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GENETICS

(19UBT02)

By

Dr. B.Jayanthi, M.Sc., Ph.D.,

Mrs. T. Subha, M.Sc., M.Phil.,

HISTORY OF CLASSICAL AND MODERN GENETICS

GENETICS - Genetics is the scientific study of genes, heredity and variation. A hereditary determinant of a trait is called a gene. CLASSICAL GENETICS

Classical genetics was the genetics that predated molecular biology. Scientists in those days had no ability to enzymatically manipulate DNA or synthesize new DNA, so all studies had to be conducted by breeding experiments.

Once people realized that the factors that influenced traits could be physically linked to each other (which violated the assumptions of Mendelian genetics), there was a lot of time spent making purposeful crosses to conduct recombination mapping.

Classical genetics is the branch of genetics based solely on visible results of reproductive acts. It is the oldest discipline in the field of genetics, going back to the experiments on Mendelian inheritance by Gregor Mendel who made it possible to identify the basic mechanisms of heredity. Subsequently, these mechanisms have been studied and explained at the molecular level.

Classical genetics consists of the techniques and methodologies of genetics that were in use before the advent of molecular biology. A key discovery of classical genetics in eukaryotes was genetic linkage. The observation that some genes do not segregate independently at meiosis broke the laws of Mendelian inheritance, and provided science with a way to map characteristics to a location on the chromosomes. Linkage maps are still used today, especially in breeding for plant improvement.

After the discovery of the genetic code and such tools of cloning as restriction enzymes, the avenues of investigation open to geneticists were greatly broadened. Some classical genetic ideas have been supplanted with the mechanistic understanding brought by molecular discoveries, but many remain intact and in use. Classical genetics is often contrasted with reverse genetics, and aspects of molecular biology are sometimes referred to as molecular genetics.

and encoded by each gene is referred to as a trait. Many organisms possess two genes for each individual trait that is present within that particular individual. These paired genes that control the same trait is classified as an allele. In an individual, the allelic genes that are expressed can be either homozygous, meaning the same, or heterozygous, meaning different. Many pairs of alleles have differing effects that are portrayed in an offspring's phenotype and genotype. The phenotype is a general term that defines an individual's visible, physical traits. The genotype of an offspring is known as its genetic makeup. The alleles of genes can either be dominant or recessive. A dominant allele needs only one copy to be expressed while a recessive allele needs two copies (homozygous) in a diploid organism to be expressed. Dominant and recessive alleles help to determine the offspring's genotypes, and therefore phenotypes.  History  Classical genetics is often referred to as the oldest form of genetics, and began with Gregor Mendel's experiments that formulated and defined a fundamental biological concept known as Mendelian inheritance. Mendelian inheritance is the process in which genes and traits are passed from a set of parents to their offspring. These inherited traits are passed down mechanistically with one gene from one parent and the second gene from another parent in sexually reproducing organisms. This creates the pair of genes in diploid organisms. Gregor Mendel started his experimentation and study of inheritance with phenotypes of garden peas and continued the experiments with plants. He focused on the patterns of the traits that were being passed down from one generation to the next generation. This was assessed by test-crossing two peas of different colors and observing the resulting phenotypes. After determining how the traits were likely inherited, he began to expand the amount of traits observed and tested and eventually expanded his experimentation by increasing the number of different organisms he tested.  About 150 years ago, Gregor Mendel published his first experiments with the test crossing of Pisum peas. Seven different phenotypic characteristics were studied and tested in the peas, including seed color, flower color and seed shape. Mendel took peas that had differing phenotypic characteristics and test-crossed them to assess how the parental plants passed the traits down to their offspring. He started by crossing a round, yellow and round, green pea and observed the resulting phenotypes. The results of this

experiment allowed him to see which of these two traits was dominant and which was recessive based upon the number of offspring with each phenotype. Mendel then chose to further his experiments by crossing a pea plant homozygous dominant for round and yellow phenotypes with a pea plant that was homozygous recessive for wrinkled and green. The plants that were originally crossed are known as the parental generation, or P generation, and the offspring resulting from the parental cross is known as the first filial, or F1, generation. The plants of the F1 generation resulting from this hybrid cross were all heterozygous round and yellow seeds.  Classical genetics is a hallmark of the start of great discovery in biology, and has led to increased understanding of multiple important components of molecular genetics, human genetics, medical genetics, and much more. Thus, reinforcing Mendel's nickname as the father of modern genetics.  Genetic linkage.  Back then, the only way to ensure the genotype of a particular individual was to generate inbred lines for the traits you wanted (ensuring they were homozygous for the allele) and then performing the requisite crosses to generate the genotype you wanted.  Basically, classical genetics took forever, and really sucked. E.g. most plants reproduce once every 6 months at best, so creating a single inbred line of 6 generations took 3 years. And that was before you started experiments. You had to house and feed the organisms for all that time, keep track of hundreds of individuals, and keep them separate so they don't mate in a way you don't want them to (i.e. need to put bags on every flower in the greenhouse so they don't accidentally pollinate, then do pollination by hand...)  MODERN GENETICS In modern molecular genetics, we can easily manipulate and "read" DNA, first through restriction enzymes, then through de novo DNA synthesis and modern synthetic biology techniques like gibson assembly. We can sequence DNA via PCR and sanger sequencing from any organism, or sequence entire genomes through next-gen sequencing. This means that we no longer need to make inbred lines to know that an individual is homozygous, we can just sequence them. In model systems where we can insert in foreign DNA into the host genome, we can synthesize completely novel pieces

inheritance. In 1928, Frederick Griffith discovered the phenomenon of transformation (see Griffith's experiment): dead bacteria could transfer genetic material to "transform" other still-living bacteria. Sixteen years later, in 1944, the Avery–MacLeod–McCarty experiment identified DNA as the molecule responsible for transformation.[22]^ The role of the nucleus as the repository of genetic information in eukaryotes had been established by Hämmerling in 1943 in his work on the single celled alga Acetabularia .[23]^ The Hershey– Chase experiment in 1952 confirmed that DNA (rather than protein) is the genetic material of the viruses that infect bacteria, providing further evidence that DNA is the molecule responsible for inheritance.[24]  James Watson and Francis Crick determined the structure of DNA in 1953, using the X- ray crystallography work of Rosalind Franklin and Maurice Wilkins that indicated DNA has a helical structure (i.e., shaped like a corkscrew).[25][26]^ Their double-helix model had two strands of DNA with the nucleotides pointing inward, each matching a complementary nucleotide on the other strand to form what look like rungs on a twisted ladder.[27]^ This structure showed that genetic information exists in the sequence of nucleotides on each strand of DNA. The structure also suggested a simple method for replication: if the strands are separated, new partner strands can be reconstructed for each based on the sequence of the old strand. This property is what gives DNA its semi- conservative nature where one strand of new DNA is from an original parent strand.[28]  Although the structure of DNA showed how inheritance works, it was still not known how DNA influences the behavior of cells. In the following years, scientists tried to understand how DNA controls the process of protein production.[29]^ It was discovered that the cell uses DNA as a template to create matching messenger RNA, molecules with nucleotides very similar to DNA. The nucleotide sequence of a messenger RNA is used to create an amino acid sequence in protein; this translation between nucleotide sequences and amino acid sequences is known as the genetic code.[30]  With the newfound molecular understanding of inheritance came an explosion of research.[31]^ A notable theory arose from Tomoko Ohta in 1973 with her amendment to the neutral theory of molecular evolution through publishing the nearly neutral theory of molecular evolution. In this theory, Ohta stressed the importance of natural selection and the environment to the rate at which genetic evolution occurs.[32]^ One important

development was chain-termination DNA sequencing in 1977 by Frederick Sanger. This technology allows scientists to read the nucleotide sequence of a DNA molecule.[33]^ In 1983, Kary Banks Mullis developed the polymerase chain reaction, providing a quick way to isolate and amplify a specific section of DNA from a mixture.[34]^ The efforts of the Human Genome Project, Department of Energy, NIH, and parallel private efforts by Celera Genomics led to the sequencing of the human genome in 2003.

Genetic Terminologies (2 marks)

1. Heredity - passing of traits from parent to offspring
2. Character: A heritable feature (skin color, height etc) or any characteristic that can be passed from parent to offspring
3. Trait: variant for a character (i.e. brown, black, white etc).
4. Genotype: is the actual genetic code that alleles or genes possess or gene combination for a trait. The genotype is the genetic constitution of an organism or cell. E.g. Tt or TT or tt With respect to Plant height in peas, TT, Tt, and tt are examples of the possible genotypes for the T and t alleles. Because gametes contain only one allele of each gene, T and t are examples of genotypes of gametes. A genotype in which the members of a pair of alleles are different, as in the Tt hybrids is said to be heterozygous ; a genotype in which the two alleles are alike is said to be homozygous. A homozygous organism may be homozygous dominant (TT) or homozygous recessive (tt). The terms homozygous and heterozygous cannot apply to gametes, which contain only one allele of each gene.
5. Phenotype is the physical feature resulting from a genotype (e.g. red, white), the observable or detectable characteristics/properties of an individual organism--the observable expression of a genotype. Round seeds, wrinkled seeds, yellow seeds, and green seeds are all phenotypes. The phenotype of an organism does not necessarily tell you anything about its genotype. For example, a seed with the phenotype "round" could have the genotype AA or Aa.
6. Genotype & Phenotype in Flowers Genotype of alleles: R = red flower r = yellow flower

14. Probability: the likelihood that a specific event will occur. Probability is usually expressed as the ratio of the number of actual occurrences to the number of possible occurrences.
15. True-breed: all offspring of same variety.

TYPES OF GENETIC CROSSES Pure Cross: A cross between a true breed plant/animal with another true breeds plant/animal is called pure cross True breeding X True breeding. Eg. WW X ww MONOHYBRID CROSS - Cross involving a single trait e.g. seed color

DIHYBRID CROSS - When a cross is made between two parents differing in two characters, the cross is known as dihybrid cross. When round yellow plants were crossed with wrinkled green plants all the F 1 were Round Yellow. When F 1 were selfed the F 2 offspring produced were in the ratio of 9:3:3:1.

Cross involving two different or contrasting traits e.g. flower color & plant height (Refer Law of independent assortment for example)

BACK CROSS - Another valuable type of cross is a backcross, in which hybrid organisms are crossed with one of the parental genotypes. Backcrosses are commonly used by geneticists and by plant and animal breeders, Generation “Gap” Parental P1 Generation = the parental generation in a breeding experiment. F1 generation = the first-generation offspring in a breeding experiment. (1st filial generation) From breeding individuals from the P1 generation F2 generation = the second-generation offspring in a breeding experiment. (2nd filial generation) From breeding individuals from the F1 generation Genes and Environment Determine Characteristics Mendel‘s Pea Plant Experiments: Why peas, Pisum sativum  Can be grown in a small area  Produce lots of offspring  Produce pure plants when allowed to self-pollinate several generations  Can be artificially cross-pollinated Reproduction in Flowering Plants  Pollen contains sperm produced by the stamen  Ovary contains eggs found inside the flower  Pollen carries sperm to the eggs for fertilization  Self-fertilization can occur in the same flower  Cross-fertilization can occur between flowers Eight Pea Plant Traits  Seed shape --- Round (R) or Wrinkled (r)  Seed Color ---- Yellow (Y) or Green (y)  Pod Shape --- Smooth (S) or wrinkled (s)  Pod Color --- Green (G) or Yellow (g)  Seed Coat Color ---Gray (G) or White (g)  Flower position ---Axial (A) or Terminal (a)  Plant Height --- Tall (T) or Short (t)  Flower color --- Purple (P) or white (p)

 Such a long DNA molecule must be greatly folded to be packaged in a small space of 1. x 0.65 μm. The bacterial chromosome is folded into loops or domains which are about 100 in number. A chromosomal domain may be defined as a discrete structural entity within which supercoiling is independent of the other domains. Thus different domains can maintain different degrees of supercoiling. The DNA chain is coiled on itself to

produce supercoiling (Fig. 5.26). The ends of the loops or domains are bound in some way which does not allow rotational events to propagate from one domain to another.  If an endonuclease puts a nick in DNA strand of one domain, this loop becomes larger due to the uncoiling, but the other domains are not affected. Each domain contains about 40 kbp (13 μm) of DNA. The loops are bound by some mechanism that may involve proteins and/or RNA but the mechanism is not clearly understood. In E. coli, a number of proteins have been isolated which have some similarities with the eukaryotic chromosomal proteins. These proteins are HU, IHF (integration host factor). HI (H-NS) and R It is suspected that HU is involved in the nucleoid condensation.  The protein HI probably has effects on gene expression. The amino acid sequence of P has some similarity with the protamine‘s (DNA of certain sperms is bound with protamine‘s). However, the functions of the P protein are not known.

Organization of Prokaryotic Chromosomes

 Chromosomes in bacteria and archaea are usually circular, and a prokaryotic cell typically contains only a single chromosome within the nucleoid. Because the chromosome contains only one copy of each gene, prokaryotes are haploid. As in eukaryotic cells, DNA supercoiling is necessary for the genome to fit within the prokaryotic cell. The DNA in the bacterial chromosome is arranged in several supercoiled domains. As with eukaryotes, topoisomerases are involved in supercoiling DNA. DNA gyrase is a type of topoisomerase, found in bacteria and some archaea, that helps prevent the overwinding of DNA. (Some antibiotics kill bacteria by targeting DNA gyrase.) In addition, histone-like proteins bind DNA and aid in DNA packaging. Other proteins bind to the origin of replication, the location in the chromosome where DNA replication initiates. Because different regions of DNA are packaged differently, some regions of chromosomal DNA are more accessible to enzymes and thus may be used more readily as templates for gene expression. Interestingly, several bacteria, including Helicobacter pylori and Shigella flexneri , have been shown to induce epigenetic changes in their hosts upon infection, leading to chromatin remodeling that may cause long-term effects on host immunity.

respectively, with the pSymA replicon of S. meliloti 2011 (1.35 Mb) and the third replicon of Burkholderia lata 383 (1.4 Mb) being the largest to have been experimentally demonstrated to be nonessential and thus megaplasmids ( 34 , 35 ). It is also interesting to note that the sizes of chromosomes follow a bell-shaped distribution ( Fig. 2A ), whereas the size distributions of plasmids and megaplasmids are instead positively skewed ( Fig. 2B and C ). This is perhaps suggestive of evolutionary forces acting to limit the size of these nonessential replicons.

 RNAP: RNA polymerase H-NS: a small highly expressed chromatin associated DNA binding protein. 

determinations of the average sizes of these domains differ between 10 kb (85) to 100 kb (119), with some studies indicating intermediate values (41, 99). The variance around these average values is very large, with the number of domains in the chromosome of E. coli estimated to vary between 12 and 400. These disparities have been attributed to inaccurate measurements (85), but they could also result from superstructures of domains that would react differently to different challenges. Small ∼10-kb domains may be organized in higher order structures if some barriers between domains are stronger than others or if there are sequence determinants for such superstructures.  Gene distribution and orientation are consistent with a multiscale structure of the bacterial nucleoid imprinted in the form of genome organization. There is also experimental evidence that such superstructures exist. Based on the frequencies of intrachromosomal site specific recombination, it has been proposed that the chromosome of E. coli is organized in four large macrodomains and two largely unstructured regions. 

THE ORGANIZATION OF THE PLANT GENOME

Plant nuclear genomes The plant nuclear genome, consisting of the DNA and associated proteins, is organized into discrete chromosomes. Each unreplicated chromosome and metaphase chromatid consists of a single DNA molecule that is linear and unbroken from one end to the other. At metaphase of mitosis, the DNA is condensed into mitotic chromosomes – short, rod like bodies – while at interphase, the chromosomes are decondensed within the interphase nucleus. The study of the chromosome and its organization involves cytogenetics, and the field of molecular cytogenetics has developed to understand DNA sequence and the molecular structure of the chromosome and chromatin. Both the size of the plant genome and the number of chromosomes vary widely between species. Composition of nuclear DNA The nuclear DNA of plants consists of the single- or low-copy coding sequences, introns, promoters and regulatory DNA sequences, but also of various classes of repetitive DNA motifs that are present in hundreds or even thousands of copies in the genome. Repetitive DNA motifs include characteristic sequences at chromosome centromeres and telomeres, and the rDNA (rRNA genes and intergenic spacers) at the 45S and 5S loci. Tandemly repeated or satellite DNA consists of a motif (as short as two bases, a microsatellite or simple sequence repeat, but sometimes 10 000 bp long) that is repeated in many copies at one or more genomic locations. Satellite DNA in plants typically consists of motifs of about 180 bp, and can be seen either as deep-staining heterochromatin that does not decondense during interphase (blue condensed chromatin) or by in situ hybridization of the sequence after labelling; these satellite sequences are abundant but their function in the genome is not known. Transposable elements are the third class of repetitive DNA sequences; both class I (retrotransposons) and class II (DNA transposons) elements may amplify and the elements and recognizable degraded remnants may represent half or more of the entire DNA present in the genome. Both classes of transposable elements include sequences that encode enzymes related to their own replication and integration into the nuclear DNA. The spherical or ovoidal plant cell DNA resides in a nucleus, occasionally when situation arises develops lobes, which increases its surface area. The majority of higher plant nuclei are 5-