Molecular genetics: Is a branch of genetics that use the methods of genetics and molecular biology to study the structure

and function of genes at the molecular level. Also, it studies how the genes are transferred from generation to generation. Cell biology study structure and functions of whole cell and its organelles Biochemistry- study of all molecules (micro and macro) and their inter-conversions inside the cell (metabolism) Molecular Biology - study structure and functions of macromolecules in the cell Molecular genetics - study of the genes at their molecular levels

Physiology Cell biology Biochemistry Anatomy Molecular Biology Molecular genetics

Clinical Goals of Molecular Genetics Identify sites of genetic variation which cause or contribute to the causes of human disease Use this knowledge to understand the molecular basis of pathology Use this knowledge to provide diagnostic insight and information to patients Use this knowledge to develop treatments and cures for human disease Syllabus : Genotype and phenotype Human Genome organization: Packaging of DNA in Chromatin and Chromosome Gene, the unit of heredity. Central dogma of transferring genetic information DNA structure and functions Gene expression (From gene to protein) Regulation of gene expression Regulation of transcription initiation Post transcriptional regulation Mutation: Types and detection Forward and reverse genetics

Separation and detection of DNA and RNA Recombinant DNA technology, Polymerase chain reaction Molecular cloning, The Human Genome Project Genome analysis Clinical implications Diagnostic molecular genetics Type of genetic diseases Gene therapy gene transfer

Genotype and Phenotype: Genotype (genome) is the genetic contents of an individual. Phenotype is the observable characteristics or traits of an organism: such as its morphology, development, biochemical or physiological properties, result from the expression of an organism genotype. In genetic sense, the genotype determines the nature of phenotype. The genotype is influenced by the environment conditions, therefore Genotype + Environment Phenotype The human genome consists of 46 chromosomes Every human cell (except sex cells and mature red blood cells) contains the complete genome of an organism. Chromosomes are thread-like structures located inside the nucleus of the cell. Each chromosome is made of proteins and a single molecule of DNA Chromosomal DNA in eukaryotic cell is surrounded by proteins which help to pack the large DNA molecule tightly inside the cell, without such packaging, DNA molecules would be too long to fit inside cells. In prokaryotic cell like bacteria the genome is a single DNA molecule which is smaller in size than eukaryotic cells and therefore not surrounded by packing proteins. Most bacteria have one circular chromosome. Humans, along with other animals and plants, have linear chromosomes that are arranged in pairs within the nucleus of the cell. For example all human somatic cells contain 23 pairs of chromosomes (diploid) while sex cells (gametes) like sperms and eggs only have one copy of 23 chromosomes (haploid).

Changes in the number or structure of chromosomes in new cells after cell division may lead to serious problems. For example, in humans, one type of leukemia and some other cancers are caused by defective chromosomes made up of joined pieces from broken chromosomes (translocation due to exchange of chromosome pieces). It is also important that reproductive cells, such as eggs and sperm, contain the right number of chromosomes and that those chromosomes have the correct structure. If not, the resulting off spring may fail to develop properly. For example, people with Down syndrome have three copies of chromosome 21, instead of the two copies found in other people. Besides the linear chromosomes found in the nucleus, the cells of humans and other complex organisms carry a much smaller type of chromosome similar to those seen in bacteria. This circular chromosome is found in mitochondria, which are structures located outside the nucleus and serve as the cell powerhouses. In humans and most other complex organisms, one copy of each chromosome is inherited from the female parent and the other from the male parent. This explains why children inherit some of their traits from their mother and others from their father. The pattern of inheritance is different for the small circular chromosome found in mitochondria. Only egg cells and not sperm cells keep their mitochondria during fertilization. So, mitochondrial DNA is always inherited from the female parent. In humans, a few conditions, including some forms of hearing impairment and diabetes, have been associated with DNA found in the mitochondria. Each linear chromosome has a constricted region called the centromere. It is usually not located exactly in the center of the chromosome and, in some cases, is located almost at the chromosome's end. The regions on either side of the centromere are referred to as the chromosome's arms. Centromeres help to keep chromosomes properly aligned during the complex process of cell division. As chromosomes are copied in preparation for production of a new cell, the centromere serves as an attachment site for the two halves of each replicated chromosome, known as sister chromatids. Telomeres are repetitive stretches of DNA located at the ends of linear chromosomes and have protection function. In many types of cells, telomeres lose a bit of their DNA every time a cell divides. Eventually, when all of the telomere DNA is gone, the cell cannot replicate and dies. White blood cells and other cell types with the capacity to divide very frequently have a special enzyme that prevents their chromosomes from losing their telomeres. Because they retain their telomeres, such cells generally live longer than other cells. Telomeres also play a role in cancer. The chromosomes of malignant cells usually do not lose their telomeres, helping to fuel the uncontrolled growth that makes cancer so severe.

Male and females differ in a pair of chromosomes known as the sex chromosomes. The Female has two X chromosomes in their cells, while male has one X and one Y chromosome. Inheriting too many or not enough copies of sex chromosomes can lead to serious problems. For example, females who have extra copies of the X chromosome are usually taller than average and some have mental retardation. Males with more than one X chromosome have Klinefelter syndrome, which is a condition characterized by tall stature and, often, impaired fertility. Another syndrome caused by imbalance in the number of sex chromosomes is Turner syndrome. Women with Turner have one X chromosome only. They are very short, usually do not undergo puberty and some may have kidney or heart problems. Packaging of DNA in Chromatin and Chromosome: The human genome contains about 3 billion nucleotide pairs organized as 23 chromosomes pairs, with each base pair is equal 0.34 nanometers long .If uncoiled, the DNA content of each chromosome would measure almost one meter long. This DNA must be packaged into a cell that is only micrometers in size . Moreover, such long DNA would be subjected to break down when the DNA is replicated before the cell division is carried out to produce two daughter cells. . This is why the chromosomal DNA is wrapped into a compact structure with the help of specialized proteins called histones. The complex DNA plus histones in eukaryotic cells is called a chromatin which is arranged like beads on a string. The basic packing unit is known as a nucleosome. Each nucleosome is about 11nm in diameter. A short length of DNA (core DNA ) is twisted around a central core of eight histones molecules (an octamer) to form a single nucleosome. A second histone (H1) called the linker DNA is 20-60 base

pairs acts to connect two neighboring nucleosomes together. Histones are small, positively charged proteins which facilitates their interaction with the negative charge of the DNA backbone. Nucleosomes are usually packed together, with the aid of a histone (H1,) to form a 30nm large fiber. As a 30nm fiber, the typical human chromosome would be about 0.1cm in length and would stretch the nucleus 100 times. This suggests higher orders of packaging, to give a chromosome the compact structure seen in a typical metaphase cell.

DNA packing in eukaryotes

Evidence that DNA is the Genetic Material The key experiments that lead to the conclusion that DNA is the Genetic Material. 1. Griffiths -1928 - Heat killed cells can transform bacteria. Genes are made of DNA? 2. Avery, MacLeod, & McCarty - 1944 - DNA transforms bacteria 3. Hershey & Chase - 1952 - DNA (not protein) is the genetic material 4. The structure of DNA is a double helix, which is compatible with its role as the genetic material. Frederick Griffith in 1928. "Basic Research" with bacterium Streptococcus pneumonia (diplococcus), which causes pneumonia in humans. 2 strains: smooth (S) and rough (R). a) S - smooth colonies - virulent (causes pneumonia) injection kills mice b) R - rough colonies - non-virulent (don't cause pneumonia) mouse lives c) Heat killed S-strain cells injected into mouse mouse lives \ no cause of death. d) Heat killed S-strain cells + R-strain live cells -> dead mouse. (Whereas control mice R-strain lived) Further Griffith"s found that:: - live cells could be recovered from these dead mice -> had - S-smooth colonies - virulent upon subsequent injections Conclution: something in the S-cell debris could convert (transform) R S form 7

in a process called transformation. -"

Transforming Principle: The presence of heat-killed S cells transforms live R cells into live S cells. Notes: First, they did not need mice as the R and S phenotype was enough. Second, the same type of transformation could be done in the lab, but using a plasmid containing an antibiotic resistance marker. Avery, MacLeod & McCarty 1944 Purified cell molecules and test each for ability to transform - chemical composition of the purified agreed with a high molecular weight molecule(500,000) like that of DNA - protease treatment activity intact - treatment with ribonucleaseactivity intact - treatment with deoxyribonuclease activity lost Transforming substance must be DNA But at the time there was reluctance to accept DNA as the genetic material Because it is less complex than Proteins..

DNA is the agent transforming the R strain into virulence

Experiment of Hershey & Chase 1952 Bacteriophage (bacterial virus) T2 - very simple Protein coat outside - DNA inside Therefore the genetic material is either protein or DNA Hershey & Chase used different radioisotopes to follow each of these components during the process of bacterial infection and replication of the Phage. Radioisotopes: 32P to follow DNA; P not found in protein 35S labels protein; S not found in DNA but incorporated radioisotopes was found with the phage in separate cultures - infected E.coli with many virus per cell - after sufficient time separated phage ghosts & cells Result 9

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S protein -> ghosts - protein never entered the cell 32P DNA did enter the cells Furthermore 32P could be recovered from the phage progeny ---> For bacteriophage T2 the genetic information is in DNA not protein Conclusion: 1- DNA is the hereditary material 2- But this was still not immediately accepted as proof that genes were DNA.

The Hershey-Chase experiment DNA has a double-helix structure Suggested by J. Watson & F. Crick 1953 which is in favor of its role as the genetic material: 1.Ability for self replication through semi-conservative mechanism 2.Carrying heredity information in the form of variable nucleotides sequence 3,Subjected to alterations (mutation). 4.The potential for transferring genetic information to other molecules.

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