow equally well on E. colistrain B. Thus the wild phenotype is readily detected by its ability to grow in strain K12 (l).

If E. colistrain K12 (l) is co-infected with 2 phage carrying mutations at different positions in rIIA, you get no multiplication of the phage (except the extremely rare wild type recombinants, which occur at about 1 in 106 progeny). In the diagram below, each line represents the chromosome from one of the parental phage.

rIIA rIIB

phage 1 _|__x______|________|_

phage 2 _|_______x_|________|_

Likewise, if the two phage in the co-infection carry mutations at different positions in rIIB, you get no multiplication of the phage (except the extremely rare wild type recombinants, about 1 in 106).

rIIA rIIB

phage 3 _|_________|_x______|_

phage 4 _|_________|______x_|_

However, if one of the co-infecting phage carries a mutation in rIIAand the other a mutation in rIIB, then you see multiplication of the phage, forming a very large number of plaques on E. colistrain K12 (l).

rIIA rIIB

phage 1 _|__x______|________|_ Provides wt rIIB protein

phage 4 _|_________|______x_|_ Provides wt rIIA protein

Together these two phage provide all the phage functions - they complementeach other. This is a positive complementation test. The first two examples show no complementation, and we place them in the same complementation group. Mutants that do not complement are placed in the same complementation group; they are different mutant alleles of the same gene. Benzer showed that there were two complementation groups (and therefore two genes) at the r II locus, which he called A and B.

In the mixed infection with phage 1 and phage 4, you also obtain the rare wild type recombinants, but there are more recombinants than are seen in the co-infections with different mutant alleles. Why?

Benzer’s experiments analyzing the rIIlocus of bacteriophage T4 formalized the idea of a cis‑transcomplementation test to define a cistron, which is an operational definition of a gene. First, let’s define cis and transwhen used to refer to genes. In the cisconfiguration, both mutations are on the same chromosome. In the transconfiguration, each mutation is on a different chromosome

Mutations in the same gene will not complement in trans, whereas mutations in different genes will complement in trans(Figure 1.12). In the cisconfiguration, the other chromosome is wild type, and wild‑type will complement any recessive mutation.

The complementation group corresponds to a genetic entity we call a cistron, it is equivalent to a gene.

This test requires a diploid situation. This can be a natural diploid (2 copies of each chromosome) or a partial, or merodiploid, e.g. by conjugating with a cell carrying an F' factor. Some bacteriophage carry pieces of the host chromosome; these are called transducingphage. Infection of E. coli with a transducing phage carrying a mutation in a host gene is another way to create a merodiploid in the laboratory for complementation analysis.

Figure 1.12. The complementation test defines the cistron and distinguishes between two genes.

Recombination within genes allows construction of a linear map of mutable sites that constitute a gene

Once the recombination analysis made it clear that chromosomes were linear arrays of genes, these were thought of as "string of pearls" with the genes, or "pearls," separated by some non‑genetic material (Figure 1.13). This putative non-genetic material was thought to be the site of recombination, whereas the genes, the units of inheritance, were thought to be resistant to recombination. However, by examining the large number of progeny of bacteriophage infections, one can demonstrate that recombination can occur within a gene. This supports the second model shown in Figure 1.13. Because of the tight packing of coding regions in phage genomes, recombination almost always occurs within genes in bacteriophage, but in genomes with considerable non-coding regions between genes, recombination can occur between genes as well.

Figure 1.13: Models for genes as either discrete mutable units separate by non-genetic material (top) or as part of a continuous genetic material (bottom).

The tests between these two models required screening for genetic markers (mutations) that are very close to each other. When two markers are very close to each other, the recombination frequency is extremely low, so enough progeny have to be examined to resolve map distances of, say 0.02 centiMorgans = 0.02 map units = 0.02 % recombinants. This means that 2 out of 10,000 progeny will show recombination between two markers that are 0.02 map units apart, and obviously one has to examine at least 10,000 progeny to reliably score this recombination. That's the power of microbial genetics ‑ you actually can select or screen through this many progeny, sometimes quite easily.

An example of recombination in phage is shown in Figure 1.14. Wild type T2 phage forms small plaques and kills only E. Thus different alleles of hcan be distinguished by plating on a mixture of E. colistrains B and B/2. The phage carrying mutant hallele will generate clear plaques, since they kill both strains. Phage with the wild type h+ give turbid plaques, since the B/2 cells are not lysed but B cells are. When a mixture of E. colistrains B and B/2 are co-infected with both T2 hrand T2 h+r+, four types of plaques are obtained. Most have the parental phenotypes, clear and large or turbid and small. These plaques contain progeny phage that retain the parental genotypes T2 hrand T2 h+r+, respectively. The other two phenotypes are nonparental, i.e. clear and small or turbid and large. These are from progeny with recombinant genotypes, i.e. T2 hr+and T2 h+r. In this mixed infection, recombination occurred between two phage genomes in the same cell.

Figure 1.14. Recombination in bacteriophage

The first demonstration of recombination within a gene came from work on the rIIAand rIIBgenes of phage T4. These experiments from Seymour Benzer, published in 1955, used techniques like that diagrammed in Figure 1.14. Remember that mutations in the rgene cause rapid lysis of infected cells, i.e. the length of the lytic cycle is shorter. The difference in plaque morphology between rand r+phage is easy to see (large versus small, respectively). These two genes are very close together, and many mutations were independently isolated in each. This was summarized in the discussion on complementation above.

Consider the results of infection of a bacterial culture with two mutant alleles of gene rIIA.

T4rIIA6 _|_______________________x______|_

and T4rIIA27 _|_______x______________________|_

(x marks the position of the mutation in each allele).

Progeny phage from this infection include those with a parental genotype (in the great majority), and at a much lower frequency, two types of recombinants:

wild type T4 r+ _|______________________________|_

double mutant T4rIIA6 rIIA27 _|_______x_______________x______|_

The wild type is easily scored because it, and not any rIImutants, will grow on E. coli strain K12(l), whereas both wild type and mutant phage grow equally well on E. coli strain B. Thus you can selectfor the wild type (and you will see only the desired recombinant). Finding the double mutants is more laborious, because they are obtained only by screening through the progeny, testing for phage that when backcrossed with the parental phage result in no wild type recombinant progeny.

Equal numbers of wild type and double mutant recombinants were obtained, showing that recombination can occur within a gene, and that this occurs by reciprocal crossing over. If recombination were only between genes, then no wild type phage would result. A large spectrum of recombination values was obtained in crosses for different alleles, just like you obtain for crosses between mutants in separate genes.

Several major conclusions could be made as a result of these experiments on recombination within the rIIgenes.

  1. A large number of mutable sites occur within a gene, exceeding some 500 for the rIIA and rIIBgenes. We now realize that these correspond to the individual base pairs within the gene.
  2. The genetic maps are clearly linear, indicating that the gene is linear. Now we know a gene is a linear polymer of nucleotides.
  3. Most mutations are changes at one mutable site (point mutations). Many genes can be restored to wild type by undergoing a reverse mutation at the same site (reversion).
  4. Other mutations cause the deletionof one or more mutable sites, reflecting a physical loss of part of the rII gene. Deletions of one or more mutable site (base pair) are extremely unlikely to revert back to the original wild type.

One gene encodes one polypeptide

One of the fundamental insights into how genes function is that one gene encodes one enzyme (or more precisely, one polypeptide). Beadle and Tatum reached this conclusion based on their complementation analysis of the genes required for arginine biosynthesis in fungi. They showed that a mutation in each gene led to a loss of activity of one enzyme in the multistep pathway of arginine biosynthesis. As discussed above in the section on genetic dissection, a large number of Arg auxotrophs (requiring Arg for growth) were isolated, and then organized into a set of complementation groups, where each complementation groups represents a gene.

The classic work of Beadle and Tatum demonstrated a direct relationship between the genes defined by the auxotrophic mutants and the enzymes required for Arg biosynthesis. They showed that a mutation in one gene resulted in the loss of one particular enzymatic activity, e.g. in the generalized scheme below, a mutation in gene 2 led to a loss of activity of enzyme 2. This led to an accumulation of the substrate for that reaction (intermediate N in the diagram below). If there were 4 complementation groups for the Arg auxotrophs, i.e. 4 genes, then 4 enzymes were found in the pathway for Arg biosynthesis. Each enzyme was affected by mutations in one of the complementation groups.

Intermediates:

M ® N ® O ® P ® Arg

enzyme 1 enzyme 2 enzyme 3 enzyme 4

gene 1 gene 2 gene 3 gene 4

Figure 1.15. A general scheme showing the relationships among metabolic intermediates (M, N, O, P), and end product (Arg), enzymes and the genes that encode them.

In general, each step in a metabolic pathway is catalyzed by an enzyme (identified biochemically) that is the product of a particular gene (identified by mutants unable to synthesize the end product, or unable to break down the starting compound, of a pathway). The number of genes that can generate auxotrophic mutants is (usually) the same as the number of enzymatic steps in the pathway. Auxotrophic mutants in a given gene are missing the corresponding enzyme. Thus Beadle and Tatum concluded that one gene encodes one enzyme. Sometimes more than one gene is required to encode an enzyme because the enzyme has multiple, different polypeptide subunits. Thus each polypeptide is encoded by a gene.

The metabolic intermediates that accumulate in each mutant can be used to place the enzymes in their order of actionin a pathway. In the diagram in Figure 1.15, mutants in gene 3 accumulated substance O. Feeding substance O to mutants in gene 1 or in gene 2 allows growth in the absence of Arg. We conclude that the defects in enzyme 1 or enzyme 2, respectively, are upstream of enzyme 3. In contrast, feeding substance O to mutants in gene 4 will not allow growth in the absence of Arg. Even though this mutant can convert substance O to substance P, it does not have an active enzyme 4 to convert P to Arg. The inability of mutants in gene 4 to grow on substance O shows that enzyme 4 is downstream of enzyme 3.

Imagine that you are studying serine biosynthesis in a fungus. You isolate serine auxotrophs, do all the pairwise crosses of the mutants and discover that the auxotrophs can be grouped into three complementation groups, called A, B and C. You also discover that a different metabolic intermediate accumulates in members of each complementation group - substance A in auxotrophs in the A complementation group, substance B in the B complementation group and substance C in the C complementation group. Each of the intermediates is fed to auxotrophs from each of the three complementation groups as tabulated below. A + means that the auxotroph was able to grow in media in the absence of serine when fed the indicated substance; a - denotes no growth in the absence of serine.

Fed:

mutant in complementation group A

mutant in complementation group B

mutant in complementation group C

substance A

-

+

+

substance B

-

-

-

substance C

-

+

-

In the biosynthetic pathway to serine in this fungus, what is the order of the enzymes encoded in the three complementation groups? Enzyme A is encoded by the gene that when altered generates mutants that fall into complementation group A, etc.

The gene and its polypeptide product are colinear

Once it was determined that a gene was a linear array of mutable sites, that genes are composed of a string of nucleotides called DNA (see Chapter 2), and that each gene encoded a polypeptide, the issue remained to be determined how exactly that string of nucleotides coded for a particular amino acid sequence. This problem was studied along several avenues, culminating in a major achievement of the last half of the 20th century – the deciphering of the genetic code. The detailed assignment of particular codons (triplets of adjacent nucleotides) will be discussed in Chapter 13. In the next few sections of this chapter, we will examine how some of the basic features of the genetic code were deciphered.

A priori, the coding units within a gene couldencode both the composition and the address for each amino acid, as illustrated in Model 1 of Figure 1.17. In this model, the coding units could be scrambled and still specify the same protein. In such a situation, the polypeptide would not be colinear with the gene.

Figure 1.16.Alternative models for gene and codon structure.

In an alternative model (Model 2 in Figure 1.16), the coding units only specify the composition, but not the position, of an amino acid. The "address" of the amino acid is derived from the position of the coding unit within the gene. This model would predict that the gene and its polypeptide product would be colinear - e.g. mutation in the 5th coding unit would affect the 5th amino acid of the protein, etc.

Charles Yanofsky and his co-workers (1964) tested these two models and determined that the gene and the polypeptide product are indeed colinear. They used recombination frequencies to map the positions of different mutant alleles in the gene that encodes a particular subunit of the enzyme tryptophan synthase. They then determined the amino acid sequence of the wild type and mutant polypeptides. As illustrated in Figure 1.17, the position of a mutant allele on the recombination map of the gene corresponds with the position of the amino acid altered in the mutant polypeptide product. For instance, allele A101 maps to one end of the gene, and the corresponding Glu ® Val replacement is close to the N terminus of the polypeptide. Allele A64maps close to the other end of the gene, and the corresponding Ser ® Leu replacement is close to the C terminus of the polypeptide. This correspondence between the positions of the mutations in each allele and the positions of the consequent changes in the polypeptide show that Model 1 can be eliminated and Model 2 is supported.

Figure 1.17.The polypeptide is colinear with the gene.

Mutable sites are base pairs along the double helix

The large number of mutable sites found in each gene, and between which recombination can occur, leads one to conclude that the mutable sites are base pairs along the DNA. Sequence determination of the wild type and mutant genes confirms this conclusion.

Single amino acids are specified by three adjacent nucleotides, which are a codons

This conclusion requires three pieces of information.

First of all, adjacent mutable sites specify amino acids. Reaching this conclusion required investigation of the fine structure of a gene, including rare recombination between very closely linked mutations within a gene. Yanofsky and his colleagues, working with mutations the trpA gene of E. coli, encoding tryptophan synthase, showed that different alleles mutated in the same codon could recombine (albeit at very low frequency). (This is the same laboratory and same system that was used to show that a gene and its polypeptide product are colinear.) Thus recombination between two different alleles can occur within a codon, which means that a codon must have more than one mutable site. We now recognize that a mutable site is a nucleotide in the DNA. Thus adjacent mutable sites (nucleotides) encode a single amino acid.

Let’s look at this in more detail (Figure 1.18). Yanofsky and colleagues examined two different mutant alleles of trpA, each of which caused alteration in amino acid 211 of tryptophan synthase. In the mutant allele A23, wild type Gly is converted to mutant Arg. In the mutant allele A46, wild type Gly is converted to mutant Glu.

GGA (Gly 211) --> AGA (Arg 211) mutant allele A23

GGA (Gly 211) --> GAA (Glu 211) mutant allele A46

A23 ´ A46 AGA ´ GAA ® GGA (wild type Gly 211 in 2 out of 100,000 progeny)

Figure 1.18.Recombination can occur between two mutant alleles affecting the same codon.

Alleles A23and A46are not alternative forms of the same mutable site, because recombination to yield wild type occurs, albeit at a very low frequency (0.002%; the sites are very close together, in fact in the same codon!). If they involved the same mutable site, one would never see the wild-type recombinant.

The second observation is that the genetic code is non-overlapping. This was shown by demonstrating that a mutation at a single site alters only one amino acid. This conflicts with the predictions of an overlapping code (see Figure 1.19), and thus the code must be non-overlapping.

Figure 1.19. Predictions of the effects of nucleotide substitutions, insertions or deletions on polypeptides encoded by an overlapping, a punctuated, or a nonoverlapping, nonpunctuated code.

The third observation is that the genetic code is read in tripletsfrom a fixed starting point. This was shown by examining the effect of frameshift mutations. As shown in Figure 1.19, a code lacking punctuation has a certain reading frame. Insertions or deletions of nucleotides are predicted to have a drastic effect on the encoded protein because they will change that reading frame. The fact that this was observed was one of the major reasons to conclude that the mRNA molecules encoded by genes are read in successive blocks of three nucleotides in a particular reading frame.

For the sequence shown in Figure 1.20, insertion of an A shifts the reading frame, so all amino acids after the insertion differ from the wild type sequence. (The 4th amino acid is still a Gly because of degeneracy in the code: both GGC and GGG code for Gly.) Similarly, deletion of a U alters the entire sequence after the deletion.

Figure 1.20. Frameshift mutations show that the genetic code is read in triplets.

These observations show that the nucleotide sequence is read, or translated, from a fixed starting point without punctuation. An alternative model is that the group of nucleotides encoding an amino acid (the codon) could also include a signal for the end of the codon (Model 2 in Figure 1.19). This could be considered a "comma" at the end of each codon. If that were the case, insertions or deletions would only affect the codon in which they occur. However, the data show that all codons, including and after the one containing the insertion or deletion, are altered. Thus the genetic code is not punctuated, but is read in a particular frame that is defined by a fixed starting point (Model 3 in Figure 1.19). That starting point is a particular AUG, encoding methionine. (More about this will be covered in Chapter 13).

The results of frame-shift mutations are so drastic that the proteins are usually not functional. Hence a screen or selection for loss-of-function mutants frequently reveals these frameshift mutants. Simple nucleotide substitutions that lead to amino acid replacements often have very little effect on the protein, and hence have little, or subtle, phenotypes.

A double mutant generated by crossing over between the insertion (+) and deletion (‑) results in an (almost) normal phenotype, i.e. reversion of insertion or deletion.

A gene containing three closely spaced insertions(or deletions) of single nucleotides will produce a functional product. However, four or five insertions or deletions do not give a functional product (Crick, Barnett, Brenner and Watts‑Tobin, 1961). This provided the best evidence that the genetic code is read in groups of three nucleotides(not two or four). Over the next 5 years the code was worked out (by 1966) and this inference was confirmed definitively.

Central Dogma: DNA to RNA to protein

A few years after he and James Watson had proposed the double helical structure for DNA, Francis Crick (with other collaborators) proposed that a less stable nucleic acid, RNA, served as a messenger RNA that provided a transient copy of the genetic material that could be translated into the protein product encoded by the gene. Such mRNAs were indeed found. These and other studies led Francis Crick to formulate this “central dogma” of molecular biology (Figure 1.21).

This model states that DNA serves as the repository of genetic information. It can be replicated accurately and indefinitely. The genetic information is expressed by the DNA first serving as a template for the synthesis of (messenger) RNA; this occurs in a process called transcription. The mRNA then serves as a template, which is read by ribosomes and translatedinto protein. The protein products can be enzymes that catalyze the many metabolic transformations in the cell, or they can be structural proteins.

Figure 1.21.The central dogma of molecular biology.

Although there have been some additional steps added since its formulation, the central dogma has stood the test of time and myriad experiments. It provides a strong unifying theme to molecular genetics and information flow in cell biology and biochemistry.

Although in many cases a gene encodes one polypeptide, other genes encode a functional RNA. Some genes encode tRNAsandrRNAsneeded for translation, others encode other structural and catalytic RNAs. Genes encode some product that is used in the cell, i.e. that when altered generates an identifiable phenotype. More generally, genes encode RNAs, some of which are functional as transcribed (or with minor alterations via processing) such as tRNAs and rRNAs, and others are messengers that are then translated into proteins. These proteins can provide structural, catalytic and regulatory roles in the cell.

Note the static role of DNAin this process. Implicit in this model is the idea that DNA does not provide an active cellular function, but rather it encodes macromolecules that are functional. However, the expression of virtually all genes is highly regulated. The sites on the DNA where this control is exerted are indeed functional entities, such as promoters and enhancers. In this case, the DNA is directly functional (cis‑regulatory sites), but the genes being regulated by these sites still encode some functional product (RNA or protein).

Studies of retroviruses lead Dulbecco to argue that the flow of information is not unidirectional, but in fact RNA can be converted into DNA (some viral RNA genomes are converted into DNA proviruses integrated into the genome). Subsequently Temin and Baltimore discovered the enzyme that can make a DNA copy of RNA, i.e. reverse transcriptase.

Transcription and mRNA structure

Several aspects of the structure of genes can be illustrated by examining the general features of a bacterial gene as now understood.

A gene is a string of nucleotides in the duplex DNA that encodes a mRNA, which itself codes for protein. Only one strand of the duplex DNA is copied into mRNA (Figure 1.22). Sometimes genes overlap, and in some of those cases each strand of DNA is copied, but each for a different mRNA. The strand of DNA that reads the same as the sequence of mRNA is the nontemplate strand. The strand that reads as the reverse complement of the mRNA is the template strand.

Figure 1.22.Only one strand of duplex DNA codes for a particular product.

NOTE: The term "sense strand" has two oppositeuses (unfortunately). Sidney Brenner first used it to designate the strand that served as the template to make RNA (bottom strand above), and this is still used in many genetics texts. However, now many authors use the term to refer to the strand that reads the same as the mRNA (top strand above). The same confusion applies to the term "coding strand" which can refer to the strand encoding mRNA (bottom strand) or the strand "encoding" the protein (top strand). Interestingly, "antisense" is used exclusively to refer to the strand that is the reverse complement of the mRNA (bottom strand).

Figure 1.22 helps illustrate the origin of terms used in gene expression. Copying the information of DNA into RNA stays in the same "language" in that both of these polymers are nucleic acids, hence the process is called transcription. An analogy would be writing exercises where you had to copy, e.g. a poem, from a book onto your paper - you transcribed the poem, but it is still in English. Converting the information from RNA into DNA is equivalent to converting from one "language" to another, in this case from one type of polymer (the nucleic acid RNA) to a different one (a polypeptide or protein). Hence the process is called translation. This is analogous to translating a poem written in French into English.

Figure 1.23 illustrates the point that a gene may be longer than the region coding for the protein because of 5' and/or 3' untranslated regions.

Figure 1.23.Genes and mRNA have untranslated sequences at both the 5’ and 3’ ends.

Eukaryotic mRNAs have covalent attachment of nucleotides at the 5' and 3' ends, and in some cases nucleotides are added internally (a process called RNA editing). Recent work shows that additional nucleotides are added post‑transcriptionally to some bacterial mRNAs as well.

Regulatory signals can be considered parts of genes

In order to express a gene at the correct time, the DNA also carries signals to start transcription (e.g. promoters), signals for regulating the efficiency of starting transcription (e.g. operators, enhancers or silencers), and signals to stop transcription (e.g. terminators). Minimally, a gene includes the transcription unit, which is the segment of DNA that is copied into RNA in the primary transcript. The signals directing RNA polymerase to start at the correct site, and other DNA segments that influence the efficiency of this process are regulatory elements for the gene. One can also consider them to be part of the gene, along with the transcription unit.

A contemporary problem - finding the function of genes

Genes were originally detected by the heritable phenotype generated by their mutant alleles, such as the white eyes in the normally red-eyed Drosophilaor the sickle cell form of hemoglobin (HbS) in humans. Now that we have the ability to isolate virtually any, and perhaps all, segments of DNA from the genome of an organism, the issue arises as to which of those segments are genes, and what is the function of those genes. (The genomeis all the DNA in the chromosomes of an organism.) Earlier geneticists knew what the function of the genes were that they were studying (at least in terms of some macroscopic phenotype), even when they had no idea what the nature of the genetic material was. Now molecular biologists are confronted with the opposite problem - we can find and study lots of DNA, but which regions are functions? Many computational approaches are being developed to guide in this analysis, but eventually we come back to that classical definition, i.e. that appropriate mutations in any functional gene should generate a detectable phenotype. The approach of biochemically making mutations in DNA in the laboratory and then testing for the effects in living cells or whole organisms is called "reverse genetics."

Additional Readings

  • Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M. (1993) An Introduction to Genetic Analysis, Fifth Edition (W. H. Freeman and Company, New York).
  • Cairns, J., Stent, G. S. and Watson, J. D., editors (1992) Phage and the Origins of Molecular Biology, Expanded Edition (Cold Spring Harbor Laboratory Press, Plainview, NY).
  • Brock, T. D. (1990) The Emergence of Bacterial Genetics (Cold Spring Harbor Laboratory Press, Plainview, NY).
  • Benzer, S. (1955) Fine structure of a genetic region in bacteriophage. Proceedings of the National Academy of Sciences, USA 47: 344-354.
  • Yanofsky, C. (1963) Amino acid replacements associated with mutation and recombination in the A gene and their relationship to in vitro coding data. Cold Spring Harbor Symposia on Quantitative Biology 18: 133-134.
  • Crick, F. (1970) Central dogma of molecular biology. Nature 227:561-563

Questions

Question 1.5. Calculating recombination frequencies

Corn kernels can be colored or white, determined by the alleles C(colored, which is dominant) or c(white, which is recessive) of the coloredgene. Likewise, alleles of the shrunkengene determine whether the kernels are nonshrunken (Sh, dominant) or shrunken (sh, recessive). The geneticist Hutchison crossed a homozygous colored shrunken strain (CC shsh) to a homozygous white nonshrunken strain (cc ShSh) and obtained the heterozygous colored nonshrunken F1. The F1 was backcrossed to a homozygous recessive white shrunken strain (cc shsh). Four phenotypes were observed in the F2 progeny, in the numbers shown below.

Phenotype Number of plants

colored shrunken 21,379

white nonshrunken 21,096

colored nonshrunken 638

white shrunken 672

a) What are the predicted frequencies of these phenotypes if the coloredand shrunkengenes are not linked?

b) Are these genes linked, and if so, what is the recombination frequency between them?

Question 1.6.Constructing a linkage map:

Consider three genes, A, B and C, that are located on the same chromosome. The arrangement of the three genes can be determined by a series of three crosses, each following two of the genes (referred to as two-factor crosses). In each cross, a parental strain that is homozygous for the dominant alleles of the two genes (e.g. AB/AB) is crossed with a strain that is homozygous for the recessive alleles of the two genes (e.g. ab/ab), to yield an F1 that is heterozygous for both of the genes (e.g. AB/ab). In this notation, the slash (/) separates the alleles of genes on one chromosome from those on the homologous chromosome. The F1 (AB/ab) contains one chromosome from each parent. It is then backcrossed to a strain that is homozygous for the recessive alleles (ab/ab) so that the fates of the parental chromosomes can be easily followed. Let's say the resulting progeny in the F2 (second) generation showed the parental phenotypes (AB and ab) 70% of the time. That is, 70% of the progeny showed only the dominant characters (AB) or only the recessive characters (ab), which reflect the haploid genotypes AB/aband ab/ab, respectively, in the F2 progeny. The remaining 30% of the progeny showed recombinant phenotypes (Aband aB) reflecting the genotypes Ab/aband aB/abin the F2 progeny. Similar crosses using F1's from parental AC/ACand ac/acbackcrossed to a homozygous recessive strain (ac/ac) generated recombinant phenotypes Acand aCin 10% of the progeny. And finally, crosses using F1's from parental BC/BCand bc/bcbackcrossed to a homozygous recessive strain (bc/bc) generated recombinant phenotypes Bcand bCin 25% of the progeny.

a. What accounts for the appearance of the recombinant phenotypes in the F2 progeny?

b. Which genes are closer to each other and which ones are further away?

c. What is a linkage map that is consistent with the data given?

Question 1.7.Why are the distances in the previous problem not exactly additive, e.g. why is the distance between the outside markers (A and B) not 35 map units (or 35% recombination)? There are several possible explanations, and this problem explores the effects of multiple crossovers. The basic idea is that the further apart two genes are, the more likely that recombination can occur multiple times between them. Of course, two (or any even number of) crossover events between two genes will restore the parental arrangement, whereas three (or any odd number of) crossover events will give a recombinant arrangement, thereby effectively decreasing the observed number of recombinants in the progeny of a cross.

For the case examined in the previous problem, with genes in the order A___C_______B, let the term abrefer to the frequency of recombination between genes Aand B, and likewise let acrefer to the frequency of recombination between genes Aand C, and cbrefer to the frequency of recombination between genes Cand B.

a) What is the probability that when recombination occurs in the interval between Aand C, an independent recombination event also occurs in the interval between Cand B?

b) What is the probability that when recombination occurs in the interval between Cand B, an independent recombination event also occurs in the interval between Aand C?

c) The two probabilities, or frequencies, in a and b above will effectively lower the actual recombination between the outside markers Aand Bto that observed in the experiment. What is an equation that expresses this relationship, and does it fit the data in problem 3?

d. What is the better estimate for the distance between genes Aand Bin the previous problem?

Question 1.8 Complementation and recombination in microbes.

The State College Bar Association has commissioned you to study an organism, Alcophila latrobus, which thrives on Rolling Rock beer and is ruining the local shipments. You find three mutants that have lost the ability to grow on Rolling Rock (RR).

a) Recombination between the mutants can restore the ability to grow on RR. From the following recombination frequencies, construct a linkage map for mutations 1, 2, and 3.

Recombination between Frequency

1- and 2- 0.100

1- and 3- 0.099

2- and 3- 0.001

b) The following diploid constructions were tested for their ability to grow on RR. What do these data tell you about mutations 1, 2, and 3?

Grow on RR?

1) 1- 2+ / 1+ 2- yes

2) 1- 3+ / 1+ 3- yes

3) 2- 3+ / 2+ 3- no

Question 1.9 Using recombination frequencies and complementation to deduce maps and pathways in phage.

A set of four mutant phage that were unable to grow in a particular bacterial host (lets call it restrictive) were isolated; however, both mutant and wild type phage will grow in another, permissive host. To get information about the genes required for growth on the restrictive host, this host was co-infected with pairs of mutant phage, and the number of phage obtained after infection was measured. The top number for each co-infection gives the total number of phage released (grown on the permissive host) and the bottom number gives the number of wild-type recombinant phage (grown on the restrictive host). The wild-type parental phage gives 1010 phage after infecting either host. The limit of detection is 102 phage.

Phenotypes of phage, problem 1.9:

Assays after co-infection with mutant phage:

Results of assays, problem 1.9:

Number of phage

mutant 1 mutant 2 mutant 3 mutant 4

mutant 1 total <102

recombinants <102

mutant 2 total 1010 <102

recombinants 5x106 <102

mutant 3 total 1010 1010 <102

recombinants 107 5x106 <102

mutant 4 total 105 1010 1010 <102

recombinants 105 5x106 107 <102

a) Which mutants are in the same complementation group? What is the minimum number of genes in the pathway for growth on the restrictive host?

b) Which mutations have the shortest distance between them?

c) Which mutations have the greatest distance between them?

d) Draw a map of the genes in the pathway required for growth on the restrictive host. Show the positions of the genes, the positions of the mutations and the relative distances between them.

Question 1.10. One of the classic experiments in bacterial genetics is the fluctuation analysisof Luria and Delbrück (1943, Mutations of bacteria from virus sensitivity to virus resistance, Genetics 28: 491-511). These authors wanted to determine whether mutations arose spontaneouslywhile bacteria grew in culture, or if the mutations were inducedby the conditions used to select for them. They knew that bacteria resistant to phage infection could be isolated from infected cultures. When a bacterial culture is infected with a lytic phage, initially it “clears” because virtually all the cells are lysed, but after several hours phage-resistant bacteria will start to grow.

Luria and Delbrück realized that the two hypothesis for the source of the mutations could be distinguished by a quantitative analysis of the number of the phage-resistant bacteria found in many infected cultures. The experimental approach is outlined in the figure below. Many cultures of bacteria are grown, then infected with a dose of phage T1 that is sufficient to kill all the cells, except those that have acquired resistance. These resistant bacteria grow into colonies on plates and can be counted.

a.What are the predictions for the distribution of the number of resistant bacteria in the two models? Assume that on average, about 1 in 107 bacteria are resistant to infection by phage T1.

b. What do results like those in the figure and table tell you about which model is correct?

Figure for question 1.10.

The actual results from Luria and Delbrück are summarized in the following table. They examined 87 cultures, each with 0.2 ml of bacteria, for phage resistant colonies.

Number of resistant bacteria

Number of cultures

0

29

1

17

2

4

3

3

4

3

5

2

6-10

5

11-20

6

21-50

7

51-100

5

101-200

2

201-500

4

501-1000

0

Interested students may wish to read about the re-examination of the origin of mutations by Cairns, Overbaugh and Miller (1988, The origin of mutants. Nature 335:142-145). Using a non-lethal selective agent (lactose), they obtained results indicating both pre-adaptive (spontaneous) mutations as well as some apparently induced by the selective agent.


Morphogenesis

Morphogenesis (from the Greek morphê shape and genesis creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of developmental biology along with the control of tissue growth and patterning of cellular differentiation.

The process controls the organized spatial distribution of cells during the embryonic development of an organism. Morphogenesis can take place also in a mature organism, such as in the normal maintenance of tissue homeostasis by stem cells or in regeneration of tissues after damage. Cancer is an example of highly abnormal and pathological tissue morphogenesis. Morphogenesis also describes the development of unicellular life forms that do not have an embryonic stage in their life cycle. Morphogenesis is essential for the evolution of new forms.

Morphogenesis is a mechanical process involving forces that generate mechanical stress, strain, and movement of cells, [1] and can be induced by genetic programs according to the spatial patterning of cells within tissues.


Genetic Material: Properties and Evidence | Cell Biology

A living cell is composed of several inorganic and organic components. Among them, one will obviously act as genetic material responsi­ble for controlling hereditary characters. Iden­tification of this genetic material remained con­troversial for a long time.

Now if any component is to be genetic mate­rial, it must fulfil a number of basic properties:

i. Genotypic function or replication or auto-synthesis.

ii. Phenotype function or expression or hetero catalysis.

The first property states that the genetic material must be capable of storing hereditary information and replicate with high efficiency in successive cell generations forming the basis for transmission of hereditary characteristics it controls.

The second property is a fundamental one involved in gene action which through a series of chemical reactions results in the ultimate expression of the characteristics within the organism. The third property states that the genetic material does undergo occasional heritable changes called mutation.

It creates variations among the organisms besides recombination. Variations, on the other hand, are the important source of raw materials for evolution.

Besides the above-mentioned important properties of genetic material, the gene substance also shows the following additional properties:

a. To control the innumerable diversities in the characteristics of organism available in nature, the genetic material must show a very wide diversity in form.

b. Since phenotype character is the final ex­pression of a chain of reactions initiated at the gene level, obviously the genetic mate­rial must be a chemically unique entity.

Before 1900 several biologists proposed that hereditary material must be in the chromosome of the cell nucleus. In 1903, Sutton and Bovery postulated that genes were located in chro­mosome. In eukaryotic system, chromosomes are made of mainly protein and nucleic acid (DNA and RNA) and one of them obviously constitute the genetic material.

But which one would be the most suitable candidate for the position of genetic material remained contro­versial for a long time.

Early molecular biol­ogists have assigned the properties of genetic material to the chromosomal proteins because they found nucleic acid too simple to carry genetic information. Besides this, nucleic acid is made of monotonous chemical components like sugar, phosphate and base.

On the other hand, protein showed a highly complex struc­ture composed of a variety of amino acid. So a wide range of diversities is possible in protein structure to fulfill the diversity required in the genetic material for controlling the countless diversities in the characteristic of organism.

The controversy about the assignment of gene substance either to chromosomal protein or to nucleic acid, existed up to 1950 when finally it was unanimously accepted that the genetic information resides in the nucleic acids rather than in proteins.

More specifically, several elegant experiments showed that DNA is the genetic material of most microorganisms and higher organisms. Later on, RNA was found to be the genetic material of some viruses where DNA is absent.

Evidence of Genetic Material:

The concept that DNA or RNA is the genetic material of most organisms has been developed and supported by following evidence:

I. Direct Evidence:

(a) Transformation in Pneumococcus:

The first direct evidence showing that the genetic material is DNA rather than protein or RNA was published by O. T. Avery, C. M. Macleod and M. McCarthy in 1944. They discovered that the substance of the cell respon­sible for the phenomenon of transformation in the bacterium Diplococcus pneumoniae is DNA.

Transformation is the mode of exchange or transfer of genetic information (recombination) from one strain of bacterium to another strain of bacterium without involving any direct con­tact between them. The process of transforma­tion was first discovered by Frederick Griffith in 1928.

This was called as Griffith’s ef­fect. The experiment of Griffith demonstrated transformation but he could not recognise the transforming principle.

Different strains of Pneumococci shows the genetic variability that can be recognised by existence of different phenotypes. Griffith ini­tially conducted his experiment on two strains of pneumococci which were phenotypically dis­tinct.

When they are grown artificially on nutrient agar medium, they form two types of colonies:

The cells of strains forming smooth (S) colonies have a smooth glittering appearance due to presence of strain-specific polysaccharides (a polymer of glucose and glucuronic acid) capsule. Such strains are able to produce pneumonia and are called virulent.

The polysaccharide capsule is required for virulence since it protects the bacterial cell against phagocytosis by leucocytes. But the cells of stain lack this capsule and they produce dull rough (R) colonies. Such stains are termed as avirulent since they cannot produce pneumonia.

Therefore smooth (S) and rough (R) phenotypic characteristic are directly related to the presence or absence of the capsule and this trait is known to be genetically determined.

Both S and R forms occur in several subtypes and are designated as S I, S II, S III, etc. and R I, R II, R III, etc., respectively, on the basis of antigen properties of the polysaccha­rides present in their capsule. This property ultimately depends on the genotype of the cell.

The experiments of Griffith (Fig. 12.1) are briefly described below stepwise on the basis of his observation:

Griffith injected live cells of the viru­lent type III S into mice, all the mice died due to pneumonia and live type III S cells were recovered from the serum of blood of the dead bodies of mice.

When live cells of the avirulent type II R were injected into a separate group of mice, none of the mice died and live type II R were isolated from the serum of blood of all mice.

When mice were injected with heat-killed virulent type III S pneumococci alone, again none of the mice died, showing that virulence is lost after heat-killing.

When mice were injected heat-killed type III S pneumococci (virulent when alive) plus live type II R pneumococci (non-virulent), some of the mice died due to pneumonia pneumococci cells isolated from the dead mice were of the type III S.

Since it is known that non-capsulate type R cells can mutate back to virulent encapsulated type S cells, the resulting cell will be type II S, not type III S. Thus the transformation of non- virulent type II R cells to virulent type III S cells cannot be explained by mutation, rather, some component of the dead type III S cells (the “transforming principle”) must convert living type II R cells to type III S.

This leads to a change in the trait of cells and helps to bring some new characters in the transformed cell. Hence the transforming principle must contain some genetic material.

(b) Transforming Principle is DNA:

Avery, Macleod and McCarthy experimentally proved that the transforming principle was DNA. They showed that if DNA extract from type IIS pneumococci was mixed with type IIR pneumococci in vitro, some of the pneumococci were transformed to type III S.

But DNA extract from type III S may be contaminated with a few molecules of proteins, RNA and this contaminating protein and RNA may be responsible for transformation from type II R to type III S. So Avery, Macleod and McCarthy demonstrated the most definitive experiment using bacterial culture system and specific enzymes that degrade DNA, RNA and protein.

In separate experiments (Fig. 12.2) DNA extract from type III S cells was treated with:

i. DNAse which degrades DNA.

ii. RNAse which degrades RNA.

iii. trypsin, a protease which degrades protein and then tested the treated DNA extract for its ability to transform type II R pneu­mococci to type III S.

iv. They observed that the treatment with RNAse or trypsin had no effect on the abil­ity of the DNA extract to transform type II R to type III S. But DNAse treatment destroyed the transforming activity of the DNA preparation and II R cells were not transformed into III S cells. This established beyond any doubt that DNA is the transforming principle.

But these findings of Avery and co-workers was not able to explain the molecular mechanism of transformation. So some biologists were unable to appreciate the significance of these findings and they were hesitant in accepting them as an incontrovertible evidence for DNA being the genetic material.

(c) The Experiment of Hershey-Chase:

Another direct evidence indicating that DNA is the genetic material was demonstrated by A. D. Hershey and M. Chase in 1952. They first studied the life cycle of T2 bacteriophages of Escherichia coli. T2 bacteriophages are composed of hexagonal box-like head coat and tail made of protein. The DNA is packed inside the proteinaceous head coat.

Bacteriophages are acellular and do not contain cytoplasm, organelles and nucleus. The DNA is present in high pure form and is not associated with RNA and protein. Bacteriophage are obligate parasite since they can reproduce only within bacterium using as host cell.

Hershey and Chase showed that, during the reproduction of bacteriophages, the DNA of the phage entered the host cell whereas most of the protein head and tail portion remained absorbed on the outside of the cell. Hence it is strongly implied that the genetic information necessary for viral reproduction was present in DNA.

DNA con­tains phosphorus (P) but no sulphur (S), while proteins of head and tail contains sulphur (S) but no phosphorus.

Hershey and Chase were able to specifically label the phage DNA by its growth in a medium containing the radioactive isotope of phosphorus, i.e., 32 P in place of normal phosphorus. Similarly, in another group of phage, the protein coats were labelled by growth in a medium containing radioactive sulphur 35 S in place of normal sulphur.

E. coli cells were then infected with 32 P labelled T2 bacteriophage and, after being allowed 10 minutes for infection, they were agitated in a blender which sheared off the phase coats. The phase coats and the infected cells were then separated by centrifugation (Fig. 12.3).

Radioactivity was then measured of the sed­iment and in phage coat suspension. Most of the radioactivity was found in the cells. When the same experiment was done using phage with 35 S-labelled protein coat, most of the radioactivity was found in the suspension of phage coats very little entered the host cells.

Since phage reproduction (both DNA synthesis) occurs inside the infected cells, and, since only the phage DNA enters the host cell, the DNA—not the protein—must carry the genetic information. As a result of the findings of Hershey and Chase led to the universal acceptance of DNA as the genetic material.

(d) Bacterial Conjugation:

Another direct evidence for DNA as the genetic material comes from the phenomenon of conju­gation of bacteria. Conjugation was discovered by J. Lederberg and E. I.

Tatum in 1946. During conjugation DNA is transferred from a donor bacterial cell to a recipient bacterial cell through conjugation tube that forms between them. The donor cell—also called male—contains a F factor or fertility factor whereas recipient cells—or fe­male cells, lack F factor, i.e., F – cell (Fig. 12.4).

In male, the F factor can exist in two dif­ferent states:

(2) Integrated state (Fig. 12.4) where the F factor is inserted with main DNA and thus the male become Hfr male (Fig. 12.5).

The F factor is a mini-circular DNA molecule. Beadle and Tatum observed that when a F + male E. coli cell conjugated with a F – female E. coli cell, an unidirectional transfer of F + factor of male cell to F – or female cell took place, so that the latter was covered into a F + or male strain.

The F factor is actually a fragment of DNA molecule that replicates during transfer. Thus mixing a population of F + or Hfr cells with a population of F + cells results in virtually all the cells in the new population becoming F + or Hfr (Fig. 12.6).

Ii. Indirect Evidence:

The fact that DNA is the genetic material of higher organisms has also been supported by some indirect evidences:

The genetic substance should have a fixed location within the cell. If it has no fixed location, then the genes are not able to function properly. It is known that the DNA, as a gene sub­stance, is always located primarily within the chromosome in the nucleus of the eukaryotic cell.

The specific location of DNA can be stud­ied in situ by the Feulgen reaction—which is re­garded as the most specific one for DNA. Feul­gen staining stains chromosome magenta colour against the clear cytoplasmic background. This technique has shown that DNA entirely remains restricted to the chromosome and it forms the major component of chromosomes.

Various macromolecules present within the cell are continuously being anabolised and catabolized. But this is not desirable for a genetic substance containing valuable hereditary information. If it happens, the genetic function will be lost. Of all the macro- molecules in the cell, DNA is the metabolically stable.

(c) Sensitivity to Mutagens:

Mutation is an important characteristic feature of the genetic material. The agents capable of inducing mutation are called mutagens. Different types of radiation (UV-ray, X-ray, y-ray) and a variety of chemical compounds acts as mutagens. When the cells of an organism are treated with mutagens, they cause a change in the structure of gene.

Since genes are DNA segments, the gene mutation include changes in the number and arrangement of nucleotide. Sometimes muta­tion causes the breaks in the DNA molecule. The changes in the DNA structure ultimately reflect the changes of the organism’s hereditary character. Therefore sensitivity of DNA to mutagens is an indirect evidence for DNA being the genetical materials.

One of the striking features of the genetic ma­terial is the correlation between DNA content and the number of chromosome sets. Various quantitative assay methods have shown that diploid cells contain twice as much DNA as do haploid cells of the same species (Table 12.1).

Similarly, tetraploid and octaploid cells con­tains four times and eight times DNA as com­pared with DNA content of the haploid cells. Even the DNA content of sperm cells shows a correlation with the same or different tissues of different organisms (Table 12.2).

Thus the parallelism of behaviour in DNA and chromosome indirectly indicates that DNA is the genetic material.

(e) RNA as Genetic Material:

The genome of viruses may be DNA or RNA. Most of the plant viruses have RNA as their hereditary material. Fraenkel-Conrat (1957) conducted experiments on tobacco mosaic virus (TMV) to demonstrate that in some viruses RNA acts as genetic material.

TMV is a small virus composed of a single molecule of spring-like RNA encapsulated in a cylindrical protein coat. Different strains of TMV can be identified on the basis of differences in the chemical composition of their protein coats. By using the appropriate chemical treatments, proteins and RNA of RNV can be separated.

Moreover, these processes are reversible by missing the protein and RNA under appropriate conditions—reconstitution will occur yielding complete infective TMV particles. Fraenkel-Conrat and Singer took two differ­ent strains of TMV and separated the RNAs from protein coats, reconstituted hybrid viruses by mixing the proteins of one strain with the RNA of the second strain, and vice versa.

When the hybrid or reconstituted viruses were rubbed into live tobacco leaves, the progeny viruses produced were always found to be phenotypically and genotypically identical to the parental type from where the RNA had been isolated (Fig. 12.7). Thus the genetic information of TMV is stored in the RNA and not in the protein.


9 Most Important Properties of Genetic Code | Biology

The nucleotides of mRNA are arranged as a linear sequence of codons, each codon consisting of three successive nitrogenous bases, i.e., the code is a triplet codon. The concept of triplet codon has been supported by two types of point mutations: frame shift mutations and base substitutions.

(i) Frameshift mutations:

Evidently, the genetic message once initiated at a fixed point is read in a definite frame in a series of three letter words. The framework would be disturbed as soon as there is a deletion or addition of one or more bases.

When such frame shift mutations were intercrossed, then in certain combinations they produce wild type normal gene. It was concluded that one of them was deletion and the other an addition, so that the disturbed order of the frame due to mutation will be restored by the other (Fig. 38.26).

If in a mRNA molecule at a particular point, one base pair is replaced by another without any deletion or addition, the meaning of one codon containing such an altered base will be changed. In consequence, in place of a particular amino acid at a particular position in a polypeptide, another amino acid will be incorporated.

Image Courtesy : wolfson.huji.ac.il/expression/vector/genetic_code.jpg

For example, due to substitution mutation, in the gene for tryptophan synthetase enzyme in E. coli, the GGA codon for glycine becomes a missence codon AGA which codes for arginine. Missence codon is a codon which undergoes an alteration to specify another amino acid.

A more direct evidence for a triplet code came from the finding that a piece of mRNA containing 90 nucleotides, corresponded to a polypeptide chain of 30 amino acids of a growing haemoglobin molecule. Similarly, 1200 nucleotides of “satellite” tobacco necrosis virus direct the synthesis of coat protein molecules which have 372 amino acids.

2. The code is non-overlapping:

In translating mRNA molecules the codons do not overlap but are “read” sequentially (Fig. 38.27). Thus, a non-overlapping code means that a base in a mRNA is not used for different codons. In Figure 38.28, it has been shown that an overlapping code can mean coding for four amino acids from six bases.

However, in actual practice six bases code for not more than two amino acids. For example, in case of an overlapping code, a single change (of substitution type) in the base sequence will be reflected in substitutions of more than one amino acid in corresponding protein. Many examples have accumulated since 1956 in which a single base substitution results into a single amino acid change in insulin, tryptophan synthelase, TMV coat protein, alkaline phosphatase, haemoglobin, etc.

However, it has been shown that in the bacteriophage ɸ × l74 there is a possibility of overlapping the genes and codons (Barrel and coworkers, 1976 Sanger, et al., 1977).

3. The code is commaless:

The genetic code is commaless, which means that no codon is reserved for punctuations. It means that after one amino acid is coded, the second amino acid will be automatically, coded by the next three letters and that no letters are wasted as the punctuation marks (Fig. 38.29).

4. The code is non-ambiguous:

Non-ambiguous code means that a particular codon will always code for the same amino acid. In case of ambiguous code, the same codon could have different meanings or in other words, the same codon could code two or more than two different amino acids. Generally, as a rule, the same codon shall never code for two different amino acids.

However, there are some reported exceptions to this rule: the codons AUG and GUG both may code for methionine as initiating or starting codon, although GUG is meant for valine. Likewise, GGA codon codes for two amino acids glycine and glutamic acid.

5. The code has polarity:

The code is always read in a fixed direction, i.e., in the 5’→3′ direction. In other words, the codon has a polarity. It is apparent that if the code is read in opposite directions, it would specify two different proteins, since the codon would have reversed base sequence:

6. The code is degenerate:

More than one codon may specify the same amino acid this is called degeneracy of the code. For example, except for tryptophan and methionine, which have a single codon each, all other 18 amino acids have more than one codon. Thus, nine amino acids, namely phenylalanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid and cysteine, have two codons each. Isoleucine has three codons. Five amino acids, namely valine, proline, threonine, alanine and glycine, have four codons each. Three amino acids, namely leucine, arginine and serine, have six codons each (see Table 38.5).

The code degeneracy is basically of two types: partial and complete. Partial degeneracy occurs when first two nucleotides are identical but the third (i.e., 3′ base) nucleotide of the degenerate codons differs, e.g., CUU and CUC code for leucine, Complete degeneracy occurs when any of the four bases can take third position and still code for the same amino acid (e.g., UCU, UCC, UCA and UCG code for serine).

Degeneracy of genetic code has certain biological advantages. For example, it permits essentially the same complement of enzymes and other proteins to be specified by microorganisms varying widely in their DNA base composition. Degeneracy also provides a mechanism of minimising mutational lethality.

7. Some codes act as start codons:

In most organisms, AUG codon is the start or initiation codon, i.e., the polypeptide chain starts either with methionine (eukaryotes) or N- formylmethionine (prokaryotes). Methionyl or N-formylmethionyl-tRNA specifically binds to the initiation site of mRNA containing the AUG initiation codon. In rare cases, GUG also serves as the initiation codon, e.g., bacterial protein synthesis. Normally, GUG codes for valine, but when normal AUG codon is lost by deletion, only then GUG is used as initiation codon.

8. Some codes act as stop codons:

Three codons UAG, UAA and UGA are the chain stop or termination codons. They do not code for any of the amino acids. These codons are not read by any tRNA molecules (via their anticodons), but are read by some specific proteins, called release factors (e.g., RF-1, RF-2, RF-3 in prokaryotes and RF in eukaryotes). These codons are also called nonsense codons, since they do not specify any amino acid.

The UAG was the first termination codon to be discovered by Sidney Brenner (1965). It was named amber after a graduate student named Bernstein (= the German word for ‘amber’ and amber means brownish yellow) who help in the discovery of a class of mutations. Apparently, to give uniformity the other two termination codons were also named after colours such as ochre for UAA and opal or umber for UGA. (Ochre means yellow red or pale yellow opal means milky white and umber means brown). The existence of more than one stop codon might be a safety measure, in case the first codon fails to function.

9. The code is universal:

Same genetic code is found valid for all organisms ranging from bacteria to man. Such universality of the code was demonstrated by Marshall, Caskey and Nirenberg (1967) who found that E. coli (Bacterium), Xenopus laevis (Amphibian) and guinea pig (mammal) amino acyl-tRNA use almost the same code. Nirenberg has also stated that the genetic code may have developed 3 billion years ago with the first bacteria, and it has changed very little throughout the evolution of living organisms.

Recently, some differences have been discovered between the universal genetic code and mitochondrial genetic code (Table 38.6).

Table 38.6. Differences between the ‘universal genetic code’ and two mitochondrial genetic codes:

Codon Mammalian mitocondrial code Yeast mitochondrial code” “Universal Code
1. UGA Trp * Trp Stop
2. AUA Met Met Lie
3. CUA Leu Thr Leu
4. AGA Stop Arg Arg
5. AGG

* Italic type indicates that the code differs from the ‘universal’ code.


Contents

Pseudogenes are usually characterized by a combination of homology to a known gene and loss of some functionality. That is, although every pseudogene has a DNA sequence that is similar to some functional gene, they are usually unable to produce functional final protein products. [1] Pseudogenes are sometimes difficult to identify and characterize in genomes, because the two requirements of homology and loss of functionality are usually implied through sequence alignments rather than biologically proven.

  1. Homology is implied by sequence identity between the DNA sequences of the pseudogene and parent gene. After aligning the two sequences, the percentage of identical base pairs is computed. A high sequence identity means that it is highly likely that these two sequences diverged from a common ancestral sequence (are homologous), and highly unlikely that these two sequences have evolved independently (see Convergent evolution).
  2. Nonfunctionality can manifest itself in many ways. Normally, a gene must go through several steps to a fully functional protein: Transcription, pre-mRNA processing, translation, and protein folding are all required parts of this process. If any of these steps fails, then the sequence may be considered nonfunctional. In high-throughput pseudogene identification, the most commonly identified disablements are premature stop codons and frameshifts, which almost universally prevent the translation of a functional protein product.

Pseudogenes for RNA genes are usually more difficult to discover as they do not need to be translated and thus do not have "reading frames".

Pseudogenes can complicate molecular genetic studies. For example, amplification of a gene by PCR may simultaneously amplify a pseudogene that shares similar sequences. This is known as PCR bias or amplification bias. Similarly, pseudogenes are sometimes annotated as genes in genome sequences.

Processed pseudogenes often pose a problem for gene prediction programs, often being misidentified as real genes or exons. It has been proposed that identification of processed pseudogenes can help improve the accuracy of gene prediction methods. [2]

Recently 140 human pseudogenes have been shown to be translated. [3] However, the function, if any, of the protein products is unknown.

There are four main types of pseudogenes, all with distinct mechanisms of origin and characteristic features. The classifications of pseudogenes are as follows:

Processed Edit

In higher eukaryotes, particularly mammals, retrotransposition is a fairly common event that has had a huge impact on the composition of the genome. For example, somewhere between 30–44% of the human genome consists of repetitive elements such as SINEs and LINEs (see retrotransposons). [6] [7] In the process of retrotransposition, a portion of the mRNA or hnRNA transcript of a gene is spontaneously reverse transcribed back into DNA and inserted into chromosomal DNA. Although retrotransposons usually create copies of themselves, it has been shown in an in vitro system that they can create retrotransposed copies of random genes, too. [8] Once these pseudogenes are inserted back into the genome, they usually contain a poly-A tail, and usually have had their introns spliced out these are both hallmark features of cDNAs. However, because they are derived from an RNA product, processed pseudogenes also lack the upstream promoters of normal genes thus, they are considered "dead on arrival", becoming non-functional pseudogenes immediately upon the retrotransposition event. [9] However, these insertions occasionally contribute exons to existing genes, usually via alternatively spliced transcripts. [10] A further characteristic of processed pseudogenes is common truncation of the 5' end relative to the parent sequence, which is a result of the relatively non-processive retrotransposition mechanism that creates processed pseudogenes. [11] Processed pseudogenes are continually being created in primates. [12] Human populations, for example, have distinct sets of processed pseudogenes across its individuals. [13]

Non-processed Edit

Non-processed (or duplicated) pseudogenes. Gene duplication is another common and important process in the evolution of genomes. A copy of a functional gene may arise as a result of a gene duplication event caused by homologous recombination at, for example, repetitive sine sequences on misaligned chromosomes and subsequently acquire mutations that cause the copy to lose the original gene's function. Duplicated pseudogenes usually have all the same characteristics as genes, including an intact exon-intron structure and regulatory sequences. The loss of a duplicated gene's functionality usually has little effect on an organism's fitness, since an intact functional copy still exists. According to some evolutionary models, shared duplicated pseudogenes indicate the evolutionary relatedness of humans and the other primates. [14] If pseudogenization is due to gene duplication, it usually occurs in the first few million years after the gene duplication, provided the gene has not been subjected to any selection pressure. [15] Gene duplication generates functional redundancy and it is not normally advantageous to carry two identical genes. Mutations that disrupt either the structure or the function of either of the two genes are not deleterious and will not be removed through the selection process. As a result, the gene that has been mutated gradually becomes a pseudogene and will be either unexpressed or functionless. This kind of evolutionary fate is shown by population genetic modeling [16] [17] and also by genome analysis. [15] [18] According to evolutionary context, these pseudogenes will either be deleted or become so distinct from the parental genes so that they will no longer be identifiable. Relatively young pseudogenes can be recognized due to their sequence similarity. [19]

Unitary pseudogenes Edit

Various mutations (such as indels and nonsense mutations) can prevent a gene from being normally transcribed or translated, and thus the gene may become less- or non-functional or "deactivated". These are the same mechanisms by which non-processed genes become pseudogenes, but the difference in this case is that the gene was not duplicated before pseudogenization. Normally, such a pseudogene would be unlikely to become fixed in a population, but various population effects, such as genetic drift, a population bottleneck, or, in some cases, natural selection, can lead to fixation. The classic example of a unitary pseudogene is the gene that presumably coded the enzyme L-gulono-γ-lactone oxidase (GULO) in primates. In all mammals studied besides primates (except guinea pigs), GULO aids in the biosynthesis of ascorbic acid (vitamin C), but it exists as a disabled gene (GULOP) in humans and other primates. [20] [21] Another more recent example of a disabled gene links the deactivation of the caspase 12 gene (through a nonsense mutation) to positive selection in humans. [22]

It has been shown that processed pseudogenes accumulate mutations faster than non-processed pseudogenes. [23]

Pseudo-pseudogenes Edit

The rapid proliferation of DNA sequencing technologies has led to the identification of many apparent pseudogenes using gene prediction techniques. Pseudogenes are often identified by the appearance of a premature stop codon in a predicted mRNA sequence, which would, in theory, prevent synthesis (translation) of the normal protein product of the original gene. There have been some reports of translational readthrough of such premature stop codons in mammals. As alluded to in the figure above, a small amount of the protein product of such readthrough may still be recognizable and function at some level. If so, the pseudogene can be subject to natural selection. That appears to have happened during the evolution of Drosophila species.

In 2016 it was reported that 4 predicted pseudogenes in multiple Drosophila species actually encode proteins with biologically important functions, [24] "suggesting that such 'pseudo-pseudogenes' could represent a widespread phenomenon". For example, the functional protein (an olfactory receptor) is found only in neurons. This finding of tissue-specific biologically-functional genes that could have been classified as pseudogenes by in silico analysis complicates the analysis of sequence data. In the human genome, a number of examples have been identified that were originally classified as pseudogenes but later discovered to have a functional, although not necessarily protein-coding, role. [25] [26] As of 2012, it appeared that there are approximately 12,000–14,000 pseudogenes in the human genome, [27] A 2016 proteogenomics analysis using mass spectrometry of peptides identified at least 19,262 human proteins produced from 16,271 genes or clusters of genes, with 8 new protein-coding genes identified that were previously considered pseudogenes. [28]

Drosophila glutamate receptor. The term "pseudo-pseudogene" was coined for the gene encoding the chemosensory ionotropic glutamate receptor Ir75a of Drosophila sechellia, which bears a premature termination codon (PTC) and was thus classified as a pseudogene. However, in vivo the D. sechellia Ir75a locus produces a functional receptor, owing to translational read-through of the PTC. Read-through is detected only in neurons and depends on the nucleotide sequence downstream of the PTC. [24]

siRNAs. Some endogenous siRNAs appear to be derived from pseudogenes, and thus some pseudogenes play a role in regulating protein-coding transcripts, as reviewed. [29] One of the many examples is psiPPM1K. Processing of RNAs transcribed from psiPPM1K yield siRNAs that can act to suppress the most common type of liver cancer, hepatocellular carcinoma. [30] This and much other research has led to considerable excitement about the possibility of targeting pseudogenes with/as therapeutic agents [31]

piRNAs. Some piRNAs are derived from pseudogenes located in piRNA clusters. [32] Those piRNAs regulate genes via the piRNA pathway in mammalian testes and are crucial for limiting transposable element damage to the genome. [33]

microRNAs. There are many reports of pseudogene transcripts acting as microRNA decoys. Perhaps the earliest definitive example of such a pseudogene involved in cancer is the pseudogene of BRAF. The BRAF gene is a proto-oncogene that, when mutated, is associated with many cancers. Normally, the amount of BRAF protein is kept under control in cells through the action of miRNA. In normal situations, the amount of RNA from BRAF and the pseudogene BRAFP1 compete for miRNA, but the balance of the 2 RNAs is such that cells grow normally. However, when BRAFP1 RNA expression is increased (either experimentally or by natural mutations), less miRNA is available to control the expression of BRAF, and the increased amount of BRAF protein causes cancer. [34] This sort of competition for regulatory elements by RNAs that are endogenous to the genome has given rise to the term ceRNA.

PTEN. The PTEN gene is a known tumor suppressor gene. The PTEN pseudogene, PTENP1 is a processed pseudogene that is very similar in its genetic sequence to the wild-type gene. However, PTENP1 has a missense mutation which eliminates the codon for the initiating methionine and thus prevents translation of the normal PTEN protein. [35] In spite of that, PTENP1 appears to play a role in oncogenesis. The 3' UTR of PTENP1 mRNA functions as a decoy of PTEN mRNA by targeting micro RNAs due to its similarity to the PTEN gene, and overexpression of the 3' UTR resulted in an increase of PTEN protein level. [36] That is, overexpression of the PTENP1 3' UTR leads to increased regulation and suppression of cancerous tumors. The biology of this system is basically the inverse of the BRAF system described above.

Potogenes. Pseudogenes can, over evolutionary time scales, participate in gene conversion and other mutational events that may give rise to new or newly functional genes. This has led to the concept that pseudogenes could be viewed as potogenes: potential genes for evolutionary diversification. [37]

Sometimes genes are thought to be pseudogenes, usually based on bioinformatic analysis, but then turn out to be functional genes. Examples include the Drosophila jingwei gene [38] [39] which encodes a functional alcohol dehydrogenase enzyme in vivo. [40]

Another example is the human gene encoding phosphoglycerate mutase [41] which was thought to be a pseudogene but which turned out to be a functional gene, [42] now named PGAM4. Mutations in it cause infertility. [43]

Pseudogenes are found in bacteria. [44] Most are found in bacteria that are not free-living that is, they are either symbionts or obligate intracellular parasites. Thus, they do not require many genes that are needed by free-living bacteria, such as gene associated with metabolism and DNA repair. However, there is not an order to which functional genes are lost first. For example, the oldest pseudogenes in Mycobacterium laprae are in RNA polymerases and the biosynthesis of secondary metabolites while the oldest ones in Shigella flexneri and Shigella typhi are in DNA replication, recombination, and repair. [45]

Since most bacteria that carry pseudogenes are either symbionts or obligate intracellular parasites, genome size eventually reduces. An extreme example is the genome of Mycobacterium leprae, an obligate parasite and the causative agent of leprosy. It has been reported to have 1,133 pseudogenes which give rise to approximately 50% of its transcriptome. [45] The effect of pseudogenes and genome reduction can be further seen when compared to Mycobacterium marinum, a pathogen from the same family. Mycobacteirum marinum has a larger genome compared to Mycobacterium laprae because it can survive outside the host, therefore, the genome must contain the genes needed to do so. [46]

Although genome reduction focuses on what genes are not needed by getting rid of pseudogenes, selective pressures from the host can sway what is kept. In the case of a symbiont from the Verrucomicrobia phylum, there are seven additional copies of the gene coding the mandelalide pathway. [47] The host, species from Lissoclinum, use mandelalides as part of its defense mechanism. [47]

The relationship between epistasis and the domino theory of gene loss was observed in Buchnera aphidicola. The domino theory suggests that if one gene of a cellular process becomes inactivated, then selection in other genes involved relaxes, leading to gene loss. [48] When comparing Buchnera aphidicola and Escherichia coli, it was found that positive epistasis furthers gene loss while negative epistasis hinders it.


Top 3 Fundamental Laws of Genetics

The following points highlight the three fundamental laws of genetics proposed by Mendel. The laws are: 1. Law of Segregation 2. Law of Dominance 3. Law of Independent Assortment and Di-Hybrid Cross.

1. Law of Segregation:

According to Altenburg, this law may be defined as “Non-mixing of alleles i.e., the allele for tallness does not mix with the allele for dwarfness in the hybrids.” Offspring’s arising from two parents receive contributions of hereditary characteristics from them through gametes. These gametes are the connecting links between successive generations.

The contrasting characters such as tall and dwarf stems of peas are determined by something that is transmitted from the parents to the offspring through the gametes are called factors or genes. The important point is that different factors such as those for tallness and dwarfness (D and d) do not blend, contaminate or mix with each other while they remain together in the hybrid.

Instead, the different factors separate or segregate pure and uncontaminated passing to two different gametes produced by the hybrid and then transmit to the different individuals or the offspring’s of the hybrid. Each gamete carries one of the two members of a pair of contrasting or alternative factors i.e., either for tallness or dwarfness (D or d) and never both.

D d (F1 hybrid tall) → factor D and d remain together pure

The simplest conventional or custom method of denoting these Mendelian factors is to give each a letter, the dominant factor being represented by capital letter and recessive by small letter. In the cross of pure bred tall and dwarf plants let D stand for the gene for tallness and d for alternate form of this gene which results in dwarfness of the stem. D and d are called alleles or allelomorphs.

Since an individual develops from the union of two gametes produced by the male and female parents. It receives two alleles D and d. The true breeding tall plant may be represented as DD and its gamete as D and the true breeding dwarf plant as dd and its gamete as d.

When the two plants are crossed an egg (D) is fertilized by the male gamete (d) or vice- versa. The resulting hybrid zygote will have both D and d. Thus the two alleles of a gene are represented by the same gene symbol and are differentiated from each other by their first letter being in capital or small (D or d).

A gene can be represented by a symbol derived from the name of the character it governs. The gene controlling length of stem as dwarf in pea may be represented by the small letter ‘d’ and the symbol for the allele producing the dominant form of character is the same as that for the recessive allele, but the first letter of this symbol is in capital. For example, tall stem is dominant and is assigned D

According to the principle of segregation the alleles borne by the heterozygous tall plant (Dd) do not mix, fuse, blend or contaminate with each other, despite the fact that the phenotype of the F1 hybrid shows only the tall character, and it fails to give any visible indication of the presence of the gene (d) in the genotype. The alleles segregate when the hybrid organism produces gametes so that approximately half of the gametes will carry D and the other half d.

In fertilization the gametes combines at random. There is an equal opportunity for the different types of gametes to unite with each other. The male gamete may unite or fuse with female gamete with either D or d. The other kind of male gamete ‘d’ may also have an equal opportunity to unite or fuse with the female gamete D or d. Hence four recombination’s occur. One fourth (1/4) of them are homozygous tall plants having only the allele for tallness (DD).

The other half of them (two out of four) are heterozygous having both the alleles D and d. Since D is dominant over d, these plants are tall. One fourth (1/4) of them are homozygous plants having only the allele for dwarfness (dd). In F2 generation, tall and dwarf plants appear in the ratio 3 : 1 (3/4 tall and 1/4 dwarf plants).

Mendel tested the validity of factor hypothesis by applying further strict method by means of which it could be confirmed or disproved. In the F2 of his cross of tall plants with dwarf plants there were tall and dwarf plants approximately in the ratio of 3:1. Mendel’s interpretation of these results by means of the law of segregation shows that there are two kinds of F2 tall plants.

About 1/3 of them should be genotypically homozygous for tallness (DD). About 2/3 should be heterozygous (Dd) carrying both the dominant and recessive alleles (D and d). The validity of these predictions can be tested in actual experiments. The homozygous dwarf plants should breed true through all subsequent generations if self- fertilized or crossed with other.

All the plants although they look alike would not behave in the same way. About 1/3 of them homozygous with the genetic formula (DD) should breed true. But 2/3 of the F2 tall plants, the heterozygotes (Dd) should breed exactly like the F1 hybrid plants. They should produce tall and dwarf plants in the phenotypic ratio 3: 1 and the genotypic ratio 1:2:1. This is what Mendel obtained in his experiments. Thus the law of segregation has been confirmed in actual experiments.

Characters become separated or segregated in the second filial (F2) generation. Thus the factors responsible for hereditary characters are independent units, which although enter the crosses together but segregate out again as distinct characters. This law is by far the most important of Mendel’s discoveries. This law is some times called as the law of purity of gametes or law of the splitting of hybrids.

(Law of segregation means that when a pair of allelomorphs are brought together in the hybrid (F1), they remain together in the hybrid without blending and in F2 generation they separate complete and pure during gamete formation. This law is also known as law of the purity of gametes).

(The two alleles present in the F1 are able to separate and pass in to separate gametes in their original form producing two different types of gametes in equal frequencies this is known as segregation).

Main facts about segregations:

To summarize Mendel’s monohybrid cross experiment, following cardinal points are notable:

The hereditary differences among the individuals depend upon the difference in cellular units of genes or factors. These genes are hereditary units, control a particular character and are present at a fixed place in the chromosomes called loci. Thus genes for tall character in the peas shown by ‘D’ in chromosome is at a fixed locus and genes for dwarf character ‘d’ is at the same locus in the other chromosome.

Law of segregation itself shows the purity of gametes and their freedom from mixing or blending with each other. The gametes contain only one factor or gene and are pure for a particular trait or character governed by the same factor or gene of gamete.

3. Non-mixing of alleles in hybrids:

These genes or factors of heredity, whatever the nature may be, unite when derived from different parental sources in the hybrids from which they may be separated out during successive or subsequent generation and unmodified with the presence of other alleles in hybrids.

In summary, the cross between tall and dwarf pea is as follows:

The original tall and dwarf variety of pea constitute the first parental generation (P1). The hybrids produced by their cross constitute first filial generation (F1) and offspring of the hybrids constitute second filial or F2 generation.

Johansen (1911) proposed the following four terms to distinguish individuals among themselves:

An organism or hybrid or zygote in which both members of a pair of genes are alike (DD or dd) are referred to as homozygous (Greek: Homos = alike = zygos, yoke (bond or under bondage of another).

Individuals having identical genes (DD or dd) are called homozygous. Homozygous are always pure.

An organism or hybrid or zygote in which both members of a pair of genes are unlike (Dd) are termed as heterozygous (heteros = dissimilar). Heterozygous individuals are always hybrid. In the F2 generation, there is a ratio of 3 tall and 1 dwarf plant apparently but genetically, this ratio is 1 DD tall: 2 Dd tall: 1 dd dwarf.

3. Genotype and Phenotype:

Genotype is the term used to denote genetic constitution of an organism. It represents the total hereditary possibilities within the individual. In the monohybrid cross experiments, the hybrid plant of F1 generation is phenotypically tall but genetically it is a hybrid (Dd).

The external morphological feature of an organism constitute its phenotype or it is the term used to denote the visible characteristics of an organism or individual. It represents the sum total of all apparent characteristics of an organism regardless of it genetic make up or genotype.

In the F2 generation, 3 out of 4 (3/4) are phenotypically tall but genotypically one third (1/3) of them is pure tall and two third (2/3) hybrid tall with two contrasting allele.

What we observe or which is visible or otherwise measurable are called phenotypes. While the genetic factors responsible for creating the phenotype are called genotype. Phenotype is determined by the dominant alleles.

Monohybrid Back cross or Test Cross:

The cross between the F1 hybrid (Dd) to one of its parents (DD or dd) is called back cross while cross between F1 hybrid (Dd) and homozygous recessive parent (dd) is called test cross since it confirms the purity of gametes.

(i) The above cross between homozygous dominant (DD) and hybrid (Dd) is called dominant back cross and (ii) Cross between homozygous recessive (dd) and hybrid (Dd) is called recessive back cross. This recessive back cross has great importance in experimentation because phenotypic and genotypic ratios are identical. Hence recessive back cross is termed test cross to identify or test gamete nature or whether an individual is homozygous or heterozygous as shown below.

In case of Back cross:

Diagram showing Monohybrid back cross between F1 hybrid and dominant homozygous parent

Phenotype – 2 Tail: 2 dwarf (50% tall and 50% dwarf)

Genotype – 2 Tall: 2 dwarf (50% tall and 50% dwarf)

Diagram showing Monohybrid test cross between F1 hybrid and recessive homozygous parent (1 : 1).

2. Law of Dominance:

Mendel’s first experiments were crosses between pea varieties differing in only one visible character. These are monohybrid cross experiments.

A heterozygote (F1 hybrid) contains two contrasting genes, but only one of the two is able to express itself, while the other remain hidden. The gene which is able to express itself in F1 hybrid is known as dominant gene, while the other gene which is unable to express itself in presence of the dominant gene is the recessive gene. No doubt recessive gene is unable to express itself, but is transmitted to the next generation without change.

When Mendel crossed true breeding tall peas, with true breeding dwarf peas the first offspring’s formed were all tall plants.

The dwarf character appears to have been suppressed and tallness seems to dominate. Such characters like tallness, redness, roundness of seeds, yellow coloured cotyledons, inflated seed pods, green unripe pods and axial flowers, were called dominants and their respective alleles as dwarfness, whiteness, wrinkledness of seeds, green coloured cotyledons, constricted seed pods, yellow unripe pods and terminal flowers were called recessives.

The law of dominance, thus states that out of a pair of a allelomorphic characters (= alternative or contrasting characters) one is dominant and other recessive. Mendel found this fact to be true between all the seven pairs of characters studied by him. The pair of contrasting or alternative characters are called allelic pair or allelomorphic pair and each member of the pair may be regarded the allele of the other.

Thus the tallness and dwarfness are alleles of each other. The hereditary units which are responsible for the appearance of character in the offsprings or progenies have been called factors or determiners. Now these are called genes.

Four types of dominance are seen:

The phenomenon in which both alleles are expressed in the hybrid (F1) is called co-dominance. Blood group antigens of man is one of the best example of Co-dominance. It produces 1:2:1 ratio in F2.

2. Complete dominance or simple dominance:

It is the ability of one allele to mask or inhibit the presence of another allele at the same locus in the heterozygote or F1 hybrid.

3. Incomplete dominance:

If the F1 hybrids or heterozygotes are phenotypically intermediate between both homozygous type.

4. Over dominance:

The superiority of heterozygote or hybrid over its both homozygotes or parents (DD and dd) is termed as over dominance. Unlike complete, partial and co-dominance, over-dominance is not the characteristics of an allele but is the consequence of the heterozygous condition of the related gene.

3. Law of Independent Assortment and Di-Hybrid Cross:

Mendel discovered not only crosses in which the parent differed in single pair or characters, but also others in which the parent differed in two pairs. Such a cross, which includes two pair of contrasting characters at a time is called di-hybrid cross. The law of independent assortment is applicable to the inheritance of two or more pair of characters.

For a di-hybrid experiment, Mendel crossed two pea plants, one of which was homozygous for yellow and round seeds and the other for green and wrinkled seeds. Genes for yellow and round characters were dominant over the green and wrinkled characters described by the Mendel. The F1 hybrids produced as a result of this cross were yellow round which were heterozygous for both the alleles known as Di-hybrid.

Genotypes and Phenotypes of F2 offsprings:


The above phenotypic ratio, which Mendel obtained may be thought of as a monohybrid phenotypic ratio 3 : 1 multiplied algebraically by 3 : 1 that means (3: 1) x (3: 1) = 9: 3: 3: 1.

Although Mendel was not aware with the behaviour of chromosomes during meiosis even then he assumed that the members of each two pairs of factors (WW, ww) for the two pairs of contrasting characters (round/wrinkled) are separated independently or freely of the members of the other pair.

In brief, according to Mendel at the time of reduction division during gamete formation, the members of each chromosome (= genes or factors) pair segregate (or separate) from one another.

They do not dilute or affect the other pair and behave independently. The separation of chromosomes or genes belonging to one pair without reference to those belonging to the other pair at reduction division is known as independent assortment (or separation) of genes.

The dihybrid (GgWw) produces four kinds of gametes (parental or non-parental types or crossover or non-crossover types) namely GW, Gw, gW, gw which by self fertilization produced F2 generation in 16 possible ways. Since G (Yellow) and W (round) are dominant characters so whatever genes (G or W) will be, the seeds will show dominant characters.

Genotypically, typical di-hybrid will show following ratio:

1GGWW : 2 GgWW : 2 GGWw : 4 GgWw : 1 ggWW : 2 ggWw : 1 GGww : 2 Ggww : 1 ggww. Their phenotypic ratio will be 9 Yellow round: 3 Yellow wrinkled: 3 Green round: 1 Green wrinkled.

Fractional Method of Calculated Ratio:

The checker board method of determining Mendelian ratio given by Punnet is useful in certain aspects. It represents graphically all the essential steps like formation of gametes, their union to form zygotes and resulting phenotypes. But its disadvantage is that it is time consuming and many other errors may come in it. Therefore, M.D. Jones (1947) described fractional method to determine ratios which is algebraic in nature.

(ii) F2 di-hybrid phenotypes:

The genotypic ratios may be obtained by dividing the dominants in to homo and heterozygotes i.e.,

If we cross the di-hybrid (GgWw) with the homozygous recessive parent (ggww) then di-hybrid will produce four types of gametes (GW, Gw, gW, gw) while green wrinkled seeds will form only one type of gamete (gw).

This gamete becomes fused with four types of gametes thus producing four classes of offsprings as follows:

1 Yellow round: 1 Yellow wrinkled: 1 Green round: 1 Green wrinkled

Thus, a dihybrid test cross will give a genotypic and phenotypic ratio of 1: 1: 1: 1 because four different types of gametes will be produced by the F1 hybrid in equal numbers.

In case of di-hybrid cross, Mendel demonstrated the independent assortment (or segregation) of factors or genes. Likewise, tri-hybrid experiments were carried out by Mendel involving three pairs of characters.

For instance, he took yellow round grey seeds and crossed them with green wrinkled white seeds, the F1 progeny will be heterozygous for three genes and will phenotypically resemble the dominant parent. Each of these F1 progeny will produce 8 types of gametes and therefore 64 combinations of F2 progeny.

Results of tri-hybrid cross worked out by the forked line method:

Genotypes of F2 and their relative proportions:

Phenotypes of F2 and their relative proportions:

A tri-hybrid test cross will give a phenotypic and genotypic ratio of 1: 1: 1: 1: 1: 1: 1: 1, because 8 different types of gametes and in equal numbers will be produced by the F1 hybrid. Test crosses are of great importance since they yield or produce same genotypic and phenotypic ratios.

It is obvious from foregoing descriptions that the number of heterozygous genes involved in a cross increases the number of types of gametes and the number of types of F2 progeny.

Phenotypes GgWwCc, GgWwcc, GgwwCc, Ggwwcc, ggWwCc, ggWwcc, ggwwCc, ggwwcc.


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